本文選題：RasGRP3 + 營養(yǎng)缺乏�。� 參考：《江蘇大學》2017年碩士論文

【摘要】：【目的】檢測Ras GRP3(RAS Guanyl Nucleotide Releasing Protein 3,Ras鳥苷酸釋放蛋白3)對食管鱗狀細胞癌血管生成和轉移能力的影響,探討其促進食管鱗癌轉移的潛在機制。【方法】(1)Western blot法檢測在正常濃度血清和低血清培養(yǎng)下的正常食管上皮細胞和4種不同食管鱗癌細胞株中Ras GRP3、Ras-GTP及總Ras的表達;CCK8法檢測正常濃度血清和低血清培養(yǎng)的食管鱗癌TE-1細胞中干擾Ras GRP3后細胞的增殖能力及Ki-67表達,流式細胞術檢測正常濃度血清和低血清培養(yǎng)的食管鱗癌TE-1細胞中干擾Ras GRP3后相應的細胞凋亡情況;免疫組織化學染色法檢測70例食管癌患者癌組織與癌旁組織中Ras GRP3的表達;免疫組化與免疫熒光雙標法檢測46例食管癌患者癌組織中Ras GRP3、CD31的表達;(2)定量PCR檢測干擾Ras GRP3后TE-1細胞中VEGF-A m RNA的表達;Western blot法檢測低血清處理組TE-1細胞干擾Ras GRP3后VEGF-A蛋白的表達量;ELISA法檢測正常培養(yǎng)組和低血清處理組TE-1細胞干擾Ras GRP3后上清液中VEGF-A的表達;Transwell共培養(yǎng)實驗檢測正常培養(yǎng)組和低血清處理組TE-1細胞中干擾Ras GRP3后管腔形成能力;(3)免疫熒光檢測不同處理組TE-1細胞中上皮標志物E-cadherin和間質(zhì)標志物Vimentin表達的變化;定量PCR檢測不同處理后TE-1細胞中Slug、snail1和Hes1的m RNA表達;Notch通路抑制劑DAPT預處理TE-1細胞后,Western blot法檢測NICD、MMP9等蛋白表達變化;劃痕實驗和TranswellTM實驗進一步驗證干擾Ras GRP3對食管癌細胞系遷移能力的影響;(4)免疫組化法檢測70例腫瘤組織中EMT及轉移相關蛋白的變化;生存曲線分析Ras GRP3與腫瘤轉移及預后的關系�！窘Y果】(1)低血清培養(yǎng)的TE-1細胞中Ras GRP3蛋白表達上調(diào),但TE-1細胞的增殖、凋亡能力沒有受到影響;Ras GRP3在食管癌組織中的分布與腫瘤轉移相關:轉移的食管癌患者癌組織中Ras GRP3的表達高于未轉移的癌組織,轉移的食管癌患者癌旁組織中Ras GRP3的表達高于未轉移的癌旁組織,且Ras GRP3表達強度與病人的年齡、性別、腫瘤大小、腫瘤部位、TMN分期無關,與淋巴結轉移、遠處轉移及腫瘤復發(fā)相關;Ras GRP3染色強度在CD31高表達的腫瘤組織中明顯增加。(2)低血清培養(yǎng)的TE-1細胞中VEGF-A的表達明顯增加;干擾Ras GRP3后VEGF-A表達下降,與HUVEC細胞共培養(yǎng)的體外管腔樣結構形成減少。(3)免疫熒光顯示低血清培養(yǎng)的TE-1細胞中E-cadherin表達強度增加,Vimentin表達下降,干擾Ras GRP3直接影響E-cadherin及Vimentin的表達;Ras GRP3的表達升高能上調(diào)EMT相關轉錄因子Slug、Snail1、Hes1的表達水平;蛋白水平顯示低血清培養(yǎng)的TE-1細胞中E-Cadherin表達下調(diào),Vimentin表達上調(diào),基質(zhì)金屬蛋白酶MMP9表達上調(diào),下調(diào)Ras GRP3和DAPT預處理均能影響E-Cadherin和Vimentin的表達并抑制食管癌細胞的遷移和侵襲。(4)MMP9、Vimentin、NICD在食管癌組織的表達與Ras GRP3表達相關,而Ki-67、VEGFR的表達與Ras GRP3表達無關;K-M生存曲線表明Ras GRP3表達水平與病人預后及轉移相關�！窘Y論】Ras GRP3通過激活Notch信號通路促進食管癌血管生成及轉移的發(fā)生,表明Ras GRP3通過影響EMT和血管生成在食管癌的發(fā)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.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.1

【參考文獻】

相關期刊論文 前2條

1 Federico Coccolini;Giulia Montori;Marco Ceresoli;Simona Cima;Maria Carla Valli;Gabriela E Nita;Arianna Heyer;Fausto Catena;Luca Ansaloni;;Advanced gastric cancer: what we know and what we still have to learn[J];World Journal of Gastroenterology;2016年03期

2 Jing-Jing Zhang;San-Jun Chu;Xiao-Lei Sun;Ting Zhang;Wei-Yun Shi;;Bevacizumab modulates retinal pigment epithelial-tomesenchymal transition via regulating Notch signaling[J];International Journal of Ophthalmology;2015年02期

，

本文編號：1899207