| Background:Endometriosis is a common disease of fertile women. Though it is a benign disease, it has the ability to transfer far and implant. The rate of malignant change is0.7%-1.0%and the recurrence rate is high. The performance of this disease is diversity. It can cause dysmenorrheal, chronic pelvic pain, abnormal menstruation, infertility, dyspareunia etc, and thus influence women’s health and life seriously. So far the pathogenesis of it is unclear. Among so many theories,’the theory of retrograde menstruation’is more popular. But it can’t explain it well because the rate of retrograde menstruation is about80%-90%, much higher than the morbidity (10%-15%). On the basis of retrograde menstruation theory, Jinghe Lang offered the theory ’entopic endometrium determinism’which point out that the abonormal biological behavior leading the occurrence of endometriosis, and retrograde menstruation is only a bridge. This theory provided new idea and new direction for deep research of this disease.Although it is a benign disease, it is similar to malignant tumor with the ability of invasiveness. So we speculated that there is something different between entopic and ectopic endometrium which can make entopic endomctrial cell has the ability to invade. Paxillin is a tumor related factor which has been proved that it’s abnormal expression can induce tumor cell morphology change and distant metastasis. However there is no research pay attention to the relationship between Paxillin and endometriosis. Paxillin is an important cell conglutination factor mainly located in focal adhesion which is a bond between focal adhesion and extracellular matrix. As an adaptor protein in the cell, Paxillin can combine a series of signal proteins and structural proteins such as vinculin, tubulin, actin etc which are prerequisite in embryonic development, wound repair and tumor metastasis. Paxillin is not only involved in the assembly of focal adhesions, but also plays an important role in cell signal transduction, cell morphology, adhesion and movement. Because Paxillin is widely present in human tissues, so we speculated that it may have some correlation with invasion of endometriosis cells. To observe the different expression of Paxillin between entopic endometrial cells of endometriosis and normal endometrial cells is crucial. Baicalein is a flavonoid extracted from Scutellaria and the main active component of Radix Scutellariae, also the material basis of pharmacological action. Research shows that baicalein has effects of antitumor, antioxidant, scavenging free radicals, antiviral, anti-inflammatory, anti-coagulant, antithrombotic. Baicalein can inhibit majority of tumor cell’s proliferation, induces apoptosis, inhibit motility and migration ability of tumor cells through the change of cell morphology, so to control tumor development. There are no research between baicalein and endometrial cell invasion currently. Because of the inhibitory of baicalein to tumuor cell invasion, we speculate it can also inhibit the endometrial cell invasiveness. Due to ethical constraints, many studies can’t do in human body, therefore, to establish good experimental model is of great significance to study of endometriosis. The experimental model for endometriosis has experienced a transformation from animal model to cell model. With the development of cell culture technology, establish endometriosis cell model in vitro model, provides great convenience for the observation of cell morphology, biological activity and drug intervention, and made great contribution to the research and treatment of endometriosis.Objective:1. Establish co-culture model of endometriosis entopic endometrial and normal endometrial glandular epithelial cells, stromal cells in vitro.2. To observe the growth and the biological activity in endometriosis entopic endometrial cells and normal endometrial cells, and doing immune identification.3. To observe protein and mRNA level of Paxillin in endometriosis entopic endometrial cells and normal endometrial cells.4. To observe protein and mRNA level of Paxillin in endometriosis entopic endometrial cells and normal endometrial cells after Baicalein treatment, and to explore the correlation between the cell invasion.Method:1.25cases of patients with endometriosis (endometriosis entopic endometrium group) and20cases of no endometriosis (control group) were taken. Digesting, primary cultured endometrial glandular epithelial cells and stromal cells.2. HE staining was taken to observe cell morphological characteristics in endometriosis group and control group.3. SP immunocytochemistry method was taken to identify cell4. RT-PCR method was taken to detect differential expression of Paxillin mRNA in two groups;5. Western blot method was taken to detect differential expression of protein Paxillin in two groups;6. MTT method was taken to detect the inhibition rate of cells with different concentration of baicalein in endometriosis group cell.7. Use Iranswell to observe of the cell invasion change in endometriosis group changes after baicalein treatment.8. Using RT-PCR method to detect expression level of Paxillin mRNA in endometriosis after baicalein treatment.9. Using Western blot method to detect expression level of protein Paxillin in endometriosis group;10. Observe the correlations between expression of mRNA and protein Paxillin and invasiveness.Result:1. Endometriosis was successful in22cases, the control group was successful in18cases. The successful establishment of endometrial glandular epithelial cells and stromal cells in co-culture models were established successfully and identified by HE staining and immunocytochemical method.2. The cell’s morphological characteristics were similar in two groups but the speed of cell growth in endometriosis group was faster than that of control group.3. The results of RT-PCR showed, the relative expression levels of endometrial cells in Paxillin mRNA (0.608±0.021) was significantly higher than that in control group (0.291±0.023), the difference was statistically significant (P<0.05).4. Western blot results showed that, the relative expression levels of Endometriosis Endometrium cell protein Paxillin (0.523±0.025) was significantly higher than the control group (0.226±0.022),the difference was statistically significant (P<0.05).5. MTT method showed baicalein could inhibit the growth of endometriosis entopic endometrial cells, and with the increase of the concentration of baicalin, the extension of time, the inhibitory rate increased, in a time-and dose-dependent.6. Oumol/L,20umol/L,40umol/L and80umol/L of baicalein solution were added in the entopic endometrium of endometriosis cells, after48h of treatment, detected by RT-PCR, displayed baicalein could reduce expression of Paxillin mRNA in entopic endometrial cells, Paxillin mRNA relative expression in each group were respectively0.606±0.019,0.528±0.018,0.41910.021,0.336±0.014, in a dose-dependent manner, with a significant difference between groups (P<0.05).7.Oumol/L,20umol/L,40umol/L and80umol/L of baicalein solution were added in the entopic endometrial cells of endometriosis, after48h of treatment, detected by Western-blot, displayed that baicalein could reduce expression of Paxillin protein in entopic endometrial cells of endometriosis. Protein Paxillin relative expression in each group were respectively0.520±0.014,0.438±0.026,0.376±0.021,0.298±0.015, in a dose-dependent manner, with a significant difference between groups (P<0.05).8. Oumol/L,20umol/L,40umol/L and80umol/L of baicalein solution were added in entopic endometrial cells of endometriosis. The invasiveness was detected after48h, the invasion cell number were separately8.80±0.548,6.40±1.140,3.80±0.837,2.20±0.836of these four groups, and the invasion cell number in normal group was0.20±0.447. Different between the groups was statistically significant (P<0.05). The cell’s invasiveness of endometriosis group was significantly higher than that of normal endometrial cells. With the increase of baicalein concentration, endometriosis group cell invasiveness was reduced, in a dose-dependent manner.Conclusion:1.This experiment successfully established co-culture model of endometriosis entopic endometrial cells in vitro and observed the similarities and differences of morphology and biological activity between entopic endometrial of endometriosis and normal endometrial cells.2.The expression of Paxillin mRNA, protein was higher in endometriosis group than those in the control group.3.The invasiveness of endometrial cells was higher in endometriosis group than that of control group.4.Baicalein can reduce the expression of Paxillin mRN’A and protein, and inhibit the invasion of entopic endometrial cells of endometriosis, in a dose-dependent manner. High expression of Paxillin gene and protein may be a cause of invasiveness in entopic endometrial cells of endometriosis.5.Baicalin can inhibit the invasiveness of endometriosis entopic endometrial cell, provides new ideas and methods for traditional Chinese medicine in the treatment of endometriosis. |
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