人端粒酶逆转录酶启动子（hTERTpromoter）介导的ODC、SAMDC反义腺病毒的构建及其靶向抗肿瘤研究

发布时间：2018-04-16 11:06

本文选题：鸟氨酸脱羧酶 + S-甲硫氨酸脱羧酶　；参考：《山东大学》2009年博士论文

【摘要】：多胺(polyamine)广泛存在于生物体内的各组织细胞中,是重要的代谢调控物质。在肿瘤组织中多胺的含量明显升高。鸟氨酸脱羧酶(ODC)是多胺生物合成的第一个关键酶,在肿瘤的发生发展过程中起重要作用。1992年Auvinen等首次在Nature上发表论文,认为ODC活性增高能促进细胞转化。S-腺苷甲硫氨酸脱羧酶(SAMDC)是多胺合成的第二个限速酶,可以促进细胞的的恶性转化。文献报道SAMDC较ODC是更为有效的细胞转化的诱导因子。抑制多胺代谢途径成为治疗肿瘤的有效途径。相应的化学抑制剂如二氟甲基鸟氨酸(DFMO)、APA、CGP48664和AMA虽然在体内外的抑癌作用良好,但其严重的毒副作用极大的防碍了临床应用。寻找一种更高效更低副作用的抑制多胺合成途径的方法势在必行。如今,美国已启动600多个基因治疗的临床实验,其中60%是关于癌症治疗的。其中腺病毒介导的基因转染途径在癌症的基因治疗中特别引人关注,因为腺病毒的DNA不能整合到宿主基因组中,也不能感染卵细胞,具有很高的生物安全性。迄今为止,基因治疗的临床实例中近30%采用的是腺病毒载体。国内外研究证明反义核酸技术是比较理想的一种基因治疗方法,构建能转录反义RNA的重组载体在细胞内不断地转录出反义RNA,抵消酶解作用,发挥较持久的治疗效应。基因治疗在当今临床已经产生令人振奋的研究结果,但它的靶向安全性仍然是人们关注的焦点。许多肿瘤组织特异性启动子已被人类所认识,hTERT基因启动子更是备受关注。体外转基因实验中分别采用hTERT启动子与CMV启动子来转染不同的肿瘤细胞系,包括人肺癌细胞系(CH1299和A549),结肠癌细胞系(DLD1和LoVo),人宫颈癌细胞Hela及两种正常细胞系,结果显示:对于肿瘤细胞,hTERT启动子的转基因表达效率较CMV高2-3倍;而就正常细胞来说,CMV转基因的表达比hTERT高500多倍,这充分展示了hTERT启动子在肿瘤细胞中转基因表达的高效性与特异性。此后进行的体内直接转基因实验也证实了这一点。据此,本课题拟将ODC、SAMDC的反义基因分别构建入pAdEasy1系统,由人端粒酶逆转录酶启动子(hTERTp)启动表达,检测重组病毒对肿瘤细胞的特异抑制作用,为肿瘤疾病的靶向性基因治疗奠定基础。第一部分细胞端粒酶活性检测及外源hTERT启动子活性检测【研究目的】检测本实验中拟使用的五株细胞中端粒酶的活性,并检测端粒酶催化亚基启动子在肿瘤细胞系和正常细胞系中的启动活性,为下一步人端粒酶逆转录酶启动子启动的ODC、SAMDC反义病毒载体的构建奠定基础。【研究方法】 (1)体外稳定培养肿瘤细胞系(人肺癌细胞系A549、人肝癌细胞系Bel-7402、HepG2)及正常细胞系(人胚肺成纤维细胞HELF、人肝细胞LO2)。 (2)提取各株细胞端粒酶蛋白,利用TRAP法进行端粒酶活性的检测,聚丙烯酰胺凝胶电泳检测结果。即将10μlTRAP产物与含溴酚蓝的6×凝胶载样缓冲液2μl混合,在120V的电压下,进行12%非变性聚丙烯酰胺凝胶电泳,直至电流使溴酚蓝色带泳出凝胶为止。固定银染显色后,出现4条以上间隔6bp的梯度条带者为端粒酶活性阳性。 (3)将带有由人端粒酶逆转录酶启动子(hTERTp)启动的指示基因luc的重组质粒(pGL3-hTERT-luc,由美国肯塔基大学微生物教研室惠赠),转入各株培养细胞。 (4)通过双荧光素酶法检测指示基因luc的表达活性,从而反映hTERT启动子在肿瘤细胞和正常细胞中的启动活性。【实验结果】 (1)利用TRAP法进行端粒酶活性的检测,经聚丙烯酰胺凝胶电泳检测显示:对于肿瘤细胞系(A549、Bel-7402和HepG2),均具有大于4条以上的阳性带;而对于正常细胞系(HELF和LO2),均未见阳性条带出现。 (2)双荧光素酶法检测结果显示,在肿瘤细胞系中,hTERT启动子的比活性分别是:A549细胞为4.5±0.25;Bel-7402细胞为5.1±0.24;HepG2细胞为4.9±0.3。而在正常细胞中,hTERT启动子的比活性分别是:HELF细胞为0.16±0.02;LO2细胞为0.11±0.01。【结论】本实验中拟使用的三株肿瘤细胞(A549、Bel-7402和HepG2),其端粒酶活性为阳性;而对应的两株正常细胞(HELF和LO2),其端粒酶活性为阴性。荧光素酶活性检测显示:hTERT启动子在肿瘤细胞中具有较强启动活性而在正常细胞中未见有明显活性。第二部分hTERT启动子介导的人ODC、SAMDC反义腺病毒载体的构建【研究目的】分别构建由人端粒酶逆转录酶启动子hTERTp介导ODC和SAMDC反义RNA表达的腺病毒载体,为在肿瘤细胞中靶向性抑制多胺合成提供新工具和手段。【研究方法】 (1)应用PCR方法扩增出ODC mRNA翻译起始位点区120bp的基因片断,和SAMDC mRNA翻译起始位点区205bp的基因片断。PCR扩增产物和带有hTERT启动子的质粒pGL3-hTERT-luc均经XbaI、NcoI双酶切,回收质粒酶切长片段和PCR酶切产物片段。 (2)将ODC和SAMDC回收片段分别与pGL3-hTERT-luc酶切长片段(-pGL3-hTERT-)进行连接,分别构成了pGL3-hTERT-ODC和pGL3-hTERT-SAMDC重组质粒。 (3) KpnI和SalI双酶切pGL3-hTERT-ODC和pGL3-hTERT-SAMDC重组质粒,回收短片段(-hTERT-ODC-和-hTERT-SAMDC-)与经KpnI和SalI双酶切的腺病毒穿梭质粒pAdTrack长片段连接。分别构成pAdTrack-hTERT-ODC和pAdTrack-hTERT-SAMDC重组质粒。 (4)重组质粒经PmeI酶切线性化后,转入Adeasy-1细菌与pAdeasy-1质粒发生同源重组。挑选阳性重组质粒pAdeasy-hTERT-ODC和pAdeasy-hTERT-SAMDC,经PacI酶切后,转染293细胞包装和扩增出腺病毒颗粒。荧光显微镜和PCR的方法对重组腺病毒进行鉴定。【实验结果】 (1)采用PCR方法扩增出120bp、205bp的ODC、SAMDC基因片断,经电泳鉴定正确。 (2) pGL3-hTERT-ODC和pGL3-hTERT-SAMDC重组质粒经XbaI、NcoI双酶切分别得到120bp、205bp小片段和4.5kb大片段,经测序鉴定正确。 (3) pAdTrack-hTERT-ODC和pAdTrack-hTERT-SAMDC重组质粒经PCR扩增分别得到长120bp的ODC片断和长205bp的SAMDC片断,经测序鉴定此序列正确。 (4)重组质粒pAd-hTERT-ODC和pAd-hTERT-SAMDC分别经PacⅠ酶切得4.5kb和35kb两个片段。 (5)重组质粒pAd-hTERT-ODC和pAd-hTERT-SAMDC分别转染293细胞进行包装扩增,可见明显病毒空斑形成,荧光显微镜下可见绿色荧光蛋白在293细胞中表达。扩增后腺病毒滴度为2×10~9pfu/ml,PCR证实两个重组质粒基因组中分别含有目的基因ODC和SAMDC。【结论】成功构建了由人端粒酶逆转录酶启动子hTERTp介导ODC反义RNA表达的腺病毒载体rAd-hTERT-ODC和SAMDC反义RNA表达的腺病毒载体rAd-hTERT-SAMDC,为肿瘤疾病的靶向性基因治疗和预防提供了必要的侯备工具。第三部分两组反义腺病毒载体靶向性抑制肿瘤细胞增殖作用的研究【研究目的】研究两组重组腺病毒rAd-hTERT-ODC/SAMDC(rAd-hTERT-ODC和rAd-hTERT-SAMDC)对五株细胞(A549、Bel-7402、HepG2、HELF和LO2)中ODC、SAMDC基因表达的下调作用,并观察其对肿瘤细胞增殖以及侵袭能力的靶向抑制作用。【研究方法】 (1)将两组重组腺病毒载体分别转染入上述五株细胞,通过MTS法测定不同MOI的腺病毒对各株细胞的转染效率,以找到不同细胞的最适感染滴度。rAd-hTERT-ODC/SAMDC分别以最适感染滴度感染人肺癌细胞系A549、人肝癌细胞系Bel-7402、HepG2及正常细胞系人胚肺成纤维细胞HELF、人肝细胞LO2。 (2)采用Western-blot技术检测重组腺病毒感染细胞后,ODC和SAMDC蛋白表达情况。 (3)采用MTS实验检测重组腺病毒对各株细胞增殖的抑制作用。 (4)采用流式细胞术检测重组腺病毒对各细胞周期分布的影响。 (5)采用Matrigel侵袭实验分析重组腺病毒对肿瘤细胞侵袭活性的影响。【实验结果】 (1)腺病毒对A549细胞的最适感染滴度为50MOI,对其它细胞为25MOI。分别以该MOI的重组腺病毒感染A549、Bel-7402和HepG2肿瘤细胞可明显抑制其生长增殖,最大抑制率分别为65%、62%和55%;但对HELF和LO2正常细胞抑制生长作用不明显。 (2)腺病毒感染肿瘤细胞后,可明显抑制ODC和SAMDC基因表达。rAd-hTERT-ODC对A549、Bel-7402和HepG2肿瘤细胞中ODC的抑制分别达50%、60%、50%,rAd-hTERT-SAMDC对SAMDC的抑制可达40%、50%、40%。两种重组腺病毒对正常细胞内的ODC和SAMDC均未见有明显抑制作用。 (3)流式细胞DNA含量分析显示,两种重组腺病毒可引起肿瘤细胞周期阻滞于G_0-G_1期,但并未引起明显凋亡。对于正常细胞,rAd-hTERT-ODC和rAd-hTERT-SAMDC组与rAd-GFP组、PBS组未见明显改变。 (4) Matrigel侵袭实验显示,重组腺病毒可显著抑制体外肿瘤细胞的侵袭能力。【结论】两组重组腺病毒rAd-hTERT-ODC、rAd-hTERT-SAMDC分别能有效抑制肿瘤细胞中ODC和SAMDC基因的表达,使细胞周期阻滞于G_0-G_1期,抑制肿瘤细胞的增殖活性,并明显抑制肿瘤细胞的侵袭活力。而对于正常细胞却没有明显抑制作用。从而初步证明rAd-hTERT-ODC、rAd-hTERT-SAMDC通过靶向性的抑制肿瘤细胞中多胺的合成,目的性抑制肿瘤的生长;重组病毒对于正常细胞却是相对安全的。
[Abstract]:Polyamines (polyamine) exists widely in the organism in various tissues, metabolic regulation is important substances. In tumor tissue polyamine content was significantly increased. Ornithine decarboxylase (ODC) is the first key enzyme of polyamine biosynthesis, in the process of tumor development plays an important role in the.1992 Auvinen for the first time published in the Nature, that ODC activity can promote cell transformation of.S- S-adenosylmethionine decarboxylase (SAMDC) is the second polyamine synthesis rate limiting enzyme, can promote cell malignant transformation. SAMDC reported that ODC is more effective than the revulsive factor.
Inhibition of polyamine metabolism is an effective way to treat cancer. The corresponding chemical inhibitors such as two DFMO (DFMO), APA, CGP48664 and AMA as tumor suppressor function in vivo is good, but its serious side effects greatly hinder the clinical application.
It is imperative to find a more efficient method to lower the side effects of inhibition of polyamine biosynthesis. Now, the United States has launched more than 600 clinical trials of gene therapy, which is about 60% in the treatment of cancer. The gene transfected by adenovirus mediated gene therapy for cancer in particular concern, because adenovirus DNA can not be integrated into the host genome, can infect the eggs, has high biological security. So far, with nearly 30% clinical examples of gene therapy of adenovirus vector.
Studies at home and abroad have proved that antisense nucleic acid technology is an ideal gene therapy. A recombinant vector that transcripts antisense RNA can be constructed, and antisense RNA can be transcribed continuously in the cell to counteract the enzymolysis and exert a lasting therapeutic effect.
Gene therapy has produced encouraging results in clinical, but its targeted security is still the focus of attention. Many tumor tissue specific promoter has been recognized by humans, the hTERT gene promoter is of concern. In vitro transgenic experiments using hTERT promoter and CMV promoter in tumor cell lines to different transfection, including human lung cancer cell lines (CH1299 and A549), colon cancer cell lines (DLD1 and LoVo), human cervical carcinoma cell Hela and two normal cell lines, the results showed that the tumor cells, 2-3 times as high as hTERT promoter in transgenic expression efficiency than CMV and normal cells; the expression of CMV, transgenic hTERT higher than 500 times, which fully demonstrated the efficiency and specificity of hTERT promoter in tumor cells of transgenic expression. After in vivo direct transgenic experiments also confirmed this point.
Accordingly, the purpose of this study is to construct antisense genes of ODC and SAMDC into pAdEasy1 system, and to start the expression by human telomerase reverse transcriptase promoter (hTERTp), to detect the specific inhibitory effect of recombinant virus on tumor cells, and lay the foundation for targeted gene therapy of cancer.
Detection of telomerase activity in part one and detection of the activity of exogenous hTERT promoter
[purpose]
This experiment intends to use telomerase activity in five cell strains, and the detection of hTERT promoter in tumor cell lines and normal cell lines, the next step for the hTERT promoter ODC SAMDC constructed Antisense Vector to lay the foundation.
[research methods]
(1) stable tumor cell line (human lung cancer cell line A549, human hepatoma cell line Bel-7402, HepG2) and normal cell line (human embryonic lung fibroblast HELF, human hepatocyte LO2) were cultured in vitro.
(2) protein from each cell line was detected telomerase, telomerase activity was analyzed by TRAP method, the detection results of polyacrylamide gel electrophoresis. The 10 lTRAP product with bromophenol blue gel loading buffer 6 * 2 l mixed in the voltage of 120V, 12% non denaturing polyacrylamide gel electrophoresis, until the current bromophenol blue with a gel electrophoresis so far. Fixed silver staining, a gradient of more than 4 with the interval of 6BP were positive for telomerase activity.
(3) a recombinant plasmid containing Luc, a promoter of human telomerase reverse transcriptase promoter (hTERTp), was transferred to each culture cell by pGL3-hTERT-luc, donated by Microbiology Department of University of Kentucky.
(4) the expression activity of the indicator gene Luc was detected by the double luciferase method, thus reflecting the promoter activity of the hTERT promoter in the tumor cells and normal cells.
[experimental results]
(1) detection of telomerase activity by TRAP method. Polyacrylamide gel electrophoresis showed that for tumor cell lines (A549, Bel-7402 and HepG2), there were more than 4 positive bands, but no positive bands appeared in normal cell lines (HELF and LO2).
(2) the detection results of dual luciferase assay showed that in tumor cell lines, hTERT promoter activity were A549 cells was 4.5 + 0.25; Bel-7402 cells was 5.1 + 0.24; 4.9 + 0.3. and HepG2 cells in normal cells, hTERT promoter activity were HELF cells for 0.16 + 0.02 LO2 0.11 + 0.01. cells;
[Conclusion]
The three tumor cell lines used in this experiment (A549, Bel-7402 and HepG2), the telomerase activity was positive; and the two normal cell lines (HELF and LO2), the telomerase activity was negative. According to the detection of luciferase activity: the hTERT promoter has a strong promoter activity but not in normal cells significantly the activity in tumor cells.
Construction of a human ODC, SAMDC antisense adenovirus vector mediated by the second part of hTERT promoter
[purpose]
Adenovirus vectors mediated by human telomerase reverse transcriptase promoter hTERTp, which mediate the antisense RNA expression of ODC and SAMDC, were constructed, providing new tools and means for targeted inhibition of polyamine synthesis in tumor cells.
[research methods]
(1) the application of PCR amplified 120bp gene fragment ODC mRNA translation initiation site area, and 205bp SAMDC mRNA translation initiation site gene fragment.PCR amplified with plasmid pGL3-hTERT-luc and hTERT promoter were identified by XbaI, NcoI double enzyme digestion, enzyme digestion and plasmid recovery long fragment digested products of PCR fragments.
(2) the fragments of ODC and SAMDC were connected to pGL3-hTERT-luc fragment (-pGL3-hTERT-) respectively, which constituted pGL3-hTERT-ODC and pGL3-hTERT-SAMDC recombinant plasmids respectively.
(3) KpnI and SalI digested pGL3-hTERT-ODC and pGL3-hTERT-SAMDC recombinant plasmid, recovery of short fragments (-hTERT-ODC- and -hTERT-SAMDC-) and by KpnI and SalI double digested Adenovirus Shuttle Plasmid pAdTrack long segments. Respectively consisting of pAdTrack-hTERT-ODC and pAdTrack-hTERT-SAMDC plasmid.
(4) the recombinant plasmid was digested with PmeI, Adeasy-1 and pAdeasy-1 into the bacterial plasmid homologous recombination. Select positive recombinant plasmids pAdeasy-hTERT-ODC and pAdeasy-hTERT-SAMDC were digested with PacI and transfected into 293 cells for packaging and amplification of recombinant adenovirus particles. Fluorescence microscopy and PCR were used to identify the recombinant adenovirus.
[experimental results]
(1) PCR method was used to amplify 120bp, 205bp ODC, SAMDC gene fragment, and it was correctly identified by electrophoresis.
(2) the recombinant plasmids of pGL3-hTERT-ODC and pGL3-hTERT-SAMDC were obtained by XbaI and NcoI double enzyme digestion to obtain 120bp, 205bp fragment and 4.5kb fragment respectively, and were correctly identified by sequencing.
(3) pAdTrack-hTERT-ODC and pAdTrack-hTERT-SAMDC recombinant plasmids were amplified by PCR for long 120bp ODC fragments and long 205bp SAMDC fragments. The sequence was correctly identified by sequencing.
(4) the recombinant plasmids pAd-hTERT-ODC and pAd-hTERT-SAMDC respectively by Pac I and 35kb 4.5kb enzyme cut two fragments.
(5) the recombinant plasmid pAd-hTERT-ODC and pAd-hTERT-SAMDC were transfected into 293 cells to package the amplification of the virus plaque formation is visible, under a fluorescence microscope and the expression of green fluorescent protein in 293 cells. After amplification of adenovirus titer was 2 * 10~9pfu/ml, PCR, ODC and SAMDC. respectively showed that the target gene containing two recombinant plasmid genome
[Conclusion]
The adenovirus vector rAd-hTERT-SAMDC expressing adenovirus vector rAd-hTERT-ODC and SAMDC antisense RNA, which is mediated by human telomerase reverse transcriptase promoter hTERTp, has been successfully constructed. It provides a necessary tool for targeted gene therapy and prevention of tumor diseases. It is an adenovirus vector rAd-hTERT-SAMDC expressing rAd-hTERT-ODC and SAMDC antisense ODC.
The study of the third part of two groups of antisense adenovirus vectors targeting the proliferation of tumor cells
[purpose]
Two groups of recombinant adenovirus rAd-hTERT-ODC/SAMDC (rAd-hTERT-ODC and rAd-hTERT-SAMDC) were used to down regulate the expression of ODC and SAMDC genes in five cell lines (A549, Bel-7402, HepG2, HELF and LO2), and to observe their targeted inhibition on tumor cell proliferation and invasion.
[research methods]
(1) two groups of recombinant adenovirus vectors were transfected into the five cell lines, the transfection efficiency was measured by different MOI adenovirus on each cell strain by MTS method, to find the most suitable cell infection titer.RAd-hTERT-ODC/SAMDC respectively to the most appropriate infection titer infected human lung cancer cell line A549, human hepatocellular carcinoma cell line Bel-7402 human embryonic lung fibroblast HELF cell line HepG2 and normal human liver cells, LO2.
(2) Western-blot technique was used to detect the expression of ODC and SAMDC protein in the recombinant adenovirus infected cells.
(3) the inhibitory effect of recombinant adenovirus on cell proliferation was detected by MTS test.
(4) the effect of recombinant adenovirus on the cell cycle distribution was detected by flow cytometry.
(5) the effect of recombinant adenovirus on the invasive activity of tumor cells was analyzed by Matrigel invasion test.
[experimental results]
(1) adenovirus in A549 cells the infection titer was 50MOI, the other cells were 25MOI. with the recombinant adenovirus MOI infection of A549, Bel-7402 and HepG2 could significantly inhibit the growth of tumor cell proliferation, the maximum inhibition rate were 65%, 62% and 55%; but for HELF and LO2 normal cell inhibition the growth effect is not obvious.
(2) adenovirus infection of tumor cells, can inhibit ODC and SAMDC expression of.RAd-hTERT-ODC gene of A549, inhibition of Bel-7402 and HepG2 in tumor cells of ODC was 50%, 60%, 50%, rAd-hTERT-SAMDC inhibition of SAMDC was 40%, 50%, two 40%. recombinant adenovirus on normal cells in ODC and SAMDC there was no obvious inhibitory effect.
(3) flow cytometry analysis of DNA showed that two kinds of recombinant adenovirus could cause tumor cell cycle arrest in G_0-G_1 phase, but did not cause significant apoptosis. For normal cells, there was no significant change in PBS and rAd-hTERT-ODC group and rAd-hTERT-SAMDC group.
(4) the Matrigel invasion experiment showed that the recombinant adenovirus could significantly inhibit the invasion ability of the tumor cells in vitro.
[Conclusion]
Two groups of recombinant adenovirus rAd-hTERT-ODC rAd-hTERT-SAMDC can effectively inhibit the expression of ODC and SAMDC gene in tumor cells, the cell cycle arrest in G_0-G_1 phase, inhibit tumor cell proliferation activity, and inhibit the invasion of tumor cells and normal cells for energy. There is no obvious inhibitory effect. It showed that rAd-hTERT-ODC, rAd-hTERT-SAMDC through the target to inhibit the tumor cell of the polyamine synthesis and to inhibit tumor growth; recombinant virus for normal cells is relatively safe.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R346

【参考文献】