The Research Of Mouse Endometrial Stem Cell

Part I The existence of endometrial stem cell in mouse uterusObjective:To study the existence of endometrial stem cell and its distribution in mouse uterus．Methods:Selected female Kunming white mice after born three days, subcutaneous injected BrdU50ug/g in experiment group and 0.9%NS in control group twice daily andtotally injected 3 d. Put mice death after injected 2h,1w,2w,4w和8w, fixed the uterus forimmunohistochemistry and pathology, 5 mice for each phase.Results:The expression of LRCs in stroma and gland cells after 2h were 45.1% and 57.4%respectively and which were gradually degraded. Only few LRCs in the gland bottom in 8wand 1.5% LRCs in stroma cells mainly located around the blood vessel andendometrial-myometrial junction. LRCs in different phases of stroma and gland cells weresignificance(P<0.05).Conclusions:There were endometrial stem cells in the uterus of Kunming white mice and mainly locatedaround the blood vessel and endometrial-myometrial junction. Part II The cloning efficiency of mouse endometrium in vitro primarycultureObjective:To study the cloning efficiency of mouse endometrium in vitro primary culture by limiteddilution technology.Methods:Selected the female kuming mice after born 3 days, sheared and digested the uterus by0.125% pancreatic enzyme, counting the living cells and inoculating them to culture flask.Observed the characteristics of cell clones during 12days and measured the sizes of clonesand calculated the rate of cloning efficiency. At the same time, cultured the uterus cells ofadult female mice as control. Identification the category of the cells by keratin and vimentinby immunohistochemistry and appearance of the cells,the origin of tissue. Detected theproliferation index of Ki-67 in gland clones by immunofluorescence.Results:There were stroma and gland clones divided by the cell origin, large and little clonesdivided by size and growth characteristics. The sizes of large and little gland clones wererespectively 3.64±1.1 and 0.53±0.24mm, the cloning efficiency was respectively 0.051%±0.013 and 0.11±0.07%；The sizes of large and little stroma clones was respectively 2.82±0.5 and 0.41±0.13 mm, the cloning efficiency was respectively 0.008±0.001% and0.43±0.03%. There was no cell growth in the adult uterus by limited dilution technology.The difference of the cloning efficiency rates of four kids of clones were all significant(P<0.05).Compared the sizes of two kinds of large clones or the two kinds of little clones,the differences were not significan(t P＞0.05), The PI of Ki-67 was higher in the nucleus oflarge clones.Conclusion:The endometrium cells can form monocell clones in vitro primary culture which includedlarge and little clones. The proliferation efficiency of the large gland clone was higher. Part III The effects of EGF and 17β-Estradiol to the growth of cellclonesObjective:To study the effects of EGF and 17β-Estradiol on the growth of cell clones.Methods:Added 10ng/ml EGF or 5×10-8 mol/L 17β-Estradiol into the medium, cultured the cell asthe methods of the part II and observed the cell appearance,measured the sizes of clonesand calculated the rate of cloning efficiency.Results:The rates of cloning efficiency in the EGF groups were higher than the control group, thedifference were not significant(P>0.05). The cloning efficiency rates of little clones in17β-Estradiol groups were higher than the control group, while the cloning efficiency ratesof large clones were lower, the difference were not significan(t P>0.05). The large clones in17β-Estradiol groups were larger than the control group and the difference were significant(P<0.05).The difference of the cloning efficiency rates and the sizes of the clones werenot significance in the EGF groups and 17β-Estradiol groups(P>0.05).Conclutions:when added 10ng/ml EGF or 5×10-8 mol/L 17β-Estradiol into the medium, the rates ofcloning efficiency were not higher. 17β-Estradiol could promote the differentiation of thegland clones.Part IV The experiment research of mesenchymal stem cellsdifferentiation to the endometriumObjective:To study the immigration and differentiation of mesenchymal stem cells to endometrium. Methods:1. Cultured the male MSCs and labeling the Brdu in vitro, then identified by CD34 andCD29; the positive rate of Brdu was examined by immunohistochemistry.2. After radiotherapy, the MSCs were injected through the tail vein of female mouse astransplantation group. The other groups were radiotherapy group and normal control group.Each groups had 8 mice.3. Observe the general condition of mice and put them death before died or living longer>60d, taken one uterus for immunohistochemistry and pathology the others for PCR and.Results:1.The MSCs was CD29(+)and CD34(-)and the Brdu positive rate was roughly 80%.2. 7/8 mice died in 14 days; 4 mice lived <20d; 2 mice survived longer >30d; 2 mice livedlonger >60d in the radiotherapy group. The Brdu was negative in radiotherapy,normalcontrol groups and early died mice in experimental group. In the long-living andshort-living mice of transplantation group, Brdu expressed on the endothelial cells of bloodcapillary and few stroma cells. The SRY gene was negative in radiotherapy,normal controlgroups and early died mice in experimental group, but it was positive in the uterus oflong-living and short-living mice.Conclusion:The mouse MSCs can immgrate to the uterus and maybe one of the origin ofendometrium.