衰老间质成纤维细胞对人结肠癌LoVo细胞生物学特性的影响
发布时间：2018-09-06 07:36
【摘要】：研究目的:通过前期建立的间质衰老模型,模拟肿瘤微环境中的肿瘤相关成纤维细胞(Tumor associated fibroblast,TAFs),观察衰老间质对人结肠癌LoVo细胞上皮-间质转化、皮下成瘤与转移等生物学作用的影响。研究方法:应用Transwell共培养模型将LoVo细胞分别与衰老间质成纤维细胞和正常间质成纤维细胞共培养(A组:LoVo+LV-GALC,B组:LoVo+LV-NC),通过Western Blot检测LoVo细胞中上皮-间质样表型转换(Epithelial mesenchymal transition,EMT)相关分子标志物表达的改变情况,并分别将LoVo细胞与LV-GALC和LV-NC细胞制成混合细胞悬液,接种于裸鼠皮下,观察其成瘤情况。通过流式细胞仪检测与间质细胞共培养后的LoVo细胞肿瘤干细胞(Cancer stem cell,CSC)相关指标的表达情况。并进行转录组学分析筛选两组中差异表达的蛋白并在荷瘤小鼠肿瘤组织中进行该蛋白的表达验证。结果:与B组相比,A组Lo Vo细胞上皮相关标记物E-cadherin表达下降,但无统计学差异,间质相关标记物Vimentin在48h及72h均明显升高(p0.05)。并且A组小鼠肿瘤体积与肿瘤重量明显大于B组小鼠(p0.05)。A组与B组LoVo细胞CD44均表达阳性且未见明显差异,而CD24、CD133均未见明显表达。转录组学分析发现:A组LoVo细胞中转录激活因子3(Activating transcription factor 3,ATF3)表达明显上调。并且A组小鼠肿瘤组织内ATF3表达量明显高于B组小鼠(p0.05)。结论:衰老间质成纤维细胞可能通过分泌相关调节因子,引起人结肠癌LoVo细胞ATF3的激活表达从而促进其发生EMT并提高其成瘤能力。
[Abstract]:Objective: to study the epithelial-interstitial transformation of human colon cancer (LoVo) cells by simulating tumor associated fibroblasts (Tumor associated fibroblast,TAFs) in tumor microenvironment by using the model of interstitial senescence. The biological effects of subcutaneous tumorigenesis and metastasis. Methods: using Transwell co-culture model, LoVo cells were co-cultured with senescent stromal fibroblasts and normal fibroblasts (group A: LoVo LV-GALC,B: LoVo LV-NC). Epithelial was detected by Western Blot in LoVo cells. Changes in the expression of molecular markers associated with mesenchymal transition,EMT, The mixed cell suspension of LoVo cells with LV-GALC and LV-NC cells were inoculated into nude mice subcutaneously to observe the tumorigenesis. The expression of (Cancer stem cell,CSC in tumor stem cells of LoVo cells after co-culture with mesenchymal cells was detected by flow cytometry. The differentially expressed proteins in the two groups were screened by transcriptome analysis and verified in tumor tissues of tumor-bearing mice. Results: compared with group B, E-cadherin expression of epithelial-associated markers of Lo Vo cells in group A decreased, but there was no statistical difference. Vimentin, the interstitial marker, increased significantly at 48h and 72h (p0.05). The tumor volume and tumor weight in group A were significantly higher than those in group B (p0.05). The positive expression of CD44 in LoVo cells of group A and group B was not significantly different, but no significant expression of CD24,CD133 was found. Transcriptome analysis showed that the expression of transcriptional activator 3 (Activating transcription factor 3 (ATF3) was up-regulated in LoVo cells of group A. The expression of ATF3 in tumor tissue of group A was significantly higher than that of group B (p0. 05). Conclusion: senescent mesenchymal fibroblasts may induce the activation and expression of ATF3 in human colon cancer LoVo cells by secreting related regulatory factors, thus promoting the development of EMT and enhancing its tumorigenesis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.3
[Abstract]:Objective: to study the epithelial-interstitial transformation of human colon cancer (LoVo) cells by simulating tumor associated fibroblasts (Tumor associated fibroblast,TAFs) in tumor microenvironment by using the model of interstitial senescence. The biological effects of subcutaneous tumorigenesis and metastasis. Methods: using Transwell co-culture model, LoVo cells were co-cultured with senescent stromal fibroblasts and normal fibroblasts (group A: LoVo LV-GALC,B: LoVo LV-NC). Epithelial was detected by Western Blot in LoVo cells. Changes in the expression of molecular markers associated with mesenchymal transition,EMT, The mixed cell suspension of LoVo cells with LV-GALC and LV-NC cells were inoculated into nude mice subcutaneously to observe the tumorigenesis. The expression of (Cancer stem cell,CSC in tumor stem cells of LoVo cells after co-culture with mesenchymal cells was detected by flow cytometry. The differentially expressed proteins in the two groups were screened by transcriptome analysis and verified in tumor tissues of tumor-bearing mice. Results: compared with group B, E-cadherin expression of epithelial-associated markers of Lo Vo cells in group A decreased, but there was no statistical difference. Vimentin, the interstitial marker, increased significantly at 48h and 72h (p0.05). The tumor volume and tumor weight in group A were significantly higher than those in group B (p0.05). The positive expression of CD44 in LoVo cells of group A and group B was not significantly different, but no significant expression of CD24,CD133 was found. Transcriptome analysis showed that the expression of transcriptional activator 3 (Activating transcription factor 3 (ATF3) was up-regulated in LoVo cells of group A. The expression of ATF3 in tumor tissue of group A was significantly higher than that of group B (p0. 05). Conclusion: senescent mesenchymal fibroblasts may induce the activation and expression of ATF3 in human colon cancer LoVo cells by secreting related regulatory factors, thus promoting the development of EMT and enhancing its tumorigenesis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.3
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