【摘要】：研究目的:通過前期建立的間質(zhì)衰老模型,模擬腫瘤微環(huán)境中的腫瘤相關(guān)成纖維細(xì)胞(Tumor associated fibroblast,TAFs),觀察衰老間質(zhì)對(duì)人結(jié)腸癌LoVo細(xì)胞上皮-間質(zhì)轉(zhuǎn)化、皮下成瘤與轉(zhuǎn)移等生物學(xué)作用的影響。研究方法:應(yīng)用Transwell共培養(yǎng)模型將LoVo細(xì)胞分別與衰老間質(zhì)成纖維細(xì)胞和正常間質(zhì)成纖維細(xì)胞共培養(yǎng)(A組:LoVo+LV-GALC,B組:LoVo+LV-NC),通過Western Blot檢測LoVo細(xì)胞中上皮-間質(zhì)樣表型轉(zhuǎn)換(Epithelial mesenchymal transition,EMT)相關(guān)分子標(biāo)志物表達(dá)的改變情況,并分別將LoVo細(xì)胞與LV-GALC和LV-NC細(xì)胞制成混合細(xì)胞懸液,接種于裸鼠皮下,觀察其成瘤情況。通過流式細(xì)胞儀檢測與間質(zhì)細(xì)胞共培養(yǎng)后的LoVo細(xì)胞腫瘤干細(xì)胞(Cancer stem cell,CSC)相關(guān)指標(biāo)的表達(dá)情況。并進(jìn)行轉(zhuǎn)錄組學(xué)分析篩選兩組中差異表達(dá)的蛋白并在荷瘤小鼠腫瘤組織中進(jìn)行該蛋白的表達(dá)驗(yàn)證。結(jié)果:與B組相比,A組Lo Vo細(xì)胞上皮相關(guān)標(biāo)記物E-cadherin表達(dá)下降,但無統(tǒng)計(jì)學(xué)差異,間質(zhì)相關(guān)標(biāo)記物Vimentin在48h及72h均明顯升高(p0.05)。并且A組小鼠腫瘤體積與腫瘤重量明顯大于B組小鼠(p0.05)。A組與B組LoVo細(xì)胞CD44均表達(dá)陽性且未見明顯差異,而CD24、CD133均未見明顯表達(dá)。轉(zhuǎn)錄組學(xué)分析發(fā)現(xiàn):A組LoVo細(xì)胞中轉(zhuǎn)錄激活因子3(Activating transcription factor 3,ATF3)表達(dá)明顯上調(diào)。并且A組小鼠腫瘤組織內(nèi)ATF3表達(dá)量明顯高于B組小鼠(p0.05)。結(jié)論:衰老間質(zhì)成纖維細(xì)胞可能通過分泌相關(guān)調(diào)節(jié)因子,引起人結(jié)腸癌LoVo細(xì)胞ATF3的激活表達(dá)從而促進(jìn)其發(fā)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.3

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