RasGRP3调控食管鳞癌血管新生及转移相关分子机制的初步研究

发布时间：2018-05-17 01:05

本文选题：RasGRP3 + 营养缺乏　；参考：《江苏大学》2017年硕士论文

【摘要】：【目的】检测Ras GRP3(RAS Guanyl Nucleotide Releasing Protein 3,Ras鸟苷酸释放蛋白3)对食管鳞状细胞癌血管生成和转移能力的影响,探讨其促进食管鳞癌转移的潜在机制。【方法】(1)Western blot法检测在正常浓度血清和低血清培养下的正常食管上皮细胞和4种不同食管鳞癌细胞株中Ras GRP3、Ras-GTP及总Ras的表达;CCK8法检测正常浓度血清和低血清培养的食管鳞癌TE-1细胞中干扰Ras GRP3后细胞的增殖能力及Ki-67表达,流式细胞术检测正常浓度血清和低血清培养的食管鳞癌TE-1细胞中干扰Ras GRP3后相应的细胞凋亡情况;免疫组织化学染色法检测70例食管癌患者癌组织与癌旁组织中Ras GRP3的表达;免疫组化与免疫荧光双标法检测46例食管癌患者癌组织中Ras GRP3、CD31的表达;(2)定量PCR检测干扰Ras GRP3后TE-1细胞中VEGF-A m RNA的表达;Western blot法检测低血清处理组TE-1细胞干扰Ras GRP3后VEGF-A蛋白的表达量;ELISA法检测正常培养组和低血清处理组TE-1细胞干扰Ras GRP3后上清液中VEGF-A的表达;Transwell共培养实验检测正常培养组和低血清处理组TE-1细胞中干扰Ras GRP3后管腔形成能力;(3)免疫荧光检测不同处理组TE-1细胞中上皮标志物E-cadherin和间质标志物Vimentin表达的变化;定量PCR检测不同处理后TE-1细胞中Slug、snail1和Hes1的m RNA表达;Notch通路抑制剂DAPT预处理TE-1细胞后,Western blot法检测NICD、MMP9等蛋白表达变化;划痕实验和TranswellTM实验进一步验证干扰Ras GRP3对食管癌细胞系迁移能力的影响;(4)免疫组化法检测70例肿瘤组织中EMT及转移相关蛋白的变化;生存曲线分析Ras GRP3与肿瘤转移及预后的关系。【结果】(1)低血清培养的TE-1细胞中Ras GRP3蛋白表达上调,但TE-1细胞的增殖、凋亡能力没有受到影响;Ras GRP3在食管癌组织中的分布与肿瘤转移相关:转移的食管癌患者癌组织中Ras GRP3的表达高于未转移的癌组织,转移的食管癌患者癌旁组织中Ras GRP3的表达高于未转移的癌旁组织,且Ras GRP3表达强度与病人的年龄、性别、肿瘤大小、肿瘤部位、TMN分期无关,与淋巴结转移、远处转移及肿瘤复发相关;Ras GRP3染色强度在CD31高表达的肿瘤组织中明显增加。(2)低血清培养的TE-1细胞中VEGF-A的表达明显增加;干扰Ras GRP3后VEGF-A表达下降,与HUVEC细胞共培养的体外管腔样结构形成减少。(3)免疫荧光显示低血清培养的TE-1细胞中E-cadherin表达强度增加,Vimentin表达下降,干扰Ras GRP3直接影响E-cadherin及Vimentin的表达;Ras GRP3的表达升高能上调EMT相关转录因子Slug、Snail1、Hes1的表达水平;蛋白水平显示低血清培养的TE-1细胞中E-Cadherin表达下调,Vimentin表达上调,基质金属蛋白酶MMP9表达上调,下调Ras GRP3和DAPT预处理均能影响E-Cadherin和Vimentin的表达并抑制食管癌细胞的迁移和侵袭。(4)MMP9、Vimentin、NICD在食管癌组织的表达与Ras GRP3表达相关,而Ki-67、VEGFR的表达与Ras GRP3表达无关;K-M生存曲线表明Ras GRP3表达水平与病人预后及转移相关。【结论】Ras GRP3通过激活Notch信号通路促进食管癌血管生成及转移的发生,表明Ras GRP3通过影响EMT和血管生成在食管癌的发展中起重要作用。
[Abstract]:[Objective] to detect the effect of Ras GRP3 (RAS Guanyl Nucleotide Releasing Protein 3, Ras guanosine releasing protein 3) on angiogenesis and metastasis of squamous cell carcinoma of the esophagus and explore its potential mechanism for promoting metastasis of esophageal squamous cell carcinoma. [Methods] Western blot method was used to detect normal esophagus in normal serum and low serum culture Expression of Ras GRP3, Ras-GTP and total Ras in epithelial cells and 4 different esophageal squamous cell carcinoma cells; CCK8 assay was used to detect the proliferation and Ki-67 expression of Ras GRP3 cells in normal serum and low serum cultured TE-1 cells of esophageal squamous cell carcinoma, and flow cytometry was used to detect the normal concentration serum and low serum culture of esophageal squamous cell carcinoma TE-1 cells. The expression of Ras GRP3 in 70 patients with esophageal cancer was detected by immunohistochemical staining, and the expression of Ras GRP3 in cancer tissues and adjacent tissues of 70 patients with esophageal cancer was detected by immunohistochemistry. The expression of Ras GRP3, CD31 in the cancer tissues of 46 patients with esophageal cancer was detected by immunohistochemistry and immunofluorescence, and (2) quantitative PCR detection interfered with Ras GRP3 in TE-1 cells. The expression of A and the expression of VEGF-A protein after Ras GRP3 were detected by TE-1 cells in low serum treatment group by Western blot method; ELISA method was used to detect the expression of VEGF-A in the supernatant of the normal culture group and the low serum treatment group after the Ras GRP3, and the co culture test was used to detect the interference of the normal culture group and the low serum treatment group. 3 posterior chamber formation ability; (3) changes in the expression of epithelial markers E-cadherin and interstitial marker Vimentin in TE-1 cells of different treatment groups by immunofluorescence; quantitative PCR was used to detect the m RNA expression of Slug, Snail1 and Hes1 in TE-1 cells after different treatments. Changes in white expression; scratch test and TranswellTM test to further verify the effect of interfering Ras GRP3 on the migration ability of esophageal cancer cell lines; (4) immunohistochemical method was used to detect the changes of EMT and metastasis related proteins in the tumor tissues; the relationship between Ras GRP3 and tumor metastasis and preconditioning was analyzed by the survival curve. [results] (1) TE-1 fine in low serum culture The expression of Ras GRP3 protein in the cell was up-regulated, but the proliferation and apoptosis of TE-1 cells were not affected; the distribution of Ras GRP3 in esophageal cancer tissues was related to tumor metastasis: the expression of Ras GRP3 in metastatic esophageal cancer tissues was higher than that of non metastatic carcinoma tissue. The expression of Ras GRP3 in metastatic esophageal cancer patients was higher than that of non metastasis. The expression intensity of Ras GRP3 was not related to age, sex, tumor size, tumor location, TMN staging, lymph node metastasis, distant metastasis and tumor recurrence, and Ras GRP3 staining intensity increased significantly in CD31 high expression of tumor tissue. (2) the expression of VEGF-A in low serum cultured TE-1 cells increased significantly; Ras interfered with Ras. The expression of VEGF-A decreased after GRP3 and decreased in vitro with the co culture of HUVEC cells. (3) immunofluorescence showed that the expression of E-cadherin in the low serum cultured TE-1 cells increased, the expression of Vimentin decreased, and the interference of Ras GRP3 directly affected the expression of E-cadherin and Vimentin. The expression level of lug, Snail1, Hes1, protein level showed that the expression of E-Cadherin in low serum cultured TE-1 cells was down regulated, the expression of Vimentin was up regulated, the expression of matrix metalloproteinase MMP9 was up-regulated. The down-regulation of Ras GRP3 and DAPT preconditioning could affect the expression of E-Cadherin and Vimentin and inhibit the migration and invasion of esophageal cancer cells. (4) The expression of Ras GRP3 was related to the expression of Ki-67 and VEGFR, and the expression of Ki-67, VEGFR was not related to the expression of Ras GRP3. The K-M survival curve showed that the GRP3 expression level of Ras was related to the prognosis and metastasis of the patients. [Conclusion] Ras GRP3 can promote the angiogenesis and metastasis of esophageal cancer by activating the Notch signal pathway. Angiogenesis plays an important role in the development of esophageal cancer.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

【参考文献】