【作者】 郭玉霞；

【导师】 徐酉华；

【作者基本信息】 重庆医科大学 ， 儿科学， 2008， 博士

【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(P>0.5)。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(P<0.05)。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%(P<0.05) ;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(P<0.05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(P>0.05);感染Ad5-HA117组细胞与感染Ad5-mdr1组相比,耐药性无显著差异(P>0.05)。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。更多还原

【Abstract】 ObjectiveTo select spectra of HA117 gene expressing with tissue chip combined with in-situ hybridization. To full-length cDNA seguence of HA117 gene, through RACE in renal tissue with expressing HA117 gene highly according to expression spectra of HA117 gene. To obtain K562 cells and Jurkat cells expressed HA117 gene highly with recombinant adenovirus mediated, and to study multi-drug resistant fouction of HA117 gene.Methods1 Selection of tissue spectra selected 31cases of malignant tumor and 17 cases of normal tissues to make tissue chip, detected the HA117 gene expression in every kinds of tissues of tissue chip with mRNA in-situ hybridization.2 Clone of total HA117 gene sequence renal tissue had a high HA117 gene expression on bais of tissue spectra result. So collected the renal tissue samples and extracted their total mRNA. Firstly, Validated the HA117 gene expression in renal tissue with RT-PCR; Then using total HA117 gene sequence which had been konwn as templete, designed idio-primers and cloned total cDNA sequence of HA117 gene through RACE in renal tissue expressing HA117 highly.3 Construction of recombined adenovirus taking alone total cDNA sequence of HA117 gene After cut plasmid vector pAdTrack-CMV and HA117 gene with restricted enzyme, linked vector and HA117 gene with T4 DNA ligase. Then selected and validated the recombined plasimid pAdTrack- HA117 containing HA117 gene. Transducted the plasimid pAdTrack- HA117 which had been validated to be correct into adenoviral homologous frame plasimid BJ-Adeasy prepared to produce adenoviral plasimid Adeasy-HA117. The recombined adenoviral plasimid Adeasy-HA117 was verified with enzyme cutting. Transfected Adeasy-HA117 into virus-producing incasing cells HEK293 with liposome mediated, and gained recombined adenoviral Ad5-HA117 with high titer using ping-pong infection. Verified the HA117 gene expression in HEK293 cells which was infected recombined adenoviral Ad5-HA117 with RT-PCR;4 Recomined adenovial Ad5-HA117 infected K562 cells and Jurkat cells in vitro: K562 cells and Jurkat cells were infected Ad5-HA117 in vitro. Detected the HA117 expression in K562 cells using flow cytometry and RT-PCR before and after infected;5 Detecting the drug-resistant change of highly HA117 gene expressing leukemia cells because of being infected Ad5-HA117. In expreiments, cells were classed into four groups: K562 cells group, K562/Ad5-HA117 group, K562/Ad5-GFP group and K562/Ad5-mdr1 group. Groups of Jurkat cells were like to K562 cells’。Studied the drug-resistant fouction and mechanism of HA117 gene through detecting the cytoactivty, cytomorph , anticancer drugs sensitive experiment with MTT and excreting daunorubicin experiment before and after HA117 highly expressed in experimental cells.Results1 The new gene HA117 mRNA expressed in normal tissues near malignancy, benign tumor and malignancy. The positive expression rate of malignacy was higher than benign tumor and normal tissues near malignancy(p>0.05). The positive expression rate of squamous carcinoma was 61.54% and higher than adencarcinoma’s 16.67%(p<0.05). The positive expression rate of metastatic malignancy tissue was 70%, obviously higher than non-metastatic malignancy’s 20% (p<0.05).2 Products of HA117 gene’s 3′RACE PCR and 5′RACE PCR were about 400bp and about 950bp respectivly. The full-length cDNA sequence which was 1110bp was obtained after spliced HA117gene’s 3′RACE, sequence and 5’RACE sequence, with HA117 sequenc which had been konwn.3 The recombined adenoviral plasimid was constructed successfly per enzyme cutting. Ad5-HA117 was infected in succeed and expressed well in cells incasing cells. After quicly amplified in incasing cells, recombined adenovirus Ad5-HA117 with high tite was gained. The tite of adenvirus solution obtained reached to 1.5×1011pfu/ml.4 Exogenous HA117 gene was transfected into K562 cells and Jurkat cells with recombined adenovirus Ad5-HA117. After transfected 48 hours, with infecting plural increasing, the infection rates of adenovirus to K562 cells and Jurkat cells were raising too. When MOI was 100, both infected cells’survival rate and infection rate were fairly well. The infection rates of K562 cells and Jurkat cells can reach to 39.72% and 17.10% respectivly5 Resistance to VCR、AD5M、Vp-16、DNR、MMC、CTX of K562 cells and Jurkat cells which were transfected in HA117 gene was higher 2-7 times(P<0.05 or p<0.01) than the drug-resisrance of untransfected K562 cells and Jurkat cells which had negative HA117 gene expression.Compare with cells in uninfected group, the transfected blank vector cells’drugresistance had no significant deviation(p>0.05). Compare with cells in infected Ad5-mdr1 group, drugresistance of cells in transfected Ad5-HA117 group had no significant deciation (p>0.05). The DNR extrusion test results of cells transfected in HA117 gene and cells transfected in mdr1 gene indicated that cells transfected in HA117 were no conspicuous drug-excreting pumping function.conclusions1 Tentative confirmed that HA117 gene may express in normal tissue near carcinoma, benign tumor tissue and malignancy tissue. The positive expression rate of malignacy was higher than the positive expression rate of benign tumor and normal tissues near malignancy. Whether the positive expression is relate to the histological type and metastasis of malignancy.The genes may be an index in monitoring the status and evaluating the prognosis of the malignant tumor.2 Full-length of HA117 gene sequence were cloned successfully in renal tissue with RACE, and it’s full-length is 1110bp3 Constructed the recombined adenovirus Ad-HA117 using Adeasy-1 adenovirus vector system with molecular biological mathod. Validated with GFP expression under fluorescence microscope , Ad5-HA117 was transfected successfully and expressed well in incasing cells. After amplified in incasing cells quickly, the recombined adenovirus with high titer was gained.4 The exogenous HA117 gene could be transferred into K562 and Jurkat cells efficiently by vecter of adenovirus in vitro, and the stable and efficient expression of HA117 gene in cells could be tested. These data provide a foundation and evidence for the further research on multi-drug resistant of HA117 in vitro .5 Multi-drug resistant function of HA117 gene was verified, Mechanism of Multi-drug resistant function may not follow the classic excretion pump of mdr1.更多还原