本文選題：ACSS2　切入點：電離輻射　出處：《江蘇大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：【目的】檢測乙酰輔酶A合成酶2(Acyl-coenzyme A synthetase short-chain family member 2,ACSS2)對人食管鱗癌細(xì)胞放療敏感性的影響,探討其影響食管鱗癌放療敏感性的相關(guān)分子機制�！痉椒ā�(1)Western blot法檢測食管鱗癌細(xì)胞(TE-1,TE-10,ECA-109,,KYSE-150)和正常食管上皮細(xì)胞HET-1A在正常培養(yǎng)(含10%胎牛血清的DMEM)與營養(yǎng)缺乏處理(含1%胎牛血清的DMEM培養(yǎng)基)后ACSS2的表達(dá)變化情況;(2)熒光定量PCR檢測營養(yǎng)缺乏處理不同時間(24 h,48 h,7 day)的食管鱗癌細(xì)胞中ACSS2的mRNA含量;Western blot法檢測食管鱗癌細(xì)胞在營養(yǎng)缺乏不同時間(0 day,1 day,2 day,7 day,30 day)后ACSS2的表達(dá)情況;CCK-8細(xì)胞增殖實驗檢測不同處理組(對照組,營養(yǎng)缺乏組,干擾組,營養(yǎng)缺乏加干擾組)細(xì)胞增殖情況;Western blot法檢測不同處理組Ki67的表達(dá)情況;(3)流式細(xì)胞術(shù)檢測不同處理組(對照組,營養(yǎng)缺乏組,干擾組,營養(yǎng)缺乏加干擾組)的細(xì)胞周期分布情況;Western blot法檢測不同處理組Akt,p-Akt,mTOR,p-m TOR的表達(dá)情況;Western blot法檢測不同處理組(對照組,氯喹組,營養(yǎng)缺乏組,營養(yǎng)缺乏加干擾組)LC3B-II,ATG5,ATG7的表達(dá)情況;(4)將實驗組分為對照組,營養(yǎng)缺乏組,干擾組,營養(yǎng)缺乏加干擾組,8 Gy照光,流式細(xì)胞術(shù)檢測不同處理組的細(xì)胞凋亡率,Western blot法檢測不同處理組cleaved caspase-3,Bcl-2,Bax的表達(dá)情況;按相同分組,細(xì)胞克隆實驗檢測食管鱗癌細(xì)胞在0 Gy、2 Gy、4 Gy、6 Gy、8 Gy五種不同放射劑量下的存活分?jǐn)?shù);(5)免疫組織化學(xué)染色法檢測60例食管鱗癌病理切片癌組織和癌旁組織中ACSS2的表達(dá)情況,并檢測ACSS2的表達(dá)與臨床病理特征的相關(guān)性�！窘Y(jié)果】(1)ACSS2在營養(yǎng)缺乏處理后表達(dá)上調(diào)。ACSS2在正常營養(yǎng)條件下,在四種人食管鱗癌細(xì)胞中都有表達(dá),在正常食管細(xì)胞HET-1A中幾乎不表達(dá);而在營養(yǎng)缺乏處理后,在食管鱗癌細(xì)胞中表達(dá)都明顯上調(diào),在正常食管細(xì)胞中變化不明顯;同時ACSS2的mRNA和蛋白的表達(dá)程度隨著營養(yǎng)缺乏處理短期內(nèi)升高,長期處理可以保持較高表達(dá)水平;(2)ACSS2的表達(dá)變化對食管鱗癌細(xì)胞的細(xì)胞增殖和細(xì)胞周期沒有影響。CCK-8細(xì)胞增殖實驗檢測發(fā)現(xiàn)不同處理組在同時間段內(nèi)的細(xì)胞增殖無明顯區(qū)別;流式細(xì)胞術(shù)檢測不同處理組細(xì)胞周期分布,差異無統(tǒng)計學(xué)意義;Western blot檢測Ki-67,在不同處理組中表達(dá)無差異;(3)營養(yǎng)缺乏后,下調(diào)ACSS2可以促進(jìn)Akt/mTOR信號通路的激活和抑制自噬的發(fā)生。營養(yǎng)缺乏組的p-Akt,p-mTOR,LC3B-II,ATG5和ATG7相對于對照組明顯上調(diào),干擾ACSS2后可以抑制上述蛋白的表達(dá)變化;(4)營養(yǎng)缺乏后,ACSS2升高抑制了由電離輻射引起的凋亡發(fā)生。流式細(xì)胞術(shù)及細(xì)胞克隆實驗均顯示,電離輻射處理后,營養(yǎng)缺乏誘導(dǎo)的ACSS2表達(dá)升高促進(jìn)了食管鱗癌細(xì)胞的Bcl-2的表達(dá)上調(diào)同時抑制cleaved caspase 3的表達(dá),從而抑制凋亡發(fā)生;下調(diào)ACSS2表達(dá)可以逆轉(zhuǎn)這一趨勢,促進(jìn)凋亡出現(xiàn)。表明營養(yǎng)缺乏條件下的ACSS2累積促進(jìn)了放療抵抗的形成;(5)食管鱗癌組織中高表達(dá)的ACSS2影響患者預(yù)后。60例食管癌組織中,食管鱗癌組織病理切片中ACSS2有較強的表達(dá),尤其在腫瘤細(xì)胞密集區(qū)域表達(dá)更明顯,顯著高于癌旁組織(p0.05);ACSS2表達(dá)強度與病人的年齡、性別、腫瘤部位、腫瘤大小、TMN分期無關(guān),與五年生存期和淋巴轉(zhuǎn)移相關(guān)(p0.05)。【結(jié)論】營養(yǎng)缺乏誘導(dǎo)ACSS2表達(dá)上調(diào),進(jìn)一步調(diào)控Akt/mTOR通路促進(jìn)食管鱗癌細(xì)胞自噬的發(fā)生,從而導(dǎo)致了以抑制凋亡為主的放療抵抗形成。
[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.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.1

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