雌激素调节猪炎性子宫内膜细胞的分子机制

发布时间：2018-05-28 18:55

本文选题：雌激素 + 猪　；参考：《西南大学》2017年硕士论文

【摘要】：母猪子宫内膜炎是指母猪子宫黏膜的粘液性或化脓性炎症,可造成母猪发情异常,是造成规模化养猪生产中母猪高淘汰率的最重要疾病之一,给养猪业造成巨大损失。有调查显示,在养猪业中,患有子宫内膜炎的分娩母猪高达20%。子宫内膜炎主要由病毒、细菌及寄生虫等引起。目前,对子宫内膜炎的治疗主要采用抗生素、中药、生物制剂等进行治疗,虽然具有较好的效果,但是,以上治疗方法并不能彻底治疗该病,且存在一定的副作用,严重影响患畜病后的繁殖能力。因此,对于家畜子宫内膜炎,应该防重于治,在弄清其发病机制的基础上,采取更加有效的防治手段,最终使该病得到有效控制。雌激素主要成分是17β-雌二醇(17β-E2),子宫是雌激素作用的重要靶器官。研究表明,多种子宫疾病均与雌激素有密切联系,雌激素分泌情况的异常通常会导致某些子宫疾病。国内外研究证明,雌激素可以调节子宫内膜炎。本实验利用体外培养的猪子宫内膜上皮细胞,探究17β-E2对母猪子宫内膜上皮细胞炎性因子的调控机制。其主要目标为:(1)?体外培养并纯化猪子宫内膜上皮细胞;(2)建立在体外条件下猪子宫内膜上皮细胞的炎症模型;(3)探究17β-E2对子宫内膜上皮细胞NO、TNF-α、IL-1表达的调控作用;(4)探究雌激素α受体拮抗剂在17β-E2调控子宫内膜上皮细胞炎性因子表达过程中的作用;(5)探究JNK信号通路在17β-E2调控子宫内膜上皮细胞炎性因子表达过程中的作用。研究如下:(1)实验选取健康母猪正常子宫,将其子宫内膜剥离,进行原代培养、纯化和传代培养,最终获得体外子宫内膜上皮细胞,经过角蛋白免疫荧光染色鉴定,子宫内膜上皮细胞的纯化率达95%以上,为后续研究提供了高纯度、符合要求的细胞;(2)为了探究制备猪子宫内膜上皮细胞炎症模型需要的LPS剂量,实验选取1μg/mL,10μg/mL,100μg/mL的脂多糖刺激体外培养的子宫内膜上皮细胞12h。通过MTT试验测定细胞的活性,并通过NO试剂盒、ELISA、RT-PCR方法检测细胞培养液中NO、TNF-α、IL-1和NOS、TNF-α、IL-1的mRNA的表达量。结果显示,10μg/mL的LPS能显著提高子宫内膜上皮细胞的活性且能增加NO、TNF-α、IL-1和NOS、TNF-α、IL-1的mRNA的表达量,与对照组相比,差异显著(P0.05);LPS的浓度和炎性因子的分泌量呈剂量相关性,但由于100μg/mL的LPS显著降低了细胞活性,因此后期实验选取10μg/mL的LPS对子宫内膜上皮细胞进行刺激,制作炎症模型;(3)为了探究17β-E2对子宫内膜上皮细胞炎性因子的影响,采用10μg/mL的LPS刺激子宫内膜上皮细胞12h后,分别选取10-6M,10-7M,10-8M的17β-E2处理激发培养的子宫内膜上皮细胞,通过NO试剂盒、荧光定量PCR和ELISA分别检测在2h、6?h、12?h、24?h的NO、TNF-α、IL-1和NOS、TNF-α、IL-1的mRNA表达水平。结果显示,三种浓度的17β-E2均不同程度的降低了炎性因子的表达量,且这种降低程度与17β-E2的浓度呈正比。(4)为了探究雌激素受体α在17β-E2调控子宫内膜上皮细胞炎性因子中的作用,实验用10μg/mL LPS激发培养12h后,加入含浓度为10-6M 17β-E2、10-6M17β-E2+三氧苯胺(TAM)的培养液,继续培养24h,通过NO试剂盒、ELISA、荧光定量PCR检测NO、NOS、TNF-α、IL-1的表达水平。结果发现,LPS+17β-E2组炎性因子的分泌显著降低,而LPS+17β-E2+TAM组炎性因子的分泌与LPS+17β-E2组差异显著,与LPS组差异不显著,表明雌激素受体α在17β-E2调节炎性因子表达过程中发挥了重要作用。(5)为了了解JNK信号转导通路在17β-E2对子宫内膜细胞炎性因子调节过程中的作用,实验测定了LPS组、LPS+17β-E2组以及LPS+TAM+17β-E2的P-JNK和JNK蛋白的表达量,发现LPS组的P-JNK水平显著提高,而加入17β-E2后则显著降低了P-JNK的表达水平,加入TAM后P-JNK的蛋白表达量比LPS+17β-E2组显著提高,与LPS组无显著差异。表明雌激素可能通过与雌激素受体α作用,抑制JNK路径,从而抑制LPS刺激下的细胞因子的表达。综上所述,该实验可以得到以下结论:1.本实验成功培养了猪子宫内膜上皮细胞并建立了该细胞炎症模型。2.雌激素可抑制LPS引起的子宫内膜上皮细胞的炎性因子的NO、TNF-α、IL-1分泌和NOS、TNF-α、IL-1 mRNA的表达。3.雌激素通过与子宫内膜上皮细胞上的受体α结合,抑制NO、TNF-α、IL-1的分泌和NOS、TNF-α、IL-1 mRNA的表达。4.雌激素通过与雌激素受体α作用,可能经过抑制JNK路径,从而抑制LPS刺激下的细胞因子的表达。
[Abstract]:Sow endometritis is a mucous or suppurative inflammation of the sow's uterine mucosa, which can cause abnormal oestrus in the sow, one of the most important diseases that cause the high elimination rate of sows in the production of large-scale pig production, causing huge losses to the pig industry. Meningitis is mainly caused by viruses, bacteria and parasites. At present, the treatment of endometritis mainly adopts antibiotics, traditional Chinese medicine, biological agents and so on, although it has good effect, but the treatment methods can not completely treat the disease, and there are certain side effects, which seriously affect the reproductive ability after the disease. Therefore, In the case of domestic animal endometritis, we should prevent weight from treatment. On the basis of making clear the mechanism of its disease, we should take more effective control methods, and finally make the disease effective control. The main ingredient of estrogen is 17 beta estradiol (17 beta -E2). The uterus is an important target organ for estrogen, and the study shows that various uterine diseases are closely related to estrogen. Association, abnormal estrogen secretion usually leads to some uterine diseases. Domestic and foreign studies have shown that estrogen can regulate endometritis. This experiment uses cultured porcine endometrium epithelial cells to explore the regulatory mechanism of 17 beta -E2 on the inflammatory factors of the endometrium epithelial cells in sows. The main objectives are: (1) in vitro culture. And purify the epithelial cells of porcine endometrium; (2) to establish an inflammatory model of the endometrium epithelial cells in vitro, and (3) to explore the regulation of 17 beta -E2 on the expression of NO, TNF-, and IL-1 in endometrial epithelial cells; (4) to explore the role of estrogen receptor antagonist in the regulation of the expression of inflammatory factors in endometrial epithelial cells by 17 beta -E2; 5) explore the role of JNK signaling pathway in the regulation of the expression of inflammatory factors in endometrial epithelial cells by 17 beta -E2. (1) the study was as follows: (1) the experiment selected healthy sow's normal uterus, stripped the endometrium, carried out the primary culture, purified and passed the culture, and finally obtained the endometrial epithelial cells in vitro, and passed the keratin immunofluorescence staining. The purification rate of endometrium epithelial cells was over 95%, which provided high purity and conforming to the required cells for follow-up study. (2) in order to explore the LPS dose needed to prepare the inflammatory model of porcine endometrial epithelial cells, the experiment selected 1 mu g/mL, 10 mu g/mL, and 100 micron g/mL to stimulate the endometrial epithelial cells of the endometrium in vitro through MTT test. Test the activity of cells and detect the expression of NO, TNF-, IL-1 and NOS, TNF- a, IL-1 in cell culture solution by NO kit, ELISA and RT-PCR. The results show that the 10 mu g/mL LPS can significantly increase the activity of endometrium epithelial cells and increase the expression of alpha, alpha and alpha, compared with the control group, The difference was significant (P0.05); the concentration of LPS and the secretion of inflammatory factors were dose-dependent, but the 100 u g/mL LPS significantly reduced the cell activity, so the later experiment selected 10 mu g/mL to stimulate the endometrium epithelial cells and make the inflammatory model. (3) to explore the effect of 17 beta -E2 on the inflammatory factors of endometrium epithelial cells. The endometrial epithelial cells of the endometrium were stimulated by 10 g/mL LPS, and 10-6M, 10-7M, 10-8M 17 beta -E2 were selected to excite cultured endometrium epithelial cells. Through NO kit, fluorescence quantitative PCR and ELISA were detected in 2H, 6, h, 12, 24, respectively. The results showed that three concentrations of 17 were 17. Beta -E2 decreased the expression of inflammatory factors in varying degrees, and the degree of reduction was proportional to the concentration of 17 beta -E2. (4) in order to explore the role of estrogen receptor alpha in the regulation of inflammatory factors in endometrial epithelial cells by 17 beta -E2, the experiment was stimulated by 10 mu g/mL LPS to add a concentration of 10-6M 17 beta -E2,10-6M17 beta -E2+ three oxygen benzene. The expression level of NO, NOS, TNF- alpha and IL-1 in the medium of amine (TAM) was continued by NO kit, ELISA, and fluorescence quantitative PCR. The secretion of inflammatory factors in LPS+17 beta -E2 group decreased significantly, and the secretion of inflammatory factors in the LPS+17 beta group was significantly different from those of the group, which showed that the difference was not significant, indicating that the estrogen receptor alpha was not significant. 17 beta -E2 played an important role in regulating the expression of inflammatory factors. (5) in order to understand the role of the JNK signal transduction pathway in the regulation of inflammatory cytokines in endometrial cells by 17 beta -E2, the expression of P-JNK and JNK proteins in group LPS, LPS+17 beta -E2 and LPS+TAM+17 beta -E2 was measured, and the P-JNK level in LPS group was significantly improved. After adding 17 beta -E2, the expression level of P-JNK was significantly reduced. After adding TAM, the protein expression of P-JNK was significantly higher than that of the LPS+17 beta -E2 group, and there was no significant difference from the LPS group. It was suggested that estrogen may inhibit the JNK path through the action of estrogen receptor alpha and inhibit the expression of cytokines under the stimulation of LPS. To the following conclusions: 1. the porcine endometrium epithelial cells were successfully cultured and the inflammatory model.2. was established to inhibit the inflammatory factors of the endometrium epithelial cells induced by LPS, NO, TNF- a, IL-1 secretion and NOS, TNF- a, and IL-1 mRNA expression of.3. estrogens through binding to receptor alpha on endometrial epithelial cells. The secretion of NO, TNF- alpha, IL-1 and the expression of NOS, TNF- a, IL-1 mRNA, the expression of.4. estrogens through the action of estrogen receptor alpha, may inhibit the JNK path, thus inhibiting the expression of cytokines under LPS stimulation.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28

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