本文選題：膀胱癌細(xì)胞　切入點(diǎn)：IL-2基因　出處：《蘭州大學(xué)》2014年碩士論文

【摘要】：目的：探討已經(jīng)構(gòu)建成功的五種共表達(dá)自殺基因聯(lián)合多細(xì)胞因子基因的泌尿組織特異性真核表達(dá)載體pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-α、 pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT、pBud-UPII-TK/CMV-ITR對人膀胱癌細(xì)胞5637的抑制作用。 方法：將已經(jīng)構(gòu)建并行測序、PCR及限制性核酸內(nèi)切酶酶切證明的雙啟動子載體pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-α pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT、pBud-UPII-TK/CMV-ITR分別轉(zhuǎn)染至膀胱癌細(xì)胞5637,通過抗性標(biāo)志篩選出抗zeocin克隆,應(yīng)用westenblot檢測五種共表達(dá)載體IL-2、TNF-α、TRAIL蛋白質(zhì)的表達(dá)量,將其與未轉(zhuǎn)染膀胱癌細(xì)胞5637以及轉(zhuǎn)染空載體pBud4.1的靶細(xì)胞做比較；PCR分析轉(zhuǎn)染前后以及轉(zhuǎn)染不同載體后IL-2、TRAIL、TNF-a mRNA的表達(dá),設(shè)未轉(zhuǎn)染膀胱癌細(xì)胞5637作為對照,對比其mRNA的表達(dá)量；MTT的方法檢測五種靶細(xì)胞與未轉(zhuǎn)染以及轉(zhuǎn)染空載體pBud4.1的5637細(xì)胞抑制率的區(qū)別,并觀察加入25ug/ml GCV后五種載體對膀胱癌細(xì)胞5637細(xì)胞抑制率的影響；使用流式細(xì)胞儀檢測五種靶細(xì)胞與未轉(zhuǎn)染膀胱癌細(xì)胞5637的凋亡率,將其結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果：westen blot結(jié)果顯示,轉(zhuǎn)染五種共表達(dá)載體后,靶細(xì)胞相應(yīng)的細(xì)胞因子蛋白的表達(dá)明顯高于未轉(zhuǎn)染以及空載體5637細(xì)胞；RT-PCR證實(shí)轉(zhuǎn)染后5637細(xì)胞中IL-2、TRAIL、TNF-a的mRNA表達(dá)量明顯增加；流式細(xì)胞儀結(jié)果提示轉(zhuǎn)染五種共表達(dá)載體后,細(xì)胞凋亡率明顯高于轉(zhuǎn)染空白載體組和未轉(zhuǎn)染組(P0.01)；MTT結(jié)果亦顯示轉(zhuǎn)染共表達(dá)載體后,細(xì)胞增殖受到明顯抑制(P0.01),加入25ug/ml GCV后細(xì)胞抑制率明顯增高(P0.01)。 結(jié)論：包含UPⅡ啟動子驅(qū)動的HSV-TK基因、并分別搭配IL-2、TNF-α、TRAIL、 IL-2-TNF-α (IT)、IL-2-TRAIL (ITR)的五種雙啟動子重組真核表達(dá)質(zhì)粒,可有效加快膀胱癌細(xì)胞5637的凋亡,在GCV的作用下,膀胱癌細(xì)胞5637的細(xì)胞抑制率明顯增加,為進(jìn)一步研究細(xì)胞因子聯(lián)合及自殺基因聯(lián)合多細(xì)胞因子的泌尿組織特異性真核表達(dá)載體應(yīng)用于膀胱癌的治療奠定了基礎(chǔ)。
[Abstract]:Objective: To evaluate the construction of five successful co expression gene combined with cytokines gene Dutch act in urinary tissue specific eukaryotic expression vector pBud-UPII-TK/CMV-IL-2, pBud-UPII-TK/CMV-TNF- alpha, pBud-UPII-TK/CMV-TRAIL, pBud-UPII-TK/CMV-IT, pBud-UPII-TK/CMV-ITR on the inhibition of human bladder cancer cell 5637.
Methods: constructed parallel sequencing, dual promoter vector pBud-UPII-TK/CMV-IL-2 PCR and restriction endonuclease proof, pBud-UPII-TK/CMV-TNF- alpha pBud-UPII-TK/CMV-TRAIL, pBud-UPII-TK/CMV-IT, pBud-UPII-TK/CMV-ITR were transfected into bladder cancer cell 5637, the resistance signs were resistant to zeocin cloning, application of westenblot detection of five kinds of co expression vector IL-2, TNF- alpha, TRAIL expression the protein, compared with non transfected bladder cancer cells transfected with vector pBud4.1 and 5637 target cells; PCR analysis before and after transfection and transfection of different carriers after IL-2, TRAIL, TNF-a expression of mRNA, a non transfected bladder cancer cells 5637 as control, the expression of mRNA of MTT; difference detection method five kinds of target cells and non transfected 5637 cells transfected with vector and the inhibition rate of pBud4.1, and to observe the 25ug/ml GCV after five The effect of carriers on the inhibition rate of bladder cancer cell 5637 was analyzed. The apoptosis rate of five target cells and 5637 cells without transfected bladder cancer cells was detected by flow cytometry, and the results were statistically analyzed.
Results: westen blot showed that transfection of five co expression vector after cytokine protein expression was significantly higher than that of the corresponding target cells without transfection and 5637 vector transfected IL-2 cells; RT-PCR confirmed, TRAIL 5637 cells, TNF-a mRNA expression was significantly increased; flow cytometry showed that transfection of five co expression after the carrier, the apoptosis rate was significantly higher than that of empty vector transfection group and non transfection group (P0.01); MTT results also showed that co expression vector after transfection, cell proliferation was significantly inhibited (P0.01), GCV after adding 25ug/ml cell inhibition rate was significantly higher (P0.01).
Conclusion: II contains the UP promoter and HSV-TK gene driven, and collocation of TNF- alpha, IL-2, TRAIL, IL-2-TNF- alpha (IT), IL-2-TRAIL (ITR) five kinds of dual promoter recombinant eukaryotic expression plasmid, can effectively accelerate the apoptosis of bladder cancer cell 5637, under the action of GCV cells of bladder cancer 5637 the cell inhibition rate increased significantly, which laid the foundation for the further study of eukaryotic expression of specific cytokines and cytokine Dutch act gene combined with urinary bladder cancer tissue treatment applied to the carrier.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.14

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 謝鑫;姜慶;梁培禾;王志剛;鄭元義;譚小軍;喬娟;;攜HSV_1-TK自殺基因超聲微泡造影劑聯(lián)合前藥更昔洛韋對前列腺癌細(xì)胞的抑制[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2012年13期

2 石向華;譚萬龍;朱文輝;梁中錕;杜躍軍;;腺病毒介導(dǎo)TK/GCV基因系統(tǒng)聯(lián)合腫瘤壞死因子對膀胱癌細(xì)胞的體外殺傷作用[J];南方醫(yī)科大學(xué)學(xué)報(bào);2008年05期

3 殷祥瑞;唐偉;喻備;王亞榮;;雙歧桿菌介導(dǎo)HSV-tk/GCV系統(tǒng)治療大鼠膀胱癌的療效[J];中國生物制品學(xué)雜志;2013年04期

4 朱宏建,張智清,郭應(yīng)祿;尿路上皮細(xì)胞分化特異性糖蛋白與膀胱癌[J];中華泌尿外科雜志;2003年09期

5 謝文練,黃健,韓金利,林天歆,許可慰;逆轉(zhuǎn)錄病毒介導(dǎo)HSV-TK基因結(jié)合更昔洛韋對裸鼠膀胱癌模型的抑癌作用[J];中華泌尿外科雜志;2005年11期

6 潘s，

本文編號：1661893