本文選題：膜聯(lián)蛋白A11 + 胃癌�。� 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:近年來,雖然全球范圍內(nèi)的胃癌發(fā)病率處于下降趨勢,但我國的胃癌發(fā)病率卻在逐年上升。胃癌的發(fā)病率與死亡率均僅次于肺癌,位于第二位。以手術(shù)為主的綜合治療是目前胃癌的主要治療手段,但我國早期胃癌的診斷率不足10%,大多數(shù)患者就診時往往失去了根治性手術(shù)的機(jī)會,這就使化療在胃癌的綜合治療中占據(jù)重要的地位。自5-氟尿嘧啶(5-fluorouracil,5-Fu)問世以來,已過去50多年,但其仍是當(dāng)前胃癌化療方案的重要組成。由于腫瘤細(xì)胞對此類藥物原發(fā)性和繼發(fā)性耐藥,導(dǎo)致其單藥化療有效率不到20%。因此,闡明5-Fu的耐藥機(jī)制,緩解甚至逆轉(zhuǎn)其耐藥,成為當(dāng)今亟待解決的問題。膜聯(lián)蛋白家族(Annexins)是廣泛表達(dá)于除酵母之外的真核細(xì)胞的細(xì)胞質(zhì)及細(xì)胞核膜上的鈣調(diào)節(jié)磷脂結(jié)合蛋白,其通過調(diào)節(jié)細(xì)胞膜表面蛋白與其他位于細(xì)胞內(nèi)、細(xì)胞核膜、細(xì)胞外基質(zhì)的蛋白之間相互作用而發(fā)揮功效。膜聯(lián)蛋白家族共有A、B、C、D、E五個組,A組發(fā)現(xiàn)于哺乳動物,包括12個成員(A1-A11和A13)。目前研究表明膜聯(lián)蛋白A11(Annexin A11,ANXA11)與腫瘤的發(fā)生及發(fā)展有著密切關(guān)系。既往在胃癌中研究發(fā)現(xiàn)膜聯(lián)蛋白A11在胃癌組織中的陽性表達(dá)率和表達(dá)強(qiáng)度較正常胃組織中明顯增高。ANXA11在轉(zhuǎn)移的淋巴結(jié)中的陽性表達(dá)率和表達(dá)強(qiáng)度也較胃癌原發(fā)灶中明顯增高�？梢夾NXA11與胃癌的發(fā)生、發(fā)展及轉(zhuǎn)移密切相關(guān)。但目前關(guān)于ANXA11在不同胃癌細(xì)胞株中的表達(dá),及其與胃癌細(xì)胞5-Fu耐藥的關(guān)系尚未報道。本研究以人胃癌細(xì)胞株SGC7901為研究對象,應(yīng)用RNA干擾技術(shù)抑制SGC7901細(xì)胞中ANXA11的表達(dá),檢測不同組對5-Fu敏感性,發(fā)現(xiàn)轉(zhuǎn)染組較陰性對照組、空白對照組敏感性提高,并對其相關(guān)機(jī)制進(jìn)行初步探討。為闡明胃癌5-Fu耐藥機(jī)制進(jìn)一步提供理論基礎(chǔ),對于緩解乃至逆轉(zhuǎn)胃癌的5-Fu耐藥性及新治療靶點(diǎn)的發(fā)掘具有重要意義。方法:培養(yǎng)人胃癌細(xì)胞株SGC7901、人胃癌細(xì)胞株BGC-823,應(yīng)用實(shí)時熒光定量聚合酶鏈反應(yīng)(quantitative real time polymerase chain reaction,q PCR)、蛋白免疫印跡(Western Blot)檢測ANXA11 m RNA及蛋白在兩種細(xì)胞株中的表達(dá),并選取ANXA11高表達(dá)的人胃癌細(xì)胞株SGC7901。設(shè)計并合成針對ANXA11的特異性干擾序列si RNA及其陰性對照序列,應(yīng)用Lipofectamine 2000轉(zhuǎn)染試劑將ANXA11-si RNA及陰性對照序列瞬時轉(zhuǎn)染入SGC7901細(xì)胞,轉(zhuǎn)染后24h、48h、72h應(yīng)用q PCR檢測ANXA11 m RNA的表達(dá),應(yīng)用Western Blot檢測蛋白的表達(dá)情況,檢測干擾效果。應(yīng)用CCK-8法檢測轉(zhuǎn)染后各組細(xì)胞對5-Fu的藥物敏感性改變并計算出各組IC50(half maximal inhibitory concentration)與IC20值(考慮IC50濃度的5-Fu對細(xì)胞殺傷力較大,遂后續(xù)實(shí)驗(yàn)采用IC20濃度的5-Fu作用于ANXA11-si RNA-1轉(zhuǎn)染組細(xì)胞)。實(shí)驗(yàn)分組為:ANXA11-si RNA-1轉(zhuǎn)染組(以下簡稱為轉(zhuǎn)染組)、ANXA11-si RNA-1轉(zhuǎn)染組聯(lián)合IC20濃度的5-Fu組(以下簡稱為聯(lián)合組)、陰性對照組及空白對照組。應(yīng)用流式細(xì)胞術(shù)檢測各組凋亡率,應(yīng)用q PCR、Western Blot檢測中胸腺嘧啶脫氧核苷合成酶(Thymidine synthetase,TS)、Bax和Bcl-2 m RNA及蛋白水平表達(dá)的改變。結(jié)果:1 ANXA11 m RNA在人胃癌細(xì)胞株SGC7901、人胃癌細(xì)胞株BGC-823細(xì)胞中的相對表達(dá)量分別為:0.045±0.0012、0.022±0.004。ANXA11蛋白在人胃癌細(xì)胞株SGC7901、人胃癌細(xì)胞株BGC-823細(xì)胞中的相對表達(dá)量分別為:1.410±0.180、0.261±0.041。ANXA11 m RNA及蛋白表達(dá)量在人胃癌細(xì)胞株SGC7901中的表達(dá)顯著高于人胃癌細(xì)胞株BGC823(P=0.033,P=0.000)。2 ANXA11-si RNA轉(zhuǎn)染人胃癌細(xì)胞株SGC7901 24、48、72小時后,通過q PCR在m RNA水平檢測ANXA11的抑制效果。結(jié)果發(fā)現(xiàn),與陰性對照組及空白對照組相比,3條ANXA11-si RNA(ANXA11-si RNA-1、ANXA11-si RNA-2、ANXA11-si RNA-3)及3條ANXA11-si RNA的混合(ANXA11-si RNA123)對ANXA11的表達(dá)均有不同程度的抑制作用,其中轉(zhuǎn)染后48小時ANXA11-si RNA-1的抑制效果最好(F=28.631,P=0.000)。采用不同濃度(20n M、40n M、60n M、80n M、100n M)的ANXA11-si RNA-1進(jìn)行轉(zhuǎn)染,24、48、72小時后,通過Western Blot在蛋白水平檢測ANXA11的抑制效果。與陰性對照組及空白對照組相比,其對ANXA11的蛋白表達(dá)均有不同程度的抑制作用,其中轉(zhuǎn)染后48小時40n M濃度的抑制效果最好(F=24.887,P=0.000)。3 CCK-8實(shí)驗(yàn)中,采用1μg/ml、10μg/ml、20μg/ml、40μg/ml、80μg/ml的5-Fu作用于轉(zhuǎn)染后不同組人胃癌細(xì)胞株SGC7901 24、48、72、96h,其中48h時轉(zhuǎn)染組與陰性、空白對照組OD值有明顯差異(P0.05)。在48小時組,人胃癌細(xì)胞株SGC7901轉(zhuǎn)染ANXA11-si RNA-1后對5-Fu的IC50為:6.827±0.770μg/ml,陰性對照組為:11.487±0.669μg/ml,空白對照組為12.053±0.720μg/ml。相應(yīng)的IC20分別為0.086±0.011μg/ml、0.191±0.0188μg/ml、0.189±0.0153μg/ml。轉(zhuǎn)染ANXA11-si RNA-1后人胃癌細(xì)胞株SGC7901對5-Fu的敏感性顯著高于陰性對照組及空白對照組(F=47.500,P=0.000;F=45.523,P=0.000)。4人胃癌細(xì)胞株SGC7901轉(zhuǎn)染ANXA11-si RNA-1后細(xì)胞凋亡率為(18.747±2.064)%,聯(lián)合組為(30.153±2.344)%,陰性對照組為(6.657±0.976)%,空白對照組為(6.510±1.509)%。轉(zhuǎn)染組、聯(lián)合組凋亡率較陰性對照組及空白對照組顯著升高(F=118.403,P=0.000),且聯(lián)合組凋亡率較轉(zhuǎn)染組凋亡率明顯升高(P=0.000)。5人胃癌細(xì)胞株SGC7901轉(zhuǎn)染ANXA11-si RNA-1后ANXA11蛋白的相對表達(dá)量為:0.523±0.091,聯(lián)合組為0.167±0.060,陰性對照組為:0.887±0.075,空白對照組對照組為0.863±0.125。聯(lián)合組及轉(zhuǎn)染組蛋白表達(dá)顯著降低(F=41.623,P=0.000)。聯(lián)合組與轉(zhuǎn)染組之間差異也具有統(tǒng)計學(xué)意義(P=0.001)。6應(yīng)用q PCR檢測轉(zhuǎn)染ANXA11-si RNA-1及轉(zhuǎn)染ANXA11-si RNA聯(lián)合5-Fu對Bax、Bcl-2及TS m RNA表達(dá)的改變,發(fā)現(xiàn)與陰性對照組及空白對照組相比,轉(zhuǎn)染組與聯(lián)合組Bax的m RNA表達(dá)上調(diào),而Bcl-2、TS的m RNA表達(dá)顯著下調(diào)(F=35.608,P=0.000;F=30.048,P=0.000;F=22.874,P=0.000)。應(yīng)用Western Blot檢測上述基因蛋白表達(dá)的改變,發(fā)現(xiàn)與陰性對照組及空白對照組相比,轉(zhuǎn)染組與聯(lián)合組Bax的蛋白表達(dá)上調(diào),而Bcl-2、TS的蛋白表達(dá)顯著下調(diào)(F=24.396,P=0.000;F=33.890,P=0.000;F=29.630,P=0.000)。聯(lián)合組與轉(zhuǎn)染組之間差異也具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:1 ANXA11在人胃癌細(xì)胞株SGC7901中表達(dá)較高。抑制ANXA11表達(dá)可提高人胃癌細(xì)胞株SGC7901的凋亡率。2抑制ANXA11表達(dá)可提高5-Fu的殺傷力,繼而增加人胃癌細(xì)胞株SGC7901對5-Fu敏感性,ANXA11可能是參與人胃癌細(xì)胞株SGC79015-Fu耐藥的新的基因。3抑制ANXA11表達(dá)提高人胃癌細(xì)胞株SGC7901 5-Fu敏感性可能與抑制TS的表達(dá)有關(guān),同時也與下調(diào)抑凋亡基因Bcl-2、上調(diào)促凋亡基因Bax的表達(dá)從而促使細(xì)胞凋亡有關(guān)。
[Abstract]:Objective: in recent years, although the incidence of gastric cancer in the world is declining, the incidence of gastric cancer in China is increasing year by year. The incidence and mortality of gastric cancer are second only to lung cancer, which is located in the second place. Comprehensive treatment based on surgery is the main treatment for gastric cancer at present, but the diagnosis rate of early gastric cancer in China is less than 10%. Most patients often lose the opportunity for radical surgery, which makes chemotherapy play an important role in the comprehensive treatment of gastric cancer. Since the advent of 5- fluorouracil (5-fluorouracil, 5-Fu), it has been over 50 years ago, but it is still an important component of the current chemotherapy regimen for gastric cancer. As a result of sexual resistance, the effective rate of single drug chemotherapy is less than 20%., so it is an urgent problem to clarify the mechanism of 5-Fu's resistance to drug resistance and even reverse its resistance. The membrane associated protein family (Annexins) is the cytoplasm and calcium regulating phospholipid binding protein of the eukaryotic cells, which are widely expressed in addition to the yeast, and it is regulated by the regulation of the protein. Cell membrane surface proteins interact with other proteins in cells, cell nuclear membranes, and extracellular matrix proteins. The annexin family consists of five groups of A, B, C, D, E. The A group is found in mammals, including 12 members (A1-A11 and A13). The present study shows the occurrence and development of the membrane associated protein A11 (Annexin A11, ANXA11) and tumor. The positive expression rate and expression intensity of annexin A11 in gastric cancer tissues were significantly higher than that in normal gastric tissue. The positive expression rate and expression intensity of.ANXA11 in the metastatic lymph nodes were also significantly higher than those in the primary gastric cancer. The occurrence, development and metastasis of ANXA11 and gastric cancer were also found. But at present, the expression of ANXA11 in different gastric cancer cell lines and the relationship with the drug resistance of gastric cancer cell 5-Fu have not been reported. In this study, the human gastric cancer cell line SGC7901 was used as the research object. The expression of ANXA11 in SGC7901 cells was suppressed by RNA interference technique, and the sensitivity of different groups to 5-Fu was detected, and the transfection group was found to be more than the negative control group. The sensitivity of the blank control group is improved and its related mechanism is preliminarily discussed. It provides a theoretical basis for clarifying the mechanism of 5-Fu resistance to gastric cancer, and is of great significance for alleviating and reversing the 5-Fu resistance of gastric cancer and the discovery of new therapeutic targets. Methods: the cultivation of human gastric cancer cell line SGC7901, human gastric cancer cell line BGC-823, is used in real time. Fluorescence quantitative polymerase chain reaction (quantitative real time polymerase chain reaction, Q PCR) and protein immunoblotting (Western Blot) were used to detect the expression of ANXA11 m and protein in two cell lines. The Lipofectamine 2000 transfection reagent was used to transfect ANXA11-si RNA and negative control sequence into SGC7901 cells instantaneously, and 24h, 48h and 72h were used to detect the expression of ANXA11 m RNA, and the expression of protein was detected by Q PCR, and the effect of interference was detected by using Q PCR. IC50 (half maximal inhibitory concentration) and IC20 values were calculated in each group. (5-Fu for IC50 concentration was more lethal than 5-Fu, then IC20 concentration 5-Fu was used in ANXA11-si RNA-1). Group 5-Fu group with IC20 concentration (hereinafter referred to as joint group), negative control group and blank control group. Flow cytometry was used to detect the apoptosis rate of each group. Q PCR, Western Blot detection of thymidine deoxynucleoside synthetase (Thymidine synthetase, TS), Bax and Bcl-2 m, and protein level expression changes. Results: 1 The relative expression of human gastric cancer cell line SGC7901 and human gastric cancer cell line BGC-823 cells are: 0.045 + 0.0012,0.022 + 0.004.ANXA11 protein in human gastric cancer cell line SGC7901, and the relative expression amount in human gastric cancer cell line BGC-823 cells is: 1.410 + 0.180,0.261 + 0.041.ANXA11 m RNA and protein expression in human gastric cancer cell strain SGC7 The expression in 901 was significantly higher than that of human gastric cancer cell line BGC823 (P=0.033, P=0.000).2 ANXA11-si RNA transfected to human gastric cancer cell line SGC7901 24,48,72 hours after SGC7901 24,48,72 hours, and the inhibitory effect was detected by Q PCR in M RNA. -si RNA-3) and the mixture of 3 ANXA11-si RNA (ANXA11-si RNA123) have different degrees of inhibition on the expression of ANXA11, and the inhibitory effect of ANXA11-si RNA-1 after 48 hours after transfection is the best (F=28.631, P=0.000). Blot was used to detect the inhibitory effect of ANXA11 at the protein level. Compared with the negative control group and the blank control group, the inhibition effect on the protein expression of ANXA11 was different. The inhibitory effect of 40n M at 48 hours after transfection was the best (F=24.887, P=0.000).3 CCK-8 experiment, using 1 Mu g/ml, 10 mu g/ml, 20 mu g/ml, 40 micron, 80 micron. SGC7901 24,48,72,96h in different groups of human gastric cancer cell line after transfection, in which 48h transfection group and negative control group were significantly different (P0.05). In the 48 hour group, the IC50 of human gastric cancer cell line SGC7901 transfected to 5-Fu was 6.827 + 0.770 mu g/ml, negative control group was 11.487 + 0.669 mu g/ml, and the blank control group was 1. The corresponding IC20 of 2.053 + 0.720 g/ml. was 0.086 + 0.011 mu g/ml, 0.191 + 0.0188 g/ml and 0.189 + 0.0153 mu g/ml. transfected to ANXA11-si RNA-1. The sensitivity of SGC7901 to 5-Fu was significantly higher than that of negative control group and blank control group (F=47.500, P=0.000; F=45.523, 0) The apoptosis rate was (18.747 + 2.064)%, the combined group was (30.153 + 2.344)%, the negative control group was (6.657 + 0.976)%, and the blank control group was (6.510 + 1.509)%. The apoptosis rate of the combined group was significantly higher than that of the negative control group and the blank control group (F=118.403, P=0.000), and the apoptosis rate of the combined group was significantly higher than that of the transfected group (P=0.000).5 human stomach. The relative expression of ANXA11 protein in the cancer cell line SGC7901 transfected with ANXA11-si RNA-1 was 0.523 + 0.091, the combined group was 0.167 + 0.060, and the negative control group was 0.887 + 0.075. The control group of the blank control group was 0.863 + 0.125. combined group and the transfection group was significantly reduced (F=41.623, P=0.000). The difference between the combined group and the transfected group was also statistically significant. P=0.001.6 applied Q PCR to detect the transfection of ANXA11-si RNA-1 and the transfection of ANXA11-si RNA with 5-Fu to Bax, Bcl-2 and TS. .874, P=0.000). Using Western Blot to detect the changes in the expression of the above gene protein, it was found that the protein expression of Bax in the transfected group and the combined group was up regulated compared to the negative control group and the blank control group, while the protein expression of the Bcl-2, TS was significantly down (F=24.396, P=0.000; F=33.890, P= 0, F=29.630,). Statistical significance (P0.05). Conclusion: 1 ANXA11 is highly expressed in human gastric cancer cell line SGC7901. Inhibition of ANXA11 expression can increase the apoptosis rate of SGC7901 in human gastric cancer cell line,.2 inhibition of ANXA11 expression can improve the viability of 5-Fu, and then increase the sensitivity of SGC7901 to 5-Fu in human gastric cancer cell line, ANXA11 may be involved in human gastric cancer cell strain SGC79015-Fu. The inhibition of ANXA11 expression by drug resistant new gene.3 enhances the sensitivity of SGC7901 5-Fu in human gastric cancer cell line, which may be related to inhibiting the expression of TS. It is also associated with down regulation of apoptosis gene Bcl-2, up regulation of the expression of apoptotic gene Bax and inducing apoptosis.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2

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