ACSS2参与食管鳞癌细胞营养缺乏诱导的放疗抵抗形成

发布时间：2018-03-10 13:16

本文选题：ACSS2　切入点：电离辐射　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：【目的】检测乙酰辅酶A合成酶2(Acyl-coenzyme A synthetase short-chain family member 2,ACSS2)对人食管鳞癌细胞放疗敏感性的影响,探讨其影响食管鳞癌放疗敏感性的相关分子机制。【方法】(1)Western blot法检测食管鳞癌细胞(TE-1,TE-10,ECA-109,,KYSE-150)和正常食管上皮细胞HET-1A在正常培养(含10%胎牛血清的DMEM)与营养缺乏处理(含1%胎牛血清的DMEM培养基)后ACSS2的表达变化情况;(2)荧光定量PCR检测营养缺乏处理不同时间(24 h,48 h,7 day)的食管鳞癌细胞中ACSS2的mRNA含量;Western blot法检测食管鳞癌细胞在营养缺乏不同时间(0 day,1 day,2 day,7 day,30 day)后ACSS2的表达情况;CCK-8细胞增殖实验检测不同处理组(对照组,营养缺乏组,干扰组,营养缺乏加干扰组)细胞增殖情况;Western blot法检测不同处理组Ki67的表达情况;(3)流式细胞术检测不同处理组(对照组,营养缺乏组,干扰组,营养缺乏加干扰组)的细胞周期分布情况;Western blot法检测不同处理组Akt,p-Akt,mTOR,p-m TOR的表达情况;Western blot法检测不同处理组(对照组,氯喹组,营养缺乏组,营养缺乏加干扰组)LC3B-II,ATG5,ATG7的表达情况;(4)将实验组分为对照组,营养缺乏组,干扰组,营养缺乏加干扰组,8 Gy照光,流式细胞术检测不同处理组的细胞凋亡率,Western blot法检测不同处理组cleaved caspase-3,Bcl-2,Bax的表达情况;按相同分组,细胞克隆实验检测食管鳞癌细胞在0 Gy、2 Gy、4 Gy、6 Gy、8 Gy五种不同放射剂量下的存活分数;(5)免疫组织化学染色法检测60例食管鳞癌病理切片癌组织和癌旁组织中ACSS2的表达情况,并检测ACSS2的表达与临床病理特征的相关性。【结果】(1)ACSS2在营养缺乏处理后表达上调。ACSS2在正常营养条件下,在四种人食管鳞癌细胞中都有表达,在正常食管细胞HET-1A中几乎不表达;而在营养缺乏处理后,在食管鳞癌细胞中表达都明显上调,在正常食管细胞中变化不明显;同时ACSS2的mRNA和蛋白的表达程度随着营养缺乏处理短期内升高,长期处理可以保持较高表达水平;(2)ACSS2的表达变化对食管鳞癌细胞的细胞增殖和细胞周期没有影响。CCK-8细胞增殖实验检测发现不同处理组在同时间段内的细胞增殖无明显区别;流式细胞术检测不同处理组细胞周期分布,差异无统计学意义;Western blot检测Ki-67,在不同处理组中表达无差异;(3)营养缺乏后,下调ACSS2可以促进Akt/mTOR信号通路的激活和抑制自噬的发生。营养缺乏组的p-Akt,p-mTOR,LC3B-II,ATG5和ATG7相对于对照组明显上调,干扰ACSS2后可以抑制上述蛋白的表达变化;(4)营养缺乏后,ACSS2升高抑制了由电离辐射引起的凋亡发生。流式细胞术及细胞克隆实验均显示,电离辐射处理后,营养缺乏诱导的ACSS2表达升高促进了食管鳞癌细胞的Bcl-2的表达上调同时抑制cleaved caspase 3的表达,从而抑制凋亡发生;下调ACSS2表达可以逆转这一趋势,促进凋亡出现。表明营养缺乏条件下的ACSS2累积促进了放疗抵抗的形成;(5)食管鳞癌组织中高表达的ACSS2影响患者预后。60例食管癌组织中,食管鳞癌组织病理切片中ACSS2有较强的表达,尤其在肿瘤细胞密集区域表达更明显,显著高于癌旁组织(p0.05);ACSS2表达强度与病人的年龄、性别、肿瘤部位、肿瘤大小、TMN分期无关,与五年生存期和淋巴转移相关(p0.05)。【结论】营养缺乏诱导ACSS2表达上调,进一步调控Akt/mTOR通路促进食管鳞癌细胞自噬的发生,从而导致了以抑制凋亡为主的放疗抵抗形成。
[Abstract]:[Objective] to detect acetyl coenzyme A synthetase 2 (Acyl-coenzyme A synthetase short-chain family member 2, ACSS2) in human esophageal squamous cell carcinoma cells influence on radiotherapy sensitivity, to investigate its effect on the sensitivity of radiotherapy for esophageal squamous cell carcinoma and related molecular mechanism. [Methods] (1) of esophageal squamous cell carcinoma cells was measured by Western blot method (TE-1, TE-10, ECA-109, KYSE-150) and normal esophageal epithelial cells in normal cultured HET-1A (DMEM containing 10% fetal bovine serum) and nutrient deficiency treatment (containing 1% FBS DMEM) expression changes after ACSS2; (2) fluorescence quantitative PCR detection of nutritional deficiency of different treatment time (24 h, 48 h, 7 day mRNA) the content of ACSS2 in esophageal squamous cell carcinoma; esophageal squamous cell carcinoma cell line Western blot detection method in the lack of nutrition in different time (0 day, 1 day, 2 day, 7 day, 30 day) expression after ACSS2; CCK-8 cell proliferation assay to detect the different treatment groups (control Group, nutrition group, interference group, lack of nutrition interference group) cell proliferation; expression of Western blot was detected in different groups of Ki67; (3) detection of flow cytometry in different treatment groups (control group, nutrition group, interference group, lack of nutrition interference group) the distribution of cell cycle Western; blot was detected in different treatment groups Akt, p-Akt, mTOR, expression of P-M TOR; Western blot was used to detect the different treatment group (control group, chloroquine group, nutritional deficiency group, deficiency plus interference group) LC3B-II, ATG5, ATG7 expression; (4) in the experimental group were divided into control group. The lack of nutrition, lack of nutrition interference group, plus interference group, 8 Gy light, to detect the apoptosis rate of different treatment groups by flow cytometry, detection of different treatment groups cleaved Caspase-3, Western blot Bcl-2, the expression of Bax; in the same group, esophageal squamous cell carcinoma cell cloning test In 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, five different doses of radiation under the survival fraction; (5) immunohistochemical expression of ACSS2 was detected in 60 cases of esophageal squamous cell carcinoma pathological sections in carcinoma and adjacent tissues, and the correlation analysis of ACSS2 expression and clinical pathological features. [results] (1) the upregulation of the expression of.ACSS2 in normal nutrition condition in ACSS2 deficiency after treatment in four human esophageal squamous cell carcinoma are expressed almost no expression in normal esophageal epithelial cells in HET-1A; and in the lack of nutrition after the treatment, the expression was significantly up-regulated in esophageal squamous cell carcinoma and in normal esophageal epithelial cells no significant change in the expression level of mRNA protein; at the same time and ACSS2 with nutrient deficiency treatment increased in the short term, long-term treatment can maintain a high level of expression; (2) the expression of ACSS2 in esophageal squamous cell carcinoma cell proliferation and cell cycle had no effect on.CCK- 8 cell proliferation assays showed no significant difference between cell proliferation in different groups in the same period of time; the distribution of cell cycle was detected by flow cytometry, the difference was not statistically significant; Western blot detection of Ki-67 expression, no difference in different groups; (3) the lack of nutrition, the down-regulation of ACSS2 can promote Akt/mTOR the signal pathway activation and inhibition of autophagy. The lack of nutrition group p-Akt, p-mTOR, LC3B-II, ATG5 and ATG7 was significantly increased compared with the control group, the expression of ACSS2 could inhibit the protein interference; (4) the lack of nutrition, elevated ACSS2 inhibited apoptosis caused by ionizing radiation. Flow cytometry and cell cloning experiments showed that ionizing radiation treatment, nutritional deficiency induced increased expression of ACSS2 promote the expression of esophageal squamous cell carcinoma cell line Bcl-2 and inhibited cleaved expression of caspase 3, thus inhibiting For the occurrence of apoptosis; expression of ACSS2 can reverse this trend, promoting apoptosis. That lack of nutrition under the conditions of the accumulation of ACSS2 promoted the resistance to radiation formation; (5) high expression in esophageal squamous cell carcinoma ACSS2 affect the prognosis of patients with.60 cases of esophageal carcinoma, the ACSS2 pathological scales of esophageal cancer tissue has a strong the expression, especially in dense regions of tumor cells expressed more obviously, significantly higher than that in paracancerous tissues (P0.05); the expression of ACSS2 and the patient's age, gender, tumor location, tumor size, TMN stage, and five year survival and lymph node metastasis (P0.05). [Conclusion] the nutritional deficiency induced up regulation of ACSS2 expression. To promote the further regulation of the Akt/mTOR pathway in esophageal squamous cell carcinoma cell autophagy, which leads to the inhibition of apoptosis is the main resistance to radiotherapy.

【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

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