降钙素基因相关肽对平滑肌细胞增殖的影响

发布时间：2019-01-12 07:22

【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~(-7)mol/L 和5×10~(-6)mol/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
[Abstract]:Objective: to investigate the effects of calcitonin gene-related peptide (CGRP) on the proliferation of vascular smooth muscle cells (VSMCs), and to provide theoretical basis for clarifying the relationship between neurologic factors and the regulation of smooth muscle cell proliferation. Methods: vascular smooth muscle cells (VSMC) were cultured in vitro from the aorta of human embryo induced by 5 months of gestational age. The purity of the cells was identified by 伪-SM-actin immunocytochemical staining. The experiment was divided into A: diamine induced group; B: diamine CGRP group; C:CGRP group; D: normal control group. The action concentrations of CGRP and diamine were 2 脳 10 ~ (-7) mol/L and 5 脳 10 ~ (-6) mol/L., respectively. The cell proliferation was detected by cell count and MTT, the expression of cyclin D _ 1C _ (1) E and matrix metalloproteinase-2 (TIMP-2) mRNA was detected by RT-PCR assay, and the cell cycle was detected by flow cytometry. Results: the primary cultured cells of fetal abdominal aorta were typical "peak and valley" growth. More than 98% of cultured vascular smooth muscle cells were positive by 伪-SM-actin immunocytochemical staining. From the cell growth curve, we can see that day 3-5 is the logarithmic growth period of cells. As a mercapto oxidant, diamine could induce the proliferation of vascular smooth muscle cells stimulated by lipid peroxidation. CGRP inhibited the proliferation of normal vascular smooth muscle cells after 24 hours. However, the inhibitory effect of CGRP on the proliferation of vascular smooth muscle cells induced by amine was more obvious. CGRP could significantly down-regulate the expression of cyclin D1 mRNA and up-regulate the expression of TIMP-2 mRNA. The results suggest that CGRP can regulate the proliferation and migration of vascular smooth muscle cells. Conclusion: 1. The primary culture of human embryonic vascular smooth muscle cells was identified by 伪-SM-actin immunocytochemical staining and the purity was over 98%. The 3-5 days of cell growth was the logarithmic phase of cell growth. 2Diamine, as a mercaptooxidant, could be used as an inducer for the proliferation of vascular smooth muscle cells. 3CGRP could down-regulate the proliferation of vascular smooth muscle cells. The effect of CGRP on the proliferation of smooth muscle cells induced by amines is more obvious, which suggests that CGRP can regulate the proliferation of smooth muscle cells, and it is necessary to further study its mechanism. 4CGRP can up-regulate the expression of TIMP-2 mRNA. The results suggest that smooth muscle cell migration can be regulated. 1111
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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