| Background:Uterus play an irreplaceable role in the establishment and maintenance of pregnancy.If trauma or infection damages the basal layer of endometrium,it can cause endometrial repair disorder and form thin endometrium,so that embryos can not be implanted in the endometrium,which is an important cause of infertility.Therapeutic methods for thin endometrium include drug therapy,scratch stimulation therapy and intrauterine injection of granulocyte colony stimulating factor.Although there are many methods,the overall effect is not good,which has become a major difficulty in reproductive medicine.Stem cells are undifferentiated cell populations with self-renewal and multidirectional differentiation potential.It has been reported that stem cells have been successfully used to repair endometrium and pregnancy.Recent studies by Chan RW have shown that there are stem cells with high proliferation,self-renewal and differentiation potential in the endometrial basal layer,and there are three types of stem cells: epithelial cells,mesenchymal cells and endothelial cells.Endometrial stem cells have the potential to become the ideal seed cells for the treatment of infertility caused by endometrial injury.At present,endometrial stem cells are either derived from endometrium of patients undergoing total hysterectomy or obtained by artificial abortion,but there are some shortcomings such as limited source and great damage.Musina et al.found that endometrial exfoliated cells contained in menstrual blood can be isolated and cultured in vitro.They have the advantages of rapid replication,wide source,easy access to materials,high content and non-invasive access.At the same time,because menstrual blood is human waste,sensitive medical ethical issues can be avoided.Because of ethical problems,experimental treatment and pathological mechanism of endometrial injury are difficult to be directly carried out in patients.It is particularly necessary to establish animal models of endometrial injury.At present,heat injury and mechanical injury are two commonly used methods to establish mouse endometrial injury models.Qu Junying et al.used the method of hydrothermal injury to build the animal model of endometrial injury,mainly using the hot water flowing through the uterine catheter to damage the endometrial tissue of mice to establish the animal model of endometrial injury.Hu Xiaoxiao et al.constructed an animal model of endometrial injury by electrothermal injury model method.Puncture method was used to insert a uterine guide wire and point discharge was used to coagulate the endometrial tissue of mice to establish an animal model of endometrial injury.Feryal established the mouse model of Asherman's syndrome endometrial injury by mechanical injury to the endometrium of C57BL/6 female mice.All the mice used in the above animal models were immune normal mice,and the transplanted stem cells were mouse embryonic stem cells or bone marrow stem cells.In this study,the mechanism of repairing endometrial injury by human menstrual blood-derived endometrial stem cells was studied.The transplanted cells were human menstrual blood-derived endometrial stem cells.In order to reduce immune rejection,nude mice were used to construct endometrial injury model.However,the study on nude mice with endometrial injury has not been reported yet.Song Tao et al.confirmed that after transplantation of rat embryonic stem cells into the uterine cavity of rat model of endometrial injury,it can be seen that rat embryonic stem cells colonized in the uterine cavity,which is conducive to the repair of endometrial injury in rats.However,whether human menstrual blood-derived endometrial stem cells can be transplanted into the uterine cavity of nude mice model of endometrial injury to repair endometrial injury has not been reported at home and abroad.Green fluorescent protein(GFP)is a kind of cell marker,which has the advantages of convenient detection,stable expression,non-toxicity and no influence on cell function.However,the enhanced green fluorescent protein labeled stem cells have the advantages of simple operation and intuitive results.They have been widely used in the tracing,quantitative analysis and detection of differentiation status of transplanted stem cells.Therefore,in this study,enhanced green fluorescent protein was used to label human menstrual blood-derived endometrial stem cells to observe the colonization of human menstrual blood-derived endometrial stem cells in the uterine cavity of nude mice.Keratin is a marker protein of epithelial cells and plays an important role in maintaining the integrity of epithelial cells.The expression level of keratin can be used to observe the proliferation of endometrial glandular epithelial cells after transplantation of human menstrual blood-derived endometrial stem cells.Vimentin is mainly expressed in the skeleton structure of mesenchymal cells.Its function is to maintain the stability of cell structure and participate in cell mitosis,differentiation and proliferation,signal transduction and so on.Vascular endothelial growth factor(VEGF)is a highly specific mitogen of vascular endothelial cells and is the main regulator of angiogenesis.Its expression level reflects the level of proliferation and migration of vascular endothelial cells,and also reflects the level of vascular construction.The expression level of vascular endothelial growth factor is low in thin endometrium.The changes of expression levels of keratin,vimentin and vascular endothelial growth factor before and after treatment can be used to judge the effect of endometrial injury repair after human endometrial stem cell transplantation and to explore the mechanism of endometrial injury repair by human endometrial stem cell transplantation.Research objective:Human endometrial stem cells were isolated from human menstrual blood and their biological characteristics were studied.By establishing a nude mouse model of endometrial injury,the ability and mechanism of human blood derived endometrial stem cells to repair the damaged endometrial were studied,providing theoretical and experimental basis for clinical treatment of human endometrial injury.Method: ?.Isolation,culture and identification of human menstrual blood-derived endometrial stem cellsFrom March 2016 to April 2017,menstrual blood was collected from healthy female volunteers(ages 20-35).The volunteers had normal menstruation with a menstrual cycle of 24-35 days(an average of 28 days)and no complications.Human menstrual blood-derived endometrial stem cells were isolated by density gradient centrifugation,then primary cells were cultured and their morphological characteristics were observed.Freeze and resuscitate cells,detect cell proliferation capacity and plot cell growth curve.Immunohistochemistry was used to detect the expression of Vimentin and identify the types of endometrial stem cells.The expression of CD146 and CD90 were detected by flow cytometry to identify endometrial stem cells.Colony formation experiment was used to detect the clone formation rate of cells.?.Study on the repairing of endometrium injury by human menstrual blood-derived endometrial stem cellsWe established a nude mouse model of bilateral endometrial mechanical injury,and transfected Ad-eGFP(enhanced green fluorescent protein adenovirus)into human menstrual blood-derived endometrial stem cells and detected the transfection rate.The endometrial stem cells were then transplanted into the right uterine cavity of the nude mice with endometrial injury,and the left uterine cavity was taken as a negative control.The green fluorescence of bilateral endometrium,the thickness of bilateral endometrium,the expression levelsof vimentin,keratin and vascular endothelial growth factor in bilateral endometrium were detected 7 days after transplantation to explore the mechanism of repairing endometrial injury by human menstrual blood-derived endometrial stem cells.To observe the repairing effect of human menstrual blood-derived endometrial stem cells on endometrial injury,nude mice models of bilateral endometrial mechanical injury were randomly divided into transplantation group and control group.The recovery of endometrial embryo implantation function after human menstrual blood-derived endometrial stem cells transplantation was judged by observing the pregnancy rate of female and male mice after mating.Results: ?.Isolation,culture and identification of humanmenstrual blood-derived endometrial stem cells1.Endometrial stem cells can be isolated and cultured from human menstrual blood by density gradient centrifugation.After 7 days of culture,most of the cells were spindle-shaped with clear outline,abundant and transparent cytoplasm,and oval and centered nuclei under inverted microscope;2.Cell growth curve showed that the incubation period of cell culture was 1-3 days with slow growth,the logarithmic period was 4-7 days with rapid growth,and the growth rate was slow after the logarithmic proliferation period ended;3.Immunohistochemical test showed that the isolated cells were positive for Vimentin,suggesting that endometrial stem cells isolated from human menstrual blood were endometrial mesenchymal stem cells;4.Flow cytometry analysis showed that: CD90-PE positive cells accounted for 90.2%,and CD146-FITC positive cells accounted for 0.014%,suggesting thatthe isolated cells are endometrial stem cells and the origin of human menstrual blood-derived endometrial stem cells is bone marrow derived stem cells;5.The clone formation rates were 14.8%,19% and 20% respectively,suggesting that the human menstrual blood-derived endometrial stem cells have strong cloning ability.?.Study on the repairing of endometrium injury by human menstrual blood-derived endometrial stem cells1.Ad-eGFP(enhanced green fluorescent protein adenovirus)was transfected into human menstrual blood-derived endometrial stem cells,and the transfection rate was up to 60% by flow cytometry 24 hours after transfection;2.After Endometrial stem cells with Ad-eGFP transfected into the right uterine cavity of nude mice with endometrial injury for 7 days,green fluorescence was detected in frozen section of endometrium of the transplanted side under fluorescence microscope,while no green fluorescence was detected in frozen section of endometrium of the control side under fluorescence microscope;3.Endometrial thickness on the transplanted side was increased 7 days after transplantation compared with that on the control side,but there was no statistical difference(P>0.05);4.The expression levels of vimentin,keratin and VEGF(vascular endothelial growth factor)in endometrium of the transplanted side were significantly higher than those of the control side 7 days after transplantation,and the difference was statistically significant(P< 0.05);5.Pregnancy rate of nude mice with endometrial injury after transplantation of human menstrual blood-derived endometrial stem cells was significantly higher than that of control group,and the difference was statistically significant(P< 0.01);Conclusion:Our study shows that endometrial stem cells can be successfully isolated and cultured from human menstrual blood.Endometrial stem cells isolated and cultured from menstrual blood are endometrial mesenchymal stem cells,which have strong cloning ability and mainly originate from bone marrow-derived stem cells.Endometrial stem cells isolated from human blood can be successfully implanted into damaged endometrium of nude mice with endometrial injury and promote the repair and proliferation of damaged endometrium.Endometrial mesenchymal stem cells repair endometrial injury by up-regulating the expression of vimentin,keratin and vascular endothelial growth factor in endometrium,thereby improving the embryo implantation function of damaged endometrium.This study confirmed that human endometrial stem cells can repair endometrial injury and treat infertility caused by endometrial injury,which provides a new therapeutic approach for clinical treatment of infertility. |
PDF Full Text Request