基因Nelin对人大隐静脉平滑肌细胞表型转化的影响

发布时间：2018-01-19 18:46

本文关键词： 大隐静脉细胞培养 Nelin 血管平滑肌细胞表型转化　出处：《山东大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的研究基因Nelin对血管平滑肌细胞(vascular smooth muscle cells, VSMC)表型转化的影响,探讨Nelin在原发性下肢静脉曲张发病中的作用机制。方法 (1)采用组织块贴壁结合酶消化法分离培养人大隐静脉来源的VSMC,以形态学观察、免疫荧光染色法鉴定细胞,应用台盼蓝染色法、绘制生长曲线测定细胞成活率、活性及增殖能力。 (2)利用血清饥饿法诱导VSMC由合成型向收缩型转变,恢复血清刺激诱导VSMC由向收缩型向合成型转变,获得不同表型的VSMC。 (3)收集不同条件处理的VSMC,利用逆转录聚合酶链反应(RT-PCR)技术检测不同表型VSMC的Nelin mRNA表达活性。 (4)转染Nelin-siRNA沉默VSMC中Nelin基因的表达,通过考马斯亮蓝染色法及乙酰胆碱(ACh)刺激法分别观察抑制Nelin表达VSMC细胞骨架和收缩功能的影响。结果 (1)原代培养5～7d可见VSMC从组织块边缘爬出,细胞呈梭形、多角形或不规则形,胞体较小,胞浆丰富,折光性强,细胞间常有突起相连,胞核多为卵圆形,有一个或多个核仁；传代细胞呈典型的“峰-谷”样生长,细胞突起增多,透明度增高,折光性下降；细胞免疫荧光染色可见所有VSMC胞质内α-平滑肌肌动蛋白免疫荧光染色阳性；传代细胞成活率为97%；生长曲线呈类“S”形。 (2) Nelin mRNA在不同表型VSMC中的表达差异：血清饥饿可诱导VSMC由合成型向收缩型转化。本实验观察了VSMC由合成型向收缩型转化过程中Nelin mRNA表达活性的变化。RT-PCR结果显示,血清刺激培养组、血清饥饿培养24h、48h、72h组以及恢复血清刺激24h、48h、72h组光密度扫描半定量结果分别为：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,血清饥饿组与血清刺激组差异有统计学意义(p0.05),血清刺激组间及血清饥饿组间差异无统计学意义(p＞0.05)。 (3) Nelin表达对VSMC细胞骨架的影响：血清饥饿前,VSMC细胞骨架呈模糊、淡染、稀疏的网格状,血清饥饿培养72h后,细胞骨架发生重构：结构变得清晰,网格致密,沿VSMC极性呈束状分布。应用siRNA技术沉默Nelin表达,在此基础上血清饥饿培养VSMC并观察细胞骨架的变化,发现不再出现由稀疏的网格状向束状的重构转变。相同条件下,转染阴性对照siRNA的VSMC不出现上述变化。 (4) Nelin表达与VSMC收缩功能的关系：收缩型VSMC可对兴奋剂产生收缩反应。结果显示,转染阴性对照siRNA的VSMC血清饥饿后对Ach刺激产生收缩反应,细胞长度缩短,细胞间隙变宽。而转染Nelin-siRNA的VSMC血清饥饿后不会对Ach的刺激出现收缩反应。结论 (1)体外培养的人大隐静脉平滑肌细胞纯度高,成活率高,结构、功能良好,传代生长3～6d的人大隐静脉VSMC增殖活力较强,是较佳的研究材料。 (2) Nelin参与血管平滑肌细胞骨架重塑并调节其收缩功能,对其表型转化的调节、收缩表型的维持具有重要作用。
[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 伪 -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 卤0.0971C 0.7114 卤0.1265U 0.7270 卤0.1026, respectively. 0.7841 卤0.1240g 0.3208 卤0.1389U 0.2997 卤0.1168m 0.1807 卤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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329

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