An In Vivo Study On Repair Damaged Endometrium By Exosome Derived From Marrow Mesenchymal Stem Cells

Chapter 1 Isolation,culture and identification of New Zealand white rabbit bone marrow mesenchymal stem cells and the secretion of exosomeObject:To separate and cultivate the New Zealand white rabbit bone marrow mesenchymal stem cells in vitro and extract exosome from cells,studying their biological characteristics and phenotypic features;Determinating of exosome protein content.To provide a cellular basis for the experimental study of rabbit endometrial injury.Methods:1.Bone mesenchymal stem cells were separated from the femur and the tibia of 4-week-old New Zealand rabbit which purified by whole bone marrow adherent culture method;the surface markers of BMSCs were detected by flow cytometry?FCM?,and the direction of adipocytes was induced.We used CCK8 method to detect the cell proliferation and plot growth curves to identify whether the frozen cells were different from the normal.2.Exosome were extracted with ExoQuick-TC^TM kit from the culture supernatant of pssage three?P3?BMSCs and using electron microscopy to identify its morphology.The concentration of exosome protein was determined by BCA method.Results:1.?1?Few primary cultured BMSCs were adherent and spindle-shaped after inoculation with48hours;the large number of cells are in the adherent state and spindle shaped and scattered in the distribution of cell colony formation after 6⁸days;after 14¹6 days was the first passage.We could seethe first passage cells adherent within 4 hours,after 24 hours cells value-added quickly,and more than 80percent of cells fused about 6⁷ days.After purification to the third passage,the cell morphology tended to be consistent,and fibroblast like growth.?2?The purified cells'surface molecular marker CD29 was positive?97.60%?but CD45?1.89%?,which confirmed that the cultured cells were mesenchymal stem cells;furthermore,BMSCs were induced to differentiate into fat and identified that has the ability to differentiate into adipocytes.?3?There was no significant difference in the growth curve of the third passage BMSCs compared with unfrozen cells.2.?1?Under the transmission electron microscope,we found a large number of round or oval membranous vesicles which the envelope was intact with a diameter of 40-160nm and a low electron density component in the cavity.?2?The concentration of exosome protein measured by BCA was 1.986?g/ul by 20 times dilution.Conclusion:1.BMSCs can be successfully isolated and expanded by whole bone marrow adherent cell separation method which have stable growth,strong added value,high purity,good activity and homogeneous phenotype.2.BMSCs were highly expressed specific antigen CD29,almost no expression of hematopoietic stem cell-specific antigen CD45.3.Cryopreserved BMSCs had no significant effect on their biological activity after resuscitation,and cryopreserved cells could be used for subsequent in vivo experimental studies.4.Exosome in BMSCs culture medium can be successfully extracted with Exo Quick-TC kit.Chapter 2 An in vivo experimental study of exosome derived from rabbit bone marrow mesenchymal stem cells on repair of endometrial injuryObject:To establisha stable New Zealand white rabbits model of endometrial injury for subsequent in vivo transplantation of exosome and to explore whether exosome derived from MSCs have the potential to repair damaged endometrium and the possible mechanism of action.Thus,MSC-generated exosomes may provide a novel cell-free therapy for the clinical treatment of intrauterine adhesions.Methods:Select sixty adult female New Zealand white rabbits and divided into four groups randomly,to build endometrium injury models by mechanical and infection double injury method.Group A: sham operation?control?;Group B: surgery?model group?;Group C: BMSCs;Group D: exosome.The uterine tissues were collected and the morphological changes and fibrosis were monitored at 1w,2w and 4w after treatment by HE/Masson staining.Immunohistochemistry and western blot was used to detect the expression of CK and VIM in endometrium.Results : After modeling the surface of the endometrium shedding and endometrial stroma naked or thinning.Forming an intrauterine adhesion finally.The results of HE/ Masson staining showed that the number of endometrial glands in BMSCs group and exosome group were significantly higher than that in model group?P?0.05?at 4 weeks after treatment,and the fibrosis area ratio was significantly lower than that in model group?P?0.05?;The relative expression of CK-19 protein was significantly higher than that of the model group?P?0.05?,and the VIM protein was significantly lower than that of the model group?P?0.05?.There was no significant difference between the two groups?P?0.05?.Conclusion:1.Double damage can cause irreversible damage to the rabbit endometrium,form a scar repair,and establish a stable animal model of endometrial damage.2.Bone marrow mesenchymal stem cells can play a role in the injured endometrium and promote the repair of damaged endometrium which may repair the damaged endometrium as exogenous stem cells.3.Exosomes derived from bone marrow mesenchymal stem cells have the same function as stem cells and can act in damaged endometrium and promote the repair of damaged endometrium.