本文選題：顆粒細(xì)胞　切入點：LPS　出處：《山東農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：在眾多影響動物繁殖的環(huán)境因素中,內(nèi)毒素(Endotoxin)感染和熱應(yīng)激(Heat stress)是兩個非常重要的方面。內(nèi)毒素化學(xué)本質(zhì)是脂多糖(Lipopolysaccharide,LPS)。LPS和熱應(yīng)激都能夠通過多種路徑對動物機體造成不良影響,本研究著重探討二者對動物繁殖產(chǎn)生的不良影響。已有研究結(jié)果證明,LPS或熱應(yīng)激作用于活體動物時,均能顯著抑制生殖內(nèi)分泌軸,抑制激素分泌、發(fā)情、排卵,降低受胎率和活胎率等。卵泡顆粒細(xì)胞(Granulosa Cell)是一種高度分化的體細(xì)胞,與卵母細(xì)胞關(guān)系密切,在動物繁殖活動中發(fā)揮重要作用,而關(guān)于LPS和熱應(yīng)激對顆粒細(xì)胞的直接影響及作用機理仍缺乏深入了解。因此,本研究以離體培養(yǎng)的豬顆粒細(xì)胞為模型,旨在探討LPS和熱應(yīng)激對顆粒細(xì)胞形態(tài)、增殖、凋亡和激素分泌功能的影響,并對作用路徑進行初步研究。經(jīng)過前期預(yù)實驗確定LPS實驗處理時間為48 h,熱應(yīng)激處理實驗為3 h。利用掃描電子顯微鏡分別放大1000×、5000×、10000×和15000×觀察LPS或熱應(yīng)激對細(xì)胞形態(tài)產(chǎn)生的影響。在LPS對顆粒細(xì)胞增殖、凋亡和雌激素分泌影響的研究中,利用CCK8測定細(xì)胞增殖情況;利用Annexin V-FITC/PI細(xì)胞凋亡檢測試劑盒借助流式細(xì)胞儀檢測細(xì)胞凋亡情況;利用ELISA法測定培養(yǎng)液中E2含量,利用Western Blot檢測細(xì)胞MAPKs蛋白含量,包括ERK、Phospho ERK、JNK、Phospho JNK、p38、Phospho p38 MAPK;利用qRT-PCR檢測與細(xì)胞增殖周期相關(guān)的基因FN1、IGF2、IGFBP2、Cyclin D1、Cyclin D2和P27kip的表達(dá)。針對LPS和熱應(yīng)激對顆粒細(xì)胞功能的影響檢測了基因P450arom、FSHR的表達(dá)。同時檢測了HSP70基因的表達(dá),研究發(fā)現(xiàn),LPS和熱應(yīng)激都能夠激活顆粒細(xì)胞HSP70基因,使這兩個環(huán)境不良因素對動物繁殖影響產(chǎn)生交匯點,因此利用小分子物質(zhì)HSP70抑制劑(VER 155008)和HSP70激活劑(STA-4783)針對HSP70作相應(yīng)處理,隨后分別檢測三個基因的表達(dá)和HSP70蛋白的表達(dá)。并在HSP70過表達(dá)的基礎(chǔ)上利用Western Blot和細(xì)胞免疫熒光染色實驗對Phospho Smad3進行了定量和定位。結(jié)果顯示LPS處理后,細(xì)胞飽滿,有較多微絨毛發(fā)和偽足,細(xì)胞狀態(tài)和活力均優(yōu)于對照組。1000 ng?mL-1濃度LPS即可顯著促進細(xì)胞增殖、抑制細(xì)胞凋亡、提高活細(xì)胞百分率(P0.05),而500 ng?mL-1 LPS即可促進與細(xì)胞生長增殖、周期相關(guān)基因FN1(P0.01),IGF2、IGFBP2(P0.05),Cyclin D1、Cyclin D2(P0.01)的表達(dá),抑制阻礙細(xì)胞周期的基因P27kip(P0.01)的表達(dá)。細(xì)胞MAPKs蛋白含量中,各總蛋白含量不變,各磷酸化蛋白含量增加。熱應(yīng)激處理使細(xì)胞固縮,微絨毛及偽足消失,細(xì)胞狀態(tài)比對照組差。LPS和熱應(yīng)激均可強烈抑制芳香化酶P450arom和FSHR基因的表達(dá)(P0.01),LPS刺激還能夠顯著抑制E2的分泌(P0.01)。LPS和熱應(yīng)激均可在轉(zhuǎn)錄和反應(yīng)水平上顯著地上調(diào)HSP70基因的相對表達(dá)量(P0.01)。經(jīng)HSP70抑制劑(VER155008)處理后,HSP70基因相對表達(dá)量代償性上調(diào),蛋白表達(dá)量被抑制。P450arom和FSHR基因的相對表達(dá)量顯著回調(diào)(P0.01)。在應(yīng)用HSP70激活劑(STA-4783)后,HSP70基因被極顯著激活(P0.01),基因相對表達(dá)量增加超過50倍,蛋白表達(dá)量顯著上調(diào)。P450arom和FSHR基因的表達(dá)與前者相反,出現(xiàn)極顯著下調(diào)。HSP70過表達(dá)3 h和48 h均能明顯抑制Smad3磷酸化和向核轉(zhuǎn)移,核內(nèi)熒光變暗,且Phospho Smad3蛋白量顯著降低。以上結(jié)果表明,LPS可以促進顆粒細(xì)胞增殖并抑制細(xì)胞凋亡,LPS和熱應(yīng)激抑制顆粒細(xì)胞功能。二者對顆粒細(xì)胞激素分泌功能的影響是通過HSP70實現(xiàn)的,HSP70阻礙Phospho Smad3的核轉(zhuǎn)移是其發(fā)揮作用的路徑之一。
[Abstract]:There are many factors that influence the animal breeding environment, endotoxin (Endotoxin) infection and heat stress (Heat stress) are two important aspects. The chemical nature of endotoxin is lipopolysaccharide (Lipopolysaccharide, LPS).LPS and heat stress can cause adverse effects on the animal body through various approaches, this research focuses on the adverse effects of the two on the animal breeding. The research results show that LPS or heat stress in a living animal, could significantly inhibit the reproductive endocrine axis, inhibiting hormone secretion, estrus, ovulation, reducing the pregnancy rate and live birth rate. Granulosa cells (Granulosa Cell) is a kind of highly differentiated somatic cells, and oocyte closely, play an important role in animal breeding activities, and on the direct effect of LPS and heat stress on granulosa cells and the mechanism is still a lack of understanding. Therefore, this study in vitro Cultured porcine granulosa cells as a model to investigate LPS and heat stress on the proliferation of granule cell morphology, apoptosis, and hormone secretion, and preliminary study on effect of path. After pre experiment to determine the LPS treatment time was 48 h, 3 h. heat stress experiment by scanning electron microscopy respectively amplified 1000 *, 5000 *, 10000 * and 15000 * LPS observation or heat stress effects on cell morphology. In LPS on the proliferation of granulosa cells, apoptosis and estrogen secretion, cell proliferation was measured by CCK8; the cell apoptosis was detected by flow cytometry using Annexin V-FITC/PI cell apoptosis detection kit; determination the content of E2 in the culture medium by ELISA method, using Western Blot to detect MAPKs protein content, including ERK, Phospho ERK, JNK, Phospho JNK, p38 Phospho, p38 MAPK; using qRT-PCR detection and cell Cell cycle related genes FN1, IGF2, IGFBP2, Cyclin, D1, D2 expression of Cyclin and P27kip. The effect of LPS and heat stress on granulosa cell function test of the P450arom gene, the expression of FSHR. At the same time to detect the expression of HSP70 gene, the study found, LPS and heat stress can activate the granulosa cell HSP70 gene so, these two factors have adverse environmental confluence on animal reproductive effects, therefore the use of small molecule inhibitors of HSP70 (VER 155008) and HSP70 activator (STA-4783) for the corresponding treatment according to the HSP70, then detected the expression of three genes and HSP70 protein expression. And the overexpression of HSP70 by Western on the basis of Blot and immunofluorescence staining experiments on Phospho Smad3 for the quantitative and positioning. The results showed that LPS treated cells full, more microvilli and pseudopodia of hair cell state and vitality, are better than the control group.1000 n G? ML-1 concentration of LPS could significantly promote cell proliferation, inhibit apoptosis, increase the percentage of viable cells (P0.05), and 500 ng? ML-1 and LPS can promote the growth and proliferation of cell cycle related genes (P0.01, FN1), IGF2, IGFBP2 (P0.05), Cyclin D1, Cyclin D2 (P0.01) expression of suppressor gene P27kip blocked the cell cycle (P0.01). The expression of MAPKs protein in the same cell, the total protein content, protein content increased acidification of various phosphorus. Heat stress to cell shrinkage, microvilli and pseudopodia disappeared, the cell state worse than the control group.LPS and heat stress can strongly inhibit the expression of aromatase P450arom and FSHR gene (P0.01), LPS stimulation can also significantly inhibit the secretion of E2 (P0.01) expression of.LPS and heat stress can be found in the transcription and reaction level significantly up-regulated the HSP70 gene (P0.01). The HSP70 inhibitor (VER155008) treatment, the relative expression of HSP70 gene The amount of compensatory up-regulated protein expression, relative expression of.P450arom was inhibited and FSHR gene significantly callback (P0.01). In the application of HSP70 activator (STA-4783), HSP70 gene was significantly activated (P0.01), relative gene expression increased more than 50 times, the protein expression was significantly up-regulated.P450arom expression and the former and the FSHR gene on the contrary, significant downregulation of.HSP70 expression of 3 h and 48 h inhibited the phosphorylation of Smad3 and nuclear transfer, nuclear fluorescence dimmed, and the Phospho Smad3 protein was significantly decreased. These results indicate that LPS can promote the proliferation of granulosa cells and inhibit cell apoptosis, and inhibition of LPS heat stress granules cell function. Effects of the two hormones on the secretion of granulosa cells is realized through HSP70, HSP70 block Phospho Smad3 nuclear transfer is one of the way to give full play to the role.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S828

【參考文獻】

相關(guān)期刊論文 前10條

1 張思思;蔡江暉;萬敬員;劉海林;劉穎菊;;羥基積雪草苷對LPS誘導(dǎo)小膠質(zhì)細(xì)胞增殖的抑制作用及機制研究[J];中國病理生理雜志;2015年03期

2 ;Raising on Water Stocking Density Reduces Geese Reproductive Performances via Water Bacteria and Lipopolysaccharide Contaminations in “Geese-Fish” Production System[J];Agricultural Sciences in China;2011年09期

3 施振旦;劉麗;孫愛東;;“禽-魚”高密度養(yǎng)殖對水禽生產(chǎn)性能危害的研究[J];中國家禽;2011年13期

4 ;HSP70 decreases receptor-dependent phosphorylation of Smad2 and blocks TGF-β-induced epithelial-mesenchymal transition[J];遺傳學(xué)報;2011年03期

5 劉中原;李延平;;細(xì)菌脂多糖的生物活性及作用機制[J];醫(yī)學(xué)綜述;2010年02期

6 郭萌;李冠民;黃清泉;;細(xì)菌內(nèi)毒素研究進展[J];中國實驗動物學(xué)報;2009年05期

7 田君;丘彥;;P450芳香化酶與多囊卵巢綜合征[J];國際生殖健康/計劃生育雜志;2008年01期

8 施振旦;李孝偉;田允波;王，

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