發(fā)布時(shí)間：2018-05-03 17:48

  本文選題：Annexin + A7�。� 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:原發(fā)性肝細(xì)胞癌(Hepatocellular carcinoma,HCC)是目前致死率最高的上皮來(lái)源惡性腫瘤之一,近年來(lái)在我國(guó)的發(fā)病率依然居高不下。淋巴道轉(zhuǎn)移是惡性腫瘤最早的出現(xiàn)的轉(zhuǎn)移方式,是造成肝癌高侵襲、高復(fù)發(fā)以及預(yù)后差的主要原因之一。國(guó)內(nèi)外許多研究結(jié)果表明淋巴道轉(zhuǎn)移機(jī)制受許多基因調(diào)控,它們或被激活或被抑制,或相互協(xié)調(diào)或互相拮抗,其中包括細(xì)胞增殖、粘附、遷移、細(xì)胞外基質(zhì)降解等多個(gè)階段與環(huán)節(jié)。膜聯(lián)蛋白A7(Annexin A7)是本課題組前期發(fā)現(xiàn)的潛在決定小鼠肝癌淋巴道轉(zhuǎn)移的關(guān)鍵基因之一,對(duì)各種腫瘤細(xì)胞生長(zhǎng)、分化、增殖、分泌、侵襲、遷移乃至凋亡能力均可產(chǎn)生影響。同時(shí)Annexin A7基因是ca2+磷脂結(jié)合蛋白,具有ca2+依賴(lài)膜融合活性,可促進(jìn)膜聚集、融合、粘著、運(yùn)輸?shù)?還可激活GTP酶,介導(dǎo)ca2+/GTP信號(hào)轉(zhuǎn)導(dǎo)通路。腺苷酸環(huán)化酶聯(lián)合蛋白(adenylate cyclase-associated protein 1,CAP1)最早發(fā)現(xiàn)于酵母,在其中可調(diào)節(jié)Ras/c AMP信號(hào)通路,并且與哺乳動(dòng)物CAP1具有相同作用調(diào)節(jié)肌動(dòng)蛋白的周轉(zhuǎn)與重組,對(duì)細(xì)胞遷移產(chǎn)生影響。近年來(lái)關(guān)于CAP1與腫瘤轉(zhuǎn)移關(guān)系的研究逐漸深入,但似乎在不同腫瘤細(xì)胞中對(duì)于細(xì)胞生物學(xué)功能的影響不盡相同。黏著斑激酶(focal adhesion kinase,FAK)是一種非受體型蛋白酪氨酸激酶,在許多上皮來(lái)源的腫瘤細(xì)胞中過(guò)表達(dá),如乳腺癌、結(jié)腸癌、口腔癌、甲狀腺癌、卵巢癌和胃癌等,可能在腫瘤生長(zhǎng)、增殖、侵襲、轉(zhuǎn)移及血管形成中產(chǎn)生影響。Src為SRC酪氨酸激酶家族成員,參與細(xì)胞生長(zhǎng)、分化、粘著、增殖、凋亡及細(xì)胞運(yùn)動(dòng)等過(guò)程。樁蛋白(Paxillin)是一種主要定位于黏著斑的關(guān)鍵銜接蛋白信號(hào)分子,是FAK下游重要的焦點(diǎn)粘附調(diào)節(jié)蛋白之一,其異常表達(dá)與腫瘤的發(fā)生、增殖、粘附、浸潤(rùn)、骨架重組及轉(zhuǎn)移能力的改變密切關(guān)聯(lián)。E-鈣黏蛋白(E-cadherin,CDH1)是與腫瘤轉(zhuǎn)移侵襲密切相關(guān)的一種鈣粘蛋白,可參與細(xì)胞極性、細(xì)胞形態(tài)與組織結(jié)構(gòu)完整性的維持,與腫瘤細(xì)胞的良惡性及轉(zhuǎn)移程度呈負(fù)相關(guān)。目的:通過(guò)細(xì)胞脂質(zhì)體轉(zhuǎn)染、qRT-PCR、Western Blot等技術(shù)觀(guān)察對(duì)比下調(diào)小鼠肝癌Hca-P細(xì)胞Annexin A7與CAP1基因后觀(guān)察其對(duì)細(xì)胞黏附相關(guān)分子FAK、Src、Paxillin、E-cadherin基因及蛋白表達(dá)水平的調(diào)控。通過(guò)CCK-8增殖檢測(cè)、流式細(xì)胞儀凋亡檢測(cè)、小鼠淋巴結(jié)粘附以及Transwell小室等實(shí)驗(yàn)方法觀(guān)察CAP1下調(diào)對(duì)Hca-P細(xì)胞生物學(xué)行為的影響。對(duì)比Annexin A7與CAP1下調(diào)后對(duì)彼此基因和蛋白表達(dá)水平的影響以及二者在Hca-P細(xì)胞的表達(dá)定位。方法:1.構(gòu)建PGPUP-GFP-neo-sh RNA-Annexin A7和PGPUP-GFP-neo-sh NC表達(dá)載體,穩(wěn)定轉(zhuǎn)染至小鼠Hca-P細(xì)胞,獲得Annexin A7下調(diào)表達(dá)及無(wú)關(guān)序列Hca-P細(xì)胞株,應(yīng)用qRT-PCR、Western Blot技術(shù)驗(yàn)證Annexin A7基因下調(diào)對(duì)CAP1、FAK、SRC、E-cadherin基因及蛋白表達(dá)水平的影響。2.構(gòu)建PGPUP-GFP-neo-sh RNA-CAP1和PGPUP-GFP-neo-sh NC表達(dá)載體,穩(wěn)定轉(zhuǎn)染至Hca-P細(xì)胞,獲得CAP1下調(diào)表達(dá)及無(wú)關(guān)序列Hca-P細(xì)胞株,應(yīng)用qRT-PCR、Western Blot技術(shù)驗(yàn)證CAP1基因下調(diào)對(duì)FAK、Src、Paxillin、E-cadherin基因及蛋白表達(dá)水平的影響。3.獲得CAP1下調(diào)表達(dá)及無(wú)關(guān)序列Hca-P細(xì)胞株后,應(yīng)用CCK-8增殖檢測(cè)、流式細(xì)胞儀凋亡檢測(cè)、小鼠淋巴結(jié)粘附以及Transwell小室等實(shí)驗(yàn)方法,對(duì)比CAP1下調(diào)組與對(duì)照組增殖、凋亡、粘附、侵襲、遷移能力,分析CAP1對(duì)Hca-P細(xì)胞生物學(xué)行為的影響。4.應(yīng)用qRT-PCR、Western Blot技術(shù)驗(yàn)證Annexin A7基因下調(diào)對(duì)CAP1基因及蛋白表達(dá)水平的影響。采用細(xì)胞免疫熒光技術(shù)檢測(cè)Annexin A7與CAP1表達(dá)的共定位。結(jié)果:1、在mRNA表達(dá)水平上,Annexin A7在ShRNA-A7細(xì)胞組較A7無(wú)關(guān)序列細(xì)胞組及Hca-P細(xì)胞組分別降低82.86%和82.78%(P0.05),而在A7無(wú)關(guān)序列組及Hca-P組間差異不明顯(P0.05)。FAK、Src基因在ShRNA-A7細(xì)胞組表達(dá)分別為A7無(wú)關(guān)序列組及Hca-P組的1.88倍、1.67倍和1.78倍、1.54倍(P0.05),二者在A7無(wú)關(guān)序列組及Hca-P組間差異不明顯(P0.05)。E-cadherin基因在ShRNA-A7細(xì)胞組較A7無(wú)關(guān)序列細(xì)胞組及Hca-P細(xì)胞組分別降低49.88%和43.01%(P0.05),在A7無(wú)關(guān)序列組及Hca-P組間差異不明顯(P0.05)。在蛋白質(zhì)表達(dá)水平上,Annexin A7在ShRNA-A7細(xì)胞組中蛋白表達(dá)水平較A7無(wú)關(guān)序列組及Hca-P細(xì)胞組降低62.75%和60.34%(P0.05),而在Hca-P與A7無(wú)關(guān)序列組間不存在明顯差異(P0.05)。FAK及Src在ShRNA-A7細(xì)胞組較A7無(wú)關(guān)序列組及Hca-P組蛋白表達(dá)量上升1.80倍、1.52倍及1.59、1.58倍(P0.05),E-cadherin蛋白在ShRNA-A7細(xì)胞組較A7無(wú)關(guān)序列組與Hca-P組下降54.27%和59.11%(P0.05),但三種蛋白在Hca-P與A7無(wú)關(guān)序列組間不存在明顯差異(P0.05)。2、在mRNA表達(dá)水平上,CAP1在ShRNA-CAP1細(xì)胞組較CAP1無(wú)關(guān)序列細(xì)胞組及Hca-P細(xì)胞組分別降低60.73%和65.25%(P0.05),而在CAP1無(wú)關(guān)序列組及Hca-P組間差異不明顯(P0.05)。FAK、Src、Paxillin基因在ShRNA-A7細(xì)胞組表達(dá)分別為A7無(wú)關(guān)序列組及Hca-P組的2.47倍、2.18倍和1.64倍、1.76倍及1.67倍、1.58倍,三者在CAP1無(wú)關(guān)序列組及Hca-P組間差異不明顯(P0.05)。E-cadherin基因在ShRNA-CAP1細(xì)胞組較CAP1無(wú)關(guān)序列細(xì)胞組及Hca-P細(xì)胞組分別降低40.11%和43.01%(P0.05),在CAP1無(wú)關(guān)序列組及Hca-P組間差異不明顯(P0.05)。在蛋白質(zhì)表達(dá)水平上,CAP1在ShRNA-CAP1細(xì)胞組中蛋白表達(dá)水平較CAP1無(wú)關(guān)序列組與Hca-P組降低50.83%和50.29%(P0.05),而在Hca-P與CAP1無(wú)關(guān)序列組間差異不明顯(P0.05)。FAK、SRC及Paxillin在ShRNA-CAP1細(xì)胞組較CAP1無(wú)關(guān)序列組和Hca-P組蛋白表達(dá)量分別升高1.64倍和1.60倍、1.67倍和1.61倍、1.43倍和1.39倍(P0.05),E-cadherin蛋白表達(dá)水平在ShRNA-CAP1組較CAP1無(wú)關(guān)序列組及Hca-P組均有下降,下降幅度為39.6%及48.22%(P0.05),但FAK、SRC、Paxillin及E-cadherin四者在Hca-P細(xì)胞組與CAP1無(wú)關(guān)序列細(xì)胞組之間表達(dá)量不存在明顯差異(P0.05)。3、CCK-8增殖實(shí)驗(yàn)分別檢測(cè)Hca-P細(xì)胞組、ShRNA-CAP1細(xì)胞組與CAP1無(wú)關(guān)序列細(xì)胞組在0h、24h、48h、72h、96h等時(shí)間段的吸光值,其中ShRNA-CAP1細(xì)胞組各時(shí)間點(diǎn)吸光值均數(shù)±標(biāo)準(zhǔn)差分別為0.106±0.015、0.152±0.010、0.522±0.049、0.651±0.040、0.651±0.040、0.708±0.037;CAP1無(wú)關(guān)序列細(xì)胞組各時(shí)間點(diǎn)吸光值均數(shù)±標(biāo)準(zhǔn)差分別為0.095±0.017、0.144±0.024、0.374±0.027、0.485±0.018、0.535±0.039;Hca-P細(xì)胞組各時(shí)間點(diǎn)吸光值均數(shù)±標(biāo)準(zhǔn)差分別為0.101±0.014、0.153±0.006、0.410±0.021、0.553±0.022、0.601±0.030,結(jié)果提示Hca-P、ShRNA-CAP1、CAP1無(wú)關(guān)序列三種細(xì)胞數(shù)量在24小時(shí)內(nèi)不存在顯著差異(P0.05),自48小時(shí)起開(kāi)始產(chǎn)生明顯差異,每個(gè)時(shí)間點(diǎn)差異均顯著(P0.05),且ShRNA-CAP1細(xì)胞增殖能力始終高于CAP1無(wú)關(guān)序列細(xì)胞組及Hca-P細(xì)胞組,CAP1無(wú)關(guān)序列組與Hca-P組的增殖能力在各時(shí)間段無(wú)顯著差異(P0.05)。4、流式細(xì)胞儀分別檢測(cè)Hca-P細(xì)胞組、ShRNA-CAP1細(xì)胞組與CAP1無(wú)關(guān)序列細(xì)胞組早期凋亡能力,結(jié)果顯示三組細(xì)胞早期凋亡細(xì)胞比例分別為6.33%±0.17%,3.41%±0.22%及6.43%±0.25%,提示在CAP1基因低表達(dá)后,ShRNA-CAP1細(xì)胞組早期凋亡細(xì)胞數(shù)低于CAP1無(wú)關(guān)序列及Hca-P細(xì)胞組。5、淋巴結(jié)粘附實(shí)驗(yàn)檢測(cè)Hca-P組、ShRNA-CAP1組與CAP1無(wú)關(guān)序列組細(xì)胞淋巴結(jié)粘附能力,三種細(xì)胞組粘附于淋巴結(jié)的細(xì)胞數(shù)目分別為255.67±28.43、734.33±67.50、228.33±20.03,提示在CAP1基因低表達(dá)后,ShRNA-CAP1細(xì)胞組對(duì)于小鼠淋巴結(jié)的黏附能力強(qiáng)于Hca-P細(xì)胞組與CAP1無(wú)關(guān)序列細(xì)胞組。6、Transwell小室細(xì)胞侵襲實(shí)驗(yàn)中Hca-P細(xì)胞組、ShRNA-CAP1細(xì)胞組與N2-Control細(xì)胞組穿透小室膜的細(xì)胞數(shù)目均數(shù)±標(biāo)準(zhǔn)差分別為61.00±8.51、173.00±18.03、58.00±6.40,提示ShRNA-CAP1細(xì)胞組穿過(guò)小室的細(xì)胞數(shù)目明顯多于CAP1無(wú)關(guān)序列細(xì)胞組及Hca-P細(xì)胞組,CAP1無(wú)關(guān)序列細(xì)胞組與Hca-P細(xì)胞組對(duì)比則差異不顯著(P0.05)。7、對(duì)Annexin A7與CAP1相互關(guān)系的分析提示,在Annexin A7基因表達(dá)下調(diào)后,CAP1基因與蛋白表達(dá)水平均降低,其中,ShRNA-A7細(xì)胞組與A7無(wú)關(guān)序列細(xì)胞組對(duì)比,CAP1基因下調(diào)至原水平的17.21%(P0.05),CAP1蛋白表達(dá)水平下調(diào)至原水平的46.86%(P0.05),表明Annexin A7基因低表達(dá)后,CAP1基因與蛋白水平均隨之下降。另外通過(guò)免疫熒光方法證明Annexin A7與CAP1均主要在在Hca-P細(xì)胞漿內(nèi)表達(dá),少量表達(dá)于細(xì)胞核,由圖像結(jié)果推測(cè)兩種蛋白可能在細(xì)胞漿及鄰近細(xì)胞內(nèi)膜處共定位。結(jié)論:Annexin A7與CAP1基因分別可調(diào)控FAK、SRC、Paxillin、E-cadherin的表達(dá),在mRNA和蛋白質(zhì)水平,FAK、SRC、Paxillin均隨Annexin A7與CAP1基因的下調(diào)而高表達(dá),而E-cadherin則在上述兩基因表達(dá)下調(diào)后低表達(dá)。CAP1低表達(dá)后,Hca-P細(xì)胞增殖、淋巴結(jié)黏附、侵襲能力均有所上升,早期凋亡能力下降;Annexin A7基因下調(diào)表達(dá)后,CAP1在mRNA和蛋白質(zhì)水平隨之下調(diào),且兩基因在Hca-P細(xì)胞內(nèi)表達(dá)位置基本一致,可能存在共定位。Annexin A7基因可能與CAP1基因存在一定分子機(jī)制的關(guān)聯(lián),可對(duì)細(xì)胞粘附轉(zhuǎn)移分子產(chǎn)生一致性影響,并對(duì)肝癌Hca-P細(xì)胞的生物學(xué)行為的產(chǎn)生密切相關(guān)。
[Abstract]:Background: Hepatocellular carcinoma (HCC) is one of the most fatal malignant tumors at present. In recent years, the incidence of cancer is still high in our country. Lymphatic metastasis is the earliest metastasis mode of malignant tumor, which is one of the main causes of high invasion, high recurrence and poor prognosis of liver cancer. Many studies at home and abroad have shown that the mechanism of lymphatic metastasis is regulated by many genes, they are activated or suppressed, or are coordinated or antagonistic to each other, including cell proliferation, adhesion, migration, and extracellular matrix degradation. Annexin A7 (A7) is a potential determinant of the early discovery of the group. One of the key genes of lymphatic metastasis of liver cancer can affect the growth, differentiation, proliferation, secretion, invasion, migration and even apoptosis of various tumor cells. At the same time, the Annexin A7 gene is a ca2+ phospholipid binding protein, with ca2+ dependent membrane fusion activity, which can promote membrane aggregation, fusion, adhesion, transport and so on, and can also activate GTP enzyme and mediate ca2+/GTP The signal transduction pathway, adenylate cyclase-associated protein 1 (CAP1), was first found in yeast, which regulates the Ras/c AMP signaling pathway, and has the same effect with mammalian CAP1 to regulate the turnover and recombination of actin, and the effect on cell migration. In recent years, CAP1 and tumor metastasis The study of the relationship is deepening, but it seems that the effect of focal adhesion kinase (FAK) is a kind of non receptor protein tyrosine kinase, which is expressed in many epithelial tumor cells, such as breast cancer, colon cancer, oral cancer, thyroid cancer, ovarian cancer. And gastric cancer, which may affect the.Src SRC tyrosine kinase family in tumor growth, proliferation, invasion, metastasis and angiogenesis, and participate in the process of cell growth, differentiation, adhesion, proliferation, apoptosis and cell movement. Paxillin is a key cohesive protein signal molecule located in the sticky spot, which is the important downstream of FAK. One of the focal adhesion regulatory proteins, whose abnormal expression is closely related to the changes in tumor occurrence, proliferation, adhesion, infiltration, cytoskeleton recombination and metastasis,.E- calcium mucin (E-cadherin, CDH1) is a kind of calcic protein closely related to tumor metastasis and invasion, which can be involved in cell polarity, cell morphology and structural integrity maintenance, and the maintenance of cell morphology and tissue structure. Objective: To observe and compare the regulation of cell adhesion related molecule FAK, Src, Paxillin, E-cadherin gene and protein expression through the transfection of cell liposomes, qRT-PCR, Western Blot and other techniques, and to observe and control the Annexin A7 and CAP1 genes of mouse liver cancer Hca-P cells. The effect of CAP1 down regulation on biological behavior of Hca-P cells was observed by colonization detection, flow cytometry apoptosis, mouse lymph node adhesion and Transwell compartment. The effect of Annexin A7 and CAP1 on the expression level of each other gene and protein and the expression of two in Hca-P fine cell were compared. Method: 1. construction of PGPUP-GFP-neo-s H RNA-Annexin A7 and PGPUP-GFP-neo-sh NC expression vector, stable transfection into mouse Hca-P cells to obtain Annexin A7 down-regulation and independent sequence Hca-P cell lines. QRT-PCR, Western Blot technique was used to verify the effect of the down regulation of the gene and the expression level of egg white. And PGPUP-GFP-neo-sh NC expression vector, stable transfection into Hca-P cells to obtain CAP1 down expression and unrelated sequence Hca-P cell lines. QRT-PCR, Western Blot technique was used to verify the effect of CAP1 down regulation on FAK, Src, Paxillin, gene and protein expression. CK-8 proliferation detection, flow cytometry apoptosis detection, mouse lymph node adhesion and Transwell chamber and other experimental methods, compare the proliferation, apoptosis, adhesion, invasion and migration ability of CAP1 down-regulation group and control group, analyze the effect of CAP1 on the biological behavior of Hca-P cells,.4. application qRT-PCR, Western Blot technique to verify Annexin A7 gene downregulation to CAP1 genes. The co localization of the expression of Annexin A7 and CAP1 was detected by cell immunofluorescence. Results: 1, at the level of mRNA expression, Annexin A7 decreased by 82.86% and 82.78% (P0.05) in the ShRNA-A7 cell group compared with the A7 independent sequence cell group and the Hca-P cell group respectively, but there was no significant difference between the A7 independent sequence group and the Hca-P group (P0.05). 05).FAK, the expression of Src gene in the ShRNA-A7 cell group was 1.88 times, 1.67 times and 1.78 times, 1.54 times (P0.05), respectively, in the A7 independent sequence group and the Hca-P group, and the two in the A7 independent sequence group and the Hca-P group were not significantly different (P0.05).E-cadherin gene decreased 49.88% and 43.01% respectively in the ShRNA-A7 cell group than that in the A7 independent sequence group and the cell group. The difference between the A7 independent sequence group and the Hca-P group was not obvious (P0.05). In the protein expression level, the protein expression level of Annexin A7 in the ShRNA-A7 cell group decreased by 62.75% and 60.34% (P0.05) in the A7 independent sequence group and Hca-P cell group, but there was no significant difference between the Hca-P and A7 independent sequence groups. The expression of A7 independent sequence group and Hca-P histone increased 1.80 times, 1.52 times and 1.59,1.58 times (P0.05). E-cadherin protein decreased 54.27% and 59.11% (P0.05) in ShRNA-A7 cell group than that in Hca-P group, but there was no obvious difference between Hca-P and A7 independent sequences. The 1 cell group was 60.73% and 65.25% (P0.05) less than that of the CAP1 unrelated sequence cell group and the Hca-P cell group, but the difference between the CAP1 independent sequence group and the Hca-P group was not obvious (P0.05).FAK, Src and Paxillin gene expression in the ShRNA-A7 cell group were 2.47 times, 2.18 and 1.64 times, 1.76 times and 1.67 times, 1.58 times, three in ShRNA-A7 cell group, respectively. The difference between the CAP1 independent sequence group and the Hca-P group was not obvious (P0.05).E-cadherin gene decreased by 40.11% and 43.01% (P0.05) in the ShRNA-CAP1 cell group than in the CAP1 unrelated sequence cell group and the Hca-P cell group, and the difference between the CAP1 independent sequence group and the Hca-P group was not obvious (P0.05). The expression level of CAP1 independent sequence group and Hca-P group decreased 50.83% and 50.29% (P0.05), while the difference between Hca-P and CAP1 independent sequences was not obvious (P0.05).FAK, SRC and Paxillin increased 1.64 times and 1.60 times, 1.67 and 1.61 times, 1.43 times and 1.39 times, respectively, in ShRNA-CAP1 cell group than CAP1 independent sequence group and Hca-P histone protein expression. The expression level of herin protein decreased in group ShRNA-CAP1 and Hca-P group, the decrease was 39.6% and 48.22% (P0.05), but there was no significant difference between the FAK, SRC, Paxillin and E-cadherin four in the Hca-P cell group and the CAP1 sequence cell group (P0.05). The absorbance of A-CAP1 cell group and CAP1 independent sequence cell group at 0h, 24h, 48h, 72h, 96h and so on, in which the mean absorption standard deviation of each time point of ShRNA-CAP1 cell group is 0.106 + 0.015,0.152 + 0.010,0.522 + 0.049,0.651 + 0.040,0.651 + 0.037, respectively. The difference is 0.095 + 0.017,0.144 + 0.024,0.374 + 0.027,0.485 + 0.018,0.535 + 0.039, and the average number of absorbance values of each time point of Hca-P cell group is 0.101 + 0.014,0.153 + 0.021,0.553 + 0.022,0.601 + 0.030 respectively. The results suggest Hca-P, ShRNA-CAP1, and there is no significant difference in the number of three cells in the CAP1 independent sequence in 24 hours. The difference (P0.05) began to produce obvious difference since 48 hours, and the difference of each time point was significant (P0.05), and the proliferation ability of ShRNA-CAP1 cells was always higher than that of CAP1 independent sequence cell group and Hca-P cell group. The proliferation ability of CAP1 independent sequence group and Hca-P group had no significant difference (P0.05).4 at each time period, and the flow cytometry was used to detect Hca-P cells respectively. The early apoptotic ability of ShRNA-CAP1 cell group and CAP1 independent sequence cell group showed that the proportion of early apoptotic cells in three groups was 6.33% + 0.17%, 3.41% + 0.22% and 6.43% + 0.25%, suggesting that the number of early apoptotic cells in the ShRNA-CAP1 cell group was lower than that of CAP1 independent sequence and Hca-P cell group.5 after the CAP1 gene was low. The cell lymph node adhesion ability of group Hca-P, ShRNA-CAP1 group and CAP1 independent sequence group was tested. The number of cells adhered to the lymph nodes in the three groups was 255.67 + 28.43734.33 + 67.50228.33 + 20.03 respectively, suggesting that the adhesion ability of ShRNA-CAP1 cell group to mouse lymph node was stronger than that of Hca-P cell group and CAP1 after the CAP1 gene was low. The number of Hca-P cells in the unrelated sequence cell group.6, the Transwell cell cell invasion experiment, the number of cells in the ShRNA-CAP1 cell group and the N2-Control cell group that penetrated the small ventricular membrane were 61 + 8.51173.00 + 6.40, respectively, indicating that the number of cells passing through the compartment in the ShRNA-CAP1 cell group was obviously more than that of the CAP1 independent sequence cell group. And Hca-P cell group, the difference of CAP1 independent sequence cell group and Hca-P cell group was not significantly different (P0.05).7. The analysis of the relationship between Annexin A7 and CAP1 suggested that the CAP1 gene and protein expression level decreased after the Annexin A7 gene expression was down, and the ShRNA-A7 cell group was down to the original. The level of 17.21% (P0.05), the expression level of CAP1 protein was down to 46.86% (P0.05) of the original level, indicating that the CAP1 gene and protein level decreased after the low expression of Annexin A7 gene. In addition, the expression of Annexin A7 and CAP1 were mainly expressed in the Hca-P cytoplasm by immunofluorescence method, which was expressed in a small amount in the nucleus and speculated two from the image results. Conclusion: Annexin A7 and CAP1 gene can regulate the expression of FAK, SRC, Paxillin, E-cadherin, respectively, in mRNA and protein levels, FAK, SRC, and Paxillin are highly expressed with the down regulation of Annexin and the down regulation of the two gene expression. After low expression, Hca-P cell proliferation, lymph node adhesion, invasion ability increased and early apoptosis decreased. After Annexin A7 gene downregulation, CAP1 was down regulated at mRNA and protein levels, and the expression of two gene in Hca-P cells was basically consistent. It may exist in CO location.Annexin A7 gene and may exist with CAP1 gene. The association of molecular mechanisms can have a consistent effect on cell adhesion and transfer molecules, and is closely related to the biological behavior of Hca-P cells.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7

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本文編號(hào)：1839474