The Expression And Significiance Of OPN & MMP9 In Endometriosis Of Rats

BackgroundThe endometrosis (EMS) is a common disease in reproductive age women, followed by clinical signes of 80% chronical pelvical pain,30% of gynecological operations and 20% of infertility.Although studys from theory and clinics show that EMS is successive homonal depended pathological changes, beginning from cells to the pelvic pain, infertility and pelvical mass in clinics. However, it's a difficult question in clinics with unclear mechanism and limited treatment. Operation is the first choise for its treatment. But it is differcult to remove all the signs and it is not superise to be recurrance. Operation combined medicine treatment can inhabbit the rest of the disease, delay the reocurance, and it shows that the medicine for anti-adherisve, anti-invasive, ant-angiogenesis is the next target for the endometriosis. Therefore, further study for the mechanism of ectope adhersion and invasion as well as effective signal molecule will be useful to research the medicine target and latter medicine treatment,and instruction the cilincal treatment and follow-up.Osteopontin (OPN) functions as both a cell attachment protein and a cytokine that signals through two cell adhesion molecules avb3-integrin and CD44, is an acidic hydrophili glycophosphoprotein. Potential steps that may involve OPN include tumor cell attachment to basement membrane through cell-surface adhesion molecules, proteolytic degradation of the extracellular matrix (ECM) and tumor cell migration through the ECM. Studys from Zhu Yaokui and Ma Chaihong has identified that OPN protein appeared higher in ectopic and uctopic endometrium in patients with endometriosis;Matrix metalloproteinase-9(MMP-9), the largest enzyme for degradation of extra cellular matrix, is playing an important role involved in adherision,implantation and growth of ectopic endometrium cells. Investigators has showed that MMP-9 plays a direct role in OPN-induced cell migration, invasion and tumor growth and that demonstrates(see the film attached). Although endometrosis is a benige desiease, it has clear malignant behavior such as invasion, transfer and recurance. However, it is nover to be reported about OPN and MMP-9 in endometriosis.Experiments confirmed:OPN gene 5'upstream side of the area pregnant hormone regulatory elements. Progesterone receptor levels can be adjusted OPN. The MMP-9 upstream of estrogen binding region. Or side by estrogen pathway factor regulating the level of MMP-9. Our previous study also showed that OPN and MMP9 were expressed in patients with endometriosis and normal eutopic and ectopic endometrium. And ectopic endometrium was significantly higher than the normal control group. Prompted the development of both play an important role in the EMS. But also by the regulation of ovarian estrogen and progesterone. Thus, we infer whether estrogen and progesterone by regulating the expression of OPN and MMP9 play a role in cell adhesion?GnRH-A, high progesterone preparations gestrinone and its receptor antagonist mifepristone are the clinical treatment of endometriosis in first-line drugs.Studies have shown that these drugs through the regulation and inhibition of the hypothalamus - pituitary - ovarian axis function competitive inhibition of the local receptor binding reduce the concentration of estrogen in the local lesions and cause ectopic disease atrophy. These drugs can also directy effect on the local lesions, transforming the molecular biology environment of Pathological changes inhibiting cell apoptosis and increaseing cell apoptosis ability.But the exact mechanism is unclearAnd the different mechanisms of drug action.In particular, whether the change in drug treatment in the molecular mechanism of membrane is not clear.The topic to be selected the changed expression of OPN/MMP9 when leuprolide, gestrinone other hormone-related drugs treating ectopic and eutopic for research purposes. Through the establishment and evaluation of rat endometriosis model. Using experimental methods such as RT-PCR. Discussion of changes in drug intervention organization OPN/MMP9 protein and mRNA. Further revealed endometriosis in the OPN and the relationship between the development of EMS. The possible mechanism of drug action,The drug of choice for clinical treatment of a reasonable and reliable basis and more indicators of prognosis. Part OneEstablishment and Estimate of an Endometriosis Model by Openning- method in RatsObjectiveTo research the reliability, estimate index and achievement ratio of an endometriosis model by openning-method in rats compared with transfering-method..MethodFemale virginal rats were operated with openning-method (lefe uterus) and transfering-method (right uterus). In left side of the uterine, endometrum was stick to the uterosacro ligament or omentum majus while right side of the uterine was taken and the endometrium was implanted to the right abdominal wall. Second operation was taken 4 weeks after implantation. Samples from the rats were examined by OPN antibody and MMP9 antibody.Results1.The achievement ratio in both mothed model:The achievement ratio of openning-mothed model is 81.08%, lower than that of 83.78% in transpering-method model without statistical significiance.2.The different signs in both mothed model:Clear vesicle is main gross appearance with 64.8% of all rats, the dark bule nodus are the second appearance with 10.81% in transfer-method model.However, the dark bule nodus is the main in openning-method model with 35.13% while Clear vesicle is the second with 24.3%. Extensive adherent rate in transfering-method model is 18.9%, significient lower that of 64.86% in openning-method model (P<0.01).There is no different in both intestinal obstruction and infection rate between two groups (P>0.05).3.The comparion between clinical manifestation and microscope features:the typical and atypical gland and endometrial cells could be found in 72,7% nodules or vesicles, and 68.4% flame signes with a strong staining of OPN and MMP9 in the ectopic endometrium.Conclusion The appearance of animal endometriosis in openning-method model is more similar to the human endometriosis in appearance with feature of reliable, large eara, convenient and less complication. Part Two: The Expression and Significiance of OPN &MMP9 in Endometriosis of RatsObjectiveAnalysis of differences between OPN and MMP9 endometriosis eutopic and ectopic lesions in the protein, gene in rats. Discussion of OPN and MMP9 expression in ectopic endometrium features and the relationship between OPN expression and MMP9 in endometriosis.MethodSuccess of the 37 pre-built model rats, the second surgery laparotomy after the model 28 days, the rat's reign (reigned group), ectopic endometrial tissue (ectopic group) and eutopic endometrium former model (control group), determination of the expression of intima OPN and MMP9mRNA, protein.Results1.OPN mainly highly expressed in the plasma membrane of epithelial cellsand a small amount of expression of interstitial.But MMP9 was mainly expressed in the cytoplasm of glandular epithelial cells and no expression in the nucleus;2. Immunohistochemistry showed that OPN expression in ectopic endometrium IOD of 53735.4±4896. Group was significantly higher than that of eutopic 33733.8±5322 and a control group of 37291.2±4588. (T reign-ectopic=17.010; P=0.000; t control-ectopic=9.122; P=0.000). Control group and the reign of group no significant difference in IOD (t=1.973, P=,0.52);3. The IOD of MMP9 expressed in ectopic endometrium group was 32645±9967. Higher than that of eutopic endometrium and control groups(T reign-ectopic= 8.559; P=0.000; t control-ectopic=5.386; P=0.000), while in office and control groups showed no significant difference (t=0.198, P=,0.844).4. The expression of OPN and MMP9 significant positive normal relationship. (R reign=0.511, P=0.036; r ectopic=0.600, P=0.015).Conclusion 1.The expression of OPN and MMP9 in eutopic and ectopic endometrium in rats are highly;2.The expression of OPN and MMP9 correlated with positive relationship. They may exist in the regulation of cell information between cascade regulation. Part ThreeThe Impact of Estrogen-related Drugs on the Expression of OPN/MMP9 in Rat Endometrium of EndometriosisObjectiveAnalysis of the relationship between estrogen and progesterone-related drugs and the expression of OPN/MMP9 in rat endometrium of endometriosis. Explore the possible mechanism of the relationship between Estrogen and progesterone and OPN/MMP9 and clinical drug treatment. Individual drugs for clinical therapy.Methods1.24 cases of successful model rats were randomly divided into four groups.There are 6 cases in groupⅠof Wagner Ruil:The dose of 7.5ug/250g was done by subcutaneous injection for every day till one month. There are 7 cases in groupⅡ:The mifepristone group:0.62mg/250g/day for a month. There are 7 cases in groupⅢ:gestrinone group:0.062mg/250g/day twice a week for one month. Control group, There are 6 cases in groupⅣ:yeast tablets.2..Rats were killed after one month, take eutopic and ectopic endometrium, RT-PCR, and immunohistochemical determination of OPN and MMP9 protein, gene expression.Results1. The diameter in each group after drug:The diameter of the cysts in each group after drug intervention reduced than before. But there was no difference between treatment.2. The OPN protein expression before and after drug intervention in eutopic and ectopic endometrium:Before the intervention, OPN expression in eutopic endometrium in each group were lower than in ectopic endometrium (P<0.05). The OPN express in drug intervention group eutopic and ectopic endometrium difference disappeared. However, the expression of the control group remained below eutopic endometriotic tissues (P<0.05). OPN expression of each group after the intervention and control group at eutopic and ectopic endometrium significantly lower than before intervention. (P intervention<0.01, P were<0.05). Before the intervention, eutopic and ectopic endometrium in each group showed no significant difference between two(F reign=0.161, P=0.921, F ectopic=2.550, P=0.082). After the intervention, eutopic endometrium (F=3.45 P=0.034) higher than the control group compared mifepristone group and GnRH groups. Ectopic endometrium in the control group was higher than the other three groups. Between GnRH group and Gestrinone mifepristone group and the group no significant difference.3. The MMP9 express in eutopic and ectopic endometrium after and before drug intervention:Each group before drug intervention MMP9 expression in eutopic endometrium was significantly lower than in ectopic endometrium (P=0.000). Drug intervention group, MMP9 expression in eutopic and ectopic endometrium was no difference (P> 0.05). Ectopic endometrium in the control group, the expression of MMP9 expression is still higher than in eutopic endometrium (P<0.05). MMP9 expression in each group before the intervention in place, there were no differences in ectopic endometrium. (F reign=2.827, P=0.062; F ectopic=2.790 P=0.064) Mifepristone group, gestrinone group and GnRH after the interventiond eutopic and ectopic endometrium in MMP9 expression was significantly lower than control group. MMP9 in mifepristone group eutopic and ectopic endometrium was ower than the Gestrinone group. Gestrinone group compared with the GnRH group was no difference in the eutopic endometrium. However, MMP9 in ectopic endometrium was 13291±2914, higher than the GnRH group,3951±265 (P=0.00).4.The expression differences at before and after intervention in eutopic and ectopic endometrium4. OPNmRNA, MMP9mRNA expression in endometrium:4.1 OPNmRNA expression:Before the intervention, OPNmRNA expression in eutopic endometrium wa significantly lower than ectopic endometrium. (P<0.001). However, compared with the reign and the reign of each group,eutopic and ectopic no difference (F reign=9.365, P=0.001; F ectopic=25.249, P=0.000); After the intervention of any two control groups in eutopic and ectopic expression in OPNmRNA higher than the other three groups.However the mifepristone group, gestrinone group, GnRH group had no difference in the three groups. 4.2 MMP9mRNA expression:Before the intervention, eutopic and ectopic endometrium compared P<0.001. the expression of MMP9 mRNA in the intervention group eutopic and ectopic endometrium was no difference. The control group significantly (p<0.001).After the intervention, MMP9mRNA expressed in eutopic and ectopic endometrium were lower than the control group. In gestrinone group ware lower than in the mifepristone group and the GnRH group (P<0.001) and no differences among other groups.Conclusion1.Drug intervention changed the environment for local lesion of the molecular biology, however, the size of the cysts had little effect.2.By changing the focus of drug intervention of local expression of OPN and MMP9. Play the treatment by adjusting adhesion and invasion of the ectopic cell.3.In the drugs inhibit the expression of OPN/MMP9 in the local lesions, GnRH-a group better than the mifepristone group and gestrinone group, the ectopic endometrium better than the eutopic endometrium.