【作者】 张蕾蕾；

【导师】 许文荣；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2008， 硕士

【摘要】 目的观察ING4基因在肿瘤发生中转录水平的变化和突变情况;克隆人骨髓间质干细胞ING4(inhibitor of growth famility,member4)基因,改造慢病毒表达载体并构建ING4基因慢病毒表达载体PNL-ING4方法培养多种肿瘤细胞株,包括肺腺癌细胞株(A549)、宫颈癌细胞株(Hela)、结肠癌细胞株(SW480)、小细胞肺癌细胞株(LTEP-a-2)、前列腺癌细胞株(PC3)、白血病干细胞(LSC)及hMSC来源肿瘤细胞(F6)和胃癌组织中分离的间质干细胞(GC-MSC),以成人骨髓间质干细胞(hBM-MSC)、人脐静脉内皮细胞(UVEC-304)为对照,收集白血病患者骨髓和胃癌患者癌组织,采用RT-PCR和Real-Time PCR技术研究各种细胞、白血病患者骨髓和胃癌组织中ING4基因的表达水平变化;从hBM-MSC、LSC、SW480、F6和慢性粒细胞白血病骨髓单个核细胞cDNA中克隆ING4基因并测序观察ING4基因的突变情况;经RT-PCR扩增出hBM-MSC来源的全长ING4cDNA,克隆、鉴定并测序后与重组改造慢病毒表达载体PNL-ires连接,构建慢病毒表达载体PNL-ING4,经双酶切、基因测序进行鉴定。采用脂质体方法转染293T细胞,通过RT-PCR检测ING4基因的表达。结果RT-PCR表明在LTEP-a-2、SW480、LSC、Hela、PC3、F6、GC-MSC、hBM-MSC、UVEC-304、急性淋巴细胞白血病(ALL)和慢性粒细胞白血病(CML)患者骨髓中有不同水平ING4基因表达,其中LSC、ALL和CML患者骨髓中表达量较低,而在A549和胃癌患者癌组织中表达量极低。定量PCR显示,ING4基因在A549、LTEP-a-2、SW480、GC-MSC、PC3、Hela、UVEC-304、hBM-MSC和F6中均有表达,表达量以A549最低,F6最高,而UVEC-304和hBM-MSC居中。全长测序显示,在hBM-MSC第七代和第九代、LSC、SW480、F6、CML患者骨髓中均发现缺失及错配,缺失大部分发生在NLS域内,错义突变和移码突变也有发现。且随着骨髓间质干细胞传代数目的增多,点突变和缺失的频率也越来越高。从hBM-MSC中克隆ING4基因全长并改造慢病毒表达载体PNL-ires,构建了PNL-ING4慢病毒表达载体,双酶切和基因测序正确。包装293T细胞后可见绿色荧光,转染效率可达80%以上,并可检测出ING4基因的表达。结论ING4基因在不同肿瘤细胞中呈不同水平的表达,部分表现出表达产物发生突变。这些因素都是通过改变ING4的表达水平或生物活性,参与肿瘤的发生,特别值得关注的是,在MSC体外突变致瘤中,ING4表达突变是其中一个重要原因。成功构建ING4慢病毒表达载体PNL-ING4,为研究ING4基因在肿瘤发生中的作用与肿瘤基因治疗奠定基础。更多还原

【Abstract】 Objective To observe the ING4 gene express in transcriptional level and mutation in tumorigenesis. To clone the gene of human ING4 drived from human bone marrow-drived mesenchymal stem cells (hBM-MSC). To recombinate the lentiviral vector PNL-EGFP and construct PNL-ING4.Methods The total RNA were extracted from A549, HeLa, SW480, LTEP-a-2, PC3, hBM-MSC, UVEC-304, GC-MSC(MSC isolated from gastric tumor tissue ), F6, leukemia patient bone marrow mononuclearcell and gastric tumor tissue. The expression of ING4 gene in each cell was detected by RT-PCR and Real-time PCR. ING4 gene was cloned respectly from hBM-MSC, LSC, SW480, LTEP-a-2, F6 and CML patient bone marrow mononuclearcell cDNA. The cloned ING4 gene was sequenced. Lentiviral vector PNL-ires was recombinated by restriction enzyme NheI and BsrgI. The cDNA of ING4 was amplified by RT-PCR from the total RNA extracted from hBM-MSC. The PCR product was inserted into PMD19-T vector. The positive recombinant clone was analyzed by digestion of restriction endonuclease and DNA sequencing. The correct sequence was subcloned into lentiviral vector. The identification was performed by analysis of restricting enzyme digestion and DNA sequence. The 293T cells were transfected by lentiviral vectors using lipofectamine 2000. The expression of ING4 gene was observed by RT-PCR.Results RT-PCR showed that LTEP-a-2, SW480, LSC, Hela, PC3, F6, GC-MSC, hBM-MSC, UVEC-304, bone marrow mononuclearcells of patients with ALL and CML express different levels of ING4. The expression level of ING4 in LSC, bone marrow mononuclearcells of patients with ALL and CML was lower than others. And nearly no expression of ING4 gene was detected in A549 and gastric tumor patient tissues. Real-time PCR results showed that A549, LTEP-a-2, SW480, GC-MSC, PC3, Hela, F6, UVEC-304 and hBM-MSC all express ING4. The expression level of ING4 was lowest in A549 and highest in F6. DNA sequencing showed deletion and mismatch were found in hBM-MSC, LSC, SW480, F6 and bone marrow mononuclearcells of patients with CML. Most of these deletion happened in NLS area. Missense mutation and frameshift mutation also be found. And with the increasing passage numbers of hBM-MSC, the frequency of point mutation and deletion were more and more higher. The recombination of the lentiviral vector PNL-ires was successful which increased the multiple clone sites. The cDNA of ING4 was amplified by RT-PCR using the total RNA extracted from hBM-MSC. Restriction enzyme digestion and DNA sequencing revealed that ING4 cloning was successful. The virus particles were produced in 293T cells and green fluorescence which can be observed about 80% or more percents in the visual fields. The expressions of ING4 gene were observed by RT-PCR.Conclusions Our research showed that the expression level of ING4 was different in various tumor cells. And mutation could be detected in some of these tumor cells. These factors participate tumorigenesis by altering the expression level of ING4 gene or changing the biological activity of ING4 proten. The most needed to point out is that the mutation of ING4 is one of the key points in tumorigenesis of MSC invitro. The results demonstrate that the PNL-ING4 lentiviral vector was obtained. And it lays the foundation for further research on the fuction of ING4 in tumorigenesis and the development of new gene therapy method.更多还原