The Research Of Spermatogonial Stem Cell Transplantation To Treat Sterile Rats

Research background and purpose The diagnosis and treatment of male infertility is a tough problem in the field of andriatria,at present,the diagnosis and treatment of infertility level is far away from the requirements of infertility patients,and the incidence of infertility has a trend of increasing.In recent years,with the technology of spermatogonial transplantation,is will to explore a new method of treatment.Spermatogonial stem cells(SSCs)are the only can replicable potential diploid cell in the spermatogenic epithelium of mammalian' testis,in vitro it can abstraction,purification,culture,cryopreservatation and autograft or allograft.In 1994,Ralph Brinster laboratory first reported the transplantion of SSCs,and proved to be a technical breakthrough in the application of study the relatationship among the stem cells,support cell and germ cell.Use of this technology can not only successfully transplanted the SSCs from one animal to another animal with the sanme species(homogeneity gene transplantation),but also can transplanted into animals of different species(heterologous gene transfer).with the application of SSCs's abstraction,culture,enrichment and cryopreservatation,transplantation of SSCs will provide a new approach for the treatment of male sterility,produce of transgenic animals and for infertile patients select gene therapy.This technology will be applied to basic science,the humanities in medicine and breeding of livestock and wildlife.SSCs has the potentiality can be immortalized and multi-directional differentiation,is the precursor cells of the sperm formed,has the ability of proliferation and differentiation in the testicular micro-environment.SSCs transplantation opens a new gate for the treatment of male infertility.In this experiment,we will explore the suitable methods to estabalsh a animal receptor modeling for spermatogonial stem cells transplantation,and explore the spermatogonial stem cell isolation and purification methods and the methods of spermatogonial stem cells in allogeneic transplantion and evaluate the effect of transplantation.Through animal experiments to provide the theory and technical support for the future clinical applications.MethodExplore suitable methods to make modelRandomly divide SD rats into 7 groups,one group have ten mice,respectively using cobalt-60 9Gy,8Gy,7Gy,6Gy,6Gy dose radiation one-week intervals twice and busulfan 40mg/kg by a single intraperitoneal injection,in addition a group of normal control group.Observed after the intervention of a three-month mortality,get the testicles to do pathology respectively after the intervention.Through mortality and testicular pathology to determine the model results of different doses of cobalt60 irradiation and busulfan by intraperitoneal injection,for follow-up of the spermatogonial stem cell transplantation provide a suitable animal model of receptor. Set up the animal model of infertility for transplantionSelected 15 8-week-old SD rats,weight 200±20g,cobalt60 6Gy dose for total body irradiation,the same dose again after a week for total body irradiation,4 weeks later for transplantation.Isolated and cultured spermatogonial stem cellsCheck 9-10 day-old testis of SD rats,using mechanical method+two-step enzymatic digestion+differential attachment method extract spermatogonial stem cells in petri dishes.Rats were killed off neck act,sterile collection of bilateral testes,placed in the disc containing HBSS,carefully stripping fat pad,epididymal,testicular capsule,and softly use HBSS to blow.Testicular tissue will be transferred to a special small containers,the dish be filled with a small amount of HBSS,with ophthalmic scissors to cut the organization to be sludge-like,the sludge-like organization will be transferred to the bottle of penicillin which has been placed blender.Add the equivalent of 10 times the volume of organization 1mg/ml typeⅣcollagenase solution at 37℃digestion 15min,and stirring softly on the magnetic stirrer.Standing 10min,draw-off the supernatant and discard,add compound enzyme solution containing 0.5mg/mlDNasel+0.25%trypsin+0.02%EDTA to the penicillin bottle,at 37℃digestion 10min,stirring softly on magnetic stirrer.FBS added to terminate the trypsin digestion,add HBSS to dilute,and blow uniform.The digested cell suspension will be filtered with 200 mesh,400 mesh grit respectively,collected the cell suspension into the centrifuge tube,1000r/min centrifuge 3min.Discarded supernatant,add the complete culture medium fresh prepared,adjusting the cell density of 3×10⁶piece/ml.Cells inoculated to the25cm² cell culture bottles manufactured with optical transparent pure polystyrene,at 37℃,5%CO₂ incubator incubated 4h,collected the suspension cells had not been affixed to the walls yet,1000r/min centrifuge 3min,add culture medium,re-adjusted cell density of 3×10⁶piece/ml.Spermatogonial stem cells transplantated to animal receptorSelect SD rats 8-week-old weigh around 200g as receptor animals,using cobalt60 irradiation twice intervals a week doses of 6Gy,a month later spermatogonial stem cell transplantated.(1) Rats after weighing,using 10%chloral hydrate in accordance with 300mg/kg (0.3ml/100g) body weight for intraperitoneal injection to anesthesia.(2) Rat in supine position,disinfection lower perineal area,make a incision about 2cm from the middle of the scrotum,discissio the scrotum,extrude both sides of the testicles placed to carbasus.(3) In accordance with the method of literature,using 1ml syringe puncture one side of rete testis,inleted spermatogonial stem cell suspension,the other side of the testis for self-control.Rearing in open cleaning environment post-transplant.Evaluation of transplantation2 months after transplantation,cut the testicles,fixed 24h with Bouin's fluid(saturated picric acid in aqueous solution 75ml,formalin 25ml,glacial acetic acid 5ml).Using hematoxylin and eosin staining,observed the testicular pathology performance of the transplantation group and control group in light microscope,SABC immunohistochemical assayed the expression of c-kit.Results Group 9Gy died all within 15 days after radiotherapy,the death rate was 100%;8 Gy group died four within three months,the mortality rate was 40%,testicular size compared with the control group decreased,testicular spermatogenic cells at all levels decreased significantly;7 Gy and 6 Gy groups had two died respectively within three months,the death rate was 20%,the testicular size had no significant difference with the control group,only see part of spermatogenic cell layer reduction,some cells with disorder;the group of 6Gy twice a week intervals irradiation had one died in irradiation 5 days later and another died in irradiation 15 days later,the death rate was 20%,testicular size compared with the control group decreased apparently,testicular spermatogenic cells decrease significantly at all levels,seminiferous tubule was "vacuoles";intraperitoneal injection of busulfan group spermatogenic cells layer reduced and disordered,but still we can see several layers of spermatogenic cells. Choose cobalt60 6Gy irradiation twice a week intervals as the receptor animal making model method.Selected 6 Gy dose irradiation twice a week interval as the way to make receptor model,started transplantation a month after the second irradiation,before transplantation had two rats dead,before transplantation randomly selected two rats's testis to do pathological section,spermatogenic cells lacking at all levels,only could see a small number of spermatogonia around the seminiferous tubules.Observed the stem cells after separation under a light microscope,the cell inequality of size,suspended in culture medium to cultivate four hours later,because of the adherent speed difference,supporting cells and Leydig'adhere fast,and spermatogonial stem cells adherent later,change culture medium,no obvious differences in cell size can be seen,round,spermatogonial stem cells adherent growth after cultured 48 hours,immunohistochemical staining shows that perinuclear cytoplasmic membrane c-kit andα₆-integrin positive expression,proved to be spermatogonial stem cells. Transplanted 10 SD rats in all,each testicles injected cell suspension or the same dose of transplant medium 0.2±0.05ml,post-transplant penicillin 400,000 units/piece.day by intramuscular injection,continuing for three days to prevent wound infection,the first day after transplantation had one mouse died,ten days after transplantation had two mice died,after no death.2 months post-transplant,the transplant group testicular size similar to the control group 2,bigger than the control group 1.Control group 1 shows that a small minority of spermatogenic restore,most was vacuolated.Under a light microscope,all levels of spermatogenic cells can be seen in most seminiferous tubules of the transplant group,but still could see seminiferous tubules failed to restore,the expression of c-kit positive degree stronger than the control group 1.The transplant group spermatogenic epithelium restoring was superior to restore control group 1.Conclusion1 Cobalt60 6Gy irradiation twice a week interval could provide a suitable receptor animals meodel for spermatogonial stem cell transplantation.2 Spermatogonial stem cell membrane and cytoplasm have c-kit,α6-integrin positive expression,c-kit andα6-integrin can be used as surface markers of spermatogonial stem cells proliferation and differentiation stages for spermatogonial stem cells identification.3 Spermatogonial stem cells can be transplanted by testicular net injecting,spermatogonial stem cells after transplantation in the receptor could proliferate,and could differentiate to form mature sperm.Spermatogonial stem cell transplantation had opened up a new world for the breeding of transgenic animals and the treatment of male infertility.