Paxillin在子宫内膜异位症在位子宫内膜的表达及黄芩素对其表达的影响
摘要
研究背景：子宫内膜异位症(endometriosis,简称内异症)是育龄妇女的常见病、多发病,该病虽为良性疾病,但具有类似恶性肿瘤浸润、生长的恶性生物学行为,且有0.7%-1.0%恶变率和较高的复发率。其病变广泛、形态多样。该病可引起痛经、慢性性盆腔痛、月经不调、性交痛、不孕等,严重影响女性的健康和生活。迄今为止内异症的发病机理尚未十分明了,关于内异症的发病机制,纵说纷纭,其中较为盛行的是Sampson的“经血逆流学说”,但经血逆流的现象较常见,发生率约为80%-90%,远高于内异症患病率(10%15%),“经血逆流学说”不能很好地解释这种现象。郎景和在“经血逆流学说”的基础上提出“在位内膜决定论”,指出在位内膜的异常生物学行为主导内异症的发生,经血逆流只是导致内异症发生的一个桥梁。该学说为内异症病机的的深入研究提供了新思想、新方向。
内异症虽然是良性疾病,但具有与恶性肿瘤相似的侵袭性。桩蛋白(Paxillin)是研究较多的与肿瘤侵袭性相关的信号衔接蛋白,广泛地存在于各物种的细胞浆内,主要定位于黏着斑,能够和一系列的信号蛋白和结构蛋白结合,将细胞外信号传递到细胞内,在细胞信号的传导中发挥重要作用。Paxillin通过传导细胞信号,在器官发育、损伤修复、细胞运动、细胞粘附等多项细胞活动中发挥重要作用。目前未曾有对Paxillin与内异症细胞侵袭性进行相关性研究。因Paxillin广泛存在于人体组织中,且其表达异常和细胞的运动有一定的相关性,因此我们推测其可能与内异症细胞的侵袭性有一定的相关性
黄芩素(baicalein)是从黄芩中提取的黄酮类化合物,是黄芩的主要有效成分,也是其发挥药理作用的物质基础。研究表明黄芩索具有抗凝、抗血栓、抗肿瘤、抗氧化、清除自由基、抗炎、抗病毒等作用。黄芩索具有抑制大多数肿瘤细胞增殖、诱导肿瘤细胞凋亡的作用,还可以通过改变细胞形态,抑制肿瘤细胞的运动和迁移能力,从而控制肿瘤发展。目前尚未有关于黄芩素和内异症在位内膜细胞侵袭性之间的研究。由于黄芩索对肿瘤细胞侵袭力的抑制性,我们推测其对内异症在位内膜细’胞的侵袭力亦有一定的抑制作用。
由于伦理学因素的限制,很多研究无法在人身上进行,因此,建立良好的实验模型对内异症的研究具有重要意义。内异症实验模型经历了从动物模型到细胞模型的转变,随着细胞培养技术的兴起,内异症细胞体外模型的建立,为观察细胞生长形态、生物学活性及药物干预提供了极大的便利,为内异症病因病机的研究及治疗措施的探索做出了巨大的贡献。
研究目的：
1.分别建立内异症在位子宫内膜及正常子宫内膜腺上皮细胞、基质细胞体外共培养模型。
2.观测内异症在位子宫内膜细胞及正常子宫内膜细胞的生长情况及生物学活性,并对其进行免疫鉴定。
3.观察内异症在位内膜细胞与正常内膜细胞中Paxillin mRNA及蛋白达水平的差异。
4.黄芩素干预后,检测内异症在位内膜细胞中Paxillin mRNA及蛋白达水平，并探讨其与细胞侵袭力之间的相关性。
方法：
1.取25例子宫内膜异位症患者分泌期在位子宫内膜(内异症组)和20例非子宫内膜异位症患者分泌期子宫内膜(对照组)组织,消化、离心洗涤、去除杂质后,混合培养子宫内膜腺上皮细胞与基质细胞。
2.HE染色观察内异症组与对照组细胞形态学特征
3.SP免疫细胞化学鉴定法鉴定细胞。
4.RT-PCR法检测Paxillin mRNA在两组细胞中的表达差异
5.Western blot法检测Paxillin蛋白在两组细胞中的表达差异
6.以不同浓度黄芩素作用于内异症组细胞,用MTT法测算细胞抑制率
7.应用transwell小室观测黄芩素干预后内异症组细胞侵袭力的改变。
8.RT-PCR法检测黄芩素干预后内异症组细胞Paxillin mRNA表达水平的变化情况。
9.Western blot法检测黄芩素干预后内异症组细胞Paxillin蛋白表达水平的变化。
10.观测Paxillin mRNA及蛋白表达水平和侵袭性的相关性
结果：
1.内异症组成功22例,对照组成功18例。成功建立子宫内膜腺上皮和间质细胞体外共培养模型,经HE染色及免疫组化鉴定培养的细胞的确为子宫内膜细胞。
2.内异症组与对照组腺细胞形态学特征相似,但内异症组细胞生长速度较对照组快。
3.RT-PCR结果显示,内异症在位内膜细胞Paxillin mRNA的相对表达水平(0.608±0.021)明显高于对照组内膜细胞Paxillin mRNA的相对表达水平(0.291±0.023),两组比较有统计学意义(P<0.05)。
4.Western blot结果显示,内异症组在位内膜细胞Paxillin蛋白的相对表达水平(0.523±0.025)明显高于对照组内膜细胞Paxillin蛋白表达水平(0.226±0.022),两者差异有统计学意义(P<0.05)。
5.MTT法显示黄芩素可以抑制内异症在位内膜细胞的生长,并随着黄芩素浓度的升高、作用时间的延长,其抑制率增加,呈时间-剂量依赖型。
6.将0umol/L.20umol/L.40umol/L.80umol/L的黄芩素溶液分别加入内异症在位内膜细胞中,干预48h后,经RT-PCR检测显示,黄芩素能够降低内异症患者在位内膜细胞Paxillin mRNA的表达水平,各组Paxillin mRNA的相对表达量分别为0.606±0.019、0.528±0.018、0.41910.021、0.336±0.014,呈剂量依赖性,组间比较有统计学意义(P<0.05)。
7.将0umol/L、20umol/L、40umol/L、80umol/I的黄芩素溶液分别加入内异症在位内膜细胞中,干预48h后,经western-blot检测显示,黄芩素能够降低内异症患者在位内膜细胞Paxillin蛋白的表达水平,各组Paxillin蛋白的相对表达量分别为0.520±0.014、0.438±0.026、0，376±0.021、0.298±0.015,呈剂量依赖性,组间比较有统计学意义(P<0.05)。
8.取0umol/L、20umol/L、40umol/L.80umol/L黄芩素干预48h后的内异症在位内膜细胞进行侵袭性检测,内异症未加药组、低浓度组、中浓度组、高浓度组及正常组侵袭细胞个数分别8.80±0.548、6.40±1.140、3.80±0.837、2.20±0.836、0.20±0.447,各组间差异有统计学意义(P<0.05)。内异症组细胞侵袭力均明显高于正常子宫内膜细胞,随着黄芩素浓度的升高,内异证组细胞侵袭力均降低,呈剂量依赖性
结论：
1、本实验成功建立了子宫内膜异位症在位子宫内膜细胞体外共培养模型,在细胞水平观测了内异症在位子宫内膜细胞和正常子宫内膜细胞形态及生物活性上的异同点。
2、内异症组位内膜细胞中Paxillin mRNA、蛋白的表达水平均高于于正常内膜细胞。
3、内异症组细胞侵袭力高于对照组细胞。
4、黄芩素可以降低内异症组细胞Paxillin mRNA及蛋白表达水平,并抑制内异症组细胞侵袭力,呈剂量依赖性。Paxillin基因及蛋白的高表达可能是引起内异症在位子宫内膜侵袭性增高的一个原因。
5、黄芩素能够抑制内异症在位子宫内膜细胞侵袭力,为中药治疗子宫内膜异位症提供了新的思路和方法。
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 cell
4. 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.
内异症虽然是良性疾病,但具有与恶性肿瘤相似的侵袭性。桩蛋白(Paxillin)是研究较多的与肿瘤侵袭性相关的信号衔接蛋白,广泛地存在于各物种的细胞浆内,主要定位于黏着斑,能够和一系列的信号蛋白和结构蛋白结合,将细胞外信号传递到细胞内,在细胞信号的传导中发挥重要作用。Paxillin通过传导细胞信号,在器官发育、损伤修复、细胞运动、细胞粘附等多项细胞活动中发挥重要作用。目前未曾有对Paxillin与内异症细胞侵袭性进行相关性研究。因Paxillin广泛存在于人体组织中,且其表达异常和细胞的运动有一定的相关性,因此我们推测其可能与内异症细胞的侵袭性有一定的相关性
黄芩素(baicalein)是从黄芩中提取的黄酮类化合物,是黄芩的主要有效成分,也是其发挥药理作用的物质基础。研究表明黄芩索具有抗凝、抗血栓、抗肿瘤、抗氧化、清除自由基、抗炎、抗病毒等作用。黄芩索具有抑制大多数肿瘤细胞增殖、诱导肿瘤细胞凋亡的作用,还可以通过改变细胞形态,抑制肿瘤细胞的运动和迁移能力,从而控制肿瘤发展。目前尚未有关于黄芩素和内异症在位内膜细胞侵袭性之间的研究。由于黄芩索对肿瘤细胞侵袭力的抑制性,我们推测其对内异症在位内膜细’胞的侵袭力亦有一定的抑制作用。
由于伦理学因素的限制,很多研究无法在人身上进行,因此,建立良好的实验模型对内异症的研究具有重要意义。内异症实验模型经历了从动物模型到细胞模型的转变,随着细胞培养技术的兴起,内异症细胞体外模型的建立,为观察细胞生长形态、生物学活性及药物干预提供了极大的便利,为内异症病因病机的研究及治疗措施的探索做出了巨大的贡献。
研究目的：
1.分别建立内异症在位子宫内膜及正常子宫内膜腺上皮细胞、基质细胞体外共培养模型。
2.观测内异症在位子宫内膜细胞及正常子宫内膜细胞的生长情况及生物学活性,并对其进行免疫鉴定。
3.观察内异症在位内膜细胞与正常内膜细胞中Paxillin mRNA及蛋白达水平的差异。
4.黄芩素干预后,检测内异症在位内膜细胞中Paxillin mRNA及蛋白达水平，并探讨其与细胞侵袭力之间的相关性。
方法：
1.取25例子宫内膜异位症患者分泌期在位子宫内膜(内异症组)和20例非子宫内膜异位症患者分泌期子宫内膜(对照组)组织,消化、离心洗涤、去除杂质后,混合培养子宫内膜腺上皮细胞与基质细胞。
2.HE染色观察内异症组与对照组细胞形态学特征
3.SP免疫细胞化学鉴定法鉴定细胞。
4.RT-PCR法检测Paxillin mRNA在两组细胞中的表达差异
5.Western blot法检测Paxillin蛋白在两组细胞中的表达差异
6.以不同浓度黄芩素作用于内异症组细胞,用MTT法测算细胞抑制率
7.应用transwell小室观测黄芩素干预后内异症组细胞侵袭力的改变。
8.RT-PCR法检测黄芩素干预后内异症组细胞Paxillin mRNA表达水平的变化情况。
9.Western blot法检测黄芩素干预后内异症组细胞Paxillin蛋白表达水平的变化。
10.观测Paxillin mRNA及蛋白表达水平和侵袭性的相关性
结果：
1.内异症组成功22例,对照组成功18例。成功建立子宫内膜腺上皮和间质细胞体外共培养模型,经HE染色及免疫组化鉴定培养的细胞的确为子宫内膜细胞。
2.内异症组与对照组腺细胞形态学特征相似,但内异症组细胞生长速度较对照组快。
3.RT-PCR结果显示,内异症在位内膜细胞Paxillin mRNA的相对表达水平(0.608±0.021)明显高于对照组内膜细胞Paxillin mRNA的相对表达水平(0.291±0.023),两组比较有统计学意义(P<0.05)。
4.Western blot结果显示,内异症组在位内膜细胞Paxillin蛋白的相对表达水平(0.523±0.025)明显高于对照组内膜细胞Paxillin蛋白表达水平(0.226±0.022),两者差异有统计学意义(P<0.05)。
5.MTT法显示黄芩素可以抑制内异症在位内膜细胞的生长,并随着黄芩素浓度的升高、作用时间的延长,其抑制率增加,呈时间-剂量依赖型。
6.将0umol/L.20umol/L.40umol/L.80umol/L的黄芩素溶液分别加入内异症在位内膜细胞中,干预48h后,经RT-PCR检测显示,黄芩素能够降低内异症患者在位内膜细胞Paxillin mRNA的表达水平,各组Paxillin mRNA的相对表达量分别为0.606±0.019、0.528±0.018、0.41910.021、0.336±0.014,呈剂量依赖性,组间比较有统计学意义(P<0.05)。
7.将0umol/L、20umol/L、40umol/L、80umol/I的黄芩素溶液分别加入内异症在位内膜细胞中,干预48h后,经western-blot检测显示,黄芩素能够降低内异症患者在位内膜细胞Paxillin蛋白的表达水平,各组Paxillin蛋白的相对表达量分别为0.520±0.014、0.438±0.026、0，376±0.021、0.298±0.015,呈剂量依赖性,组间比较有统计学意义(P<0.05)。
8.取0umol/L、20umol/L、40umol/L.80umol/L黄芩素干预48h后的内异症在位内膜细胞进行侵袭性检测,内异症未加药组、低浓度组、中浓度组、高浓度组及正常组侵袭细胞个数分别8.80±0.548、6.40±1.140、3.80±0.837、2.20±0.836、0.20±0.447,各组间差异有统计学意义(P<0.05)。内异症组细胞侵袭力均明显高于正常子宫内膜细胞,随着黄芩素浓度的升高,内异证组细胞侵袭力均降低,呈剂量依赖性
结论：
1、本实验成功建立了子宫内膜异位症在位子宫内膜细胞体外共培养模型,在细胞水平观测了内异症在位子宫内膜细胞和正常子宫内膜细胞形态及生物活性上的异同点。
2、内异症组位内膜细胞中Paxillin mRNA、蛋白的表达水平均高于于正常内膜细胞。
3、内异症组细胞侵袭力高于对照组细胞。
4、黄芩素可以降低内异症组细胞Paxillin mRNA及蛋白表达水平,并抑制内异症组细胞侵袭力,呈剂量依赖性。Paxillin基因及蛋白的高表达可能是引起内异症在位子宫内膜侵袭性增高的一个原因。
5、黄芩素能够抑制内异症在位子宫内膜细胞侵袭力,为中药治疗子宫内膜异位症提供了新的思路和方法。
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 cell
4. 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.
引文
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