本文選題：溶瘤腺病毒　切入點(diǎn)：等溫組裝　出處：《蘇州大學(xué)》2015年碩士論文

【摘要】：目的本實(shí)驗(yàn)通過(guò)等溫組裝法構(gòu)建重組溶瘤腺病毒RCAd C68.p IX-EGFP,并通過(guò)比較RCAd C68.p IX-EGFP與復(fù)制缺陷型重組腺病毒Ad C68-ΔE1-EGFP對(duì)結(jié)腸癌細(xì)胞的殺傷作用,初步觀察RCAd C68.p IX-EGFP對(duì)結(jié)腸癌細(xì)胞增殖的影響。方法以黑猩猩型腺病毒Ad C68為模板,利用等溫組裝法構(gòu)建腺病毒衣殼蛋白p IX展示EGFP的重組溶瘤腺病毒質(zhì)粒,HEK293細(xì)胞中包裝成完整病毒并擴(kuò)增、純化,獲得具有溶瘤效應(yīng)可表達(dá)EGFP的重組溶瘤腺病毒載體,命名為RCAd C68.p IX-EGFP。以不同的病毒劑量(100、500、2500、12500vp/cell)感染結(jié)腸癌細(xì)胞HCT 116、NCI-H508、HT-29及人胚腎細(xì)胞HEK-293,分別于感染后第3、5、7天用MTT法檢測(cè)RCAd C68.p IX-EGFP對(duì)不同細(xì)胞的抑制率,結(jié)晶紫染色實(shí)驗(yàn)檢測(cè)RCAd C68.p IX-EGFP對(duì)不同細(xì)胞的殺傷作用,熒光顯微鏡與相差顯微鏡觀察細(xì)胞形態(tài)學(xué)變化和病變情況。結(jié)果重組溶瘤腺病毒載體RCAd C68.p IX-EGFP構(gòu)建成功。RCAd C68.p IX-EGFP對(duì)結(jié)腸癌細(xì)胞HCT116、NCI-H508、HT-29的增殖抑制作用與病毒感染劑量及感染時(shí)間成正比,以500vp/cell病毒感染劑量感染細(xì)胞7天后,RCAd C68.p IX-EGFP對(duì)HCT 116、NCI-H508、HT-29、HEK293細(xì)胞的抑制率分別為86.70%、96.85%、99.70%、99.99%,Ad C68-ΔE1-EGFP的細(xì)胞抑制率分別為6.57%、11.46%、9.11%、99.99%,RCAd C68.p IX-EGFP與Ad C68-ΔE1-EGFP對(duì)結(jié)腸細(xì)胞增殖抑制作用的差異有統(tǒng)計(jì)學(xué)意義(P0.05),而對(duì)HEK-293細(xì)胞增殖抑制作用的差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論本研究成功構(gòu)建重組溶瘤腺病毒載體RCAd C68.p IX-EGFP,RCAd C68.p IX-EGFP能顯著抑制結(jié)腸癌細(xì)胞HCT 116、NCI-H508、HT-29的增殖,初步顯示出RCAd C68.p IX-EGFP作為溶瘤病毒載體治療腫瘤的潛能。
[Abstract]:Objective to construct recombinant adenovirus RCAd C68.p IX-EGFP by isothermal assembly, and to compare the cytotoxicity of RCAd C68.p IX-EGFP and replication-deficient recombinant adenovirus Ad C68- 螖 E1-EGFP on colon cancer cells. The effect of RCAd C68.p IX-EGFP on the proliferation of colon cancer cells was studied. Methods the chimpanzee adenovirus Ad C68 was used as a template. Adenovirus capsid protein (PIX) was constructed by isothermal assembly method. The recombinant adenovirus plasmid pIX, which displayed EGFP, was packaged into a complete virus, amplified and purified, and a recombinant adenovirus vector expressing EGFP was obtained. It was named RCAd C68.p IX-EGFP.Colon cancer cell line HCT 116nCI-H508 HT-29 and human embryonic kidney cell line HEK-293were infected with different doses of RCAd C68.p IX-EGFP.The inhibitory rates of RCAd C68.p IX-EGFP on different cells were detected by MTT method on the 3rd day after infection. The cytotoxicity of RCAd C68.p IX-EGFP to different cells was detected by crystal violet staining. Results the recombinant adenovirus vector RCAd C68.p IX-EGFP was successfully constructed to inhibit the proliferation of human colon cancer cell line HCT116NCI-H508H508HT-29. Results the inhibitory effect of recombinant adenovirus vector RCAd C68.p IX-EGFP on the proliferation of human colon cancer cell line HCT116NCI-H508h-29 was in direct proportion to the dose and time of infection. The inhibition rates of RCAd C68.p IX-EGFP on HCT 116NCI-H508h-29HEK293 cells were 86.70 and 96.96.858.70- 螖 E1-EGFP respectively after 7 days of 500vp/cell virus infection. The inhibitory rates of RCAd C68.p IX-EGFP and Ad C68- 螖 E1-EGFP on the proliferation of colon cells were significantly different from those of Ad C68- 螖 E1-EGFP, respectively (6.5711.469.115p), while the inhibitory rates of RCAd C68.p IX-EGFP and Ad C68- 螖 E1-EGFP on the proliferation of colon cells were significantly different from those of RCAd C68.p IX-EGFP and Ad C68- 螖 E1-EGFP. Conclusion the recombinant adenovirus vector RCAd C68.p IX-EGFPGFP RCAd C68.p IX-EGFP can significantly inhibit the proliferation of colon cancer cell line HCT 116NCI-H508H508h-29, and there is no significant difference in the inhibition of proliferation of HEK-293 cells by P0.055.Conclusion the recombinant adenovirus vector RCAd C68.p IX-EGFPFP-RCAd C68.p IX-EGFP can significantly inhibit the proliferation of colon cancer cells. The potential of RCAd C 68 p IX-EGFP to treat tumor as a vector of oncolytic virus was preliminarily demonstrated.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.3

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本文編號(hào)：1686206