【摘要】：目的：克隆人IL-24基因，并構(gòu)建其真核表達(dá)載體；分離Hep-2喉癌細(xì)胞株中CD133陽性細(xì)胞（CD133+Hep-2），通過研究人IL-24基因在CD133+Hep-2細(xì)胞中的表達(dá)，探討IL-24基因?qū)D133+Hep-2細(xì)胞的作用。 方法：分離正常人外周血單個核細(xì)胞，應(yīng)用Trizol法提取單個核細(xì)胞的總RNA，將總RNA應(yīng)用RT-PCR法反轉(zhuǎn)錄為cDNA，設(shè)計引物P1、P2，擴(kuò)增人IL-24基因，瓊脂糖凝膠電泳及基因測序正確后將其TA克隆入pMD19-T simple vector載體；通過NheI、XhoI雙酶切人IL-24基因和真核表達(dá)載體pIRES2-ZsGreen1，構(gòu)建人IL-24基因的真核表達(dá)載體pIRES2-ZsGreen1-hIL-24并應(yīng)用酶切及基因測序驗證；流式細(xì)胞儀（FCM）從Hep-2細(xì)胞中分選CD133+Hep-2細(xì)胞，將驗證正確的pIRES2-ZsGreen1-hIL-24在脂質(zhì)體2000介導(dǎo)下轉(zhuǎn)入CD133+Hep-2細(xì)胞，并驗證其表達(dá)情況；MTT、FCM檢測人IL-24基因?qū)D133+Hep-2細(xì)胞生長的影響；裸鼠移植瘤實驗檢測轉(zhuǎn)染人IL-24基因的CD133+Hep-2細(xì)胞的成瘤能力。 結(jié)果：在人外周血單個核細(xì)胞中成功克隆了621bp的人IL-24基因，基因序列測定結(jié)果與GenBank中報道一致；pMD19-T-hIL-24、pIRES2-ZsGreen1-hIL-24重組表達(dá)質(zhì)粒酶切、基因測序驗證正確；用脂質(zhì)體介導(dǎo)法將重組質(zhì)粒pIRES2-ZsGreen1-hIL-24成功轉(zhuǎn)染入CD133+Hep-2細(xì)胞，經(jīng)檢驗人IL-24基因整合入轉(zhuǎn)染細(xì)胞并穩(wěn)定表達(dá)；MTT法檢測結(jié)果顯示轉(zhuǎn)入IL-24基因?qū)嶒灲M的細(xì)胞生長與未轉(zhuǎn)染及空載體對照組相比明顯受到抑制（P0.05); FCM檢測細(xì)胞凋亡的結(jié)果顯示實驗組與未轉(zhuǎn)染及空載體對照組相比凋亡率明顯增加（P0.05);裸鼠移植瘤實驗證實轉(zhuǎn)入hIL-24基因的實驗組CD133+Hep-2細(xì)胞裸鼠移植瘤的體積、重量明顯低于未轉(zhuǎn)染及空載體對照組移植瘤（P0.05)。 結(jié)論：人IL-24基因的表達(dá)能夠抑制CD133+Hep-2細(xì)胞的增殖，促進(jìn)其凋亡，，抑制CD133+Hep-2細(xì)胞裸鼠移植瘤的生長。
[Abstract]:Objective: to clone human IL-24 gene and construct its eukaryotic expression vector. CD133 positive cells (CD133 Hep-2) were isolated from Hep-2 laryngeal carcinoma cell lines. The effect of IL-24 gene on CD133 Hep-2 cells was studied by studying the expression of human IL-24 gene in CD133 Hep-2 cells. Methods: normal human peripheral blood mononuclear cells (PBMC) were isolated. Total RNA, of mononuclear cells was extracted by Trizol method. Total RNA was reverse transcribed into cDNA, designed primer P1CP2to amplify human IL-24 gene. The TA was cloned into pMD19-T simple vector after correct agarose gel electrophoresis and gene sequencing. The eukaryotic expression vector pIRES2-ZsGreen1-hIL-24 of human IL-24 gene was constructed by NheI,XhoI double enzyme digestion of human IL-24 gene and eukaryotic expression vector pIRES2-ZsGreen1, and verified by enzyme digestion and gene sequencing. Flow cytometry (FCM) (FCM) was used to separate CD133 Hep-2 cells from Hep-2 cells. The correct pIRES2-ZsGreen1-hIL-24 was transferred into CD133 Hep-2 cells mediated by liposome 2000 and its expression was confirmed. The effect of human IL-24 gene on the growth of CD133 Hep-2 cells was detected by MTT,FCM, and the tumorigenic ability of CD133 Hep-2 cells transfected with human IL-24 gene was detected by tumor transplantation assay in nude mice. Results: the human IL-24 gene of 621bp was cloned successfully in human peripheral blood mononuclear cells, and the results of gene sequencing were consistent with those reported in GenBank. The recombinant plasmid pIRES2-ZsGreen1-hIL-24 was successfully transfected into CD133 Hep-2 cells by liposome-mediated transfection, and the human IL-24 gene was integrated into the transfected cells and expressed stably. The results of MTT assay showed that the growth of the cells transferred into the IL-24 gene group was significantly inhibited compared with the untransfected and empty vector control group (P0.05). The results of FCM showed that the apoptotic rate of the experimental group was significantly higher than that of the untransfected and empty vector control group (P0.05). Tumor transplantation in nude mice confirmed that the volume and weight of transplanted tumor of CD133 Hep-2 cells transferred to hIL-24 gene in nude mice were significantly lower than those in untransfected and empty vector control group (P0.05). Conclusion: the expression of human IL-24 gene can inhibit the proliferation of CD133 Hep-2 cells, promote their apoptosis and inhibit the growth of xenografts of CD133 Hep-2 cells in nude mice.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.65

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 吳黎明;程彩濤;陳先祥;王江華;劉海燕;田林;;肝細(xì)胞癌組織CD133與VEGF及CD34表達(dá)預(yù)后意義分析[J];中華腫瘤防治雜志;2013年13期

2 王志強(qiáng);;sFRP、WIF-1、CD_(133)、CD_(44)在肺癌組織中的表達(dá)及其臨床意義[J];實用心腦肺血管病雜志;2014年02期

3 侯紅麗;李莉;王贊宏;曹濤;王真;;IL-24對宮頸癌細(xì)胞株Siha細(xì)胞生長和凋亡的影響[J];現(xiàn)代婦產(chǎn)科進(jìn)展;2009年08期

4 程涵蓉;文飛球;李博;陳麗;溫愛惠;;IL-24體外誘導(dǎo)兒童急性白血病骨髓單個核細(xì)胞凋亡研究[J];中國實驗血液學(xué)雜志;2011年01期

5 程涵蓉;文飛球;李博;黃進(jìn)潔;;兒童急性白血病血清白細(xì)胞介素24的測定及意義[J];中國小兒血液與腫瘤雜志;2010年06期

6 楊耿兵;何倩;黃青紅;胡徐龐;劉立;錢程;駱菁菁;;雙靶向溶瘤腺病毒聯(lián)合奧沙利鉑對腫瘤細(xì)胞凋亡的研究[J];浙江理工大學(xué)學(xué)報;2011年05期

7 溫瑩浩;殷正豐;康曉燕;李瑾;錢海華;陳瑋;;腺病毒介導(dǎo)IL-24聯(lián)合化療藥物增強(qiáng)對肝癌細(xì)胞PLC/PRF/5增殖的抑制[J];中國腫瘤生物治療雜志;2007年04期

8 潘s

本文編號：2411307