本文選題：鳥氨酸脫羧酶 + S-甲硫氨酸脫羧酶�。� 參考：《山東大學(xué)》2009年博士論文

【摘要】：多胺(polyamine)廣泛存在于生物體內(nèi)的各組織細(xì)胞中,是重要的代謝調(diào)控物質(zhì)。在腫瘤組織中多胺的含量明顯升高。鳥氨酸脫羧酶(ODC)是多胺生物合成的第一個關(guān)鍵酶,在腫瘤的發(fā)生發(fā)展過程中起重要作用。1992年Auvinen等首次在Nature上發(fā)表論文,認(rèn)為ODC活性增高能促進細(xì)胞轉(zhuǎn)化。S-腺苷甲硫氨酸脫羧酶(SAMDC)是多胺合成的第二個限速酶,可以促進細(xì)胞的的惡性轉(zhuǎn)化。文獻(xiàn)報道SAMDC較ODC是更為有效的細(xì)胞轉(zhuǎn)化的誘導(dǎo)因子。 抑制多胺代謝途徑成為治療腫瘤的有效途徑。相應(yīng)的化學(xué)抑制劑如二氟甲基鳥氨酸(DFMO)、APA、CGP48664和AMA雖然在體內(nèi)外的抑癌作用良好,但其嚴(yán)重的毒副作用極大的防礙了臨床應(yīng)用。 尋找一種更高效更低副作用的抑制多胺合成途徑的方法勢在必行。如今,美國已啟動600多個基因治療的臨床實驗,其中60%是關(guān)于癌癥治療的。其中腺病毒介導(dǎo)的基因轉(zhuǎn)染途徑在癌癥的基因治療中特別引人關(guān)注,因為腺病毒的DNA不能整合到宿主基因組中,也不能感染卵細(xì)胞,具有很高的生物安全性。迄今為止,基因治療的臨床實例中近30%采用的是腺病毒載體。 國內(nèi)外研究證明反義核酸技術(shù)是比較理想的一種基因治療方法,構(gòu)建能轉(zhuǎn)錄反義RNA的重組載體在細(xì)胞內(nèi)不斷地轉(zhuǎn)錄出反義RNA,抵消酶解作用,發(fā)揮較持久的治療效應(yīng)。 基因治療在當(dāng)今臨床已經(jīng)產(chǎn)生令人振奮的研究結(jié)果,但它的靶向安全性仍然是人們關(guān)注的焦點。許多腫瘤組織特異性啟動子已被人類所認(rèn)識,hTERT基因啟動子更是備受關(guān)注。體外轉(zhuǎn)基因?qū)嶒炛蟹謩e采用hTERT啟動子與CMV啟動子來轉(zhuǎn)染不同的腫瘤細(xì)胞系,包括人肺癌細(xì)胞系(CH1299和A549),結(jié)腸癌細(xì)胞系(DLD1和LoVo),人宮頸癌細(xì)胞Hela及兩種正常細(xì)胞系,結(jié)果顯示:對于腫瘤細(xì)胞,hTERT啟動子的轉(zhuǎn)基因表達(dá)效率較CMV高2-3倍;而就正常細(xì)胞來說,CMV轉(zhuǎn)基因的表達(dá)比hTERT高500多倍,這充分展示了hTERT啟動子在腫瘤細(xì)胞中轉(zhuǎn)基因表達(dá)的高效性與特異性。此后進行的體內(nèi)直接轉(zhuǎn)基因?qū)嶒炓沧C實了這一點。 據(jù)此,本課題擬將ODC、SAMDC的反義基因分別構(gòu)建入pAdEasy1系統(tǒng),由人端粒酶逆轉(zhuǎn)錄酶啟動子(hTERTp)啟動表達(dá),檢測重組病毒對腫瘤細(xì)胞的特異抑制作用,為腫瘤疾病的靶向性基因治療奠定基礎(chǔ)。 第一部分細(xì)胞端粒酶活性檢測及外源hTERT啟動子活性檢測 【研究目的】 檢測本實驗中擬使用的五株細(xì)胞中端粒酶的活性,并檢測端粒酶催化亞基啟動子在腫瘤細(xì)胞系和正常細(xì)胞系中的啟動活性,為下一步人端粒酶逆轉(zhuǎn)錄酶啟動子啟動的ODC、SAMDC反義病毒載體的構(gòu)建奠定基礎(chǔ)。 【研究方法】 (1)體外穩(wěn)定培養(yǎng)腫瘤細(xì)胞系(人肺癌細(xì)胞系A(chǔ)549、人肝癌細(xì)胞系Bel-7402、HepG2)及正常細(xì)胞系(人胚肺成纖維細(xì)胞HELF、人肝細(xì)胞LO2)。 (2)提取各株細(xì)胞端粒酶蛋白,利用TRAP法進行端粒酶活性的檢測,聚丙烯酰胺凝膠電泳檢測結(jié)果。即將10μlTRAP產(chǎn)物與含溴酚藍(lán)的6×凝膠載樣緩沖液2μl混合,在120V的電壓下,進行12%非變性聚丙烯酰胺凝膠電泳,直至電流使溴酚藍(lán)色帶泳出凝膠為止。固定銀染顯色后,出現(xiàn)4條以上間隔6bp的梯度條帶者為端粒酶活性陽性。 (3)將帶有由人端粒酶逆轉(zhuǎn)錄酶啟動子(hTERTp)啟動的指示基因luc的重組質(zhì)粒(pGL3-hTERT-luc,由美國肯塔基大學(xué)微生物教研室惠贈),轉(zhuǎn)入各株培養(yǎng)細(xì)胞。 (4)通過雙熒光素酶法檢測指示基因luc的表達(dá)活性,從而反映hTERT啟動子在腫瘤細(xì)胞和正常細(xì)胞中的啟動活性。 【實驗結(jié)果】 (1)利用TRAP法進行端粒酶活性的檢測,經(jīng)聚丙烯酰胺凝膠電泳檢測顯示:對于腫瘤細(xì)胞系(A549、Bel-7402和HepG2),均具有大于4條以上的陽性帶;而對于正常細(xì)胞系(HELF和LO2),均未見陽性條帶出現(xiàn)。 (2)雙熒光素酶法檢測結(jié)果顯示,在腫瘤細(xì)胞系中,hTERT啟動子的比活性分別是:A549細(xì)胞為4.5±0.25;Bel-7402細(xì)胞為5.1±0.24;HepG2細(xì)胞為4.9±0.3。而在正常細(xì)胞中,hTERT啟動子的比活性分別是:HELF細(xì)胞為0.16±0.02;LO2細(xì)胞為0.11±0.01。 【結(jié)論】 本實驗中擬使用的三株腫瘤細(xì)胞(A549、Bel-7402和HepG2),其端粒酶活性為陽性;而對應(yīng)的兩株正常細(xì)胞(HELF和LO2),其端粒酶活性為陰性。熒光素酶活性檢測顯示:hTERT啟動子在腫瘤細(xì)胞中具有較強啟動活性而在正常細(xì)胞中未見有明顯活性。 第二部分hTERT啟動子介導(dǎo)的人ODC、SAMDC反義腺病毒載體的構(gòu)建 【研究目的】 分別構(gòu)建由人端粒酶逆轉(zhuǎn)錄酶啟動子hTERTp介導(dǎo)ODC和SAMDC反義RNA表達(dá)的腺病毒載體,為在腫瘤細(xì)胞中靶向性抑制多胺合成提供新工具和手段。 【研究方法】 (1)應(yīng)用PCR方法擴增出ODC mRNA翻譯起始位點區(qū)120bp的基因片斷,和SAMDC mRNA翻譯起始位點區(qū)205bp的基因片斷。PCR擴增產(chǎn)物和帶有hTERT啟動子的質(zhì)粒pGL3-hTERT-luc均經(jīng)XbaI、NcoI雙酶切,回收質(zhì)粒酶切長片段和PCR酶切產(chǎn)物片段。 (2)將ODC和SAMDC回收片段分別與pGL3-hTERT-luc酶切長片段(-pGL3-hTERT-)進行連接,分別構(gòu)成了pGL3-hTERT-ODC和pGL3-hTERT-SAMDC重組質(zhì)粒。 (3) KpnI和SalI雙酶切pGL3-hTERT-ODC和pGL3-hTERT-SAMDC重組質(zhì)粒,回收短片段(-hTERT-ODC-和-hTERT-SAMDC-)與經(jīng)KpnI和SalI雙酶切的腺病毒穿梭質(zhì)粒pAdTrack長片段連接。分別構(gòu)成pAdTrack-hTERT-ODC和pAdTrack-hTERT-SAMDC重組質(zhì)粒。 (4)重組質(zhì)粒經(jīng)PmeI酶切線性化后,轉(zhuǎn)入Adeasy-1細(xì)菌與pAdeasy-1質(zhì)粒發(fā)生同源重組。挑選陽性重組質(zhì)粒pAdeasy-hTERT-ODC和pAdeasy-hTERT-SAMDC,經(jīng)PacI酶切后,轉(zhuǎn)染293細(xì)胞包裝和擴增出腺病毒顆粒。熒光顯微鏡和PCR的方法對重組腺病毒進行鑒定。 【實驗結(jié)果】 (1)采用PCR方法擴增出120bp、205bp的ODC、SAMDC基因片斷,經(jīng)電泳鑒定正確。 (2) pGL3-hTERT-ODC和pGL3-hTERT-SAMDC重組質(zhì)粒經(jīng)XbaI、NcoI雙酶切分別得到120bp、205bp小片段和4.5kb大片段,經(jīng)測序鑒定正確。 (3) pAdTrack-hTERT-ODC和pAdTrack-hTERT-SAMDC重組質(zhì)粒經(jīng)PCR擴增分別得到長120bp的ODC片斷和長205bp的SAMDC片斷,經(jīng)測序鑒定此序列正確。 (4)重組質(zhì)粒pAd-hTERT-ODC和pAd-hTERT-SAMDC分別經(jīng)PacⅠ酶切得4.5kb和35kb兩個片段。 (5)重組質(zhì)粒pAd-hTERT-ODC和pAd-hTERT-SAMDC分別轉(zhuǎn)染293細(xì)胞進行包裝擴增,可見明顯病毒空斑形成,熒光顯微鏡下可見綠色熒光蛋白在293細(xì)胞中表達(dá)。擴增后腺病毒滴度為2×10~9pfu/ml,PCR證實兩個重組質(zhì)�；蚪M中分別含有目的基因ODC和SAMDC。 【結(jié)論】 成功構(gòu)建了由人端粒酶逆轉(zhuǎn)錄酶啟動子hTERTp介導(dǎo)ODC反義RNA表達(dá)的腺病毒載體rAd-hTERT-ODC和SAMDC反義RNA表達(dá)的腺病毒載體rAd-hTERT-SAMDC,為腫瘤疾病的靶向性基因治療和預(yù)防提供了必要的侯備工具。 第三部分兩組反義腺病毒載體靶向性抑制腫瘤細(xì)胞增殖作用的研究 【研究目的】 研究兩組重組腺病毒rAd-hTERT-ODC/SAMDC(rAd-hTERT-ODC和rAd-hTERT-SAMDC)對五株細(xì)胞(A549、Bel-7402、HepG2、HELF和LO2)中ODC、SAMDC基因表達(dá)的下調(diào)作用,并觀察其對腫瘤細(xì)胞增殖以及侵襲能力的靶向抑制作用。 【研究方法】 (1)將兩組重組腺病毒載體分別轉(zhuǎn)染入上述五株細(xì)胞,通過MTS法測定不同MOI的腺病毒對各株細(xì)胞的轉(zhuǎn)染效率,以找到不同細(xì)胞的最適感染滴度。rAd-hTERT-ODC/SAMDC分別以最適感染滴度感染人肺癌細(xì)胞系A(chǔ)549、人肝癌細(xì)胞系Bel-7402、HepG2及正常細(xì)胞系人胚肺成纖維細(xì)胞HELF、人肝細(xì)胞LO2。 (2)采用Western-blot技術(shù)檢測重組腺病毒感染細(xì)胞后,ODC和SAMDC蛋白表達(dá)情況。 (3)采用MTS實驗檢測重組腺病毒對各株細(xì)胞增殖的抑制作用。 (4)采用流式細(xì)胞術(shù)檢測重組腺病毒對各細(xì)胞周期分布的影響。 (5)采用Matrigel侵襲實驗分析重組腺病毒對腫瘤細(xì)胞侵襲活性的影響。 【實驗結(jié)果】 (1)腺病毒對A549細(xì)胞的最適感染滴度為50MOI,對其它細(xì)胞為25MOI。分別以該MOI的重組腺病毒感染A549、Bel-7402和HepG2腫瘤細(xì)胞可明顯抑制其生長增殖,最大抑制率分別為65%、62%和55%;但對HELF和LO2正常細(xì)胞抑制生長作用不明顯。 (2)腺病毒感染腫瘤細(xì)胞后,可明顯抑制ODC和SAMDC基因表達(dá)。rAd-hTERT-ODC對A549、Bel-7402和HepG2腫瘤細(xì)胞中ODC的抑制分別達(dá)50%、60%、50%,rAd-hTERT-SAMDC對SAMDC的抑制可達(dá)40%、50%、40%。兩種重組腺病毒對正常細(xì)胞內(nèi)的ODC和SAMDC均未見有明顯抑制作用。 (3)流式細(xì)胞DNA含量分析顯示,兩種重組腺病毒可引起腫瘤細(xì)胞周期阻滯于G_0-G_1期,但并未引起明顯凋亡。對于正常細(xì)胞,rAd-hTERT-ODC和rAd-hTERT-SAMDC組與rAd-GFP組、PBS組未見明顯改變。 (4) Matrigel侵襲實驗顯示,重組腺病毒可顯著抑制體外腫瘤細(xì)胞的侵襲能力。 【結(jié)論】 兩組重組腺病毒rAd-hTERT-ODC、rAd-hTERT-SAMDC分別能有效抑制腫瘤細(xì)胞中ODC和SAMDC基因的表達(dá),使細(xì)胞周期阻滯于G_0-G_1期,抑制腫瘤細(xì)胞的增殖活性,并明顯抑制腫瘤細(xì)胞的侵襲活力。而對于正常細(xì)胞卻沒有明顯抑制作用。從而初步證明rAd-hTERT-ODC、rAd-hTERT-SAMDC通過靶向性的抑制腫瘤細(xì)胞中多胺的合成,目的性抑制腫瘤的生長;重組病毒對于正常細(xì)胞卻是相對安全的。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R346

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本文編號：1758607