【摘要】：研究目的:本課題探討降鈣素基因相關(guān)肽(CGRP)對(duì)血管平滑肌細(xì)胞增殖的影響,為闡明神經(jīng)因素與平滑肌細(xì)胞增生調(diào)控之間的關(guān)系提供理論依據(jù)。研究方法:取五個(gè)月胎齡水囊引產(chǎn)人胚胎的主動(dòng)脈,體外原代培養(yǎng)血管平滑肌細(xì)胞,用α-SM-actin 免疫細(xì)胞化學(xué)染色鑒定細(xì)胞純度。實(shí)驗(yàn)分為A:聯(lián)胺誘導(dǎo)組;B:聯(lián)胺+CGRP 組;C:CGRP 組;D:正常對(duì)照組。CGRP 和聯(lián)胺的作用濃度分別為2×10~(-7)mol/L 和5×10~(-6)mol/L。用細(xì)胞計(jì)數(shù)法、MTT 法檢測(cè)細(xì)胞增殖,RT-PCR 法檢測(cè)細(xì)胞周期蛋白D1、E 和基質(zhì)金屬蛋白酶抑制劑-2(TIMP-2)mRNA 的表達(dá),流式細(xì)胞儀法檢測(cè)細(xì)胞周期的變化。 研究結(jié)果:本文用胎兒腹主動(dòng)脈原代培養(yǎng)的細(xì)胞呈典型“峰與谷”樣生長(zhǎng)。培養(yǎng)的血管平滑肌細(xì)胞通過(guò)α-SM-actin 免疫細(xì)胞化學(xué)染色鑒定,98%以上的細(xì)胞呈陽(yáng)性反應(yīng)。從細(xì)胞生長(zhǎng)曲線(xiàn)可以看出第3-5 天是細(xì)胞的對(duì)數(shù)生長(zhǎng)期。聯(lián)胺作為巰基氧化劑,能誘導(dǎo)血管平滑肌細(xì)胞脂質(zhì)過(guò)氧化刺激平滑肌細(xì)胞的增殖。CGRP 作用正常血管平滑肌細(xì)胞,24 小時(shí)之后對(duì)細(xì)胞的增殖呈抑制作用,但對(duì)聯(lián)胺誘導(dǎo)增生的血管平滑肌細(xì)胞抑制作用更明顯。CGRP 對(duì)周期蛋白D1 mRNA 的表達(dá)有明顯下調(diào)作用,對(duì)TIMP-2 mRNA 的表達(dá)有上調(diào)作用。提示:CGRP 對(duì)血管平滑肌細(xì)胞的增殖和遷移有調(diào)控作用。結(jié)論:1、人胚胎血管平滑肌細(xì)胞原代培養(yǎng)通過(guò)α-SM-actin 免疫細(xì)胞化學(xué)染色鑒定,純度可達(dá)98%以上。細(xì)胞生長(zhǎng)的第3-5 天是細(xì)胞生長(zhǎng)的對(duì)數(shù)期。2、聯(lián)胺作為巰基氧化劑可作為血管平滑肌細(xì)胞增殖的誘導(dǎo)劑。3、CGRP 對(duì)血管平滑肌細(xì)胞的增殖有下調(diào)作用,特別對(duì)聯(lián)胺誘導(dǎo)增生的平滑肌細(xì)胞作用更明顯,提示CGRP 對(duì)平滑肌細(xì)胞增殖有調(diào)控作用,有必要對(duì)其作用機(jī)理作進(jìn)一步研究。4、CGRP 對(duì)TIMP-2 mRNA的表達(dá)有上調(diào)作用,提示對(duì)平滑肌細(xì)胞的遷移有調(diào)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 偽-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 偽-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 偽-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
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 李洪麗;鮭魚(yú)降鈣素與降鈣素基因相關(guān)肽基因原核表達(dá)的研究[D];黑龍江大學(xué);2010年

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本文編號(hào)：2407506