新基因HA117全长cDNA克隆及其耐药功能的实验研究

发布时间：2018-06-01 09:32

本文选题：新基因HA117 + cDNA克隆　；参考：《重庆医科大学》2008年博士论文

【摘要】： 目的应用组织芯片结合原位杂交的方法筛选出HA117基因组织表达谱。然后以组织表达谱筛选出的高表达HA117基因的肾脏组织为模板,通过RACE的方法克隆基因全长cDNA,进一步应用重组腺病毒介导的方法获得高表达HA117基因的K562、Jurkat细胞,初步研究HA117基因对K562、Jurkat细胞的多药耐药(MDR)功能。方法 1组织表达谱筛选,选取石蜡包埋的31例常见肿瘤组织标本制和17例癌旁正常组织制作组织芯片,应用mRNA原位杂交技术检测HA117 mRNA在组织芯片各种组织中的表达情况; 2组织表达谱结果提示肾脏组织高表达HA117基因,收集并提取肾脏组织总mRNA,首先应用RT-PCR方法验证HA117基因在肾脏组织表达情况;然后在高表达HA117基因肾脏组织中,根据已获得的已知HA117基因序列为模板设计特异引物,通过RACE技术克隆HA117基因全长cDNA序列; 3构建携带新基因HA117全长cDNA序列的重组腺病毒,酶切质粒载体pAdTrack-CMV和目的基因HA117后,用T4DNA连接酶将载体和目的基因定向克隆连接,然后筛选和鉴定含目的基因HA117的重组质粒pAdTrack- HA117。将筛选正确的质粒pAdTrack- HA117导入已制备的腺病毒同源骨架质粒BJ-Adeasy中,产生腺病毒质粒Adeasy- HA117,进一步酶切鉴定得到的重组腺病毒质粒Adeasy- HA117。脂质体介导的方法将Adeasy- HA117转染入产病毒包装细胞HEK 293,通过乒乓感染,收获高滴度Ad5- HA117重组腺病毒;并用RT-PCR方法验证,HA117基因在重组腺并毒Ad5- HA117感染的HEK293细胞的表达情况; 4重组腺病毒Ad5- HA117体外感染K562、Jurkat细胞,将重组腺病毒Ad5- HA117体外感染K562、Jurkat细胞,应用流式细胞术及RT-PCR方法检测感染后的K562细胞HA117表达情况; 5检测感染重组腺病毒Ad5- HA117使HA117基因高表达后白血病细胞耐药性改变:实验分四组:K562细胞组,K562/ Ad5- HA117细胞组, K562/ Ad5-GFP细胞组, K562/ Ad5-mdr1细胞组,Jurkat细胞分组同K562。通过对细胞活性、细胞形态学及MTT法抗癌药物敏感实验,柔红霉素排泄实验等检测HA117基因高表达前后细胞形态及耐药性的变化,初步研究HA117基因的耐药功能及耐药机制。结果 1新基因HA117 mRNA在人体癌旁正常组织和良性肿瘤、恶性肿瘤组织中均有表达,恶性肿瘤组织中表达阳性率高于良性肿瘤及癌旁正常组织中表达阳性率(P0.5)。在鳞癌和腺癌中的阳性表达率分别是16.67%和61.54%,腺癌中的表达高于鳞癌(P0.05)。有转移的癌组织中HA117 mRNA的表达率高达70%,明显高于非转移型肿瘤组织的20%(P0.05) ; 2首先采用RT-PCR方法验证了HA117基因在肾脏组织中高表达。HA117基因的3′RACE PCR产物大小在400bp左右, 5′以RACE PCR产物大小在950bp左右,将HA117基因3′RACE序列、5’RACE扩增序列与己知的HA117基因序列拼接后,得到1110 bp的全长cDNA序列; 3经酶切鉴定成功构建腺病毒重组质粒Ad5- HA117,重组腺病毒Ad5- HA117成功转染包装细胞,在细胞内得到良好的表达,并在包装细胞中迅速扩增,获得高滴度的重组腺病毒。收获的病毒感染液滴度达1.5×1011pfu/ml;RT-PCR方法证实外源性HA117基因在感染HEK293细胞基因组中功能性表达; 4重组腺病毒Ad5-HA117成功将外源性HA117基因导入K562、Jurkat细胞,转染48小时后发现随着感染复数的增加,病毒对K562、Jurkat细胞的感染率也增加, MOI值为100时细胞的存活率和感染率均较好,转染率分别可以达到39.72%、17.10%;RT-PCR方法证实外源性HA117基因在转染K562、Jurkat细胞基因组中功能性表达; 5转染HA117基因的K562、Jurkat细胞对VCR、AD5M、Vp-16、DNR、MMC、CTX药物的耐药性均比低表达HA117基因未转染的k562、Jurkat细胞增高,分别增高2~7倍(P0.05或0.01),转染空载体组细胞耐药性跟未转染组的k562、Jurkat细胞相比无显著差异(P0.05);感染Ad5-HA117组细胞与感染Ad5-mdr1组相比,耐药性无显著差异(P0.05)。转染HA117基因的使其高表达后的K562、Jurkat细胞,与转染了MDR1基因的细胞组柔红霉素排除实验结果显示,感染Ad5-HA117组细胞未见有明显的药物外排泵功能。结论 1初步证实HA117基因,在人体癌旁正常组织和良性肿瘤、恶性肿瘤组织中均有表达,恶性肿瘤组织中表达阳性率高于良性肿瘤及癌旁正常组织中表达阳性率。HA117表达阳性率可能与恶性肿瘤的组织学类型及是否为转移瘤有关; 2肾脏组织中高表达HA117基因,采用RACE法从肾脏组织中成功克隆得到了HA117基因全长cDNA序列,为全长1110bp; 3应用分子生物学方法构建了HA117重组腺病毒Ad5- HA117。通过荧光显微镜证实,重组腺病毒Ad5- HA117成功转染包装细胞,在细胞内得到良好的表达,获得高滴度的重组腺病毒。证实外源性HA117基因在感染的HEK293细胞有效表达; 4通过腺病毒载体可在体外将外源性HA117基因有效的转K562、Jurkat细胞中,转染的HA117基因能在靶细胞中表达; 5初步证实HA117基因具有多药耐药功能;其多药耐药功能可能不是遵循mdr1经典的药物外排泵机制。
[Abstract]:objective
The tissue expression profile of HA117 gene was screened by the method of tissue microarray and in situ hybridization. Then the kidney tissue with high expression of HA117 gene screened by tissue expression spectrum was used as a template. The full length cDNA of the gene was cloned through the RACE method, and the recombinant adenovirus mediated K562, Jurkat cell, which expressed the high expression of HA117 gene, was obtained. Objective to study the multidrug resistance (MDR) function of HA117 gene on K562 and Jurkat cells.
Method
1 tissue expression profiles were screened, 31 common tumor tissue specimens embedded in paraffin were selected and 17 cases of normal tissues adjacent to cancer were fabricated. MRNA in situ hybridization was used to detect the expression of HA117 mRNA in tissue microarrays.
2 the results of tissue expression suggest that the kidney tissue is highly expressed HA117 gene, collecting and extracting the total mRNA of kidney tissue. First, RT-PCR method is used to verify the expression of HA117 gene in the kidney tissue, and then the specific primers are designed according to the known sequence of known HA117 genes in the high expression of HA117 gene, and the RACE technical gram is used. The long cDNA sequence of the long HA117 gene;
3 the recombinant adenovirus carrying the full length cDNA sequence of the new gene HA117, the plasmid vector pAdTrack-CMV and the target gene HA117 were constructed, the vector and the target gene were cloned with the T4DNA ligase, and the recombinant plasmid pAdTrack- HA117. containing the target gene HA117 would be screened and identified, and the correct plasmid pAdTrack- HA117 was screened and introduced into the system. In the adenovirus homologous skeleton plasmid BJ-Adeasy, the adenovirus plasmid Adeasy- HA117 was produced, and the recombinant adenovirus plasmid Adeasy- HA117. liposome mediated by the recombinant adenovirus plasmid, Adeasy- HA117 was transfected into the virus packaged cell HEK 293, and the high titer Ad5- HA117 recombinant adenovirus was captured by ping-pong infection; and RT-PCR prescription was used. The expression of HA117 gene in HEK293 cells infected with Ad5- HA117 was detected by the method.
4 recombinant adenovirus Ad5- HA117 infected K562, Jurkat cells in vitro, and the recombinant adenovirus Ad5- HA117 was infected with K562, Jurkat cells in vitro, and the HA117 expression of K562 cells after infection was detected by flow cytometry and RT-PCR methods.
5 detection of recombinant adenovirus Ad5- HA117 to change the drug resistance of leukemia cells after high expression of HA117 gene: the experiment was divided into four groups: K562 cell group, K562/ Ad5- HA117 cell group, K562/ Ad5-GFP cell group, K562/ Ad5-mdr1 cell group, Jurkat cells grouping with cell activity, cell morphology and anti-cancer drug sensitivity experiment, Daunorubicin excretion test was used to detect the changes of cell morphology and drug resistance before and after high expression of HA117 gene, and to preliminarily study the resistance and resistance mechanism of HA117 gene.
Result
1 new gene HA117 mRNA was expressed in normal tissues and benign tumors and in malignant tumor tissues. The positive rate of malignant tumor tissues was higher than that in benign tumor and normal tissue adjacent to cancer (P0.5). The positive expression rate in squamous and adenocarcinoma was 16.67% and 61.54%, respectively, and the expression in adenocarcinoma was higher than that of squamous carcinoma (P0.05). The expression rate of HA117 mRNA in metastatic cancer tissues was 70%, which was significantly higher than that in non metastatic tumor tissues (20%) (P0.05).
2 first, the RT-PCR method was used to verify that the size of the 3 'RACE PCR product of the high expression of the.HA117 gene in the kidney tissue was around 400bp, and the 5' was about 950bp about the size of the RACE PCR product, and the 3 'RACE sequence of the HA117 gene, the 5' RACE amplification sequence was spliced with the known sequence of the genes, and the full length sequence of 1110 was obtained.
3 the recombinant adenovirus recombinant plasmid Ad5- HA117 was successfully constructed by enzyme digestion. The recombinant adenovirus Ad5- HA117 was successfully transfected into the packed cells, and the recombinant adenovirus was successfully expressed in the cells. The recombinant adenovirus was rapidly amplified in the packaging cells and obtained the recombinant adenovirus with high titer. The titer of the harvested virus was 1.5 * 1011pfu/ml, and the RT-PCR method confirmed the exogenous HA117 gene. Functional expression in the genome of infected HEK293 cells;
4 recombinant adenovirus Ad5-HA117 succeeded in introducing exogenous HA117 gene into K562 and Jurkat cells. After 48 hours transfection, the infection rate of K562 and Jurkat cells increased with the increase of complex number of infection. The survival rate and infection rate of the cells were better when the MOI value was 100, and the transfection rate could reach 39.72% and 17.10%, respectively. RT-PCR method proved exogenous. Functional HA117 gene was expressed in K562 and Jurkat cells.
5 the K562 of HA117 gene transfected, the resistance of Jurkat cells to VCR, AD5M, Vp-16, DNR, MMC, and CTX were all higher than that of the low expression HA117 gene. There was no significant difference in the resistance between the cells and the infected Ad5-mdr1 group (P0.05). The transfection of HA117 gene to the high expression of K562, Jurkat cells, and the result of the removal of erythromycin from the cell group transfected with the MDR1 gene showed that there was no obvious drug efflux pump function in the infected Ad5-HA117 group.
conclusion
1 it was preliminarily confirmed that HA117 gene was expressed in normal tissue and benign tumor, and in malignant tumor tissues. The positive rate of expression in malignant tumor tissues was higher than that of benign tumor and normal tissue adjacent to cancer. The positive rate of.HA117 expression may be related to the histological type of malignant tumor and whether it is metastatic tumor.
2 the HA117 gene was highly expressed in renal tissue. The full-length cDNA sequence of HA117 gene was successfully cloned from renal tissue by RACE method, and the full-length 1110bp was obtained.
3 the recombinant adenovirus Ad5- HA117. of HA117 was constructed by the molecular biology method. The recombinant adenovirus Ad5- HA117 was successfully transfected into the packed cell, and the recombinant adenovirus was well expressed in the cell, and the recombinant adenovirus with high titer was obtained. The effective expression of the exogenous HA117 gene in the infected HEK293 cells was confirmed.
4 the exogenous HA117 gene can be effectively transferred into K562 and Jurkat cells by adenovirus vector in vitro. The transfected HA117 gene can be expressed in target cells.
5 it is preliminarily confirmed that HA117 gene has multidrug resistance function, and its multidrug resistance function may not follow the classical drug efflux pump mechanism of MDR1.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346

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