【作者】 李燕；

【导师】 李明远；

【作者基本信息】 四川大学 ， 病原生物学， 2007， 博士

【摘要】 研究背景：端粒酶是一种特殊的反转录酶，能以自身的RNA为模板反转录合成端粒DNA从而维持染色体末端的稳定。端粒酶在肿瘤组织中阳性率为85～90％，而在癌旁正常组织或良性肿瘤中仅为4．2％。端粒酶是维持肿瘤细胞永生化的重要分子基础，通过抑制端粒酶活性来达到治疗恶性肿瘤的目的已成为肿瘤基因治疗的热点。目前研究认为端粒酶复合体由六部分组成，其中最重要的部分是人端粒酶RNA（hTR）和人端粒酶反转录酶（hTERT）。hTR提供合成端粒DNA的模板部分5’-CUAACC CUAAC-3’，该部分能与端粒序列（TFAGGG）n互补，利用自身作为模板并在hTERT的催化作用下不断合成新的TFAGGG，以维持端粒的长度。因此，hTR对端粒酶复合体发挥其功能是必不可少的，它也可以作为端粒酶活性抑制剂的天然靶位。靶向hTR常用的方法有RNA干扰（RNAi）、反义核苷酸技术封闭、锤头状核酶切割等。其中小干扰RNA（siRNA）更是在哺乳动物细胞中显示了强大的、有效的RNA干扰能力，因而化学合成的21-23核苷酸的siRNA或质粒表达的小发夹RNA（shRNA）被常规用于基因沉默研究。这两种途径产生的siRNA具有作用短暂、转染率低等缺点，科研人员更推崇病毒载体介导的细胞内shRNA表达技术，该shRNAs在体内再被加工为siRNAs，这种方式有可能导致有效的、长期的基因沉默。表达shRNA的病毒载体正被广泛用于抗病毒、抗肿瘤和神经系统疾病的研究。腺病毒由于具有瞬时高效表达、宿主范围广、对人致病性低、在增殖和非增殖细胞中均能高效感染和外源DNA容量大等优点，而且，腺病毒对肝有明显的嗜性，所以在众多的病毒载体中脱颖而出，成为基因治疗、RNAi分子递送的重要工具，广泛受到学者的青睐。构建重组腺病毒的两种传统方法是以同源重组为基础的，但由于在HEK293细胞中同源重组率偏低：或在转化BJ5183之前克隆步骤过于复杂，使这两种方法制备腺病毒的效率偏低。研究目的：我们运用BD^TM Knockout Adenoviral RNAi System 1，在国内首次采用两种质粒（pSIREN和pAdeno-X）基础上的体外连接的方法构建腺病毒，沉默肿瘤细胞中的hTR基因表达，以观察其抑制肿瘤细胞端粒酶活性能力及对裸鼠移植瘤生长的影响，为siRNA腺病毒表达载体用于临床治疗肿瘤提供可靠的依据和有效的手段。研究方法：1 RNAi腺病毒的构建（1）穿梭质粒的构建利用BD siRNA靶序列分析设计系统，根据siRNA设计原则，经BLAST序列同源性分析后，选择针对hTR（U86046）的19nt的序列（136-154位碱基）作为靶序列，化学合成67nt寡核苷酸和它的互补链，退火、连接到BamH I／EcoRI线性化的pSIREN-Shuttle，构建重组穿梭质粒pSIREN-hTR。插入序列采用Pst I和测序的方法来证实。siRNA的阴性对照寡核苷酸被用来构建pSIREN-NT，构建方法同hTR。（2）腺病毒骨架质粒的构建重组的pSIREN-hTR经I-Ceu I和PI-SceI双酶切后，U6-RNA启动子和67bp插入片段被切出，用PCR纯化试剂盒纯化酶切产物，再与I-Ceu I／PI-Sce I线性化的pAdeno-X（32.6 kb）连接产生腺病毒的骨架质粒pAd-hTR。该质粒用PCR、酶切和测序的方法证实。（3）腺病毒的包装将pAd-hTR用Pac I酶切线性化，经PCR纯化试剂盒纯化，取4μg纯化的酶切产物包裹入10μl的脂质体（比率1：2.5）中，转染密度为90％左右的HEK293细胞，进行腺病毒Ad-hTR-siRNA的包装，并进一步扩增、纯化和滴度测定该病毒。阴性对照病毒Ad-NT-siRNA的构建策略同上。2 RNAi腺病毒沉默肿瘤细胞的hTR基因用浓度100MOI的RNAi腺病毒Ad-hTR-siRNA和对照病毒Ad-NT-siRNA感染几种肿瘤细胞系，终点分析法测定病毒对细胞的敏感性，TRAP-ELISA法测定细胞端粒酶活性、Real-time PCR测定hTR的mRNA水平、间接荧光免疫组化法测定人端粒酶反转录酶（hTERT）的蛋白变化和流式细胞术测定肿瘤细胞的凋亡率。3 RNAi腺病毒裸鼠体内抗肿瘤分析在每只裸鼠（BALB／C nu／nu）的右腋皮下注射无血清DMEM重悬的HeLa细胞0.2ml（4×10⁶／ml），构建裸鼠肿瘤模型。荷瘤成功后，将裸鼠随机分为4组，每只鼠分别瘤内多点注射0.1ml的DMEM、Ad-NT-sjRNA(10¹⁰pfu／ml)、Ad-hTR-siRNA(10¹⁰pfu／ml)或顺铂（1.20mg／ml）。以后每隔3天测量一次肿瘤体积，再注射一次对应的DMEM、病毒或顺铂，共注射6次。最后一次注射后间隔7天处死动物，剥离出肿瘤组织称重。切取瘤组织做HE染色观察肿瘤组织的形态学变化，并做TUNEL染色测定组织的凋亡指数。研究结果：1经酶切、测序和PCR鉴定证明，依次成功构建了靶向hTR的穿梭质粒pSIREN-hTR、腺病毒骨架质粒pAd-hTR和腺病毒Ad-hTR-siRNA。同时成功构建阴性对照腺病毒Ad-NT-siRNA。两病毒扩增至第4代时的滴度分别为1.0×10¹⁰pfu／ml和1.5×10¹⁰pfu／ml。2各细胞系对重组腺病毒的敏感程度依次为HeLa≥HepG2＞A549＞HL-7702。与阴性对照病毒Ad-NT-siRNA相比，感染了Ad-hTR-siRNA的肿瘤细胞的端粒酶活性明显降低，其中以HeLa细胞降低最明显，其端粒酶活性抑制率达到58.87％，hTR的mRNA表达下调70.21％，细胞凋亡率为29.7％，但hTERT的蛋白表达并不被阻断。HL-7702细胞的端粒酶活性基本不受影响。3将HeLa细胞移植到裸鼠皮下，成瘤率100％。与DMEM处理组和Ad-NT-siRNA处理组相比，用Ad-hTR-siRNA处理移植瘤动物模型，可以使肿瘤的生长受到明显抑制，肿瘤体积和重量都显著降低了45.48％和34.68％，HE染色显示大量的细胞可能经历了坏死、凋亡，TUNEL分析显示凋亡细胞量约11.8％；但是Ad-hTR-siRNA的抗肿瘤作用不及顺铂。结论：以上实验结果表明，Ad-hTR-siRNA可以高效、特异地抑制hTR基因表达，抑制宫颈癌细胞活性，体外和体内实验均显示其抗肿瘤活性。hTR-siRNA重组腺病毒在肿瘤基因治疗方面具有潜在的应用价值。迄今为止，应用体外连接方法构建RNAi腺病毒沉默端粒酶hTR基因尚未见类似报道。更多还原

【Abstract】 Background:Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of chromosomes. Telomerase has been found to be up-regulated in 85-90% of tumor cells, but only 4.2% in mormal somatic cells or benign tumor cells. Thus, telomerase is an attractive target for anti-tumor therapy. Six subunits comprising the telomerase complex have been identified. Of them, both human telomerase RNA （hTR） and human telomerase reverse transcriptase （hTERT） are very important. The template region of hTR encompasses 11 nucleotides （5’-CUAACCCUAAC-3’）, which is complementary to the human telomere sequence （TrAGGG）_n. The hTR serves as a template, and together with hTERT, synthesizes and adds new TTAGGG repeats to the ends of telomeres, thereby elongating it. The RNA component of telomerase is absolutely required for the function of telomerase complex and is therefore a natural target for anti-telomerase agents. M ny methods, such as short interfering RNA （siRNA）, hammerhead ribozymes and antisense oligonucleotides, have been designed to target hTR, especially the template region of the 11 nucleotides. Of these methods, siRNA has been shown to induce strong and efficient RNAi in mammalian cells. Thus it has been routinely used in gene silencing by transfection of synthesized 21-23 nucleotide siRNA or plasmid derived shRNA. Nevertheless, transient siRNA expression, low and variable transfection efficiency remained problems for chemically synthesized and vector derived siRNA. Some scientists prefer to use viral vectors to RNA interference. Recently, several retrovirus, lentivirus and adenovirus plasmid systems have been developed for efficient delivery of shRNA into mammalian cells, where the shRNA might be cut into siRNA by Dicer. These viral vectors have been designed to produce shRNA driven by either the U6 or the H1-RNA promoter for efficient, uniform delivery and immediate knockdown of target gene（s）. Now, the shRNA-encoding viruses have opened numerous novel opportunities for tumor, viral infection and nervous system deseases research across the field of bilolgy. Because adenoviruses have many advantages in terms of anti-tumor therapy, such as high efficiency, transient duration, targeting both proliferative and non-proliferative tumor cells, no risk of insertional mutagenesis, and liver tropism, they are popular for gene therapy trials, and extensive to be used to transfect cells with expressing hairpin-RNA. The two traditional methods of adenovirus construction are based on homologous recombination either in Escherichia coli BJ5183 or in human embryonic kidney 293 cells. But for difficult to manipulate or low efficient to recombinate, adenoviruses generation has poven difficult by using the two methods.Objective:In this study, we will utilize a commercially available adenovirus system （BDTM Knockout Adenoviral RNAi System 1） to construct an adenovirus vector against hTR template region by a ligation method in vitro, which depended on pSIREN and pAdeno-X. A series of experiments will be then conducted to evaluate the effect of the hTR-siRNA adenovirus on hTR mRNA gene silence, telomerase activity inhibition and anti-tumor in vitro and in vivo. All these results will offer reliable data and powerful way to tumor gene therapy. Furthermore, there are not similar reports about RNAi adenovirus which was constructed by ligation and used to silence hTR gene.Methods:1 Construction of RNAi adenovirus（1） construction the shuttle plasmidBased on BD siRNA designing system, principals and BLAST assay, the 67-bp oligonucleotides and complementary oligonucleotides encoding hTR-specific siRNA were synthesized. The target sequence corresponds to bases 136-154 of hTR （GenBank accession number U86046）. The oligonucleotides were annealed and ligated to the BamH I and EcoR I sites of RNAi-Ready pSIREN-Shuttle to produce pSIREN-hTR plasmid. The inserted sequences were confirmed by Pst I digestion and sequencing. The negative control siRNA annealed oligonucleotides were also introduced into pSIREN-NT as described above for hTR.（2） construction the skeleton plasmidThe U6-RNA promoter and the siRNA coding insert were cut from pSIREN-hTR with PI-Sce I and I-Ceu I, and ligated with pAdeno-X（32.6kb） digested by the same restriction enzymes. The products of ligation were transformed into E. coli （DH5a） to get the pAd-hTR. The pAd-hTR was confirmed by restriction enzyme mapping, PCR screening and sequencing.（3） package of adenovirusRecombinant adenoviral pAd-hTR was linearized with Pac I and purified using the PCR purified kit. HEK-293 cells with 90% density were used to pack this adenovirus and transfected with 4μg Pac I linearized pAd-hTR in the presence of LipofectamineTM 2000 in serum-free and non-antibiotic medium. Ad-hTR-siRNA was generated. Then this virus was harvested, concentrated, titrated, amplified and purified. Ad-NT-siRNA, which derived from pSIREN-NT as a negative virus control, was constructed and packed in a similar way.2 RNAi adenovirus silenced hTR gene in tumor cellsDifferent tumor cells and liver cell line, HL-7702, were infected with 100 MOl of the recombinant adenoviruses Ad-hTR-siRNA and Ad-NT-siRNA, respectly. End-Point Dilution Assay, TRAP-ELISA, Real-time PCR and FCM were used to analyze virus sensitivity, telomerase activity, hTR mRNA, apoptosis rate and hTERT protein expression.3 anti-tumor assay In vivo with RNAi adenovirusThis assay was performed using BALB/c nu/nu mice. HeLa cells were suspended as single cell suspension in serum-free DMEM shortly before subcutaneous inoculation into the right armpit. Each mouse received a 0.2 ml of 0.8×10⁶ HeLa cells. After successful implantation, the mice were randomized into four groups. The mice were intratumorally injected with 0.1ml of serum-free DMEM alone, or Ad-hTR-siRNA （10⁹ pfu/mouse）, or Ad-NT-siRNA （10⁹ pfu/mouse）, or 120μg of cisplatin as positive control. These different treatments were given once every 3 days for 15 consecutive days. After the last injection, mice were observed for 7 days continuously and sacrificed at the end. Tumors were excised, weighed, sectioned and assessed for morphology by HE and for apoptosis by the TUNEL assayResults:1 Restriction enzyme mapping, PCR screening and sequencing demonstrated that pSIREN-hTR, pAd-hTR and Ad-hTR-siRNA were constructed successfully. At the same time, the negative control virus Ad-NT-siRNA was obtained. The titers of the fourth passage viruses were 1.0×10¹⁰ pfu/ml and 1.5×10¹⁰pfu/ml, separately. 2 The arrangement of virus sensitivity in turn was HeLa≥HepG2＞A549＞HL-7702. As compared with Ad-NT-siRNA, Ad-hTR-siRNA reduced both hTR mRNA levels （70.21%） and telomerase activity （58.87%） of HeLa cells significantly, increased apoptosis rate （29.7%）. But the telomerase activity of HL-7702 and hTERT protein didn’t show the tendency of decrease.3 Tumors-implanted were established successfully by 100% in the right armpit with HeLa cells. As compared with DMEM- and Ad-NT-siRNA-treated mice, Ad-hTR-siRNA could slow down tumor growth, decrease tumor volume （45.48%） and tumor weight （34.68%） and push forward the apoptosis and necrosis of tumor cells. The TUNEL positive cells were about 11.8%. But the anti-tumor activity of Ad-hTR-siRNA didn’t catch on cisplatin’s.Conclusions:Taken together, our results demonstrated that adenovirus-delivered siRNA could efficiently and specifically knock-down hTR, decrease telomerase activity, and inhibit tumor cells growth in vitro and in vivo. All of these indicated the prospect of applying this siRNA expressing recombinant adenovirus system in cancer gene therapy.更多还原