【作者】 杨刚；

【导师】 吴开云；

【作者基本信息】 苏州大学 ， 人体解剖学， 2005， 硕士

【摘要】 研究目的:本课题探讨降钙素基因相关肽（CGRP）对血管平滑肌细胞增殖的影响,为阐明神经因素与平滑肌细胞增生调控之间的关系提供理论依据。研究方法:取五个月胎龄水囊引产人胚胎的主动脉,体外原代培养血管平滑肌细胞,用α-SM-actin 免疫细胞化学染色鉴定细胞纯度。实验分为A:联胺诱导组;B:联胺+CGRP 组;C:CGRP 组;D:正常对照组。CGRP 和联胺的作用浓度分别为2×10^-7mol/L 和5×10^-6mol/L。用细胞计数法、MTT 法检测细胞增殖,RT-PCR 法检测细胞周期蛋白D1、E 和基质金属蛋白酶抑制剂-2（TIMP-2）mRNA 的表达,流式细胞仪法检测细胞周期的变化。研究结果:本文用胎儿腹主动脉原代培养的细胞呈典型“峰与谷”样生长。培养的血管平滑肌细胞通过α-SM-actin 免疫细胞化学染色鉴定,98%以上的细胞呈阳性反应。从细胞生长曲线可以看出第3-5 天是细胞的对数生长期。联胺作为巯基氧化剂,能诱导血管平滑肌细胞脂质过氧化刺激平滑肌细胞的增殖。CGRP 作用正常血管平滑肌细胞,24 小时之后对细胞的增殖呈抑制作用,但对联胺诱导增生的血管平滑肌细胞抑制作用更明显。CGRP 对周期蛋白D1 mRNA 的表达有明显下调作用,对TIMP-2 mRNA 的表达有上调作用。提示:CGRP 对血管平滑肌细胞的增殖和迁移有调控作用。结论:1、人胚胎血管平滑肌细胞原代培养通过α-SM-actin 免疫细胞化学染色鉴定,纯度可达98%以上。细胞生长的第3-5 天是细胞生长的对数期。2、联胺作为巯基氧化剂可作为血管平滑肌细胞增殖的诱导剂。3、CGRP 对血管平滑肌细胞的增殖有下调作用,特别对联胺诱导增生的平滑肌细胞作用更明显,提示CGRP 对平滑肌细胞增殖有调控作用,有必要对其作用机理作进一步研究。4、CGRP 对TIMP-2 mRNA的表达有上调作用,提示对平滑肌细胞的迁移有调控作用。 1111更多还原

【Abstract】 Objective To evaluate the effect of neuropeptide on the proliferation and migration of vascular smooth muscle cells（VSMCs）, this study was to investigate whether calcitonin gene-related peptide（CGRP） can regulate the proliferation and migration of vascular smooth muscle cells reduced by diamide. Methods VSMCs of abdominal aorta were obtained fresh aborted 5-month human fetus through primary culture.The purity of cultured VSMCs was assessed by positive immuoreaction cells of anti-α-smooth-actin with immunocytochemical method.The experiment was divided into four groups:A,diamide group, B,CGRP group,C,diamide and CGRP group,D,control group. the concentration of CGRP and Diamide is 2×10^-7M and 5×10^-6M respectively. the proliferous change of VSMCs was confirmed by the MTT and cell counter method. The mRNA expression change of the cyclin D1 and the tissue inhibitor of metalloproteinase-2（TIMP-2） was detected by reverse transcription-polymerase chain raction method （RT-PCR） and the changes of the cell cycle .by flow cytometry . Results It was shown that the primary cultured VSMCs grow like a peak and valley appearance and the purity of primary cultured VSMCs was 98 %. From the curve of growth of VSMCs, it can be seen that the logarithmic growth phase of the VSMCs was in 3 to 5 day after beginning.The diamide can increase VSMCs proliferation by lipid peroxidation.CGRP can increase the VSMCs’proliferation at the beginning,but after 24h the proliferous VSMCs were inhibited by CGRP. Nevertheless,CGRP intensive inhibitory effect on the proliferous VSMCs induced by diamide. CGRP can down regulate the mRNA expression of cyclin D1 and up regulate the expression of tissue inhibitor of metalloproteinase-2.It is suggested that CGRP can regulate the proliferation and migration of VSMCs. Conclusion 1. the purity of primary culture of VSMCs obtained from fresh aborted 5-month human abdominal aorta was 98%.2.The diamide can induce the VSMCs proliferation. 3.CGRP can down regulate the proliferation of VSMCs induced by diamide . It is showed that CGRP might have regulative effect on the proliferation of VSMCs, but the mechanism need to study further . 4. CGRP can up regulate the mRNA expression of tissue inhibitor of metalloproteinase-2. 5.The reserch of CGRP’s effect on the proliferation of VSMCs is very important,which might be significant in the development of atherosclerosis.更多还原