本文選題：雌激素 + 豬��； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：母豬子宮內(nèi)膜炎是指母豬子宮黏膜的粘液性或化膿性炎癥,可造成母豬發(fā)情異常,是造成規(guī)模化養(yǎng)豬生產(chǎn)中母豬高淘汰率的最重要疾病之一,給養(yǎng)豬業(yè)造成巨大損失。有調(diào)查顯示,在養(yǎng)豬業(yè)中,患有子宮內(nèi)膜炎的分娩母豬高達(dá)20%。子宮內(nèi)膜炎主要由病毒、細(xì)菌及寄生蟲等引起。目前,對子宮內(nèi)膜炎的治療主要采用抗生素、中藥、生物制劑等進(jìn)行治療,雖然具有較好的效果,但是,以上治療方法并不能徹底治療該病,且存在一定的副作用,嚴(yán)重影響患畜病后的繁殖能力。因此,對于家畜子宮內(nèi)膜炎,應(yīng)該防重于治,在弄清其發(fā)病機制的基礎(chǔ)上,采取更加有效的防治手段,最終使該病得到有效控制。雌激素主要成分是17β-雌二醇(17β-E2),子宮是雌激素作用的重要靶器官。研究表明,多種子宮疾病均與雌激素有密切聯(lián)系,雌激素分泌情況的異常通常會導(dǎo)致某些子宮疾病。國內(nèi)外研究證明,雌激素可以調(diào)節(jié)子宮內(nèi)膜炎。本實驗利用體外培養(yǎng)的豬子宮內(nèi)膜上皮細(xì)胞,探究17β-E2對母豬子宮內(nèi)膜上皮細(xì)胞炎性因子的調(diào)控機制。其主要目標(biāo)為:(1)?體外培養(yǎng)并純化豬子宮內(nèi)膜上皮細(xì)胞;(2)建立在體外條件下豬子宮內(nèi)膜上皮細(xì)胞的炎癥模型;(3)探究17β-E2對子宮內(nèi)膜上皮細(xì)胞NO、TNF-α、IL-1表達(dá)的調(diào)控作用;(4)探究雌激素α受體拮抗劑在17β-E2調(diào)控子宮內(nèi)膜上皮細(xì)胞炎性因子表達(dá)過程中的作用;(5)探究JNK信號通路在17β-E2調(diào)控子宮內(nèi)膜上皮細(xì)胞炎性因子表達(dá)過程中的作用。研究如下:(1)實驗選取健康母豬正常子宮,將其子宮內(nèi)膜剝離,進(jìn)行原代培養(yǎng)、純化和傳代培養(yǎng),最終獲得體外子宮內(nèi)膜上皮細(xì)胞,經(jīng)過角蛋白免疫熒光染色鑒定,子宮內(nèi)膜上皮細(xì)胞的純化率達(dá)95%以上,為后續(xù)研究提供了高純度、符合要求的細(xì)胞;(2)為了探究制備豬子宮內(nèi)膜上皮細(xì)胞炎癥模型需要的LPS劑量,實驗選取1μg/mL,10μg/mL,100μg/mL的脂多糖刺激體外培養(yǎng)的子宮內(nèi)膜上皮細(xì)胞12h。通過MTT試驗測定細(xì)胞的活性,并通過NO試劑盒、ELISA、RT-PCR方法檢測細(xì)胞培養(yǎng)液中NO、TNF-α、IL-1和NOS、TNF-α、IL-1的mRNA的表達(dá)量。結(jié)果顯示,10μg/mL的LPS能顯著提高子宮內(nèi)膜上皮細(xì)胞的活性且能增加NO、TNF-α、IL-1和NOS、TNF-α、IL-1的mRNA的表達(dá)量,與對照組相比,差異顯著(P0.05);LPS的濃度和炎性因子的分泌量呈劑量相關(guān)性,但由于100μg/mL的LPS顯著降低了細(xì)胞活性,因此后期實驗選取10μg/mL的LPS對子宮內(nèi)膜上皮細(xì)胞進(jìn)行刺激,制作炎癥模型;(3)為了探究17β-E2對子宮內(nèi)膜上皮細(xì)胞炎性因子的影響,采用10μg/mL的LPS刺激子宮內(nèi)膜上皮細(xì)胞12h后,分別選取10-6M,10-7M,10-8M的17β-E2處理激發(fā)培養(yǎng)的子宮內(nèi)膜上皮細(xì)胞,通過NO試劑盒、熒光定量PCR和ELISA分別檢測在2h、6?h、12?h、24?h的NO、TNF-α、IL-1和NOS、TNF-α、IL-1的mRNA表達(dá)水平。結(jié)果顯示,三種濃度的17β-E2均不同程度的降低了炎性因子的表達(dá)量,且這種降低程度與17β-E2的濃度呈正比。(4)為了探究雌激素受體α在17β-E2調(diào)控子宮內(nèi)膜上皮細(xì)胞炎性因子中的作用,實驗用10μg/mL LPS激發(fā)培養(yǎng)12h后,加入含濃度為10-6M 17β-E2、10-6M17β-E2+三氧苯胺(TAM)的培養(yǎng)液,繼續(xù)培養(yǎng)24h,通過NO試劑盒、ELISA、熒光定量PCR檢測NO、NOS、TNF-α、IL-1的表達(dá)水平。結(jié)果發(fā)現(xiàn),LPS+17β-E2組炎性因子的分泌顯著降低,而LPS+17β-E2+TAM組炎性因子的分泌與LPS+17β-E2組差異顯著,與LPS組差異不顯著,表明雌激素受體α在17β-E2調(diào)節(jié)炎性因子表達(dá)過程中發(fā)揮了重要作用。(5)為了了解JNK信號轉(zhuǎn)導(dǎo)通路在17β-E2對子宮內(nèi)膜細(xì)胞炎性因子調(diào)節(jié)過程中的作用,實驗測定了LPS組、LPS+17β-E2組以及LPS+TAM+17β-E2的P-JNK和JNK蛋白的表達(dá)量,發(fā)現(xiàn)LPS組的P-JNK水平顯著提高,而加入17β-E2后則顯著降低了P-JNK的表達(dá)水平,加入TAM后P-JNK的蛋白表達(dá)量比LPS+17β-E2組顯著提高,與LPS組無顯著差異。表明雌激素可能通過與雌激素受體α作用,抑制JNK路徑,從而抑制LPS刺激下的細(xì)胞因子的表達(dá)。綜上所述,該實驗可以得到以下結(jié)論:1.本實驗成功培養(yǎng)了豬子宮內(nèi)膜上皮細(xì)胞并建立了該細(xì)胞炎癥模型。2.雌激素可抑制LPS引起的子宮內(nèi)膜上皮細(xì)胞的炎性因子的NO、TNF-α、IL-1分泌和NOS、TNF-α、IL-1 mRNA的表達(dá)。3.雌激素通過與子宮內(nèi)膜上皮細(xì)胞上的受體α結(jié)合,抑制NO、TNF-α、IL-1的分泌和NOS、TNF-α、IL-1 mRNA的表達(dá)。4.雌激素通過與雌激素受體α作用,可能經(jīng)過抑制JNK路徑,從而抑制LPS刺激下的細(xì)胞因子的表達(dá)。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.28

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