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目的分析共培养时大鼠血管内皮细胞(VECs)对大鼠血管平滑肌细胞(VSMCs)表型转化的影响。方法 VSMCs直接种植于培养板表面,VECs则种植于与VSMCs相对的浮胶底面。免疫荧光方法观察和鉴定VECs和VSMCs,RT-PCR和共聚焦显微镜分析表型相关基因的表达。结果原代培养的大鼠VECs呈铺路石样形态,vWF染色阳性。原代培养的大鼠VSMCs免疫荧光染色α-SMA呈阳性。RT-PCR检测结果表明,48h共培养组VSMCs的合成表型相关基因CRBP-1、Smemb的表达水平显著高于单独培养组,分别为1.4倍、1.5倍;72h达到峰值,分别为1.7倍、2.1倍,96h开始下降;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,96hSmoothelin-B却高于单独培养组。单独培养组上述各基因的变化趋势不变或保持稳定。免疫荧光结果显示SM-MHC蛋白表达在共培养组中96h后从下降转为升高(P<0.05)。结论在共培养体系中,血管内皮细胞对血管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。
Objective To analyze the effect of rat vascular endothelial cells (VECs) on the phenotypic changes of rat vascular smooth muscle cells (VSMCs) during co-culture. Methods VSMCs were directly planted on the surface of culture plates, while VECs were planted on the bottom of the floating glue opposite to VSMCs. VECs and VSMCs were observed and identified by immunofluorescence. The expression of phenotype-related genes was analyzed by RT-PCR and confocal microscopy. Results Primary cultured rat VECs showed paving-like morphology and vWF staining was positive. Primary cultured rat VSMCs immunofluorescent staining α-SMA was positive. The results of RT-PCR showed that the expression of CRBP-1 and Smemb of VSMCs in 48 h co-culture group were significantly higher than those in the culture group (1.4 and 1.5 times, respectively) and reached the peak at 72 and 1.7 times, 2.1-fold and 96h. The gene expression levels of Smoothelin-B and SM-MHC in co-culture group were significantly lower at 48h and 72h than those in the culture alone group, while 96hSmoothelin-B was higher than that in the culture alone group. The change trend of the above genes in the culture alone group remained unchanged or remained stable. The results of immunofluorescence showed that the expression of SM-MHC protein was increased from decreased to increased after 96h in co-culture group (P <0.05). Conclusion In co-culture system, the effect of vascular endothelial cells on the phenotypic transformation of vascular smooth muscle cells is to promote the transformation to synthetic type, and promote the contractile transformation after 96 hours.