小鼠乳腺癌细胞系4T1表面CCL5表达调控的研究

发布时间：2018-04-13 15:25

本文选题：小鼠乳腺癌细胞系4T1 + CCL5　；参考：《河北医科大学》2015年硕士论文

【摘要】：目的:乳腺癌作为威胁女性身心健康首位的恶性肿瘤,发病率呈逐年上升趋势。由于发病原因尚未完全阐明,因此预防、早期筛查和诊断便成为治愈的关键,这就使得分子生物技术倍受关注。CCL5趋化细胞因子是一种炎症细胞因子,趋化T细胞和单核细胞,在肿瘤生长和转移中具有多重效应,包括抑制肿瘤细胞的免疫效应、促进肿瘤的生长、新血管的生成和转移等,也是乳腺肿瘤细胞表达的主要趋化因子,与乳腺癌的进展息息相关,p38是细胞内重要的信号通路,可以调节很多细胞因子的表达。本实验拟对LPS诱导小鼠乳腺癌4T1细胞系引起CCL5的表达升高的机制展开进一步研究。方法:1用RT-PCR和Western Blot的方法检测小鼠乳腺癌细胞系4T1表面TLR4的表达及诱导表达。2 LPS诱导4T1细胞,提取总RNA,用RT-PCR检测MCP-1、VEGF和CCL5在基因水平的表达情况。同时用实时定量PCR的方法进行检测MCP-1、VEGF和CCL5在基因水平的表达情况。3用Western Blot的方法检测LPS诱导4T1细胞后MAPK中T-p38和P-p38、T-Erk和P-pErk、T-JNK和P-JNK及NF-κB中T-p65和P-p65的蛋白表达变化。4采用实时定量PCR的方法来检测使用通路抑制剂后LPS诱导的4T1细胞的CCL5的表达。5构建携带目的基因重组质粒并在小鼠巨噬细胞RAW264.7进行脂质体转染。在荧光素酶检测仪上进行荧光素酶的数据测定。6划痕实验和Transwell实验检测p38抑制剂对4T1细胞迁移及侵袭能力的影响。7用免疫印迹法来检测使用p38抑制剂后的4T1细胞EMT相关蛋白的表达。结果1 LPS诱导及未诱导的两组4T1细胞在基因和蛋白水平均有TLR4的表达。2在基因水平上,LPS诱导4T1细胞组与未诱导4T1细胞组相比较,CCL5表达明显升高,而MCP-1和VEGF的表达没有明显变化。3 LPS刺激4T1细胞后,可以导致p38、Erk、JNK和p65明显磷酸化升高。4 CCL5在使用p38和p65的阻断剂后的表达有差异,且p38阻断剂组与p65阻断剂组相比而言差异更加显著,阻断Erk、JNK后CCL5的表达无明显变化。5荧光素酶检测的结果表明,LPS在4T1细胞上导致的CCL5表达的增高是通过导致启动子表达增高实现的。6 p38抑制剂可以抑制4T1细胞的迁移能力而对侵袭能力没有影响7 p38抑制剂可以抑制EMT的相关蛋白β-catenin和Vimentin的表达,而对Snail的表达无影响。结论:1 LPS诱导4T1细胞使得CCL5表达增高,这种诱导增高是通过使得CCL5启动子的能力增强来实现的。2 p38抑制剂可以抑制4T1细胞的迁移能力和EMT相关蛋白的表达。
[Abstract]:Objective: the incidence of breast cancer is increasing year by year as the first malignant tumor threatening women's physical and mental health.Since the cause of the disease has not been fully clarified, prevention, early screening and diagnosis are the key to cure, which makes molecular biotechnology pay more attention to. CCL5 chemokine is an inflammatory cytokine, chemokine T cell and monocyte.It has multiple effects in tumor growth and metastasis, including inhibiting the immune effect of tumor cells, promoting tumor growth, and the formation and metastasis of new blood vessels. It is also the main chemokine of breast cancer cell expression.P38 is an important cellular signaling pathway that regulates the expression of many cytokines.The aim of this study was to investigate the mechanism of CCL5 expression in mouse breast cancer 4T1 cell line induced by LPS.Methods RT-PCR and Western Blot were used to detect the expression of TLR4 on the surface of mouse breast cancer cell line 4T1, and the induced expression of .2 LPS was used to induce 4T1 cells. The total RNAs were extracted, and the expression of MCP-1VEGF and CCL5 at gene level was detected by RT-PCR.Meanwhile, real-time quantitative PCR was used to detect the expression of MCP-1VEGF and CCL5 at the gene level. Western Blot was used to detect the changes of T-p38 and P-p38 T-Erk and P-pErkton-JNK and P-JNK in 4T1 cells induced by LPS and T-p65 and P-p65 in NF- 魏 B.The expression of CCL5 in 4T1 cells induced by LPS was detected by quantitative PCR. 5. The recombinant plasmid carrying the target gene was constructed and transfected into murine macrophages RAW264.7 by liposome.The effect of p38 inhibitor on the migration and invasion of 4T1 cells by Transwell assay. 7. Western blot method was used to detect 4T1 cells after p38 inhibitor was used to detect the effect of p38 inhibitor on the migration and invasion of 4T1 cells.Expression of EMT related proteins.Results 1 the expression of TLR4 was found in both LPS induced and uninduced 4T1 cells at both gene and protein levels. The expression of CCL5 in LPS-induced 4T1 cells was significantly higher than that in uninduced 4T1 cells at the gene level.However, the expression of MCP-1 and VEGF did not change significantly after 3. 3 LPS stimulated 4T1 cells, which resulted in a significant increase in phosphorylation of p38 Erkon JNK and p65 in 4T1 cells. There was a difference in the expression of 4. 4 CCL5 after the use of p38 and p65 blockers.The difference between p38 blocker group and p65 blocker group was more significant.There was no significant change in the expression of CCL5 after blocking CCL5. 5 the results of luciferase detection showed that the increase of CCL5 expression induced by LPs in 4T1 cells could inhibit the migration of 4T1 cells by inducing the increase of promoter expression.The expression of 尾 -catenin and Vimentin in EMT was inhibited by 7p38 inhibitor.However, there was no effect on the expression of Snail.Conclusion 1 LPS induces an increase in CCL5 expression in 4T1 cells. This increase is achieved by enhancing the ability of CCL5 promoters to inhibit the migration of 4T1 cells and the expression of EMT related proteins.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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