CN103756965B - A kind of method of lavation hemopoietic stem cell from placenta - Google Patents
A kind of method of lavation hemopoietic stem cell from placenta Download PDFInfo
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Abstract
The invention provides a kind of method of lavation hemopoietic stem cell from placenta, described method comprises the collection of placenta, the initial survey of placenta, the pre-sterilization of placenta, placenta and female blood examination survey, the lavation of placental hematopoietic stem cell, concentrated and purified six steps of hemopoietic stem cell, the equipment of described method and use is simple, easy to operate, wash-out hematopoietic stem cell injuries is out little, quantity is many, concentration is high, active strong, the transplanting of the hemopoietic stem cell of autologous or allosome can be applied to, and contribute to the reparation of damaged tissue.
Description
Technical field
The present invention relates to a kind of method of lavation hemopoietic stem cell from placenta.
Background technology
The method of the hematopoietic stem cell transplantation mainly taked in the treatment of international disease in the blood system at present, according to the source of cell, can be divided three classes: marrow hemopoietic stem cells is transplanted (BMT), mobilized Peripheral blood stem cell transplantation (MPST) and cord blood stem cell to transplant (UCBT).The above two source of human stem cell enrich, and general number of nucleated cells can reach 5-10 × 10
8/ Kg, CD34+ cell (a kind of surface markers of hematopoietic stem/progenitor) can reach 1-5 × 10
6/ Kg, but owing to needing HLA Strict Compliance between donor and acceptor, the success that guarantee is transplanted, otherwise donor hemopoietic stem cell is not easily lived in patient body's interplantation, even if survive, more serious graft versus host disease (GVH disease) (GVHD) also can occur.In the children of one family, only have the probability that 1/4HLA conforms to, and in the unrelated donor of non-blood relationship, this probability only has one of percentage, significantly limit BMT or MPST widespread use clinically.
Umbilical cord blood stem cell transplantation successfully applies the development facilitating umbilical cord blood hematopoietic stem cell research clinically in the recent decade.Cord blood comes from placenta, usually discarded after Delivery, find now that concentration and the marrow of its CD34+ cell are similar containing abundant hemopoietic stem cell in Cord blood, account for the 0.1-0.5% of total cell count, and more early stage hemopoietic stem cell CD 34-also comparatively marrow concentration is higher.As a kind of source of hematopoietic cell, utilizing now Umbilical Cord Blood Transplantation to perform the operation increases gradually.Compared with BMT, the advantage of UCBT resides in reduced serious graft-vs-host reaction, effectively expands the possibility those being lacked to suitable human leukocyte antigen matching family or irrelevant donor transplanting.The hemopoietic stem cell limited amount that what Umbilical Cord Blood Transplantation was main be limited in bleeding of the umbilicus.This restriction impels larger the growing up of clinical middle use 2 multiple dose unit umbilical cord blood transplantation to be help others, another kind method carries out the cultivation of Hematopoiesis in Vitro expansion of stem cells, but amplification in vitro needs spended time, expense is also high, while the more important thing is amplification, hemopoietic stem cell also breaks up, and the result of clinical application shows before and after cord blood cells amplification transplanting without much difference.
There is a large amount of hemopoietic stem cells in the mature placenta of the mankind, has more hemopoietic stem cell than Cord blood, and these placental hematopoietic stem cell can be separated before and after refrigerated storage.The activity of placental hematopoietic stem cell colony-forming unit (CFU) is determined, the transplantation experiments in Immune deficient mice has proved the potentiality of placenta cells in transplanting.These results effectively show, the mature placenta of the mankind likely becomes a kind of source of the novel hemopoietic stem cell for transplanting.And there is a large amount of hemopoietic stem cells in placenta, placental blood stem cell is comparatively early stage stem cell, various cell can be divided in vivo, containing abundant early stage hemopoietic stem cell of various stages in placental blood, its content is approximately tens times of Cord blood, hemopoietic stem cell in a placenta can be satisfied with two adult Man's Demands completely, if patient can be applied to together with cord blood cell, considerably increase the content of hemopoietic stem cell undoubtedly, make this hemopoietic stem cell can be applied to all applicable crowds completely.
Patent CN01131190.8 discloses a kind of method that method by shredding digestion obtains hemopoietic stem cell, and method is complicated, and the hemopoietic stem cell quantity of acquisition is few, purity is low, and the easy microbiological contamination of cell.
Summary of the invention
Technical problem to be solved by this invention there is provided a kind of method of lavation hemopoietic stem cell from the mature placenta of healthy puerpera.
Described method comprises the collection of placenta, the initial survey of placenta, the pre-sterilization of placenta, placenta and female blood examination survey, the lavation of placental hematopoietic stem cell, concentrated and purified six steps of hemopoietic stem cell, and each step is specially:
(1) collection of placenta: collect placenta and female blood under Operation theatre gnotobasis, deposit in aseptic placenta accumulating box, post label, bar code, the temperature of placenta accumulating box remains on 4-10 DEG C, sends to laboratory in 24 hours;
(2) initial survey of placenta: check in placenta accumulating box box, whether temperature, label, the date of service, accumulating box have seepage, whether have female blood blood sample;
(3) pre-sterilization of placenta: the fetus face of placenta is launched be positioned over the bottom that placenta rinses box, with normal saline flushing placenta surface, open the water vent that placenta rinses box bottom surface, remove the physiological saline of flushing, check placenta Surface calcification situation;
(4) placenta and female blood examination are surveyed: detect got placenta and female blood sample, and the project of detection comprises hepatitis virus detection, virus of AIDS detects, venereal disease detects, tissue matching (HLA) is detected, hematopoietic stem/progenitor qualitative detection (CFU-GM);
(5) lavation of placental hematopoietic stem cell: placenta is aseptically washed blood clot on placenta and hematocele by stroke-physiological saline solution, after adopting sterilizing agent sterilization, perfusion liquid syringe needle is inserted in placenta Umbilical artery, placental perfusion returnable bottle syringe needle inserts in umbilical vein, mosquito forceps clamps, slow unlatching constant flow pump, perfusion liquid, through sebific duct, switch, peristaltic pump, syringe needle, placental artery, placenta, placental vein, placental perfusion returnable bottle syringe needle, finally receives irrigating solution in irrigating solution returnable bottle;
(6) hemopoietic stem cell is concentrated and purified: by lavation irrigating solution out, in centrifuges with the centrifugal 15-20 minute of 1500-2000rpm, remove supernatant liquor, the liquid of collecting precipitation and lower floor, the liquid of the precipitation collected and lower floor and physiological saline are mixed to obtain mixed solution in the ratio of 2:1-1:2, then mixed solution and lymphocyte separation medium are joined in centrifuge tube respectively in the ratio of 2:1-1:2, the order of adding for first adding lymphocyte separation medium in centrifuge tube, slowly add mixed solution again, note keeping the liquid level of lymphocyte separation medium in adition process smooth, with the centrifugal 20-25 minute of 2200-2500rpm after adding, slowly slow deceleration is accelerated time centrifugal, after centrifugal end, in the middle of collecting, tunica albuginea layer is in new centrifuge tube, physiological saline mixing is added with the ratio of 2:1-1:2, the centrifugal 10-15 minute of 1200-1500rpm, supernatant liquor is removed after centrifugal end, add 10-20mL physiological saline piping and druming precipitation, the centrifugal 10-15 minute of 1200-1500rpm, after centrifugal end, removes supernatant liquor again, by the resuspended precipitation of DMEM substratum, collect hemopoietic stem cell group, resuspended cell mass is carried out cell and mycotic culture, hemopoietic stem cell detection by quantitative (CD34), hemopoietic stem cell Activity determination (Trypan Blue), hemopoietic stem cell qualitative detection (CFU-GM).
The lavation step of the hemopoietic stem cell described in aforesaid method is:
The irrigating solution used when starting lavation adds 10%FBS50mL, nitroglycerin injection (1mL:5mg) 2, heparin sodium injection (2mL:12500 unit) 2 in every 500mLDMEM substratum, the irrigating solution that mixing is obtained, with the speed lavation 2 hours of drip irrigation washing lotion p.s. 3, lavation 1000mL irrigating solution; Close constant flow pump, irrigating solution is changed in every 500mLDMEM substratum and add 10%FBS50mL, nitroglycerin injection (1mL:5mg) 2, heparin sodium injection (2mL:12500 unit) 2, AMD3100(5mg/ bottle) irrigating solution of 0.5 bottle, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s. 3, lavation 1500mL irrigating solution; Preparation lavation DMEM substratum used, 10% FBS use before be first positioned in the water-bath of 37 DEG C, be preheating to 37 DEG C in advance.The lavation step of the hemopoietic stem cell described in aforesaid method can also be:
The irrigating solution used when starting lavation adds 10%FBS50mL, nitroglycerin injection (1mL:5mg) 2, heparin sodium injection (2mL:12500 unit) 2 in every 500mLDMEM substratum, the irrigating solution that mixing is obtained, with the speed lavation 2 hours of drip irrigation washing lotion p.s. 3, lavation 1000mL irrigating solution; Close constant flow pump, irrigating solution is changed into the DMEM substratum that 50mL is 100mg/L containing the concentration of AMD3100, with the speed lavation 5 minutes of drip irrigation washing lotion p.s. 10, lavation, after lavation is complete; Close constant flow pump, after 30 minutes, irrigating solution is changed in every 500mLDMEM substratum and add 10%FBS50mL, nitroglycerin injection (1mL:5mg) 2, heparin sodium injection (2mL:12500 unit) 2, AMD3100(5mg/ bottle) irrigating solution of 0.5 bottle, open constant flow pump, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s. 3, lavation 1500mL irrigating solution, reclaims irrigating solution.
Hemopoietic stem cell described in aforesaid method is concentrated and purified can also be: by lavation irrigating solution out, centrifugal, centrifugal condition is: rotating speed 1500rpm, pick-up period 45s, deceleration time 45s, centrifugation time 20min, after centrifugal end, returnable bottle is transferred in aseptic operating platform, liquid-transfering gun siphons away supernatant liquid, hemocyte is stayed to precipitate, the physiological saline of 10-20mL is added in hemocyte precipitation, blow and beat precipitation gently, get 50mL centrifuge tube, often pipe adds the lymphocyte separation medium of 20mL, tilt containing the centrifuge tube of lymphocyte separation medium, slowly join on lymphocyte separation medium along centrifuge tube tube wall with the hemocyte that liquid-transfering gun absorption piping and druming is got up, the process added is wanted slowly, keep lymphocyte separation medium liquid level smooth, often pipe adds hemocyte 25mL, move after liquid completes, centrifugal, centrifugal condition: rotating speed 2200rpm, pick-up period 600s, deceleration time 600s, centrifugation time 20min, after centrifugal end, solution can divide four layers, with tunica albuginea layer in the middle of pipette, extract, often pipe 20mL transfers in new 50mL centrifuge tube, and then often pipe adds the physiological saline of 20mL, after mixing, centrifugal, centrifugal condition: rotating speed 1500rpm, pick-up period 45s, deceleration time 45s, centrifugation time 15min, after centrifugal end, transfer in aseptic operating platform, remove supernatant liquor, often pipe add 20mL physiological saline piping and druming precipitation, then centrifugal, centrifugal condition: rotating speed 1500rpm, pick-up period 45s, deceleration time 45s, centrifugation time 15min, after centrifugal end, remove supernatant liquor, with the DMEM substratum re-suspended cell of the 10%FBS of 20mL, collect hemopoietic stem cell group, cell mass is carried out cell and mycotic culture, hemopoietic stem cell detection by quantitative (CD34), hemopoietic stem cell Activity determination (Trypan Blue), hemopoietic stem cell qualitative detection (CFU-GM).
In hematopoietic stem cell transplantation, the content of placental hematopoietic stem cell is very important, and the placental hematopoietic stem cell adopting present method to extract, after lymphocyte separation medium is separated, uses cell counter counting, has 8 × 10
7-1.5 × 10
8mononuclearcell number, although individual difference is comparatively large, also can meet the sick human needs of all transplanting.Wherein about have 1.5-3.6% to be CD34 positive cell, external granulocyte colony cultivates (CFU-GM), the cell mass that visible colonies is huge.
The placental hematopoietic stem cell obtained by method lavation of the present invention can be applied to various malignant hematologic disease, as leukemia; Heredopathia, as congenital hemolytic anemia; Blood regeneration is benign disease not, as aplastic anemia; Radiation disease, as radioactive rays surprisingly etc.Methods for the treatment of adopts the various means such as chemotherapy or radiotherapy to destroy the ill medullary cell of patient self and immunity system, then healthy hemopoietic stem cell is inputted, the reconstruction of chemotherapy or the postoperative hematopoietic cell of radiotherapy can well be used for from the hemopoietic stem cell that placenta lavation obtains by the inventive method, graft-vs-host reaction is little, greatly improves surviving rate.
The application approach of placenta stem-cell prepared by the present invention can be intravenous injection, intra-arterial injection, also can local injection, generally inputs to patient with vein, does not get rid of the application approach adopting other modes.
Reagent used by the present invention or Pharmaceutical Compositions, as physiological saline, DMEM substratum, nitroglycerin injection, foetal calf serum (FBS), heparin sodium injection liquid and AMD3100 etc., be all aseptic, apyrogenic, be all applicable to the patient of clinical transplantation.
All operations step of the present invention, it is all the conventional practices that pharmacopeia allows, comprise and adopt aseptic technique, stroke-physiological saline solution washing, sterilizing, steril cell to concentrate and purifying etc., the reagent applied or Pharmaceutical Compositions, if physiological saline, DMEM substratum, nitroglycerin injection, foetal calf serum, heparin sodium injection liquid and AMD3100 etc. are all the requirements meeting health organ of China, do conventional viral, cytoscopy and bacterium, mycotic culture after end of operation, the placental hematopoietic stem cell only meeted the requirements just can be applied to clinical.
The advantage of the present invention preparation method required for protection:
(1) equipment of the inventive method and use is simple, and easy to operate, efficiency is high, greatly shorten from the cycle being prepared into application, and placenta abundance, donor no pain;
(2) the inventive method lavation hematopoietic stem cell injuries out from placenta is little, and quantity is many, and purity is high, active high, the transplanting of the hemopoietic stem cell of autologous or allosome can be applied to, and contribute to the reparation of damaged tissue, substantially increase the surviving rate of patient;
(3) irrigation system of the inventive method is closed system, and the external world does not contact with air, avoids microbiological contamination;
(4) the inventive method can keep the integrity of placenta, conveniently from placenta, extracts other cells;
(5) reagent of the inventive method use is few, effectively economizes on resources and cost.
Accompanying drawing explanation
Fig. 1: the flow cytomery result of the hemopoietic stem cell that the lavation from placenta of embodiment one method obtains.
Fig. 2: Colony cultivation (CFU-GM) the culture experiment result of the hemopoietic stem cell that lavation obtains from placenta.
Fig. 3: be separated hemopoietic stem cell Activity determination (Trypan Blue) detected result obtained from placenta.
Fig. 4: the flow cytomery result of the hemopoietic stem cell that the lavation from placenta of embodiment two method obtains.
In figure, Fig. 1 refers to: the flow cytomery result of the hemopoietic stem cell that the lavation from placenta of embodiment one method obtains, visible cell a considerable number of, wherein the ratio of CD34 positive cell is 1.65%, the a considerable number of of the hemopoietic stem cell that placenta lavation obtains is described, ratio is very high, very high to clinical using value.
Fig. 2 refers to: cultivated by the placental hematopoietic stem cell methylcellulose gum semisolid medium obtained, after cultivating two time-of-weeks, can observe and have obvious colony in methylcellulose gum semisolid medium, the activity of this hemopoietic stem cell obtained and multiplication capacity are all higher.
Fig. 3 refers to: placental hematopoietic stem cell is carried out Activity determination, does not observe hemopoietic stem cell and is colored, and illustrate that the activity of hemopoietic stem cell is better, and the form of cell is fine.
Fig. 4 refers to: the flow cytomery result of the hemopoietic stem cell that the lavation from placenta of embodiment two method obtains, visible cell a considerable number of, wherein the ratio of CD34 positive cell is 3.2%, the a considerable number of of the hemopoietic stem cell that placenta lavation obtains is described, ratio is very high, very high to clinical using value.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, but not as a limitation of the invention.
The technical scheme that the present invention relates to is a kind of method providing lavation hemopoietic stem cell from placenta.
The method of lavation hemopoietic stem cell from placenta of the present invention comprises the step of the collection of placenta, the initial survey of placenta, the pre-sterilization of placenta, placenta and female blood examination survey, the lavation of placental hematopoietic stem cell, the concentrated and purified of hemopoietic stem cell, and it is respectively:
(1) collection of placenta: collect placenta and female blood under Operation theatre gnotobasis, deposit in aseptic placenta accumulating box, post label, bar code, the temperature of placenta accumulating box remains on 4-10 DEG C, sends to laboratory in 24 hours;
(2) initial survey of placenta: open placenta accumulating box, checks thermometer scales in box, requires that in box, temperature remains between 4-10 DEG C; Label checks, placenta accumulating box is labelled should be complete, clear, and the date of service is before the placenta accumulating box Expiration Date, otherwise be considered as defective, will not receive; Check whether placenta accumulating box has seepage, if any seepage, will not receive; Check whether female blood blood sample, and indicated complete, if without mother's blood sample, will not receive;
(3) pre-sterilization of placenta: careful for placenta is taken out by tweezers entirety, being positioned over the placenta of having disinfected in advance gently rinses in box, launch placenta, fetus face is launched be positioned over the bottom that placenta rinses box, with normal saline flushing placenta surface, open the water vent that placenta rinses box bottom surface, remove the physiological saline of flushing, check placenta Surface calcification situation, placental calcification is caused by the focal infarct of pregnant woman's placentogenesis in late period, infarct is more, occur that calcification point is more, the comparatively high light spot showed under B ultrasonic is more, three degree can be divided into according to the placental calcification spot large young pathbreaker's calcification degree that distributes when B ultrasonic detects, i.e. I degree, II degree, III degree, the calcification situation diagnosed under B ultrasonic not necessarily conforms to actual, and make a definite diagnosis and must conclude according to postpartum examination placental calcification area, according to placenta Surface calcification situation determination calcification degree I degree, II degree, III degree, keeping records,
(4) placenta and female blood examination are surveyed: detect got placenta and female blood sample, and the project of detection comprises hepatitis virus detection, virus of AIDS detects, venereal disease detects, tissue matching (HLA) is detected, hematopoietic stem/progenitor qualitative detection (CFU-GM);
(5) lavation of placental hematopoietic stem cell: placenta is aseptically cleaned by stroke-physiological saline solution, wash the blood clot on placenta and hematocele, after adopting sterilizing agent sterilization, by the current face of placenta, fetus face and umbilical cord cross section mug, then perfusion liquid syringe needle is inserted in placenta Umbilical artery, two Umbilical artery are had in umbilical cord, only insert wherein one, mosquito forceps steps up the syringe needle inserting Umbilical artery, due to the existence of venous sinus, irrigating solution can not flow out from another Umbilical artery, placental perfusion returnable bottle syringe needle inserts in umbilical vein, the syringe needle of umbilical vein is inserted in mosquito forceps clamping, irrigating solution is placed on 32 DEG C of constant temperature electric heating sheets, the box depositing placenta is positioned in the constant temperature electric heating sheet of 32 DEG C, after temperature-stable, the switch of slow unlatching constant flow pump (will open constant flow pump before intubate, ducted air is discharged, make to be full of irrigating solution in sebific duct), by speed setting to middling speed (speed with drip irrigation washing lotion p.s. 3), guarantee to rinse path smooth, rinse the take-off rate that for some time started will note receiving end irrigating solution the moment, if stop suddenly the speed flowing out or flow out obviously to decline, close constant flow pump, open vein stanch pincers, take out returnable bottle syringe needle, look into and see if there is sludged blood blocking syringe needle, and then pin, clamping mosquito forceps, open constant flow pump, also will note the gathering speed of liquid in returnable bottle in the process of rinsing the moment, perfusion liquid, through sebific duct, switch, peristaltic pump, syringe needle, placental artery, placenta, placental vein, placental perfusion returnable bottle syringe needle, finally receives irrigating solution in irrigating solution returnable bottle,
(6) hemopoietic stem cell is concentrated and purified: by lavation irrigating solution out, with 1500rpm centrifugal 20 minutes in centrifuges, remove supernatant liquor, the liquid of collecting precipitation and lower floor, by the liquid of the precipitation collected and lower floor and physiological saline in 1: 1 ratio mix to obtain mixed solution, then by mixed solution and lymphocyte separation medium in 1: 1 ratio join respectively in centrifuge tube, the order of adding for first adding lymphocyte separation medium in centrifuge tube, slowly add mixed solution again, note keeping the liquid level of lymphocyte separation medium in adition process smooth, the heart is left 20 minutes with 2200 after adding, slowly slow deceleration is accelerated time centrifugal, after centrifugal end, centrifuge tube from top to bottom solution is divided into four layers, the first layer is plasma layer, the second layer is ring-type oyster white buffy coat, third layer is transparent parting liquid, 4th layer is red blood cell layer.Collect second layer tunica albuginea layer in new centrifuge tube, add the mixing of 10-20mL physiological saline, 1500 leave the heart 15 minutes; Supernatant liquor is removed after centrifugal end, physiological saline piping and druming precipitation, 1500 leave the heart 15 minutes again, after centrifugal end, remove supernatant liquor, by the resuspended precipitation of DMEM substratum, collect hemopoietic stem cell group, resuspended cell mass is carried out cell and mycotic culture, hemopoietic stem cell detection by quantitative (CD34), hemopoietic stem cell Activity determination (Trypan Blue), hemopoietic stem cell qualitative detection (CFU-GM).
The method of embodiment one lavation hemopoietic stem cell from placenta
Placenta comes from local obstetrics and gynecology hospital.First, obtain after pregnant woman or its family members agree to contribute placenta, consult all audit reports of pregnant woman, confirm, without any virus, the syphilis virus infection relevant with blood, then to inquire family's medical history of pregnant woman, infection and infection medical history.After determining that all normally, gather female blood 5mL, placenta splendid attire together with female blood is entered placenta accumulating box, posts label, bar code.In box in placenta storage and transport process, temperature remains on 4-10 DEG C, in 24 hours, be transported to laboratory.
Behind laboratory, bar code and label are all inputted in computer, careful for placenta is taken out by tweezers entirety, being positioned over the placenta of having disinfected in advance gently rinses in box, launch placenta, fetus face is launched be positioned over the bottom that placenta rinses box, with normal saline flushing placenta surface, open the water vent that placenta rinses box bottom surface, remove the physiological saline of flushing.Check placenta Surface calcification situation.Detect got female blood and placental samples, the project of detection comprises hepatitis virus detection, virus of AIDS detection, venereal disease detection, tissue matching (HLA) detection, hematopoietic stem/progenitor qualitative detection (CFU-GM).
By placenta aseptically with stroke-physiological saline solution cleaning, wash the blood clot on placenta and hematocele, after adopting thimerosal sterilization, by the current face of placenta, fetus face and umbilical cord cross section mug.Then perfusion liquid syringe needle is inserted in placenta Umbilical artery, two Umbilical artery are had in umbilical cord, only insert wherein one, mosquito forceps steps up, due to the existence of venous sinus, irrigating solution can not flow out from a wherein artery, placental perfusion returnable bottle syringe needle inserts in umbilical vein, mosquito forceps clamps, open perfusion system, the irrigating solution used when starting lavation adds 10%FBS50mL in every 500mLDMEM substratum, nitroglycerin injection (1mL:5mg) 2, heparin sodium injection (2mL:12500 unit) 2, the irrigating solution that mixing is obtained, with the speed lavation 2 hours of drip irrigation washing lotion p.s. 3, lavation 1000mL irrigating solution, close constant flow pump, irrigating solution is changed in every 500mLDMEM substratum and add 10%FBS50mL, nitroglycerin injection (1mL:5mg) 2, heparin sodium injection (2mL:12500 unit) 2, AMD3100(A5602,5mg/ bottle) irrigating solution of 0.5 bottle, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s. 3, lavation 1500mL irrigating solution, at receiving end, receives irrigating solution.
By the irrigating solution be recovered in 250mL returnable bottle, trim, centrifugal, centrifugal condition is: rotating speed 1500rpm, pick-up period 45s, deceleration time 45s, centrifugation time 20min; After centrifugal end, transferred to by returnable bottle in aseptic operating platform, liquid-transfering gun siphons away supernatant liquid and pours in waste liquid cylinder, stays hemocyte to precipitate, and adds the physiological saline of 20mL, blown and beaten precipitation gently according to hemocyte precipitation.Get 50mL centrifuge tube, often pipe adds the lymphocyte separation medium of 20mL, tilt containing the centrifuge tube of lymphocyte separation medium, draw with liquid-transfering gun and blow and beat the hemocyte of getting up, slowly join on lymphocyte separation medium along centrifuge tube tube wall, the process added must blunting of attention, and keep lymphocyte separation medium liquid level smooth, often pipe adds hemocyte 25mL; Move after liquid completes, centrifugal, centrifugal condition: rotating speed 2200rpm, pick-up period 600s, deceleration time 600s, centrifugation time 20min; After centrifugal end, solution can divide four layers, with tunica albuginea layer in the middle of pipette, extract, often pipe 20mL transfers in new 50mL centrifuge tube, and then often pipe adds the physiological saline of 20mL, after mixing, centrifugal, centrifugal condition: rotating speed 1500rpm, pick-up period 45s, deceleration time 45s, centrifugation time 15min; After centrifugal end, transfer in aseptic operating platform, remove supernatant liquor, often pipe adds the physiological saline of 20mL, piping and druming precipitation, then centrifugal, centrifugal condition: rotating speed 1500rpm, pick-up period 45s, deceleration time 45s, centrifugation time 15min; After centrifugal end, remove supernatant, the DMEM substratum of the 10%FBS of 20mL is joined and removes in the centrifuge tube of supernatant liquor, slowly shake centrifuge tube, precipitation is is all blown and beaten, form solution.Then from wherein getting 20ul to 1.5mL centrifuge tube, two pipes are got, for cell counting and Activity determination (Trypan Blue); And then from wherein getting the centrifuge tube of 200ul to 1.5mL respectively, three pipes, the first pipe adds the physiological saline of 1ul, second pipe adds the Isotype control PE of CD34,3rd pipe adds CD34 antibody, hatches 15 minutes, then flow cytomery by being placed on dark place after three pipe mixings.
Cell counting, cytoactive detection, flow cytomery and Colony cultivation experiment are carried out to the hemopoietic stem cell obtained.By the enchylema of a pipe 20ul on cell counter, counting cells number 1.0 × 10
8individual mononuclearcell; By the enchylema of another pipe 20ul, add the trypan blue dye liquor of 20ul concentration 0.08%, mixing, then get 4ul on slide glass, examine under a microscope, do not observe cell and be colored, explanation cytoactive is better, and the form of cell very well (see Figure of description 3); Cell quantification detect (CD34) three pipe 200ul are hatched after enchylema detect to flow cytometer, by detecting, the ratio of CD34 positive cell reaches 1.65%, this time detect CD34 positive cell and add negative control, data are more conservative, obtain in normal experiment the most high energy of ratio of CD34 positive cell reach 3.6%, this absolutely proves that the ratio of the hemopoietic stem cell that placenta lavation obtains is very high, a considerable number of, clinical using value very high (the results are shown in Figure of description 1); Methylcellulose gum semisolid medium cultivation (CFU-GM) is carried out to the hemopoietic stem cell obtained, get Tissue Culture Plate hole, methylcellulose gum semisolid medium 3mL is added in hole, then get the enchylema of 1ul, be added in methylcellulose gum semisolid medium, and separately get a hole and add 2mL distilled water, add well, build culture plate lid, be placed in carbon dioxide cell incubator, the condition 7 DEG C of incubator, 5%CO
2, humidity 90% cultivates 14 days, observes complete cell colony, this hemopoietic stem cell obtained there is multiplication capacity (the results are shown in Figure of description 2).
The method of embodiment two lavation hemopoietic stem cell from placenta
Placenta comes from local obstetrics and gynecology hospital.First, obtain after pregnant woman or its family members agree to contribute placenta, consult all audit reports of pregnant woman, confirm, without any virus, the syphilis virus infection relevant with blood, then to inquire family's medical history of pregnant woman, infection and infection medical history.After determining that all normally, gather female blood 5mL, placenta splendid attire together with female blood is entered placenta accumulating box, posts label, bar code.In box in placenta storage and transport process, temperature remains on 4-10 DEG C, in 24 hours, be transported to laboratory.
The method of reference example one is carried out the initial survey of placenta, the pre-sterilization of placenta, placenta and female blood examination and is surveyed,
The lavaging method of placental hematopoietic stem cell is changed into: the irrigating solution used when starting lavation adds 10%FBS50mL, nitroglycerin injection (1mL:5mg) 2, heparin sodium injection (2mL:12500 unit) 2 in every 500mLDMEM substratum, the irrigating solution that mixing is obtained, with the speed lavation 2 hours of drip irrigation washing lotion p.s. 3, lavation 1000mL irrigating solution; Close constant flow pump, irrigating solution is changed into the DMEM substratum that 50mL is 100mg/L containing the concentration of AMD3100, with the speed lavation 5 minutes of drip irrigation washing lotion p.s. 10, lavation, after lavation is complete; Close constant flow pump, after 30 minutes, irrigating solution is changed in every 500mLDMEM substratum and add 10%FBS50mL, nitroglycerin injection (1mL:5mg) 2, heparin sodium injection (2mL:12500 unit) 2, AMD3100(5mg/ bottle) irrigating solution of 0.5 bottle, open constant flow pump, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s. 3, lavation 1500mL irrigating solution, at receiving end, receive irrigating solution.
The method of reference example one is carried out concentrated and purified to hemopoietic stem cell, after processing sample, flow cytomery (the results are shown in accompanying drawing 4) is carried out to the hemopoietic stem cell obtained, the ratio of CD34 positive cell reaches 3.2%, illustrate that the ratio of the hemopoietic stem cell that placenta lavation obtains is very high, a considerable number of.
Claims (3)
1. the method for a lavation hemopoietic stem cell from placenta, it is characterized in that, described method comprises the step of the collection of placenta, the initial survey of placenta, the pre-sterilization of placenta, placenta and female blood examination survey, the lavation of placental hematopoietic stem cell, the concentrated and purified of hemopoietic stem cell, and it is respectively:
(1) collection of placenta: collect placenta and female blood under Operation theatre gnotobasis, deposit in aseptic placenta accumulating box, post label, bar code, the temperature of placenta accumulating box remains on 4-10 DEG C, sends to laboratory in 24 hours;
(2) initial survey of placenta: check in placenta accumulating box box, whether temperature, label, the date of service, accumulating box have seepage, whether have female blood blood sample;
(3) pre-sterilization of placenta: the fetus face of placenta is launched be positioned over the bottom that placenta rinses box, with normal saline flushing placenta surface, open the water vent that placenta rinses box bottom surface, remove the physiological saline of flushing, check placenta Surface calcification situation;
(4) placenta and female blood examination are surveyed: detect got placenta and female blood sample, and the project of detection comprises hepatitis virus detection, virus of AIDS detects, venereal disease detects, HLA tissue matching is detected, CFU-GM hematopoietic stem/progenitor qualitative detection;
(5) lavation of placental hematopoietic stem cell: placenta is aseptically washed blood clot on placenta and hematocele by stroke-physiological saline solution, after adopting sterilizing agent sterilization, perfusion liquid syringe needle is inserted in placenta Umbilical artery, placental perfusion returnable bottle syringe needle inserts in umbilical vein, mosquito forceps clamps, slow unlatching constant flow pump, perfusion liquid is through sebific duct, switch, peristaltic pump, syringe needle, placental artery, placenta, placental vein, placental perfusion returnable bottle syringe needle, finally in irrigating solution returnable bottle, receive irrigating solution, the irrigating solution used when starting lavation adds 10%FBS50mL in every 500mLDMEM substratum, 1mL:5mg nitroglycerin injection 2, 2mL:12500 units heparin sodium injection 2, the irrigating solution that mixing is obtained, with the speed lavation 2 hours of drip irrigation washing lotion p.s. 3, lavation 1000mL irrigating solution, close constant flow pump, irrigating solution is changed in every 500mLDMEM substratum and add 10%FBS50mL, 1mL:5mg nitroglycerin injection 2,2mL:12500 units heparin sodium injection 2, the irrigating solution of the AMD3100 of 0.5 bottle of 5mg/ bottle, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s. 3, lavation 1500mL irrigating solution, preparation lavation DMEM substratum used, 10% FBS use before be first positioned in the water-bath of 37 DEG C, be preheating to 37 DEG C in advance,
(6) hemopoietic stem cell is concentrated and purified: by lavation irrigating solution out, in centrifuges with the centrifugal 15-20 minute of 1500-2000rpm, remove supernatant liquor, the liquid of collecting precipitation and lower floor, the liquid of the precipitation collected and lower floor and physiological saline are mixed to obtain mixed solution in the ratio of 2:1-1:2, then mixed solution and lymphocyte separation medium are joined in centrifuge tube respectively in the ratio of 2:1-1:2, the order of adding for first adding lymphocyte separation medium in centrifuge tube, slowly add mixed solution again, note keeping the liquid level of lymphocyte separation medium in adition process smooth, with the centrifugal 20-25 minute of 2200-2500rpm after adding, slowly slow deceleration is accelerated time centrifugal, after centrifugal end, in the middle of collecting, tunica albuginea layer is in new centrifuge tube, physiological saline mixing is added with the ratio of 2:1-1:2, the centrifugal 10-15 minute of 1200-1500rpm, supernatant liquor is removed after centrifugal end, add 10-20mL physiological saline piping and druming precipitation, the centrifugal 10-15 minute of 1200-1500rpm, after centrifugal end, removes supernatant liquor again, by the resuspended precipitation of DMEM substratum, collect hemopoietic stem cell group, resuspended cell mass is carried out cell cultures, CD34 hemopoietic stem cell detection by quantitative, Trypan Blue hemopoietic stem cell Activity determination, CFU-GM hemopoietic stem cell qualitative detection.
2. the method for lavation hemopoietic stem cell from placenta according to claim 1, is characterized in that, the lavation step of described hemopoietic stem cell is:
The irrigating solution used when starting lavation adds 10%FBS50mL, 1mL:5mg nitroglycerin injection 2,2mL:12500 units heparin sodium injection 2 in every 500mLDMEM substratum, the irrigating solution that mixing is obtained, with the speed lavation 2 hours of drip irrigation washing lotion p.s. 3, lavation 1000mL irrigating solution; Close constant flow pump, irrigating solution is changed into the DMEM substratum that 50mL is 100mg/L containing the concentration of AMD3100, with the speed lavation 5 minutes of drip irrigation washing lotion p.s. 10, lavation, after lavation is complete; Close constant flow pump, after 30 minutes, irrigating solution is changed in every 500mLDMEM substratum and add 10%FBS50mL, 1mL:5mg nitroglycerin injection 2,2mL:12500 units heparin sodium injection 2, the irrigating solution of the AMD3100 of 0.5 bottle of 5mg/ bottle, open constant flow pump, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s. 3, lavation 1500mL irrigating solution, reclaims irrigating solution.
3. the method for lavation hemopoietic stem cell from placenta according to claim 1, it is characterized in that, described hemopoietic stem cell is concentrated and purified can also be: by lavation irrigating solution out, centrifugal, centrifugal condition is: rotating speed 1500rpm, pick-up period 45s, deceleration time 45s, centrifugation time 20min, after centrifugal end, returnable bottle is transferred in aseptic operating platform, liquid-transfering gun siphons away supernatant liquid, hemocyte is stayed to precipitate, the physiological saline of 10-20mL is added in hemocyte precipitation, blow and beat precipitation gently, get 50mL centrifuge tube, often pipe adds the lymphocyte separation medium of 20mL, tilt containing the centrifuge tube of lymphocyte separation medium, slowly join on lymphocyte separation medium along centrifuge tube tube wall with the hemocyte that liquid-transfering gun absorption piping and druming is got up, the process added is wanted slowly, keep lymphocyte separation medium liquid level smooth, often pipe adds hemocyte 25mL, move after liquid completes, centrifugal, centrifugal condition: rotating speed 2200rpm, pick-up period 600s, deceleration time 600s, centrifugation time 20min, after centrifugal end, solution can divide four layers, with tunica albuginea layer in the middle of pipette, extract, often pipe 20mL transfers in new 50mL centrifuge tube, and then often pipe adds the physiological saline of 20mL, after mixing, centrifugal, centrifugal condition: rotating speed 1500rpm, pick-up period 45s, deceleration time 45s, centrifugation time 15min, after centrifugal end, transfer in aseptic operating platform, remove supernatant liquor, often pipe add 20mL physiological saline piping and druming precipitation, then centrifugal, centrifugal condition: rotating speed 1500rpm, pick-up period 45s, deceleration time 45s, centrifugation time 15min, after centrifugal end, remove supernatant liquor, with the DMEM substratum re-suspended cell of the 10%FBS of 20mL, collect hemopoietic stem cell group, cell mass is carried out cell cultures, CD34 hemopoietic stem cell detection by quantitative, Trypan Blue hemopoietic stem cell Activity determination, CFU-GM hemopoietic stem cell qualitative detection.
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