| Endometriosis(EMT) is a disease defined by the presence of viable endometrial tissue(glands and stroma) outside the uterine cavit. EMT is a common disease of women of childbearing age,the main performance of which is dysmenorrhea, chronic pelvic pain and infertility. The incidence of the disease has increased significantly in recent years, accounting for about 10%- 15% of women of childbearing age, 40%-60% of women dysmenorrhea,30%- 40% combined EMT in patients with infertility and 40%- 60% EMT patients suffer from infertility.EMT is a benign disease with a capacity similar as cancer,such as metastasis, invasion and growth. Ectopic endometrium may infringe any part of the body, mainly on the The pelvic viscera and peritoneum, but also on the uterus, womb rectum concave, peritonaeum viscerale,Vaginal rectum diaphragmatic,etc,So it is called pelvic EMT.In recent years, scholars have done a lot for EMT in basic and clinical researches, but have not figured out its clear etiology and pathogenesis yet and its treatment effect is very limited. EMT current treatment methods include expectant treatment, medication(endocrine) therapy, surgery, comprehensive treatment, etc, but in addition to radical surgery(removal of the uterus and two annexes), other treatment methods when stopped treatment or after surgery the recurrence rate can be up to 50%-60%.But women with EMT who were performed radical surgery may completely lost reproductive function and endocrine function, this is highly not applicable to patients of childbearing age. And currently used drugs have the side effects of weight gain, liver dysfunction and menopause syndrome, so they can not be used long time. Therefore, to find more effective treatments of EMT become an common concern issue for scholars.Cyclooxygenase-2(COX-2) is a rate-limiting enzyme which can phospholipid arachidonic acid catalyzed synthesis of prostaglandins substances, its production of prostaglandins that have the inhibitory effect of cell apoptosis, immune surveillance, involved in inflammation and cancer and can cause pain through multiple pathways is an important class of inflammatory cytokines. COX-2 gene was first reported occurrence of gastrointestinal tumors, subsequent studies found that COX-2 gene involved in a variety of ways by a variety of solid tumors and EMT occurrence and development.Small interfering RNA(si RNA) technology is a newly developed gene therapy means, it can be removed or shut down specific genes, and with the advantages of economy, fast, efficient, high stability and diffusible, etc, so it has been widely used to explore the gene function, as well as the therapeutic of cancer and infectious diseases and the like. Establishment and development of the technology, both in theory and practice provides a new strategy for the treatment of EMT.ObjectiveThis paper is to investigate the effect of targeted interruption of COX-2 gene through RNA interference(which is triggered by si RNA) on Cyclooxygenase-2(COX-2), Vascular endothelial growth factor(VEGF),matrix metalloproteinase-9(MMP-9), the protein expression and the apoptosis of eutopic and ectopic endometrial stroma cells with endometriosis, and to discuss the feasibility of endometriosis treatment through targeted silencing COX-2 gene by si RNA.Materials and methods1. Object of study60 cases of patients with EMT(30 cases of ectopic endometrium and 30 cases of eutopic endometrium) and 10 cases of patients with other gynecological benign diseases were selected with laparoscopy or laparotomy in The Third Affiliated Hospital of Zhengzhou University between 2013.12 and 2014. 09.2. Methods⑴ 30 normal endometrial stroma cells and 30 ectopic endometrial stroma cells with endometriosis are isolated and cultured and classified into three groups: si RNA interference group, negative control group and blank control group. si RNA interference group is injected into si RNA transfection complex(including 150 pmol COX-2 si RNA and LipofectamineTM 2000 5ul) and negative control group is injected into negative control transfection complex(including 150 pmol negative control si RNA and LipofectamineTM 2000 5ul). 10 normal endometrial stroma cells without endometriosis are the normal control group.⑵ Real time RT-PCR detected the m RNA expression of COX-2, VEGF, MMP-9 of pre-transfected and post-transfected eutopic and ectopic endometrial stroma cells with endometriosis. 48 hours after transfection, one si RNA sequence with the best inhibitory effect screened by Real time RT-PCR is used in the following experients.⑶ Western Blot detected the protein expression of COX-2, VEGF, MMP-9 of pre-transfected and post-transfected eutopic and ectopic endometrial stroma cells with endometriosis.⑷ 48 hours after transfection, the apoptosis of cells is detected by Flow Cytometry.Results1. 35, 32 eutopic endometrial stroma cells and ectopic endometrial stroma cells with endometriosis are cultured respectively, with 30 successfully cultured cells for each kind. 10 normal endometrial stroma cells without endometriosis are all successfully cultured.2. 3 COX-2 si RNA sequences have certain inhibitory effect, the inhibition ratio of si RNA-2 was the best.3. The COX-2, VEGF, MMP-9 mRNA expression of the blank control group are all higher than those of the normal control group(P<0.05) and ectopic endometrium is obviously more than eutopic endometrium(P<0.05). After interruption COX-2 gene, the COX-2, VEGF, MMP-9 m RNA expression of the eutopic endometrial stroma cells and ectopic endometrial stroma cells in interference group are obviously lower than those of negative control group and blank control group. The discrepancy between the latter two groups has no statistical significance(P>0.05). Besides, the COX-2, VEGF, MMP-9, and protein expression of ectopic endometrial stroma cells decrease greatly than those of the eutopic endometrial stroma cells.4. The COX-2, VEGF, MMP-9 protein expression of the blank control group are all higher than those of the normal control group(P<0.05) and ectopic endometrium is obviously more than eutopic endometrium(P<0.05). After interruption COX-2 gene, the COX-2, VEGF, MMP-9 protein expression of the eutopic endometrial stroma cells and ectopic endometrial stroma cells in interference group are obviously lower than those of negative control group and blank control group. The discrepancy between the latter two groups has no statistical significance(P>0.05). Besides, the COX-2, VEGF, MMP-9, and protein expression of ectopic endometrial stroma cells decrease greatly than those of the eutopic endometrial stroma cells.5. Flow Cytometry reveals that the apoptosis of the eutopic endometrial stroma cells and ectopic endometrial stroma cells in si RNA interference group increases compared with those of negative control group and blank control group. Besides, the discrepancy between the latter two groups has no statistical significance(P>0.05), and the apoptosis of ectopic endometrial stroma cells is more serious than that of the eutopic endometrial stroma cells(P<0.05).Conclusion1. COX-2 gene may play a significant role in endometriosis.2. The targeted interruption COX-2 gene by si RNA can efficiently inhibit the VEGF, MMP-9 m RNA and protein expression and greatly increase the apoptosis rate of the eutopic endometrial stroma cells and ectopic endometrial stroma cells with endometriosis.3. Culture endometrial stromal cells in is an ideal experimental model to study endometriosis.4. whether the targeted interruption COX-2 gene by si RNA may be a new and feasible method in endometriosis treatment is an question. |
PDF Full Text Request