【作者】 郑改焕；

【导师】 徐酉华；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 博士

【摘要】 目的采用基因转染和RNA干扰技术,以人淋巴瘤Raji细胞株和人结肠癌CW-2细胞株作为实验细胞,研究新基因HA117的耐药功能,并初步探讨HA117基因影响CW-2细胞耐药性的机制;通过建立裸鼠结肠癌皮下移植瘤模型,进一步对HA117基因的耐药功能进行裸鼠体内实验验证。方法1构建双克隆质粒pSOS-HUS-HA117-HAi筛选靶向HA117基因的最佳siRNA。提取质粒pSOS-HUS的DNA,经酶切后与HA117基因进行克隆重组,得到重组质粒pSOS-HUS-HA117;设计合成5对靶向HA117基因的siRNA模板DNA,使其与重组质粒pSOS-HUS-HA117分别进行再重组,构建了5个双克隆质粒pSOS-HUS-HA117-HAi。把5个双克隆质粒分别转染293细胞,通过镜下观察、流式细胞仪及RT-PCR检测,筛选鉴定出对HA117基因RNA干扰效果最强的siRNA。2应用基因重组技术构建靶向HA117基因的RNA干扰载体pSES-HUS-HAi5及Ad-HAi5。提取pSES-HUS腺病毒穿梭质粒DNA,经酶切后与筛选出的最佳siRNA模板HAi5连接,转导感受态细菌DH5α,构建HA117基因的RNA干扰重组质粒pSES-HUS-HAi5;将pSES-HUS-HAi5导入腺病毒同源骨架质粒BJ-Adeasy中产生腺病毒质粒Adeasy- HAi5,脂质体介导的方法将Adeasy-HAi5转染入产病毒包装细胞HEK 293,通过乒乓感染收获高滴度HA117基因的RNA干扰重组腺病毒Ad-HAi5。3通过基因转染研究HA117基因对Raji、CW-2细胞株耐药性的影响。Raji细胞实验分3组:实验组(转染HA117基因的Raji/HA117细胞),RNA干扰组(转染HA117基因及其RNA干扰载体的Raji/ HA117/HAi细胞),空白对照组(未转染的Raji细胞); CW-2细胞分组同Raji。采用脂质体介导重组质粒及重组腺病毒直接感染的方法分别对Raji细胞和CW-2细胞进行基因转染。用荧光显微镜、流式细胞仪检测各组细胞转染率,RT-PCR检测各组细胞HA117基因表达,MTT检测各组细胞对柔红霉素(DNR)、阿霉素(ADM)、甲氨喋呤(CTX)、长春新碱(VCR)及5-氟尿嘧啶(5-FU)的敏感性。用AnnexinV-FITC和碘化丙啶(PI)双染结合流式细胞仪的方法检测各组CW-2细胞的凋亡率。4通过建立裸鼠结肠癌皮下移植瘤模型对HA117基因的耐药功进行裸鼠体内实验验证。在Balb/c裸鼠皮下接种具有HA117耐药性的CT26细胞悬液,建立Balb/c裸鼠结肠癌皮下移植瘤模型。建模7天后把荷瘤裸鼠随机分为实验组(10只)和对照组(10只)。于当日在实验组裸鼠肿瘤内直接注射Ad-HA117病毒液,对照组裸鼠肿瘤内直接注射等量的Ad-null空病毒液。首次瘤内注射病毒24h后,从裸鼠腹腔注射5-FU对两组荷瘤裸鼠进行化疗。化疗2周后用B超测量肿瘤直径计算瘤体积, RT-PCR检测瘤组织内HA117基因mRNA的表达。通过比较2组荷瘤裸鼠化疗后瘤体积,研究HA117基因对裸鼠结肠癌移植瘤耐药性的影响。结果1经两次克隆重组,成功构建了5个双克隆质粒pSOS-HUS-HA117-HAi。经荧光显微镜观察、流式细胞仪及RT-PCR检测,5对模板DNA中HAi5转录的siRNA对HA117基因的干扰效果最强2经酶切及测序鉴定,成功构建了重组腺病毒穿梭质粒pSES-HUS-HAi5,将其与Bj-Adeasy同源重组及HEK293细胞包装得到重组腺病毒Ad-HAi5。经乒乓交互感染,收获的Ad-HAi5病毒感染液滴度达2.3×1011pfu/ml。3与未转染的Raji、CW-2细胞相比,转染HA117基因的Raji/HA117、CW-2/HA117细胞对DNR、ADM、VCR、CTX和5-FU的耐药指数均增高3~7倍(P<0.05),同时转染HA117基因及其RNA干扰载体的Raji/HA117/HAi、CW-2/HA117/HAi细胞对上述五种化疗药物的耐药指数与未转染的Raji、CW-2细胞相比均无显著差异(P>0.05)。4转染HA117基因的CW-2细胞凋亡率为6.77%,未转染的CW-2细胞凋亡率11.47%(P<0.05),两者具有显著性差异。同时转染HA117基因及其RNA干扰载体的CW-2细胞凋亡率为12.06%,与未转染的CW-2细胞凋亡率差异无显著性(p>0.05)。5用CT26细胞皮下接种裸鼠7 d后,20只裸鼠接种部位均出现直径在5 mm~10mm的皮下结节,移植瘤模型建立成功。化疗前实验组和对照组组荷瘤裸鼠肿瘤的体积大小差异无显著性(P>0.05),化疗2周后,实验组裸鼠瘤体积大于对照组裸鼠瘤体积(P<0.05)。结论1从靶向HA117基因的5对siRNA模板中,成功筛选鉴定出了对HA117基因RNAi效果最强的HAi5,为应用RNAi技术成功沉默HA117基因表达奠定了基础。2成功构建了HA117基因的RNA干扰重组质粒pSES-HUS-HAi5及重组腺病毒Ad-HAi5,收获的Ad-HAi5病毒液滴度为2.3×1011pfu/ml。3高表达外源性HA117基因的CW-2、Raji细胞对DNR、ADM、VCR、CTX和5-FU的耐药性增强,外源性HA117基因表达被沉默后的CW-2、Raji细胞对上述五种药物的耐药性消失,提示外源性HA117基因可使Raji、CW-2细胞产生多药耐药,表明HA117基因具有多药耐药功能;初步探讨HA117基因可能是通过抑制细胞凋亡的途径使瘤细胞产生多药耐药性。4新基因HA117可增强裸鼠结肠癌皮下移植瘤对5-FU的耐受性,进一步在动物体内实验表明HA117基因可使肿瘤细胞产生耐药性,是一个参与调控肿瘤细胞产生耐药性的新基因。更多还原

【Abstract】 ObjectivesTo research on the drug resistant function of the novel HA117 gene in human lymphoma cell lines (Raji cells) and in human colon carcinoma cell line(CW-2 cells) through gene transfection and RNA interference. And to investigate initially the possible mechanism how the HA117 gene could affect on the multi-drug resistance of CW-2 cells. As well to investigate the drug resistant function of HA117 gene further in vivo through establishing the nude mice subcutaneou transplanted carcinoma model.Methods1 Selecting the optimal siRNA target sites of HA117 gene through constructing the double clone recombined plasmid pSOS-HUS-HA117-HAi The pSOS-HUS DNA were extracted and digested by enzymes. Then it linked with HA117 gene to get the recombined plasmid pSOS-HUS-HA117;Five pairs siRNA target sites of HA117 gene were designed and synthesized and cloned them into recombined plasmid pSOS-HUS-HA117 respectively to construct five double clone recombined plasmid pSOS-HUS-HA117-HAi. Then the five kinds of double clone plasmids were transfected into 293 cells respectively. The optimal siRNA target sites were selected and validated through detecting and comparing intensity of green fluorescence by fluorescent microscope and flow cytometry, as well expression of HA117 gene mRNA was detcted by RT-PCR.2 Constructing RNA interferential vector pSES-HUS-HAi5 and Ad- HAi5 targetting to HA117 gene by recombinant DNA technology. The pSES-HUS DNA were extracted and digested by enzymes. Then it linked with the optimal siRNA target sites to get the RNA interferential recombined vector pSES-HUS-HAi5 targetting to HA117 gene. The plasimid pSES- HUS-HAi were transducted into the adenoviral homologous frame plasimid BJ-Adeasy to produce the adenoviral plasimid Adeasy-HAi. Then the Adeasy-HAi was transfected into HEK293 cells , and the recombined adenoviral Ad-HAi5 with high titer were gained through ping-pong infection.3 Investigating the drug resistant function of HA117 gene in Raji、CW-2 cells through gene transfection. The Raji cells were divided into three groups: the experimental group ( the Raji/HA117 cells with HA117 gene transfected), the RNA interferential group(the Raji/ HA117/HAi cells with HA117 gene and it’s RNA interferential vector transfected), the blank control group (the untransfected Raji cells). The groups of CW-2 cells were same as Raji cells’. The gene transfection to Raji cells and CW-2 cells were carried out by transfected the reombind plasmid with lipidosome mediated and infected directly the recombind adenovirus, respectively. The transfection or infection efficiencies of cells in every groups were detected by fluorescence and flow cytometry. The HA117 gene mRNA expression levels of cells in every group were detected by RT-PCR. The drug sensitivities to daunorubicin(DNR), adriamycin(ADM), methopterin (CTX), vincristine (VCR), 5-fluorouracil(5-FU)of cells in each group were detected by MTT. The apoptosis rates of CW-2 cells in every group were detected by AnnexinV-FITC and flow cytometry. 4 Investigating drug resistant function of HA117 gene in vivo through establishing the nude mice subcutaneou transplanted carcinoma model. The transplanted mice colon carcinoma models were established by subcutaneous injecting CT26 cells which had HA117 gene drug resistance to the Balb/c nude mice. Seven days after injection when the diameter of the tumor reached about 5-10mm, 20 nude mice were divided randomly into exprimental group (10 mice) and control group (10 mice) . Nude mice in exprimental group were directly injected the recombined adenovirus Ad-HA117 suspension to the bearing-colon carcinomas. While the nude mice in control group were directly injected blank adenovirus Ad-null suspension to the bearing-colon carcinomas. After injected adenovirus 24 hours, nude mice in each group were received chemotherapy with intraperitoneal(i.p.) injection of 5-FU. After two weeks, the diameters of the bearing-colon carcinomas were measureed by type-B ultrasonic. The HA117 gene mRNA expressions in the bearing-colon carcinoma tissue of nude mice in each group were detected by RT-PCR. The effect of HA117 gene on the drug senstivity of mouse colon tumor cells in vivo was investigated through comparing the subcutaneou transplanted carcinomas volumes of nude mice in expremental group to of which in control group.Results1 Five double clone recombined plasmid pSOS-HUS-HA117-HAi were constructed successfully. The optimal siRNA target sites HAi5 was selected and validated from five siRNA sites targetting to HA117 gene by by fluorescent microscope , flow cytometry and RT-PCR.2 The RNA interferential recombined plasmid pSES-HUS-HAi5 targetting to HA117 gene was constructed successfully. After homologous recombined with BJ-Adeasy, it was pakaged to be the recombined adenovirus HAi5 in 293 cells. And the HA117 gene’s RNA interferential recombind adenovirus Ad-HAi5 with 2.3×1011pfu/ml were gained through ping-pong infection.3 The drug resistance to DNR、ADM、VCR、CTX and 5-FU of the Raji/HA117 and CW-2 /HA117 cells with HA117 gene transfected were increased(p<0.05) 3～7 times than of which untransfected Raji and CW-2 cells respectively. Campared to of which untransfected Raji and CW-2 cells, the drug resistance to the five kinds of drugs above-mentioned of Raji/ HA117/HAi cells and CW-2/HA117/HAi cells with HA117 gene and it’s RNA interferential vector transfected were no significant difference (P>0.05), respectively.4 The apoptosis rate of CW-2 /HA117 cells with HA117 gene transfected was 6.77% and lower than the untransfected CW-2 cells’apoptosisi rate 11.47%(P<0.05).Campared to of untransfected CW-2 cells the apoptosis rate of CW-2/HA117/HAi cells with HA117 gene and it’s RNA interferential vector transfected was 12.06%, and no significant difference (P>0.05).5 After injected CT26 cells seven days, the inoculated positions of nude mice developed subcutaneous nodules which diameters were between 5mm and 10mm, and the subcutaneou transplanted carcinoma models of nude mice were established successfully. There was no significance difference between tumor volume of carcinoma-bearing nude mice in experimental group and of that in control group before chemo(P>0.05). After used chem tow weeks , the tumor volume of carcinoma-bearing nude mice in control group exceeded than of which in control group(P<0.05). Conclusions1 The optimal siRNA target sites HAi5 that had strongest RNA interferencial effect tagetting to HA117 gene was selected and validated from five pairs siRNA target sites of HA117 gene, which pave the way for silencing HA117 gene expression successfully using RNA interference technology.2 The RNA interferential recombined plasmid pSES-HUS-HAi5 and recombined adenovirus Ad-HAi5 which express the selected optimal siRNA sites targeting to HA117 gene were constructed successfully. The titer of the abtained recombined adenovirus Ad-HAi5 was 2.3×1011pfu/ml.3 The multi-drug resistance to DNR、ADM、VCR、CTX and 5-FU of the Raji and CW-2 cells expressing highly exogenous HA117 gene increased, while the multidrug resistance of Raji and CW-2 cells were disappeared after the exogenous HA117 gene expression silenced, Which indecated that the novel gene HA117 the multi-drug resistance of Raji cells and CW-2 cells and HA117 gene might have multi-drug resistant function. And the possible mechanism how the HA117 gene could affect on the multi-drug resistance of CW-2 cells might be through inhibitting CW-2 cells apoptosis.4 The novel gene HA117 could increase the resistance of the nude mice subcutaneou transplanted carcinoma to 5-FU in vivo, which indicated further that the HA117 gene might have drug resistant function and be one of important genes which regulated development of tumor cells’drug resisitance.更多还原