本文關(guān)鍵詞： IL-13 溶血磷脂酸 肝星狀細(xì)胞 Ⅰ型膠原 Ⅲ型膠原　出處：《南昌大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的: 白細(xì)胞介素13(Interleukine-13,IL-13)是一種主要由激活的T淋巴細(xì)胞(Th2細(xì)胞)產(chǎn)生的具有多效能的免疫調(diào)節(jié)細(xì)胞因子。它可以調(diào)節(jié)炎癥、黏液的產(chǎn)生、組織重建和纖維化。IL-13在肝纖維化、腎纖維化、心臟纖維化、肺纖維化等纖維化疾病發(fā)病機(jī)制中起重要的作用,它通過與IL-13受體α1(IL-13Rα1)結(jié)合產(chǎn)生促纖維化作用,因此它是纖維化疾病治療的重要靶點(diǎn)。 溶血磷脂酸(Lysophosphatidic acid, LPA)是一種細(xì)胞膜脂類衍生物,是已知最小、最簡(jiǎn)單的甘油磷脂分子。LPA由成纖維細(xì)胞、血小板、脂肪細(xì)胞、癌細(xì)胞等分泌,是一種重要的細(xì)胞絲裂原素,它的作用多樣化。它具有促進(jìn)血管平滑肌收縮、促進(jìn)血小板聚集等作用,也可以抑制細(xì)胞的增殖和膠原的合成。 本實(shí)驗(yàn)主要檢測(cè)IL-13是否能促進(jìn)人肝星狀細(xì)胞膠原的合成,溶血磷脂酸(LPA)是否能下調(diào)人肝星狀細(xì)胞膠原的合成,為臨床上肝硬化等纖維化疾病提供治療的理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。 方法: 1.臺(tái)盼藍(lán)染色檢測(cè)肝人星狀細(xì)胞LX-2的活性。 2. HE染色觀察LX-2細(xì)胞的形態(tài)。 3.檢測(cè)LPA刺激LX-2細(xì)胞對(duì)IL-13Rα1表達(dá)的影響。分別以不同濃度、不同時(shí)間刺激LX-2細(xì)胞,并設(shè)空白對(duì)照組,RT-PCR檢測(cè)IL-13Rα1mRNA的表達(dá)。 4.檢測(cè)IL-13刺激LX-2細(xì)胞對(duì)Ⅰ型膠原(Procollagen typeⅠ,PCOLⅠ)、Ⅲ型膠原(Procollagen typeⅢ, PCOLⅢ)mRNA表達(dá)的影響;檢測(cè)LPA刺激LX-2細(xì)胞對(duì)Ⅰ型膠原(PCOLⅠ)、Ⅲ型膠原(PCOLⅢ)mRNA表達(dá)的影響,用不同濃度LPA在不同時(shí)間刺激LX-2細(xì)胞,RT-PCR檢測(cè)PCOLⅠ、PCOLⅢmRNA的表達(dá);然后分四組:空白組、IL-13組、LPA和IL-13混合組、LPA組,RT-PCR檢測(cè)PCOLⅠ、PCOLⅢmRNA的表達(dá)。 5.免疫組化檢測(cè)Ⅰ型膠原蛋白的表達(dá)。分四組:空白組、IL-13組、LPA和IL-13混合組以及LPA組。 結(jié)果: 1. LX-2細(xì)胞活性良好,而且IL-13和LPA對(duì)細(xì)胞無毒性作用。 2. HE染色觀察LX-2細(xì)胞為星狀,細(xì)胞核呈圓形或橢圓形。 3. RT-PCR檢測(cè)顯示,不同濃度LPA在不同時(shí)間刺激LX-2細(xì)胞,對(duì)LX-2細(xì)胞表達(dá)IL-13Rα1mRNA無影響。 4. RT-PCR檢測(cè)顯示,IL-13刺激LX-2細(xì)胞后,PCOLⅠ、PCOLⅢmRNA表達(dá)上調(diào)。 5. 0.1μM濃度的LPA刺激LX-2細(xì)胞6h、12h、24h時(shí),LX-2細(xì)胞PCOL Ⅰ、PCOLⅢmRNA表達(dá)與空白對(duì)照組相比無明顯變化。1μM、10μM LPA刺激24h后,LX-2細(xì)胞PCOLⅠ、PCOLⅢmRNA表達(dá)下降。分組實(shí)驗(yàn)結(jié)果顯示:IL-13組PCOLI、PCOLⅢmRNA的表達(dá)均高于空白對(duì)照組,而LPA組的表達(dá)均低于空白對(duì)照組。免疫組化檢測(cè)PCOLⅠ蛋白表達(dá)結(jié)果與RT-PCR結(jié)果一致。 結(jié)論: 1. IL-13、LPA刺激LX-2細(xì)胞對(duì)IL-13Rα1的表達(dá)沒有影響。 2. IL-13上調(diào)LX-2細(xì)胞PCOLⅠ、PCOLⅢ的表達(dá)。 3. LPA在達(dá)到一定的濃度時(shí)下調(diào)LX-2細(xì)胞PCOLⅠ、PCOLⅢ的表達(dá)。
[Abstract]:Objective:. Interleukine-13 (IL-13) is a multipotent immunomodulatory cytokine produced mainly by activated T lymphocyte Th2 cells. It regulates inflammation, mucus production, tissue remodeling, and fibrosis. IL-13 plays an important role in liver fibrosis and renal fibrosis. Heart fibrosis, pulmonary fibrosis and other fibrotic diseases play an important role in the pathogenesis of fibrosis. It is an important target for the treatment of fibrosis by combining with the IL-13 receptor 偽 1tl IL-13R 偽 1. Lysophosphatidic acid (LPA) is a kind of cell membrane lipids derivative, is the smallest known, the simplest glycerol phospholipid molecule. LPA is secreted by fibroblasts, platelets, adipocytes, cancer cells and so on, is an important cell mitogen. It can promote vascular smooth muscle contraction, platelet aggregation, and inhibit cell proliferation and collagen synthesis. The aim of this study was to investigate whether IL-13 could promote the synthesis of collagen in human hepatic stellate cells, and whether lysophosphatidic acid (LPA) could down-regulate the synthesis of collagen in human hepatic stellate cells, and provide a theoretical and experimental basis for the treatment of liver cirrhosis and other fibrosis diseases. Methods:. 1. The activity of LX-2 in hepatic human stellate cells was detected by trypan blue staining. 2. The morphology of LX-2 cells was observed by HE staining. 3. The effect of LPA stimulation on the expression of IL-13R 偽 1 in LX-2 cells was detected, and the expression of IL-13R 偽 1 mRNA in LX-2 cells was detected by RT-PCR with different concentration and time. 4. To detect the effect of IL-13 stimulation on the expression of procollagen type 鈪，

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