| Objective:1.To explore a simple and efficient method for isolation,culture and purification of endometrial stem cells in vitro,and to observe cell morphology and surface marker identification.2.To explore the feasibility of establishing a stable mouse model of thin endometrium by intrauterine injection of 95%ethanol,characterized by HE staining,immunohistochemistry and fertility assessment,and provide an ideal animal model for the study of pathological features and repair mechanisms of thin endometrium.3.HE staining,immunohistochemistry and fertility evaluation were used to explore the effect of endometrial stem cell transplantation on thin endometrium in mice,which provided a new idea for the research and clinical treatment of thin endometrium.Methods:1.Female C57BL/6J mice that were produced and weaned were selected as experimental animals for extracting endometrial stem cells,and endometrial stem cells of C57BL/6J mice were isolated and cultured in vitro by mechanical separation and two-step enzyme digestion.Endometrial stem cells were cryopreserved and resuscitated,their growth status was observed,and the growth curve was measured by CCK-8 assay to analyze the proliferation ability of cells.Flow cytometry was used to detect the expression of surface markers of endometrial stem cells.2.Eighty-four female unmated C57BL/6J mice with sexual maturity and regular estrus were used in this study.Thirty-six mice were randomly divided into three groups:the blank group,control group,and experimental group.A mouse model of thin endometrium was established by intrauterine infusion of 95%ethanol injured endometrium.The experimental mice were euthanized by cervical dislocation in the third estrus period after modeling,and uterine samples were collected.The appearance of the uterus and the thickness of endometrium were observed,and HE staining were used to observe the morphology.The regeneration of endometrial cells was evaluated by immunohistochemistry for cytokeratin,vimentin,vascular endothelial growth factor(VEGF)and estrogen receptor?(ER?).Another group of forty-eight mice were randomly divided into the blank group,control group,unilateral injury group,and the bilateral injury group.All surviving mice were simultaneously caged after modeling to analyze the effect of thin endometrium on the fertility of mice and to evaluate whether the mouse model of thin endometrium was successfully established.3.One hundred and ninety-two female unmated C57BL/6J mice with sexual maturity and regular estrus were used as experimental animals.A mouse model of unilateral thin endometrium was established by intrauterine injection of 95%ethanol into the right side of the uterus,and the left side of the uterus was used as a normal control.The model mice were randomly divided into the control group of PBS injection3d(model mice were given PBS 0.1 mL,n=24);Control group of PBS injection 7d(model mice were given PBS 0.1 mL,n=24);Control group of PBS injection 14d(model mice were given PBS 0.1 mL,n=24);Control group of PBS injection 30d(model mice were given PBS 0.1 mL,n=24);The group of Endometrial stem cells transplantation 3d(after modeling,intrauterine injection of endometrial stem cells immediately 0.1 mL,2×10~8/L~5×10~8/L,n=24);The group of Endometrial stem cells transplantation 7d(after modeling,intrauterine injection of endometrial stem cells immediately 0.1 mL,2×10~8/L~5×10~8/L,n=24);The group of Endometrial stem cells transplantation 14d(after modeling,intrauterine injection of endometrial stem cells immediately 0.1 mL,2×10~8/L~5×10~8/L,n=24);The group of Endometrial stem cells transplantation 30d(after modeling,intrauterine injection of endometrial stem cells immediately 0.1 mL,2×10~8/L~5×10~8/L,n=24).The mice were euthanized by cervical dislocation on the 3rd,7th,14th and 30th day after transplantation of endometrial stem cells/PBS,and uterine samples were collected,12 in each group.HE staining was used to detect the structure of endometrium,and measure the thickness of endometrium.The regeneration of endometrial cells was evaluated by immunohistochemistry for cytokeratin,vimentin,vascular endothelial growth factor(VEGF)and estrogen receptor?(ER?).At the same time,the remaining mice in each group were simultaneously caged at the corresponding time points of the sampling to analyze the effects of endometrial stem cells transplantation on the fertility of mice and to evaluate whether endometrial stem cells can repair thin endometrium from the functional aspects.Results:1.The endometrial stem cells obtained by mechanical separation and two-step enzymatic digestion were adherent,and the cells are mainly spindle-shaped cells,which grow in a spiral or radiant manner.All adherent cells took on typical morphologically resembled fibroblast and maintained similar morphology with passages.The growth curve was similar between P1 and P3 endometrial stem cells and were S-type,and there were certain retention,logarithmic and plateau phases.And the growth characteristics of endometrial stem cells after cryopreservation were similar to those of unfrozen cells with good growth state;Flow cytometry showed that the cell surface antigens CD29,CD73and CD105 were positive,CD45 and CD117 were negative.2.HE staining showed that the endometrium in experimental group significantly became thinner and the uterine cavity was enlarged,the number of glands was decreased,and the partial blood vessels were sparse.The results of the immunohistochemistry showed that the expressions of cytokeratin,vimentin,VEGF and ER?in the endometrial cells of the experimental group were significantly lower than those in the blank and control groups.Fertility assessment showed that mice in the blank and control groups were normally conceived after mating and that no mice in the bilateral injury group were conceived.In addition,no mice were conceived in the injured side of the uterus in the unilateral injury group,whereas mice in the uninjured side of the uterus were conceived normally.The mean pregnancy rate of the injured side of the uterus in the experimental group was significantly lower than that on the uninjured side(P<0.01),indicating that intrauterine injection of 95%ethanol damaged the endometrium,resulting in reduced fertility.3.Compared with the each PBS control group,the results of HE staining after endometrial stem cell transplantation showed that the degree of endometrial edema in each endometrial stem cell transplantation group was significantly reduced,the state was better,the intrauterine adhesions and occlusion were significantly reduced,and the number of glands and blood vessels increased;the thickness of the endometrium was significantly thicker,and the difference was statistically significant(P<0.05).The results of Immunohistochemical showed that the average optical density(AOD)of cytokeratin,vimentin,vascular endothelial growth factor(VEGF)and estrogen receptor?(ER?)in each group of endometrial stem cell transplantation was significantly higher than that in each PBS control group(P<0.05),Compared with the other group,the expression of the indexes of endometrial stem cell transplantation in the 30d group was significantly different(P<0.01),but it is still lower than the normal control group on the left side of the uterus(P<0.05).The fertility evaluation experiment showed that the normal uterus of the left side of each group was normally conceived,the right side of the uterus in PBS control group of 3d,7d and 14d was not conceived,and the average pregnancy rate of the injury side of the uterus in the endometrial stem cell transplantation group was significantly higher than that of PBS control group(P<0.01).Compared with the 3d and 7d groups of endometrial stem cell transplantation,the difference in the pregnancy rate of the right uterus in the 14d group of stem cell transplantation was statistically significant(P<0.05),while the difference in the 30d group of stem cell transplantation was significant(P<0.01),which indicates that endometrial stem cell intrauterine orthotopic transplantation has a certain repairing effect on the thin endometrium of mice and can improve fertility.Conclusion:1.The method of mechanical separation and the two-step enzymatic digestion could obtain endometrial stem cells with strong expansion ability in vitro,high purity and good growth state,it is a simple and effective method for obtaining endometrial stem cells,and the obtained cells can be used as seed cells for related research.2.We have successfully established a mouse model of thin endometrium,which can be used to study the repair mechanisms of the thin endometrium.3.Endometrial stem cells are transplanted orthotopically into the mouse models of thin endometrial,which can increase the thickness of endometrial,promote the regeneration of endometrial cells,repair endometrial tissue and improve fertility,suggesting that endometrial stem cell transplantation has a certain repair effect on the thin endometrium. |
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