本文選題：新基因HA117 + cDNA克隆��； 參考：《重慶醫(yī)科大學》2008年博士論文

【摘要】： 目的 應用組織芯片結(jié)合原位雜交的方法篩選出HA117基因組織表達譜。然后以組織表達譜篩選出的高表達HA117基因的腎臟組織為模板,通過RACE的方法克隆基因全長cDNA,進一步應用重組腺病毒介導的方法獲得高表達HA117基因的K562、Jurkat細胞,初步研究HA117基因?qū)562、Jurkat細胞的多藥耐藥(MDR)功能。 方法 1組織表達譜篩選,選取石蠟包埋的31例常見腫瘤組織標本制和17例癌旁正常組織制作組織芯片,應用mRNA原位雜交技術檢測HA117 mRNA在組織芯片各種組織中的表達情況; 2組織表達譜結(jié)果提示腎臟組織高表達HA117基因,收集并提取腎臟組織總mRNA,首先應用RT-PCR方法驗證HA117基因在腎臟組織表達情況;然后在高表達HA117基因腎臟組織中,根據(jù)已獲得的已知HA117基因序列為模板設計特異引物,通過RACE技術克隆HA117基因全長cDNA序列; 3構(gòu)建攜帶新基因HA117全長cDNA序列的重組腺病毒,酶切質(zhì)粒載體pAdTrack-CMV和目的基因HA117后,用T4DNA連接酶將載體和目的基因定向克隆連接,然后篩選和鑒定含目的基因HA117的重組質(zhì)粒pAdTrack- HA117。將篩選正確的質(zhì)粒pAdTrack- HA117導入已制備的腺病毒同源骨架質(zhì)粒BJ-Adeasy中,產(chǎn)生腺病毒質(zhì)粒Adeasy- HA117,進一步酶切鑒定得到的重組腺病毒質(zhì)粒Adeasy- HA117。脂質(zhì)體介導的方法將Adeasy- HA117轉(zhuǎn)染入產(chǎn)病毒包裝細胞HEK 293,通過乒乓感染,收獲高滴度Ad5- HA117重組腺病毒;并用RT-PCR方法驗證,HA117基因在重組腺并毒Ad5- HA117感染的HEK293細胞的表達情況; 4重組腺病毒Ad5- HA117體外感染K562、Jurkat細胞,將重組腺病毒Ad5- HA117體外感染K562、Jurkat細胞,應用流式細胞術及RT-PCR方法檢測感染后的K562細胞HA117表達情況; 5檢測感染重組腺病毒Ad5- HA117使HA117基因高表達后白血病細胞耐藥性改變:實驗分四組:K562細胞組,K562/ Ad5- HA117細胞組, K562/ Ad5-GFP細胞組, K562/ Ad5-mdr1細胞組,Jurkat細胞分組同K562。通過對細胞活性、細胞形態(tài)學及MTT法抗癌藥物敏感實驗,柔紅霉素排泄實驗等檢測HA117基因高表達前后細胞形態(tài)及耐藥性的變化,初步研究HA117基因的耐藥功能及耐藥機制。 結(jié)果 1新基因HA117 mRNA在人體癌旁正常組織和良性腫瘤、惡性腫瘤組織中均有表達,惡性腫瘤組織中表達陽性率高于良性腫瘤及癌旁正常組織中表達陽性率(P0.5)。在鱗癌和腺癌中的陽性表達率分別是16.67%和61.54%,腺癌中的表達高于鱗癌(P0.05)。有轉(zhuǎn)移的癌組織中HA117 mRNA的表達率高達70%,明顯高于非轉(zhuǎn)移型腫瘤組織的20%(P0.05) ; 2首先采用RT-PCR方法驗證了HA117基因在腎臟組織中高表達。HA117基因的3′RACE PCR產(chǎn)物大小在400bp左右, 5′以RACE PCR產(chǎn)物大小在950bp左右,將HA117基因3′RACE序列、5’RACE擴增序列與己知的HA117基因序列拼接后,得到1110 bp的全長cDNA序列; 3經(jīng)酶切鑒定成功構(gòu)建腺病毒重組質(zhì)粒Ad5- HA117,重組腺病毒Ad5- HA117成功轉(zhuǎn)染包裝細胞,在細胞內(nèi)得到良好的表達,并在包裝細胞中迅速擴增,獲得高滴度的重組腺病毒。收獲的病毒感染液滴度達1.5×1011pfu/ml;RT-PCR方法證實外源性HA117基因在感染HEK293細胞基因組中功能性表達; 4重組腺病毒Ad5-HA117成功將外源性HA117基因?qū)隟562、Jurkat細胞,轉(zhuǎn)染48小時后發(fā)現(xiàn)隨著感染復數(shù)的增加,病毒對K562、Jurkat細胞的感染率也增加, MOI值為100時細胞的存活率和感染率均較好,轉(zhuǎn)染率分別可以達到39.72%、17.10%;RT-PCR方法證實外源性HA117基因在轉(zhuǎn)染K562、Jurkat細胞基因組中功能性表達; 5轉(zhuǎn)染HA117基因的K562、Jurkat細胞對VCR、AD5M、Vp-16、DNR、MMC、CTX藥物的耐藥性均比低表達HA117基因未轉(zhuǎn)染的k562、Jurkat細胞增高,分別增高2~7倍(P0.05或0.01),轉(zhuǎn)染空載體組細胞耐藥性跟未轉(zhuǎn)染組的k562、Jurkat細胞相比無顯著差異(P0.05);感染Ad5-HA117組細胞與感染Ad5-mdr1組相比,耐藥性無顯著差異(P0.05)。轉(zhuǎn)染HA117基因的使其高表達后的K562、Jurkat細胞,與轉(zhuǎn)染了MDR1基因的細胞組柔紅霉素排除實驗結(jié)果顯示,感染Ad5-HA117組細胞未見有明顯的藥物外排泵功能。 結(jié)論 1初步證實HA117基因,在人體癌旁正常組織和良性腫瘤、惡性腫瘤組織中均有表達,惡性腫瘤組織中表達陽性率高于良性腫瘤及癌旁正常組織中表達陽性率。HA117表達陽性率可能與惡性腫瘤的組織學類型及是否為轉(zhuǎn)移瘤有關; 2腎臟組織中高表達HA117基因,采用RACE法從腎臟組織中成功克隆得到了HA117基因全長cDNA序列,為全長1110bp; 3應用分子生物學方法構(gòu)建了HA117重組腺病毒Ad5- HA117。通過熒光顯微鏡證實,重組腺病毒Ad5- HA117成功轉(zhuǎn)染包裝細胞,在細胞內(nèi)得到良好的表達,獲得高滴度的重組腺病毒。證實外源性HA117基因在感染的HEK293細胞有效表達; 4通過腺病毒載體可在體外將外源性HA117基因有效的轉(zhuǎn)K562、Jurkat細胞中,轉(zhuǎn)染的HA117基因能在靶細胞中表達; 5初步證實HA117基因具有多藥耐藥功能;其多藥耐藥功能可能不是遵循mdr1經(jīng)典的藥物外排泵機制。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R346

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