| Endometriosis is one of the common, difficult and complicated diseases in gynecology, which is defined as endometriotic tissues growing outside normal uterine cavity. Its incidence has showed increasing trend in recent years, and it has trended to be younger. The main clinical manifestations of this disease are progressive dysmenorrheal, pelvic pain, menstrual disorder, sterility, et al, which has been seriously injuring the female patients both in body and mentality. Its pathogenesis remains unclear, and curative effect is not satisfactory. Thus from basic to clinical studies are hot, and traditional Chinese medicine therapy has also become one of the hot spots of exploring.This object focused on the study of the theoretical foundation about the treatment by Guiling xiaoyi Decoction. In order to explore the mechanism of Guiling xiaoyi Decoction on endometriosis and provide theoretical basis for clinical treatment, we studied the effect of drug serum of Guiling xiaoyi Decoction on the growth of eutopic endometrial glandular epithelial cells of endometriosis and Caspase-3expression of glandular epithelial cells in vitro by established the in-vitro cultural method of human endometrial glandular epithelial cell.一、The theoretical study of the treatment of Guiling xiaoyi Decoction for endometriosisThere is no disease named endometriosis in Traditional Chinese Medicine. The main symtoms are correlated closely to "dysmenorrheal","abdominal mass","irregular menses"and"sterility"etc. Considering the function of kidney which is not sufficient is the radical factor of occurrence of this disease, and blood stasis is an important causative agent and pathological manifest of the disease, so the therapy should be based upon the concept of enhancing warming kidney and dispersing blood stasis.Guizhi fuling pill originates from Jinkui yaolue. Zhongjing Zhang, the author of the book, designed this prescription to mildly remove mass in the abdomen, disperse blood stasis with the purpose of preventing abortion which was due to be abdominal mass from endangering the fetus. If the prescription was utilized mainly to remove blood stasis and disperse the mass in the abdomen, there may be some shortage of the strength of the drugs in the prescription, and it should be added with some drugs in order to resolve blood stasis and promote blood circulation. Thus Guiling xiaoyi Decoction was designed which combines the Guizhi fuling pill with Xianlingpi to strengthen warming kidney, and Xuejie to disperse blood stasis. The vitro experimental studies on the therapy of endometriosis were carried out with Guiling xiaoyi Decoction provided a basis fof clinical rational use.二、The culture method of glandular epithelial cells established initially in vitroThe endometria were be collected from female patients who suffered from endometriosis or uterine fibroids and received operation in the First People’s Hospital of Yangzhou and Chinese Medicine Hospital from October in2010to October in2011. Glandular epithelial cells were isolated and transferred of culture generation. The cell proliferation was observed by microscope, and the cells were characterized by immunofluorescence method.The results showed that human endometrial glandular epithelial cells were successfully isolated and cultured by combination of collagenase digestion, sieve separation, selective adherence and secondary digestion method, and the success rate was75.00%. The cells could be subcultured for1-2generations, and survived for4-6weeks. It was confirmed that human endometrial glandular epithelial cells in vitro have good proliferative capacity under our experimental conditions. The glandular epithelial cells from both endometriosis and non-endometriosis can be isolated and cultured successfully(P>0.05). Because of rare endometrium, two cases of the endometrial glandular epithelial cells in proliferative early phase were few, and at last the culture failed. So we selected endometria according to preoperative endometrial thickness>5mm. And the differences on the achievement ratio of endometrial glandular epithelial cell culture both in proliferative advanced stage and secretory phase were not significant P>0.05). Based on our experiment, our effective research platform can be provided for the study on the pathogenesy and pharmacodynamics of endometriosis.三、The effect of Guiling xiaoyi Decoction on growth and Caspase-3content of endometrial glandular epithelial cellsWe prepared Guiling xiaoyi Decoctio-serum and gestrinone-serum to interpose in the eutopic glandular epithelial cells of patients suffering endometriosis, then growth suppression was detected by MTT. As a result, Guiling xiaoyi Decoctio-serum and gestrinone-serum can inhibit the growth of the eutopic glandular epithelial cells of endometriosis, and Guiling xiaoyi Decoction has better effect than gestrinone. We can presume that Guiling xiaoyi Decoction induces apoptosis, inhibits the growth of ectopic foci to promote ectopic foci to shink and disappear, and meanwhile, it plays a preventive role to promote apoptosis of potential lesions of the endometrium and inhibit the cultivation and growth.We prepared different drugs serums to interfere in eutopic glandular epithelial cells of endometriosis.Then we observed the cell morphologic changes under microscope, and meanwhile collected supernatant liquid of cell culture at24h、48h、72h,and tested the concentration of Caspase-3by the method of ELISA respectively.Through detection, we confirmed that Caspase-3is expressed in both normal endometrium and eutopic endometrium of endometriosis which are cultured in vitro. Caspase-3expression of eutopic endometrial glandular epithelial cell of endometriosis in proliferative phase is remarkably lower than that in normal endometrium(P<0.01); Caspase-3expression of eutopic endometrial glandular epithelial cell of endometriosis in secretory phase has no difference with that in normal endometrium(P>0.05). Caspase-3expression of normal endometrium in proliferative phase is lower than that in secretory phase(P<0.01); Caspase-3expression of eutopic endometrium of endometriosis in proliferative phase is also lower than that in secretory phase(P<0.01). It is explained that abnormal expression of Caspase-3is effected by different phases of menstrual cycle, and its low expression of eutopic endometrial glandular epithelial cells of endometriosis in proliferative phase may be mediated EMs. The serums of Guiling xiaoyi Decoction could promote Caspase-3expression of endometrial glandular epithelial cells in proliferative phase(P<0.01), and the effect of increase is more and more obvious with the concentration increasing of Guiling xiaoyi Decoction-serum and the time extending. The serums of Guiling xiaoyi Decoction could also promote Caspase-3expression of endometrial glandular epithelial cells in secretory phase(P<0.01), and the effect of increase is more and more obvious with the concentration increasing of Guiling xiaoyi Decoction-serum. It prompts that Guiling xiaoyi Decoction maybe induce apoptosis of the endometrial glandular epithelial cells in both proliferative and secretory phase, and can inhibit the implantation and growth of the cells. Then it may hinder the occurrence of endometriosis. But the glandular epithelial cells of endometriosis in secretory phase were interfered with the serums of Guiling xiaoyi Decoction, and promotive effect is not obvious with the time extending(P>0.05). We presumed that it may relate to higher apoptosis activities of the endometrial glandular epithelial cells in secretory phase. |
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