肿瘤相关成纤维细胞培养液上清促进肿瘤细胞增殖和血管生成的研究

Experimental research of tumor-associated cells conditioned medium promote tumor cells proliferation and angiogenesis

目的探讨在体外实验条件下，肿瘤相关成纤维细胞如何通过旁分泌机制促进肿瘤细胞的增殖和血管生成。方法分别提取原代卵巢癌相关成纤维细胞（TAFs）与正常卵巢相关成纤维细胞（NFs）的培养液上清（CM）；实验组为2mlTAFs—CM处理卵巢癌细胞（SKOV-3，1×10^5／孔），对照组为2mlNFs—CM处理卵巢癌细胞（SKOV-3，1×10^5／孔），空白组为正常培养的卵巢癌细胞（SKOV-3，1×10^5个／孔），干预组为在实验组中加入20umol转化生长因子-β（TGF-β）特异性小分子抑制剂SB-431542（10umol／ml）；应用流式细胞仪分析各组细胞生长周期；逆转录-聚合酶链反应（RT-PCR）法检测各组细胞增殖细胞核抗原（PCNA）、a-平滑肌肌动蛋白（a-SMA）、血管内皮生长因子（VEGF）mRNA的表达，Westernblot法检测各组细胞TGF-β1、a-SMA、VEGF蛋白的表达。结果实验组卵巢癌细胞增殖较对照组显著增加（细胞s期比例：实验组22．10±1．84比对照组12．77±1．43，P〈0．05）；而加入SB-431542干预后则可明显抑制促增殖作用（细胞S期比例：干预组12．33±1．12比实验组22．10±1．84，P〈0．05）；RT-PCR提示实验组PCNA、a-SMA、VEGFmRNA较其他组表达显著上调（P〈0．05），Western blot结果提示实验组TGF-β1、a—SMA、VEGF蛋白表达亦显著上调（P〈0．05），在加入SB-431542后以上基因及蛋白的表达均受到抑制（P〈0．05）。结论卵巢癌相关成纤维细胞可以通过旁分泌机制促进卵巢癌细胞的增殖，并上调血管生成相关基因及蛋白的表达，而通过抑制TGF—β信号通路作用后则可以有效抑制这类作用。

Objective To discuss how tumor-associated cells can promote the proliferation and angiogenesis of ovarian cancer cells via paracrine mechanism in vitro. Methods Tumor-associated fibroblasts （TAFs） and normal fibroblasts （NFs） were isolated and cultured, respectively, and after then collected conditioned mediums （CM） ; 2 ml TAFs-CM treated 1 × 10^5 cell/well ovarian cancer cells as experimental group; 2 ml NFs-CM treated 1 × 10^5 cell/well ovarian cancer cells as control group; complete mediums treated 1 × 10^5 cell/well ovarian cancer cells as blank group; 20 umol SB431512 （10 umol/ml） was added in experimental group as intervene group; Cell cycle was determined at every group by flow cytometry ; the proliferating cell nuclear antigen （PCNA）, a-smooth muscle actin （a-SMA）, vascular endothelial growth factor （VEGF） mRNA of every group were detected by reverse transcription-polymerase chain reaction （RT-PCR） ; the transforming growth factor （TGF）-β1, a-SMA, VEGF protein expression in every group were detected by Western blotting. Results Proliferation of OCCs in experimental group were significantly promoted compared with the control group （22. 10 ± 1.84 vs 12. 77 ± 1.43, P 〈0. 05） ; The effect of this kind of promotion was significantly inhibited in intervene group than the experimental groups （8. 20 ±0. 80 vs 11.66 ± 1.60, P 〈 0.05 ）. Compare to control group and blank group, the RT-PCR showed the PCNA, a-SMA, VEGF mRNA expression in experimental group were enhanced, and the expression of TGF-β1, a-SMA, VEGF protein in experimental group were significantly increased which showed by Western blotting （ P 〈 0.05 ）. The increased expression of mRNA and protein can be significantly inhibited while adding SB-431512 into experimental group （P 〈0. 05）. Conclusion Tumor-associated cells can promote the proliferation of ovarian cancer Cells via paracrine mechanism, and upregulated some gene and protein related to angiogenesis, and which can be effectively inhibited by inhibiting TGF-β signal pathway.