本文關(guān)鍵詞： 大隱靜脈 細(xì)胞培養(yǎng) Nelin 血管平滑肌細(xì)胞 表型轉(zhuǎn)化　出處：《山東大學(xué)》2013年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的 研究基因Nelin對(duì)血管平滑肌細(xì)胞(vascular smooth muscle cells, VSMC)表型轉(zhuǎn)化的影響,探討Nelin在原發(fā)性下肢靜脈曲張發(fā)病中的作用機(jī)制。 方法 (1)采用組織塊貼壁結(jié)合酶消化法分離培養(yǎng)人大隱靜脈來(lái)源的VSMC,以形態(tài)學(xué)觀(guān)察、免疫熒光染色法鑒定細(xì)胞,應(yīng)用臺(tái)盼藍(lán)染色法、繪制生長(zhǎng)曲線(xiàn)測(cè)定細(xì)胞成活率、活性及增殖能力。 (2)利用血清饑餓法誘導(dǎo)VSMC由合成型向收縮型轉(zhuǎn)變,恢復(fù)血清刺激誘導(dǎo)VSMC由向收縮型向合成型轉(zhuǎn)變,獲得不同表型的VSMC。 (3)收集不同條件處理的VSMC,利用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)技術(shù)檢測(cè)不同表型VSMC的Nelin mRNA表達(dá)活性。 (4)轉(zhuǎn)染Nelin-siRNA沉默VSMC中Nelin基因的表達(dá),通過(guò)考馬斯亮藍(lán)染色法及乙酰膽堿(ACh)刺激法分別觀(guān)察抑制Nelin表達(dá)VSMC細(xì)胞骨架和收縮功能的影響。 結(jié)果 (1)原代培養(yǎng)5～7d可見(jiàn)VSMC從組織塊邊緣爬出,細(xì)胞呈梭形、多角形或不規(guī)則形,胞體較小,胞漿豐富,折光性強(qiáng),細(xì)胞間常有突起相連,胞核多為卵圓形,有一個(gè)或多個(gè)核仁；傳代細(xì)胞呈典型的“峰-谷”樣生長(zhǎng),細(xì)胞突起增多,透明度增高,折光性下降；細(xì)胞免疫熒光染色可見(jiàn)所有VSMC胞質(zhì)內(nèi)α-平滑肌肌動(dòng)蛋白免疫熒光染色陽(yáng)性；傳代細(xì)胞成活率為97%；生長(zhǎng)曲線(xiàn)呈類(lèi)“S”形。 (2) Nelin mRNA在不同表型VSMC中的表達(dá)差異：血清饑餓可誘導(dǎo)VSMC由合成型向收縮型轉(zhuǎn)化。本實(shí)驗(yàn)觀(guān)察了VSMC由合成型向收縮型轉(zhuǎn)化過(guò)程中Nelin mRNA表達(dá)活性的變化。RT-PCR結(jié)果顯示,血清刺激培養(yǎng)組、血清饑餓培養(yǎng)24h、48h、72h組以及恢復(fù)血清刺激24h、48h、72h組光密度掃描半定量結(jié)果分別為：0.1940±0.0971,0.7114±0.1265,0.7270±0.1026,0.7841±0.1240,0.3208±0.1389,0.2997±0.1168,0.1807±0.0845,血清饑餓組與血清刺激組差異有統(tǒng)計(jì)學(xué)意義(p0.05),血清刺激組間及血清饑餓組間差異無(wú)統(tǒng)計(jì)學(xué)意義(p＞0.05)。 (3) Nelin表達(dá)對(duì)VSMC細(xì)胞骨架的影響：血清饑餓前,VSMC細(xì)胞骨架呈模糊、淡染、稀疏的網(wǎng)格狀,血清饑餓培養(yǎng)72h后,細(xì)胞骨架發(fā)生重構(gòu)：結(jié)構(gòu)變得清晰,網(wǎng)格致密,沿VSMC極性呈束狀分布。應(yīng)用siRNA技術(shù)沉默Nelin表達(dá),在此基礎(chǔ)上血清饑餓培養(yǎng)VSMC并觀(guān)察細(xì)胞骨架的變化,發(fā)現(xiàn)不再出現(xiàn)由稀疏的網(wǎng)格狀向束狀的重構(gòu)轉(zhuǎn)變。相同條件下,轉(zhuǎn)染陰性對(duì)照siRNA的VSMC不出現(xiàn)上述變化。 (4) Nelin表達(dá)與VSMC收縮功能的關(guān)系：收縮型VSMC可對(duì)興奮劑產(chǎn)生收縮反應(yīng)。結(jié)果顯示,轉(zhuǎn)染陰性對(duì)照siRNA的VSMC血清饑餓后對(duì)Ach刺激產(chǎn)生收縮反應(yīng),細(xì)胞長(zhǎng)度縮短,細(xì)胞間隙變寬。而轉(zhuǎn)染Nelin-siRNA的VSMC血清饑餓后不會(huì)對(duì)Ach的刺激出現(xiàn)收縮反應(yīng)。 結(jié)論 (1)體外培養(yǎng)的人大隱靜脈平滑肌細(xì)胞純度高,成活率高,結(jié)構(gòu)、功能良好,傳代生長(zhǎng)3～6d的人大隱靜脈VSMC增殖活力較強(qiáng),是較佳的研究材料。 (2) Nelin參與血管平滑肌細(xì)胞骨架重塑并調(diào)節(jié)其收縮功能,對(duì)其表型轉(zhuǎn)化的調(diào)節(jié)、收縮表型的維持具有重要作用。
[Abstract]:Purpose To study the effect of gene Nelin on the phenotypic transformation of vascular smooth muscle cells (VSMCs) in vascular smooth muscle cells (VSMCs). To investigate the role of Nelin in the pathogenesis of primary varicose veins of lower extremity. Method 1) VSMCs from human great saphenous vein were isolated and cultured by tissue mass adhesion and enzyme digestion. The cells were identified by morphological observation, immunofluorescence staining and trypan blue staining. Cell survival rate, activity and proliferation ability were measured by drawing growth curve. (2) VSMC was induced to change from synthetic to contractile by serum starvation, and VSMC was induced to change from contractile to synthetic by restoring serum stimulation, and different phenotypic VSMCs were obtained. The expression of Nelin mRNA in different phenotypes of VSMC was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of Nelin gene in VSMC was silenced by transfection of Nelin-siRNA. The effects of inhibition of Nelin expression on the cytoskeleton and contractile function of VSMC cells were observed by Coomassie brilliant blue staining and acetylcholine acetylcholine acetylcholine (ache) stimulation, respectively. Results 1) VSMC crawled out from the edge of the tissue mass in primary culture for 5 ~ 7 days. The cells were fusiform, polygonal or irregular, small in body, abundant in cytoplasm, strong in refraction, and often connected with each other. The nucleus is mostly oval with one or more nucleoli. The passage cells showed typical "peak-valley" growth, cell processes increased, transparency increased, and refractive index decreased. All VSMC cytoplasm were positive for 偽 -smooth muscle actin immunofluorescence staining. The survival rate of passage cells was 97%. The growth curve is like "S" shape. 2). The expression of Nelin mRNA in different phenotypic VSMC was different:. Serum starvation could induce the transformation of VSMC from synthetic to contractile type. In this experiment, we observed Nelin during the transformation of VSMC from synthetic to contractile type. Changes of mRNA expression activity. RT-PCR results showed. Serum stimulation group, serum starvation culture group for 24 h and 48 h for 72 h group, and recovery serum stimulation group for 24 h and 48 h. In 72 h group, the semi-quantitative results of optical density scanning were 0.1940 鹵0.0971C 0.7114 鹵0.1265U 0.7270 鹵0.1026, respectively. 0.7841 鹵0.1240g 0.3208 鹵0.1389U 0.2997 鹵0.1168m 0.1807 鹵0.0845. The difference between serum hunger group and serum stimulation group was statistically significant (p 0.05), but there was no significant difference between serum stimulation group and serum hunger group (P > 0.05). (3) the effect of Nelin expression on the cytoskeleton of VSMC cells: the cytoskeleton of VSMC cells before serum starvation was vague, light stained, sparse and reticular. After 72 hours of serum starvation, the cytoskeleton of VSMC cells was blurry. Cytoskeleton remodeling: the structure became clear, the grid became dense and distributed along the polarity of VSMC. The expression of Nelin was silenced by siRNA technique. On this basis, VSMC was cultured with serum starvation and the cytoskeleton was observed. The above changes were not observed in VSMC transfected with negative control siRNA. (4) relationship between Nelin expression and contractile function of VSMC: contractile VSMC can produce contractile response to stimulant. The VSMC serum transfected with negative control siRNA had contractile response to Ach stimulation after starvation, and the cell length was shortened. The gap widened and the VSMC serum transfected with Nelin-siRNA did not contract to Ach stimulation after starvation. Conclusion 1) the smooth muscle cells of the saphenous vein of the people's Congress were cultured in vitro with high purity, high survival rate, good structure and good function, and the proliferation activity of the VSMC of the saphenous vein of the people's Congress was stronger after the passage for 36 days. Is a better research material. 2) Nelin is involved in the remodeling of vascular smooth muscle cytoskeleton and regulates its contractile function, which plays an important role in the regulation of phenotypic transformation and maintenance of contractile phenotype.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張蕾;心肌肌聯(lián)蛋白的結(jié)構(gòu)及其生物學(xué)意義[J];國(guó)外醫(yī)學(xué)(兒科學(xué)分冊(cè));2003年01期

2 毛昱嘉,王文杰;胰酶消化法培養(yǎng)大鼠胸主動(dòng)脈平滑肌細(xì)胞[J];中國(guó)動(dòng)脈硬化雜志;2004年06期

3 于曉強(qiáng);秦士勇;陳士輝;張曙光;;新基因nelin在下肢曲張靜脈中的表達(dá)及其意義[J];中國(guó)現(xiàn)代普通外科進(jìn)展;2010年12期

4 侯樂(lè)偉;廖明芳;翁劍鋒;楊琳;鄒思力;景在平;;α-平滑肌肌動(dòng)蛋白在人胸主動(dòng)脈夾層中的表達(dá)[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2010年01期

5 韓梅,溫進(jìn)坤,鄭斌,聶磊;血清饑餓可誘導(dǎo)人血管平滑肌細(xì)胞再分化[J];中國(guó)生物化學(xué)與分子生物學(xué)報(bào);2003年02期

6 王震,劉冬青,王茵,曹慧青,丁金鳳,郭蓮軍,屈伸,孟憲敏;應(yīng)用酵母雙雜交系統(tǒng)篩選與新基因nelin相互作用的蛋白質(zhì)[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年01期

7 王震;劉冬青;王茵;屈伸;孟憲敏;;Nelin與F-actin及Filamin相互作用的鑒定[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2007年03期

8 程云會(huì),韓梅,溫進(jìn)坤,張永鋼,劉智敏;一種改良的肌細(xì)胞骨架染色方法[J];細(xì)胞生物學(xué)雜志;2004年02期

9 程云會(huì),韓梅,溫進(jìn)坤;平滑肌細(xì)胞骨架結(jié)構(gòu)及其信號(hào)調(diào)節(jié)途徑[J];細(xì)胞生物學(xué)雜志;2004年06期

10 尤昕,曹慧青,時(shí)娜,王震,趙秀文,丁金鳳,孟憲敏;人細(xì)胞骨架調(diào)節(jié)蛋白基因Nelin N端片段的原核表達(dá)[J];延邊大學(xué)醫(yī)學(xué)學(xué)報(bào);2003年04期

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