The Role Of Capn5in The Pathogenesis Of Endometriosis And The Effect Of TANⅡA

BackgroundEndometriosis is a common disease of fertile women, defined by the presence of ectopic endometrial glands and stroma, affecting approximately10-15%of fertile women,80%with pelvic pain,30-50%with algomenorrhea and50%with infertility. The incidence of endometriosis is raising recently, the disease performance diversity,so far the pathogenesis of it is unclear.Accumulated evidences suggest that abnormal expression of some genes in eutopic endometrium with endometriosis, such as the abnormal expression of apoptosis, is believed to contribute to the establishment and maintenance of the disease. Apoptosis is a type of cell death in which the cell uses specialized cellular machinery to kill itself. Cell suicide mechanism that enables metazoans to control cell number and eliminates cells that threaten the animal’s survival. An increase in cell proliferation without an equivalent change in apoptosis has been described.Calpain5is a member of the cytoplasmic cysteine protease family, a group of Ca++regulated enzymes. CAPNs in general, and in particular CAPN5, have been implicated in multiple cellular processes including cellular differentiation and apoptosis. CAPN5is expressed in human endometrium and regulated by HOXA10. The function of HOXA10in the uterus is well defined, HOXA10is a transcription factor that regulates a battery of target genes. The molecular mechanism by which HOXA10directs endometrial receptivity and decidualization includes the regulation of CAPN5in human endometrial cells. Both CAPN5and HOXA10are expressed in endometrial glandular epithelial cells and stromal cells throughout the menstrual cycle. This in turn likely regulates apoptosis of the endometrium. Endometriosis is prone to progression and invasion, exhibiting some characteristics of malignant tumor behavior, such as the invasion, distant metastasis, recurrence etc, so far the treatment measures for it is limited. The main components extracted from Salvia miltiorrhiza, a common herb, is Tan Ⅱ A. The antitumor function of Tan Ⅱ A dues to decrease in cell proliferation and regulate apoptosis.In order to research the pathogenesis of endometriosis and discuss the treatment with herb, a good model must be established, so that we can observe the differentiation and morphography of endometrial cells, to determine expression of CAPN5and HOXA10, and to discuss the effect of Tan Ⅱ A.Objective1.To establish endometriosis endometrial glandular epithelial cells and stromal cells in vitro co-culture model.2.Using the model, to observe and compare the growth and morphography of endometrial cells of endometriosis and controls.3.To determine and compare expression of CAPN5and HOXA10in each group.4.To determine and compare expression of CAPN5and HOXA10in endometriosis group after the effect of Tan Ⅱ A.5.To observe the growth and apoptosis of endometrial cells of endometriosis after the effect of Tan Ⅱ A.Methods1.Eutopic endometrium were obtained from20women with endometriosis as a group,13eutopic endometrium were obtained from women without endometriosis as controls. Isolated and culture primary glandular epithelial cells and stromal cells.2.To observe and compare the growth and morphogram of endometrial cells of endometriosis and controls with HE staining.3.Identification of endometrial cell by immunocytochemistry.4.RT-PCR was performed to determine expression of CAPN5and HOXA10 in each group.5.Western blot was performed to determine expression of CAPN5in each group.6.Annexin V-PE/7-AAD kit was performed to determine the apoptosis of each group.7.Estimate the apoptosis of endometrial cells of endometriosis with MTT cell proliferation assay after the effect of Tan II A.8.RT-PCR was performed to determine expression of Calpain5and HOXA10in endometrial cells of endometriosis after the effect of Tan II A.9.Western blot was performed to determine expression of CAPN5in endometrial cells of endometriosis after the effect of Tan II A.10.Annexin V-PE/7-AAD kit was performed to determine the apoptosis of endometrial cells after the effect of Tan II A.Results1.Both endometrial glandular epithelial cells and stromal cells grew well. the immunocytochemisty with antibodies against CK19and vimentin were positive.2.The endometrial cells obtained from controls share a similar morphogram with the eutopic endometrial cells of women with endometriosis, but grew slower.3.The result was confirmed using RT-PCR showed a decrease in HOXA10mRNA in endometriosis compared with endometrium obtained from controls (P<0.05).4.The results were confirmed using RT-PCR and western blot, that both also showed a similar decrease in CAPN5mRNA and protein, respectively, in endometriosis compared with endometrium obtained from controls (P<0.05).5.The result of apoptosis assay using Annexin V-PE/7-AAD kit showed a lower rate of eutopic endometrium in endometriosis compared with endometrium obtained from controls (P<0.05).6.MTT confirmed Tan Ⅱ A up-regulated the restriction of the eutopic endometrium cells of women with endometriosis in a time-dose manner(P<0.05).7.RT-PCR confirmed that, in the eutopic endometrium of women with endometriosis,there were significant increases in HOXA10mRNA expression after the effect of Tan Ⅱ A compared with non-dose controls (P<0.05).8.The results were confirmed using the RT-PCR, and western blot, that both showed a similar increases in CAPN5mRNA and protein after the effect of Tan Ⅱ A, respectively, in the eutopic endometrium of women with endometriosis compared with non-dose controls (P<0.05).9.The result of apoptosis assay using Annexin V-PE/7-AAD kit confirmed that Tan Ⅱ A up-regulated the apoptosis of the eutopic endometrium cells of women with endometriosis compared with non-dose controls(P<0.05).Conclusion1.Successfully established endometriosis in vitro cell co-culture model. Use this model we can get real time observation of the growth and morphography of endometrial cells from endometriosis and controls.2.In the eutopic endometrium of women with endometriosis, there were significant decreases in CAPN5mRNA and protein expression, as well as HOXA10mRNA, compared with fertile controls.3.Decreased apoptosis in the eutopic endometrium of women with endometriosis compared with endometrium obtained from controls implicate that, aberrant expression of CAPN5is associated with endometriosis.4.TanⅡA could higher the expression of CAPN5mRNA and protein, as well as HOXA10mRNA in a dose-manner.5.TanⅡA is associated with restricted proliferation and increased cell death of the eutopic endometrium of women with endometriosis, which can provide a theory of treating endometriosis with Chinese herb.