血管內(nèi)皮細胞對平滑肌細胞表型轉(zhuǎn)化的影響
發(fā)布時間：2018-09-08 17:25
【摘要】：目的血管內(nèi)皮細胞和平滑肌細胞是構(gòu)成血管壁的兩種主要細胞,為血管組織工程的關(guān)鍵種子細胞,內(nèi)皮細胞通過分泌bFGF、TGF-β等多種細胞因子對平滑肌細胞的增殖及表型轉(zhuǎn)化起到重要作用。體內(nèi)正常血管的平滑肌細胞處于收縮表型狀態(tài),維持血管的彈性和收縮功能。促進組織工程化血管的平滑肌細胞向收縮表型轉(zhuǎn)化,使其在生理功能上更接近體內(nèi)成熟的血管,是組織工程化血管面臨的一個重要問題。本實驗將大鼠外周血單個核細胞誘導分化為血管平滑肌細胞(SMCs),以鼠尾Ⅰ型膠原作為體外細胞共培養(yǎng)支架,將其與大鼠主動脈內(nèi)皮細胞(AECs)共培養(yǎng),嘗試探索AECs及渥漫青霉素(Wortmannin)對SMCs表型轉(zhuǎn)化的影響。 方法密度梯度離心法分離SD大鼠外周血中的單個核細胞,用含PDGF-BB的EGM-2-MV培養(yǎng)液將其誘導分化到血管平滑肌細胞；以組織塊培養(yǎng)法分離大鼠主動脈平滑肌細胞和內(nèi)皮細胞；根據(jù)細胞形態(tài)和特異性標記物免疫熒光染色對細胞進行鑒定。采用濃度為1.5 mg/ml鼠尾Ⅰ型膠原制備膠原浮膠細胞共培養(yǎng)體系,即單層AECs種植于浮膠底面,單層SMCs種植在與浮膠相對的培養(yǎng)板表面,加入適量培養(yǎng)液使膠原凝膠處于懸浮狀態(tài),MTS法分析共培養(yǎng)和單獨培養(yǎng)中細胞的增殖情況,利用倒置相差光鏡觀察細胞形態(tài)結(jié)構(gòu)。RT-PCR、免疫熒光顯微鏡技術(shù)分析不同表型平滑肌細胞標記物的表達情況。不同濃度的Wortmannin作用于外周血誘導分化來源的平滑肌細胞,Western blot檢測合成表型相關(guān)蛋白CRBP-1的相對表達量,MTS法測定細胞增殖能力。 結(jié)果RT-PCR和激光共聚焦顯微鏡熒光強度分析表明,在膠原浮膠細胞共培養(yǎng)體系中,血管內(nèi)皮細胞對平滑肌細胞表型轉(zhuǎn)化的作用表現(xiàn)為先促進向合成型轉(zhuǎn)化,后促進向收縮型轉(zhuǎn)化。RT-PCR檢測結(jié)果表明,48h外周血誘導分化來源的平滑肌細胞的合成表型相關(guān)基因CRBP-1、Smemb的表達水平顯著高于平滑肌細胞單獨培養(yǎng)組,分別為1.6倍、1.9倍；72h達到峰值,分別為1.9倍、2.4倍；96h開始下降,分別為1.2倍,1.5倍；共培養(yǎng)組中收縮表型標記物Smoothelin-B和SM-MHC的基因表達水平在48h、72h顯著低于單獨培養(yǎng)組,96h Smoothelin-B卻高于單獨培養(yǎng)組。在共培養(yǎng)組中收縮型標志基因表達水平由下升趨勢轉(zhuǎn)為上升,96h Smoothelin-B和SM-MHC的基因表達水平是其72h的1.6倍、1.4倍,而合成型標志基因表達水平由上升趨勢轉(zhuǎn)為降低。單獨培養(yǎng)組上述各基因的變化趨勢不變或保持穩(wěn)定。共培養(yǎng)體系中,外周血誘導分化來源的平滑肌細胞和主動脈血管壁來源的平滑肌細胞的以上基因表達并無顯著差異(P0.05)。免疫熒光對SM-MHC表達的定量分析顯示,與內(nèi)皮細胞聯(lián)合培養(yǎng)的平滑肌細胞SM-MHC蛋白表達量先降低后升高。Wortmannin可以降低合成表型相關(guān)基因CRBP-1的表達,且對其增殖能力有顯著的抑制作用,150 nmol/L濃度即可使其相對抑制率達52.6%。 結(jié)論共培養(yǎng)體系中,內(nèi)皮細胞對平滑肌細胞表型轉(zhuǎn)化的作用表現(xiàn)為先促進向合成型轉(zhuǎn)化,96h后促進向收縮型轉(zhuǎn)化。Wortmannin可以抑制合成型相關(guān)蛋白CRBP-1的表達,且顯著抑制其增殖能力。
[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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329
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