低剂量铬上调FLNA促进膀胱癌细胞增殖效应的初步探索
发布时间：2019-03-05 10:59
【摘要】：目的明确低剂量重金属铬暴露后,对膀胱癌进展产生的影响,并初步探讨其对FLNA基因表达的影响及相关的细胞生物学行为。方法采用MTT方法筛选Cr(VI)暴露T24细胞的亚致死剂量,明确低剂量Cr(VI)对T24细胞亚致死浓度;目的是筛选出对细胞增殖效应最为明显的铬浓度。在该浓度下的Cr(VI)处理T24细胞后分别进行细胞划痕实验、Transwell实验研究Cr(VI)暴露对细胞迁移能力、侵袭能力;采用免疫组化、qPCR免疫荧光和Western bolt等技术分析膀胱癌及其癌旁组织;膀胱癌细胞系中的表达水平,探讨其与临床分期的关系。利用shFLNA质粒转染T24细胞系后,利用流式细胞技术检测敲低FLNA后的T24细胞凋亡影响。利用划痕实验、Transwell实验检测敲低前后的侵袭性、迁移性的改变。用Cr(VI)刺激T24细胞后,利用免疫荧光和Western bolt检测FLNA的表达情况。结果Cr(VI)暴露可促进T24细胞增殖,并发现在铬暴露剂量为0.4uM时,其促进增殖效应最为显著(P0.05);Cr(VI)暴露T24细胞后,细胞迁移以及侵袭能力均显著增加(P0.05);而免疫组化结果显示,浸润性尿路上皮癌组织中FLNA的表达呈强阳性,而非浸润性尿路上皮癌的组织则为弱阳性,正常组织免疫组化结果为阴性,结果具有统计学意义。qPCR检测膀胱癌组织及其癌旁组织FLNA mRNA表达量表明,肌层浸润性尿路上皮癌FLNA的表达较非肌层浸润性尿路上皮癌高,而癌旁组织的表达量最低。Western blot结果表明正常人脐静脉细胞(HUVC)中FLNA的蛋白表达量低于T24细胞系qPCR结果也表明T24细胞系的FLNA表达高于HUVC细胞系。划痕实验和Transwell实验的结果表明shFLNA肿细胞的迁移能力和侵袭能力较正常未处理对照组T24细胞低。而在低剂量铬表露后Western blot结果表明FLNA的表达增高。而免疫荧光的结果同样也表明FLNA在低剂量铬暴露后在细胞质中较未经过铬刺激的细胞呈高表达。结论Cr(VI)暴露可显著影响膀胱细胞的生物学行为,促进膀胱癌进展。该作用机制是低剂量铬调控FLNA及相关信号通路的表达发挥其促进肿瘤进展的作用。
[Abstract]:Objective To study the effect of low dose of heavy metal chromium on the progression of bladder cancer and to explore its effect on the expression of FLNA gene and the related cellular biological behavior. Methods The sublethal dose of Cr (VI) exposed to T24 cells was selected by MTT method, and the sublethal concentration of low dose of Cr (VI) on T24 cells was determined. The cell scratch test was carried out after the treatment of T24 cells with Cr (VI) at this concentration, and the cell migration ability and the invasion ability of Cr (VI) were studied by Transwell. The expression level of the bladder cancer cell line was analyzed by immunohistochemistry, qPCR immunofluorescence and Western blot. The relationship between the clinical stage and the clinical stage was discussed. After the T24 cell line was transfected with the shFLA plasmid, the apoptosis of T24 cells following the knockdown of FLNA was detected by flow cytometry. Using the scratch test, the transwell's experiment was used to detect the change of the invasion and migration before and after the knock. After T24 cells were stimulated with Cr (VI), the expression of FLNA was detected by immunofluorescence and Western blot. Results Cr (VI) exposure could promote the proliferation of T24 cells, and it was found that the effect of Cr (VI) on the proliferation of T24 cells was the most significant (P0.05). After the exposure of Cr (VI) to T24 cells, the cell migration and the invasion ability were significantly increased (P0.05). The expression of FLNA in the tissues of invasive urothelial carcinoma was strongly positive, while the non-invasive urothelial carcinoma was weakly positive, and the immunohistochemical results of normal tissues were negative and the results were of statistical significance. The expression of FLNA mRNA in bladder cancer tissues and its adjacent tissues was detected by qPCR, and the expression of FLNA in the invasive urothelial carcinoma of the myometrium was higher than that of the non-myometrial invasion urothelial carcinoma, while the expression of the adjacent tissue was the lowest. The results of Western blot show that the expression of FLNA in human umbilical vein cell (HUVC) is lower than that of T24 cell line, and the expression of FLNA in the T24 cell line is higher than that of HUVC cell line. The results of the scratch test and the Transwell experiment show that the ability and the invasion ability of the shFLA swollen cells are lower than that of the control group T24. The results showed that the expression of FLNA was increased after low dose of chromium. The results of immunofluorescence also show that FLNA is highly expressed in the cytoplasm, which is not stimulated by chromium, after low-dose chromium exposure. Conclusion The exposure of Cr (VI) can significantly affect the biological behavior of bladder cells and promote the progression of bladder cancer. The mechanism is the role of low-dose chromium control FLA and related signal pathways to play a role in promoting the progression of the tumor.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14
本文编号：2434834
[Abstract]:Objective To study the effect of low dose of heavy metal chromium on the progression of bladder cancer and to explore its effect on the expression of FLNA gene and the related cellular biological behavior. Methods The sublethal dose of Cr (VI) exposed to T24 cells was selected by MTT method, and the sublethal concentration of low dose of Cr (VI) on T24 cells was determined. The cell scratch test was carried out after the treatment of T24 cells with Cr (VI) at this concentration, and the cell migration ability and the invasion ability of Cr (VI) were studied by Transwell. The expression level of the bladder cancer cell line was analyzed by immunohistochemistry, qPCR immunofluorescence and Western blot. The relationship between the clinical stage and the clinical stage was discussed. After the T24 cell line was transfected with the shFLA plasmid, the apoptosis of T24 cells following the knockdown of FLNA was detected by flow cytometry. Using the scratch test, the transwell's experiment was used to detect the change of the invasion and migration before and after the knock. After T24 cells were stimulated with Cr (VI), the expression of FLNA was detected by immunofluorescence and Western blot. Results Cr (VI) exposure could promote the proliferation of T24 cells, and it was found that the effect of Cr (VI) on the proliferation of T24 cells was the most significant (P0.05). After the exposure of Cr (VI) to T24 cells, the cell migration and the invasion ability were significantly increased (P0.05). The expression of FLNA in the tissues of invasive urothelial carcinoma was strongly positive, while the non-invasive urothelial carcinoma was weakly positive, and the immunohistochemical results of normal tissues were negative and the results were of statistical significance. The expression of FLNA mRNA in bladder cancer tissues and its adjacent tissues was detected by qPCR, and the expression of FLNA in the invasive urothelial carcinoma of the myometrium was higher than that of the non-myometrial invasion urothelial carcinoma, while the expression of the adjacent tissue was the lowest. The results of Western blot show that the expression of FLNA in human umbilical vein cell (HUVC) is lower than that of T24 cell line, and the expression of FLNA in the T24 cell line is higher than that of HUVC cell line. The results of the scratch test and the Transwell experiment show that the ability and the invasion ability of the shFLA swollen cells are lower than that of the control group T24. The results showed that the expression of FLNA was increased after low dose of chromium. The results of immunofluorescence also show that FLNA is highly expressed in the cytoplasm, which is not stimulated by chromium, after low-dose chromium exposure. Conclusion The exposure of Cr (VI) can significantly affect the biological behavior of bladder cells and promote the progression of bladder cancer. The mechanism is the role of low-dose chromium control FLA and related signal pathways to play a role in promoting the progression of the tumor.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14
【参考文献】
相关期刊论文 前1条
1 史黎薇;铬化合物对健康影响的研究进展[J];卫生研究;2003年04期
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