Relationship Between Endometrial Ectopic Transplantation And Gap Junctional Intercellular Communication

Objective: To explore the isolation and culture of human endometrial glandular epithelial cells and stromal cells , establish cell model of endometriosis in vitro.Methods: Digestion with high concentrated collagenase and DNase, filtration, adhesion purification were used to isolate, purify human endometrial glandular epithelial cells and stromal cells in 30samples. And the two kinds of cells were reproduced.Results: The endometrial cell culture was successfully established in 29 of 30 endometrium samples. From one gramme of endometrial tissue, the yields of glandular epithelial cells and stromal cells were (40~50)×106 and (40~50)×106 respectively. The yields of cells were good. The purities of glandular epithelial cells and stromal cells were 96% and 98% respectively. Stromal cells could be reproduced into several passage. However, glandular epithelial cells could not be reproduced.Conclusions: A good yield of purified human endometrial glandular epithelial cells and stromal cells can be obtained by the modified culture procedure. And just the procedure has provided a good experimental model for studying the pathogenesy of endometriosis.PART TWO Establish a Nude Mice Model of EndometriosisObjective : To establish the experimental nude mice model of endometriosis by human eutopic endometrium.Methods: Female nude mice of 6~8 weeks aged were selected . Nude mice of experimental group(n=20 ) were implanted with the endometrium of patients with endometriosis or without endometriosis into pelvic and abdominal cavities.Nude mice of control group(n=3) were implanted with the greater omentum.Nude mice were killed at 5th, 15th, and 30th day after implantation to observe the growth of endometriotic lesions. The morphological and histological changes of endometriotic lesions from diferent sources and at diferent time points of growth were examined by light microscopy.Results: The achievement ratio of transplantation was 100%. At the 5th day, endometriotic lesions were found in the pelvic and abdominal cavities, and the adhesion to mice tissues was already tight. There were nascent vascular net between endometriotic lesions and mice mesepithelium. Endometrial glands around with stroma cells wrer found under light microscope. At the 15th day, endometriotic lesions mixed together with mice tissues. Endometriotic lesions presented with parenchymatous and cystiform . There were a lot of endometrial glands and stroma cells with rich blood supply under light microscope. At the 30th day, endometriotic lesions have atrophied, and the adhension was serve partly. Endometriotic lesions presented serve fibrosis and some endometrial cells were not integrity under light microscope. In the control group, some of the greater omentum adhesion the incisal opening of abdominal wall, the others were free and atrophied or merging gradually. There was no change in the construction greater omentum except for some inflammatory reaction.Conclusions: Nude mice model of endometriosis was good for studying the endometria cell proliferation, neovascularization, hormone, immune response and so on, since the implanted endometrium remain the original morphological and histological features. It is a perfect model of endometriosis because of its significant and steady character, low cost, simple operation and short observation cycle.PART THREE Regulation of Connexin26, Connexin32 and Connexin43 Expression in Nude Mice Mode1 of Endometriosis by Estrogen and ProgesteroneObjective: To observe effects on connexin26, connexin32 and connexin43 expressioin in nude mice mode1 of endometriosis by estrogen and progesterone.Methods: Establish nude mice mode1 of endometriosis. 60 nude mice were randomly divided into three groups. Estrogen group: 17-β-oestradiol (30μg·kg, i.m.), at 5 day intervals thereafter implanted. Progesterone group: progesterone(10mg·kg, i.m.), at 5 day intervals thereafter implanted. Control group: olive oil(0.2ml, i.m.), at 5 day intervals thereafter implanted. Mice of each group were killed at 7th,14th,21th and 28th day randomly. To detect the expression of gene and protein (CX26,CX32,CX43) in endometriotic lesions by RT-PCR and Western Blot respectively.Results: The expression of connexin26 mRNA and protein was enhanced by estroge(nP<0.05), but suppressed by progesteron(eP<0.05). The expression of connexin43 mRNA and protein was strongly suppressed by estrogen(P<0.05). But progesterone had no effect on the expression of connexin43 mRNA and protein(P>0.05). The expression of connexin32 mRNA and protein was not be detected in any group.Conclusions:①Regulation of connexin26 by estrogen and progesterone was opposite.②The expression of connexin32 mRNA and protein was not be detected in any group, so its effection on gap junctional intercellular communication and endometrial ectopic transplantation was not clear.③The expression of connexin43 mRNA and protein was strongly suppressed by estrogen. But progesterone had no effect on it. So we conjectured that estrogen could down regulation connexin43, depress the function of GJIC, encourage abnormal cell proliferation and cell differentiation, enhance the ability of endometrial ectopic transplantation.PART FOUR Effect on the GJIC Function, Invasion and Proliferation of Eutopic Endometrial Glandular Epithelial Cells of Endometriosis by CX43 Gene TransfectionObjective:To explore the effection on GJIC function, invasion and proliferation of eutopic endometrial glandular epithelial cells of endometriosis by CX43 gene transfection.Methods: Establish the expression vector which can express connexin43 and enhanced green flurescent protein simultaneously. Transfect endometrial glandular epithelial cells of endometriosis with liposome. The function of GJIC was detected by fluorescence scarification test. The ability of growth was detected by soft agar clone formation test. The ability of invasion was detected by Transwell cabin test.Results: The expression of connexin43 mRNA and protein in the glandular epithelial cells was increased after connexin43 transfection. The function of GJIC was improved than the other groups(P<0.05). The ability of proliferation and invasion of the glandular epithelial cells was weaken than the other groups(P<0.05).Conclusions: Improved the expression of connexin43 gene of human eutopic endometrial glandular epithelial cells of endometriosis could recover the function of GJIC, decrease abnormal cell proliferation and cell differentiation, and maybe reduce endometrial eutopic transplantation.