血管内皮细胞对平滑肌细胞表型转化的影响
发布时间：2018-09-08 17:25
【摘要】：目的血管内皮细胞和平滑肌细胞是构成血管壁的两种主要细胞,为血管组织工程的关键种子细胞,内皮细胞通过分泌bFGF、TGF-β等多种细胞因子对平滑肌细胞的增殖及表型转化起到重要作用。体内正常血管的平滑肌细胞处于收缩表型状态,维持血管的弹性和收缩功能。促进组织工程化血管的平滑肌细胞向收缩表型转化,使其在生理功能上更接近体内成熟的血管,是组织工程化血管面临的一个重要问题。本实验将大鼠外周血单个核细胞诱导分化为血管平滑肌细胞(SMCs),以鼠尾Ⅰ型胶原作为体外细胞共培养支架,将其与大鼠主动脉内皮细胞(AECs)共培养,尝试探索AECs及渥漫青霉素(Wortmannin)对SMCs表型转化的影响。 方法密度梯度离心法分离SD大鼠外周血中的单个核细胞,用含PDGF-BB的EGM-2-MV培养液将其诱导分化到血管平滑肌细胞；以组织块培养法分离大鼠主动脉平滑肌细胞和内皮细胞；根据细胞形态和特异性标记物免疫荧光染色对细胞进行鉴定。采用浓度为1.5 mg/ml鼠尾Ⅰ型胶原制备胶原浮胶细胞共培养体系,即单层AECs种植于浮胶底面,单层SMCs种植在与浮胶相对的培养板表面,加入适量培养液使胶原凝胶处于悬浮状态,MTS法分析共培养和单独培养中细胞的增殖情况,利用倒置相差光镜观察细胞形态结构。RT-PCR、免疫荧光显微镜技术分析不同表型平滑肌细胞标记物的表达情况。不同浓度的Wortmannin作用于外周血诱导分化来源的平滑肌细胞,Western blot检测合成表型相关蛋白CRBP-1的相对表达量,MTS法测定细胞增殖能力。 结果RT-PCR和激光共聚焦显微镜荧光强度分析表明,在胶原浮胶细胞共培养体系中,血管内皮细胞对平滑肌细胞表型转化的作用表现为先促进向合成型转化,后促进向收缩型转化。RT-PCR检测结果表明,48h外周血诱导分化来源的平滑肌细胞的合成表型相关基因CRBP-1、Smemb的表达水平显著高于平滑肌细胞单独培养组,分别为1.6倍、1.9倍；72h达到峰值,分别为1.9倍、2.4倍；96h开始下降,分别为1.2倍,1.5倍；共培养组中收缩表型标记物Smoothelin-B和SM-MHC的基因表达水平在48h、72h显著低于单独培养组,96h Smoothelin-B却高于单独培养组。在共培养组中收缩型标志基因表达水平由下升趋势转为上升,96h Smoothelin-B和SM-MHC的基因表达水平是其72h的1.6倍、1.4倍,而合成型标志基因表达水平由上升趋势转为降低。单独培养组上述各基因的变化趋势不变或保持稳定。共培养体系中,外周血诱导分化来源的平滑肌细胞和主动脉血管壁来源的平滑肌细胞的以上基因表达并无显著差异(P0.05)。免疫荧光对SM-MHC表达的定量分析显示,与内皮细胞联合培养的平滑肌细胞SM-MHC蛋白表达量先降低后升高。Wortmannin可以降低合成表型相关基因CRBP-1的表达,且对其增殖能力有显著的抑制作用,150 nmol/L浓度即可使其相对抑制率达52.6%。 结论共培养体系中,内皮细胞对平滑肌细胞表型转化的作用表现为先促进向合成型转化,96h后促进向收缩型转化。Wortmannin可以抑制合成型相关蛋白CRBP-1的表达,且显著抑制其增殖能力。
[Abstract]:Objective Vascular endothelial cells (VECs) and smooth muscle cells (SMCs) are the two main cells that constitute the vascular wall. They are the key seed cells for vascular tissue engineering. It is an important problem facing tissue-engineered blood vessels to promote smooth muscle cells (SMCs) of tissue-engineered blood vessels to transform to contractile phenotype and to make them more physiological function close to the mature blood vessels in vivo. In this experiment, rat peripheral blood mononuclear cells (PBMCs) were induced to differentiate into vascular smooth muscle cells (SMCs). In order to explore the effect of AECs and Wortmannin on the phenotypic transformation of SMCs, rat tail type I collagen was used as co-culture scaffold in vitro and co-cultured with rat aortic endothelial cells (AECs).
Methods Mononuclear cells from peripheral blood of SD rats were isolated by density gradient centrifugation and differentiated into vascular smooth muscle cells by EGM-2-MV medium containing PDGF-BB. Rat aortic smooth muscle cells and endothelial cells were isolated by tissue block culture. The cells were stained by immunofluorescence according to cell morphology and specific markers. Identification. Collagen float cell co-culture system was prepared with 1.5 mg/ml rat tail type I collagen, i.e. monolayer AECs were planted on the bottom of float, monolayer SMCs were planted on the surface of the plate opposite to float, and the collagen gel was suspended by adding proper amount of culture medium. MTS method was used to analyze the proliferation of the cells in co-culture and separate culture. Reversed phase contrast microscope was used to observe the cell morphology and structure. RT-PCR and immunofluorescence microscopy were used to analyze the expression of markers of different phenotypes of smooth muscle cells. Cell proliferation ability.
Results RT-PCR and confocal laser microscopy fluorescence intensity analysis showed that vascular endothelial cells could promote the phenotypic transformation of smooth muscle cells in the collagen floating gel cell co-culture system. RT-PCR results showed that 48 h peripheral blood induced differentiated smooth muscle cells. The expression levels of CRBP-1 and Smemb genes were 1.6 and 1.9 times higher than those of SMC cultured alone, reaching the peak value at 72 hours, respectively, 1.9 and 2.4 times, and decreasing at 96 hours, respectively, 1.2 and 1.5 times; and the expression levels of Smoothelin-B and SM-MHC genes in co-cultured group were significant at 48 and 72 hours. In the co-culture group, the expression level of SMoothelin-B and SM-MHC was 1.6 and 1.4 times higher than that in the 72 h group, but the expression level of SMoothelin-B was higher than that in the single culture group. In the co-culture system, there was no significant difference in the expression of SM-MHC between smooth muscle cells derived from peripheral blood and smooth muscle cells derived from aortic vascular wall (P 0.05). The quantitative analysis of SM-MHC expression by immunofluorescence showed that SM-MHC was co-cultured with endothelial cells. The expression of MHC protein decreased first and then increased. Wortmannin could decrease the expression of CRBP-1 and inhibit the proliferation of CRBP-1. The relative inhibition rate reached 52.6% at 150 nmol/L.
Conclusion In co-culture system, endothelial cells can promote the phenotypic transformation of smooth muscle cells to synthetical type at first, and then to contractile type at 96h. Wortmannin can inhibit the expression of CRBP-1 and inhibit its proliferation.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
[Abstract]:Objective Vascular endothelial cells (VECs) and smooth muscle cells (SMCs) are the two main cells that constitute the vascular wall. They are the key seed cells for vascular tissue engineering. It is an important problem facing tissue-engineered blood vessels to promote smooth muscle cells (SMCs) of tissue-engineered blood vessels to transform to contractile phenotype and to make them more physiological function close to the mature blood vessels in vivo. In this experiment, rat peripheral blood mononuclear cells (PBMCs) were induced to differentiate into vascular smooth muscle cells (SMCs). In order to explore the effect of AECs and Wortmannin on the phenotypic transformation of SMCs, rat tail type I collagen was used as co-culture scaffold in vitro and co-cultured with rat aortic endothelial cells (AECs).
Methods Mononuclear cells from peripheral blood of SD rats were isolated by density gradient centrifugation and differentiated into vascular smooth muscle cells by EGM-2-MV medium containing PDGF-BB. Rat aortic smooth muscle cells and endothelial cells were isolated by tissue block culture. The cells were stained by immunofluorescence according to cell morphology and specific markers. Identification. Collagen float cell co-culture system was prepared with 1.5 mg/ml rat tail type I collagen, i.e. monolayer AECs were planted on the bottom of float, monolayer SMCs were planted on the surface of the plate opposite to float, and the collagen gel was suspended by adding proper amount of culture medium. MTS method was used to analyze the proliferation of the cells in co-culture and separate culture. Reversed phase contrast microscope was used to observe the cell morphology and structure. RT-PCR and immunofluorescence microscopy were used to analyze the expression of markers of different phenotypes of smooth muscle cells. Cell proliferation ability.
Results RT-PCR and confocal laser microscopy fluorescence intensity analysis showed that vascular endothelial cells could promote the phenotypic transformation of smooth muscle cells in the collagen floating gel cell co-culture system. RT-PCR results showed that 48 h peripheral blood induced differentiated smooth muscle cells. The expression levels of CRBP-1 and Smemb genes were 1.6 and 1.9 times higher than those of SMC cultured alone, reaching the peak value at 72 hours, respectively, 1.9 and 2.4 times, and decreasing at 96 hours, respectively, 1.2 and 1.5 times; and the expression levels of Smoothelin-B and SM-MHC genes in co-cultured group were significant at 48 and 72 hours. In the co-culture group, the expression level of SMoothelin-B and SM-MHC was 1.6 and 1.4 times higher than that in the 72 h group, but the expression level of SMoothelin-B was higher than that in the single culture group. In the co-culture system, there was no significant difference in the expression of SM-MHC between smooth muscle cells derived from peripheral blood and smooth muscle cells derived from aortic vascular wall (P 0.05). The quantitative analysis of SM-MHC expression by immunofluorescence showed that SM-MHC was co-cultured with endothelial cells. The expression of MHC protein decreased first and then increased. Wortmannin could decrease the expression of CRBP-1 and inhibit the proliferation of CRBP-1. The relative inhibition rate reached 52.6% at 150 nmol/L.
Conclusion In co-culture system, endothelial cells can promote the phenotypic transformation of smooth muscle cells to synthetical type at first, and then to contractile type at 96h. Wortmannin can inhibit the expression of CRBP-1 and inhibit its proliferation.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
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