本文選題：CASK　切入點(diǎn)：H1299　出處：《廣西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的肺癌是我國(guó)最常見(jiàn)的惡性腫瘤之一,而非小細(xì)胞肺癌(non-small cell lung cancer,NSCLC)約占肺癌總數(shù)的80%-85%,是臨床最常見(jiàn)的一種類(lèi)型。由于肺癌在早期缺乏明顯的特異性癥狀,同時(shí)易發(fā)生血行轉(zhuǎn)移,因此大多數(shù)患者在確診時(shí)已進(jìn)入中晚期階段,治療效果及預(yù)后均不佳。本課題組在前期通過(guò)生物信息學(xué)方法篩選出11個(gè)基因,其中人鈣/鈣調(diào)蛋白依賴(lài)性絲氨酸蛋白激酶(calcium/calmodulin-dependent serine protein kinase,CASK)在報(bào)道中與肺癌發(fā)病有重要關(guān)系。故本研究通過(guò)構(gòu)建能高效表達(dá)人CASK基因的慢病毒載體并對(duì)H1299細(xì)胞系穩(wěn)定轉(zhuǎn)染后進(jìn)行鑒定,并通過(guò)對(duì)人非小細(xì)胞肺癌H1299細(xì)胞株進(jìn)行感染后,研究CASK過(guò)表達(dá)對(duì)其增殖能力和遷移能力的影響。方法1.CASK過(guò)表達(dá)慢病毒載體的構(gòu)建采用聚合酶鏈反應(yīng)(PCR)擴(kuò)增CASK基因片段,通過(guò)重組反應(yīng)完成目的基因擴(kuò)增產(chǎn)物和線性化GV358載體的體外環(huán)化。對(duì)重組產(chǎn)物進(jìn)行轉(zhuǎn)化后,挑取單克隆進(jìn)行PCR鑒定,并對(duì)陽(yáng)性克隆進(jìn)行測(cè)序分析。最后將正確克隆菌液擴(kuò)大培養(yǎng)、抽提以獲得高純度質(zhì)粒。將重組質(zhì)粒和包裝質(zhì)粒共轉(zhuǎn)染至293T細(xì)胞。Western Blot檢測(cè)轉(zhuǎn)染后293T細(xì)胞蛋白表達(dá)水平;用酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)法進(jìn)行滴度檢測(cè)。2.細(xì)胞模型效能檢測(cè)將成功包裝的CASK過(guò)表達(dá)慢病毒LV-CASK(OE組)和陰性對(duì)照病毒CON238(NC組)分別感染人非小細(xì)胞肺癌H1299細(xì)胞,同時(shí)設(shè)置空白對(duì)照組(MOCK組),用熒光定量PCR(FQ-PCR)和Western Blot法檢測(cè)CASK表達(dá)水平。3.CASK過(guò)表達(dá)對(duì)H1299細(xì)胞生物學(xué)功能特性的影響實(shí)驗(yàn)分組:空白對(duì)照組(MOCK組)、CASK過(guò)表達(dá)組(OE組)和陰性對(duì)照組(NC組),NC組和OE組分別用CON238和LV-CASK進(jìn)行感染,MOCK組不予任何處理,分別使用MTT法和細(xì)胞劃痕實(shí)驗(yàn)觀察CASK過(guò)表達(dá)對(duì)H1299細(xì)胞增殖能力和遷移能力的影響。結(jié)果1.CASK過(guò)表達(dá)慢病毒載體的構(gòu)建通過(guò)PCR技術(shù)成功地?cái)U(kuò)增CASK基因片段并連接到GV358病毒載體上,PCR及DNA測(cè)序鑒定結(jié)果證明CASK-GV358質(zhì)粒構(gòu)建正確。重組慢病毒轉(zhuǎn)染293T細(xì)胞后可觀察到綠色熒光及蛋白表達(dá),包裝過(guò)表達(dá)慢病毒并測(cè)定其濃縮滴度為2×108 TU/m L。2.細(xì)胞模型效能檢測(cè)FQ-PCR和Western Blot檢測(cè)結(jié)果顯示,與NC組相比,OE組CASK m RNA和蛋白的表達(dá)均顯著上調(diào)(P0.05)。3.CASK過(guò)表達(dá)對(duì)H1299細(xì)胞生物學(xué)功能特性的影響MTT法結(jié)果表明,OE組細(xì)胞增殖能力與NC組相比有明顯差異(P0.05),提示CASK過(guò)表達(dá)對(duì)H1299細(xì)胞增殖能力有抑制作用;細(xì)胞劃痕實(shí)驗(yàn)結(jié)果表明OE組細(xì)胞遷移能力與NC組無(wú)明顯差異(P0.05),CASK過(guò)表達(dá)對(duì)H1299細(xì)胞遷移能力無(wú)明顯作用。結(jié)論成功構(gòu)建了CASK基因過(guò)表達(dá)慢病毒載體,可在H1299細(xì)胞中穩(wěn)定表達(dá),CASK過(guò)表達(dá)可抑制H1299細(xì)胞增殖能力,為進(jìn)一步研究CASK在肺癌中的機(jī)制和作用奠定了基礎(chǔ)。
[Abstract]:Objective Lung cancer is one of the most common malignant tumors in China, and non-small cell lung cancer (NSCLC) accounts for 80% -85% of the total number of lung cancer. Therefore, most of the patients had entered the stage of middle and late stage of diagnosis, and the therapeutic effect and prognosis were not good. 11 genes were screened out by bioinformatics in the early stage. Calcium / calmodulin-dependent serine protein kinase (CASKK) is associated with the pathogenesis of lung cancer. Therefore, a lentivirus vector expressing human CASK gene was constructed and identified after stable transfection of H1299 cell line. The effect of CASK overexpression on the proliferation and migration of human non-small cell lung cancer cell line H1299 was studied. Methods the construction of 1.CASK overexpression lentivirus vector was used to amplify the CASK gene fragment by polymerase chain reaction (PCR). In vitro cyclization of the target gene amplification product and linearized GV358 vector was completed by recombination reaction. After the recombinant product was transformed, the monoclonal was selected for PCR identification. The positive clones were sequenced. Finally, the recombinant plasmid and packaging plasmid were cotransfected into 293T cells. Western Blot was used to detect the protein expression level of 293T cells after transfection. Elisa assay was used to detect the titer of human non-small cell lung cancer H1299 cells. 2. The efficacy of the cell model was detected. The CASK overexpressed lentivirus LV-CASK(OE group and the negative control virus CON238(NC group were respectively infected with H1299 cells in human non-small cell lung cancer (NSCLC). At the same time, a blank control group was set up to detect the expression level of CASK. 3. The effect of over expression of CASK on the biological function of H1299 cells was detected by fluorescence quantitative PCR FQ-PCRR) and Western Blot. The experimental group was divided into two groups: the blank control group (mock group) and the control group (mock overexpression group) and negative group (OE group). The control group, NC group and OE group were treated with CON238 and LV-CASK respectively. The effects of CASK overexpression on the proliferation and migration of H1299 cells were observed by MTT assay and scratch assay respectively. Results the construction of 1.CASK overexpression lentivirus vector successfully amplified the CASK gene fragment and ligated to GV358 by PCR technique. The construction of CASK-GV358 plasmid was confirmed by PCR and DNA sequencing. Green fluorescence and protein expression were observed after the recombinant lentivirus was transfected into 293T cells. The lentivirus was overexpressed and its concentration titer was determined to be 2 脳 10 ~ 8 TU/m L. 2.The results of FQ-PCR and Western Blot detection showed that, Effect of CASK m RNA and protein expression on the biological function of H1299 cells in OE group compared with NC group the results of MTT assay showed that the proliferation ability of H1299 cells in OE group was significantly different from that in NC group, indicating the overexpression of CASK in H1299 cells. 3. The proliferation of H1299 cells was inhibited. The results of cell scratch test showed that there was no significant difference between OE group and NC group in cell migration ability. There was no significant effect of P0.05CSK overexpression on H1299 cell migration. Conclusion the lentivirus vector of CASK gene overexpression was constructed successfully. Overexpression of Cask in H1299 cells could inhibit the proliferation of H1299 cells, which laid a foundation for further study of the mechanism and role of CASK in lung cancer.
【學(xué)位授予單位】：廣西醫(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)】：R734.2

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