血清剥夺促进成纤维细胞向内皮细胞转分化的体外研究

发布时间：2018-10-13 14:31

【摘要】：目的模拟体内急性心肌梗死缺血缺氧微环境,探讨血清剥夺是否能激活心肌成纤维细胞向内皮细胞转分化。方法取新鲜分离的C57BL/6新生小鼠心脏组织,获取成纤维细胞进行培养,用白血病抑制因子(LIF)促进成纤维细胞自我更新、长程维持干性和抑制其向终末分化。将第3代成纤维细胞分为:对照组[DMEM+10%胎牛血清(FBS)培养]、血清剥夺24 h组(不含FBS的DMEM培养24 h)、血清剥夺48 h组(不含FBS的DMEM培养48 h)、血清剥夺72 h组(不含FBS的DMEM培养72 h)。经过不同处理后,采用q PCR法检测各组细胞谱系特异性基因的表达变化,采用体外血管新生实验和Dil-Ac-LDL吞噬实验检测血清剥夺后成纤维细胞的功能学变化,采用ELISA法检测血清剥夺后成纤维细胞分泌血管内皮生长因子(VEGF)的情况。结果血清剥夺不同时间组成纤维细胞形成血管分叉的数目与对照组相比均增加(P0.05),但均未发现诱导细胞吞噬Dil-Ac-LDL的现象。与对照组比较,血清剥夺48 h组成纤维细胞中内皮特异性基因CD31和VE-herin的表达均升高(P0.05);其中血清剥夺48 h组成纤维细胞中CD31的表达是对照组的13.7倍(P0.05),血清剥夺72 h组是对照组的2倍(P0.05)。ELISA检测结果显示,血清剥夺48h组成纤维细胞分泌的VEGF是对照组的7倍(P0.05),血清剥夺72 h组是对照组的3.7倍(P0.05)。结论血清剥夺可以激活心肌成纤维细胞向内皮细胞谱系的转分化。
[Abstract]:Aim to investigate whether serum deprivation can activate myocardial fibroblasts to differentiate into endothelial cells by simulating the microenvironment of acute myocardial infarction. Methods Fibroblast cells were obtained from freshly isolated heart tissue of C57BL/6 newborn mice. Leukemia inhibitory factor (LIF) was used to promote the self-renewal of fibroblasts and to maintain dryness and inhibit their terminal differentiation for a long time. The third passage fibroblasts were divided into three groups: control group [DMEM 10% fetal bovine serum (FBS) culture], serum deprivation 24 h group (DMEM without FBS culture 24 h), serum deprivation 48 h group) DMEM without FBS culture 48 h), serum deprivation 72 h group (FBS free DMEM culture 72 h).) After different treatments, the expression of pedigree specific genes was detected by Q PCR assay, and the functional changes of fibroblasts after serum deprivation were detected by in vitro angiogenesis test and Dil-Ac-LDL phagocytosis test. The level of vascular endothelial growth factor (VEGF) secreted by fibroblasts after serum deprivation was detected by ELISA method. Results compared with the control group, the number of vascular bifurcation of fibroblasts in different time of serum deprivation increased (P0.05), but none of them found the phenomenon of inducing cells to phagocytosis of Dil-Ac-LDL. Compared with the control group, The expression of endothelium-specific gene CD31 and VE-herin in fibroblasts of 48 h of serum deprivation increased significantly (P0.05), and the expression of CD31 in fibroblasts of 48 h of serum deprivation was 13.7 times higher than that of the control group (P0.05), and the expression of CD31 in 72 h group of serum deprivation was 2 times of that of the control group (P0.05). Times (P0.05). ELISA test results showed that, The VEGF secreted by fibroblasts after 48 hours of serum deprivation was 7 times of that of the control group (P0.05) and 3.7 times of that of the control group after 72 hours of serum deprivation (P0.05). Conclusion Serum deprivation can activate the transdifferentiation of myocardial fibroblasts into endothelial cell lineages.
【作者单位】：第二军医大学长征医院心血管内科;
【基金】：国家自然科学基金(9153910092,91539118,81473445,8151101216)~~
【分类号】：R54

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