肿瘤相关成纤维细胞通过低氧诱导因子-1通路促进结直肠癌细胞增殖

Cancer associated fibroblasts promote proliferation of colorectal cancer cell through hypoxia inducible factor-1 pathway

目的研究结直肠癌中肿瘤相关成纤维细胞促进结直肠癌细胞增殖的机制。方法体外培养原代结直肠癌组织肿瘤相关成纤维细胞(CAF)以及癌旁正常纤维细胞(NF),利用慢病毒高表达人端粒酶反转录酶(hTERT)使其永生化,收集上清处理肿瘤细胞以及将肿瘤相关成纤维细胞或癌旁正常成纤维细胞和肿瘤细胞置入Transwell共培养系统,脂质体介导低氧诱导因子-1(HIF-1α)为靶分子的小干扰RNA(siRNA)转染结肠癌细胞株SW48后,利用细胞核增殖抗原(Ki-67)流式染色、集落形成实验、流式细胞周期检测测定细胞增殖能力的变化,利用细胞计数试剂盒(CCK-8)检测生长曲线,采用蛋白质印迹法(Western blot)检测细胞中HIF-1α的表达。应用SPSS 17.0统计软件分析,计量资料以均值±标准差(Mean±SD)表示,用独立样本t检验来比较组间差异。结果siRNA 1~2组HIF-1α表达水平明显低于阴性对照组,CAF上清处理组较空白对照组克隆生成率明显增高(43.6%比12.4%,t=-12.376,P<0.01),流式细胞周期检测显示CAF共培养组、NF共培养组,HIF-1αsi+CAF共培养组G1~G0期、S期细胞比例分别为(54.6±2.5)%比(65.1±3.0)%比(67.2±1.0)%(F=6.400,P<0.05)、(30.51±2.3)%比(20.3±1.8)%比(21.6±3.8)%(F=4.690,P<0.05),CAF共培养组、NF共培养组、HIF-1αsi+CAF共培养组Ki-67阳性细胞比例分别为(98.4±0.6)%比(95.3±0.8)%比(94.6±0.5)%(F=6.800,P<0.05)。结论肿瘤相关成纤维细胞可以通过间接效应促进结直肠癌肿瘤细胞的增殖,而这一效应可能依赖HIF-1α。

Objective Investigate phenomenon and mechanism of cancer associated fibroblasts(CAF)promote proliferation of colon-rectal cancer(CRC)cells.Methods Isolation and cultivation of primary cancer associated fibroblasts,place CAF and tumor cell in Transwell co-culture system and collect supervision of CAF,The small interfering RNA(siRNA)vectors targetinghypoxia inducible factor-1gene were transfected into SW48 cells,testing capacity of proliferation of CRC cells based on plate clony-forming test,analysis cell-cycle by Flow-cytometry,detection of expression of proliferation cell nuclear antigen(Ki-67)based on cell-cytometry,the proliferation curve was depicted by cell counting kit 8(CCK-8),the expression of hypoxia inducible factor(HIF)-1αwas detected by Western blotting.t-test was used for comparison of measurement data.Results The expression of HIF-1αin siRNA1-2 group was down-regulated compared with negative control,CAF derived condition medium culture group had stronger colony-forming efficiency compared with blank group(43.6%vs.12.4%t=-12.376,P<0.01),analysis of cell-cycle indicated that the proportion of G1-G0 phase,S phase of CAF co-culture group,NF co-culture group,HIF-1αsi+CAF co-culture group is(54.6±2.5)%vs.(65.1±3.0)%vs.(67.2±1.0)%(F=6.400,P<0.05),(30.51±2.3)%vs.(20.3±1.8)%vs.(21.6±3.8)%(F=4.690,P<0.05),the proportion of Ki-67 positive cells of CAF co-culture group,NF co-culture group,HIF-1αsi+CAF co-culture group is(98.4±0.6)%vs.(94.3±0.8)%vs.(95.6±0.5)%(F=6.800,P<0.05),CAF can increase capacity of proliferation of CRC cells,and the function can be blocked by down-regulation of HIF-1αwas also confirmed by CCK-8.Conclusion CAF can promote proliferation of CRC cell,and it may depend of HIF-1αexpression.