本文關(guān)鍵詞： RGD 腺病毒 LIF IL-24 白血病細(xì)胞 生長抑制　出處：《蘇州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:構(gòu)建RGD修飾的LIF和/或IL-24單、雙基因腺病毒表達(dá)載體,研究其對人白血病MEG01細(xì)胞及裸鼠移植瘤體內(nèi)外的抑制作用及分子機(jī)制。方法:將本實(shí)驗(yàn)室構(gòu)建的LIF和/或IL-24重組轉(zhuǎn)移質(zhì)粒與RGD修飾的腺病毒骨架質(zhì)粒pAd(RGD)同源重組,經(jīng)QBI-293A細(xì)胞包裝獲得RGD修飾的Ad.RGD-LIF、Ad.RGD-IL24和Ad.RGD-LIF-IL24重組腺病毒載體。在293A細(xì)胞中擴(kuò)增并檢測病毒的效價(jià)。感染白血病K562、MEG01細(xì)胞,熒光顯微鏡和流式細(xì)胞儀檢測經(jīng)RGD修飾后的腺病毒載體對白血病細(xì)胞的感染效率。體外實(shí)驗(yàn)分為Ad.RGD-LIF、Ad.RGD-IL24、Ad.RGD-LIF-IL24腺病毒載體組和PBS對照組、Ad.RGD空病毒載體組。各實(shí)驗(yàn)組感染MEG01細(xì)胞后,Western blot檢測目的基因LIF、IL-24的表達(dá),通過CCK-8法檢測LIF和/或IL-24基因表達(dá)對MEG01細(xì)胞增殖的影響,PE-AnexinV/7-AAD雙染后,經(jīng)流式細(xì)胞儀檢測攜帶LIF和/或IL-24基因的腺病毒對MEG01細(xì)胞凋亡的影響。PI染色,流式細(xì)胞儀檢測各實(shí)驗(yàn)組細(xì)胞周期的變化。Transwell法檢測LIF和/或IL-24基因表達(dá)對MEG01細(xì)胞侵襲能力的影響。Western blot檢測細(xì)胞凋亡相關(guān)蛋白Bcl-2、Bax、Bad、P53的表達(dá),Real-time PCR檢測細(xì)胞周期及侵襲相關(guān)基因P21、E2F1、MMP-2/9的表達(dá)。體內(nèi)實(shí)驗(yàn),建立人白血病MEG01細(xì)胞裸鼠移植瘤模型,實(shí)驗(yàn)組在瘤體內(nèi)注射腺病毒Ad.RGD-LIF、Ad.RGD-IL24、Ad.RGD-LIF-IL24進(jìn)行治療,對照組注射PBS和Ad.RGD空病毒,隔日一次,共注射5次。通過觀察并測量瘤體體積、重量,研究LIF和/或IL-24基因表達(dá)對裸鼠移植瘤的生長抑制作用。移植瘤取出后,石蠟包埋切片,免疫組化檢測相關(guān)因子Bcl-2、Bax、Caspase3、P53、E2F1的表達(dá)。結(jié)果:成功構(gòu)建RGD修飾的腺病毒載體Ad.RGD-LIF、Ad.RGD-IL24和Ad.RGD-LIF-IL24。經(jīng)RGD修飾后,對白血病細(xì)胞MEG01的感染效率從30%提升到90%,K562細(xì)胞的感染效率從15%提升到50%左右。Western blot證實(shí)了腺病毒載體攜帶LIF和/或IL-24基因能夠在MEG01細(xì)胞中成功表達(dá),體外實(shí)驗(yàn)結(jié)果表明:與PBS組和空病毒組相比,Ad.RGD-LIF、Ad.RGD-IL24和Ad.RGD-LIF-IL24實(shí)驗(yàn)處理組均能夠明顯抑制MEG01細(xì)胞的增殖和誘導(dǎo)細(xì)胞凋亡,且Ad.RGD-LIF-IL24雙基因共表達(dá)組對細(xì)胞抑制作用更顯著;周期結(jié)果顯示,各實(shí)驗(yàn)組均能產(chǎn)生G2/M期阻滯作用;侵襲實(shí)驗(yàn)結(jié)果表明LIF和/或IL-24單、雙基因表達(dá)均能夠抑制MEG01細(xì)胞的侵襲轉(zhuǎn)移,且雙基因共表達(dá)的抑制作用更顯著。體內(nèi)實(shí)驗(yàn),成功建立裸鼠人白血病MEG01細(xì)胞移植瘤模型,瘤體內(nèi)注射攜帶目的基因LIF和/或IL-24的腺病毒治療后,移植瘤的生長受到明顯的抑制,且雙基因共表達(dá)組抑瘤作用更顯著。分子機(jī)制結(jié)果表明:Ad.RGD-LIF-IL24雙基因共表達(dá)能夠明顯上調(diào)Bax、P53、Caspase3、P21和下調(diào)Bcl-xl、Bcl-2、E2F1、MMP-2/9等細(xì)胞凋亡、周期及侵襲相關(guān)基因的表達(dá),且其效應(yīng)較Ad.RGD-LIF或Ad.RGD-IL24單基因組更為顯著。結(jié)論:1、成功構(gòu)建RGD修飾的腺病毒載體Ad.RGD-LIF、Ad.RGD-IL24、Ad.RGD-LIF-IL24。RGD修飾后,能夠顯著提升腺病毒載體對MEG01和K562細(xì)胞的感染效率。2、腺病毒載體攜帶LIF和/或IL-24單/雙基因表達(dá)在體外、體內(nèi)均能夠顯著抑制白血病MEG01細(xì)胞的生長和誘導(dǎo)細(xì)胞發(fā)生凋亡,且Ad.RGD-LIF-IL24雙基因共表達(dá)組具有抑癌增效作用。3、腺病毒載體攜帶LIF和/或IL-24單/雙基因在MEG01細(xì)胞中表達(dá)均能明顯上調(diào)Bax、P53、P21和下調(diào)Bcl-2、E2F1等凋亡和周期相關(guān)因子的表達(dá),從而誘導(dǎo)細(xì)胞凋亡并產(chǎn)生G2/M期阻滯;下調(diào)MMP-2/9的表達(dá)從而抑制MEG01細(xì)胞的侵襲;且Ad.RGD-LIF-IL24雙基因共表達(dá)組對凋亡、周期相關(guān)基因的調(diào)控作用更顯著,這可能是雙基因共表達(dá)發(fā)揮抑瘤增效作用的重要分子機(jī)制。
[Abstract]:Objective: to construct the RGD modified LIF and / or IL-24 single and double gene adenovirus expression vector of human leukemia MEG01 cells and xenografts in vivo inhibitory effect and molecular mechanism. Methods: the laboratory construction of adenovirus LIF and / or IL-24 recombinant transfer plasmid and RGD modified plasmid pAd (RGD) by homologous recombination, QBI-293A packaging cell RGD modified Ad.RGD-LIF, Ad.RGD-IL24 and Ad.RGD-LIF-IL24. The titer of recombinant adenovirus vector in 293A cells to amplify and detect the virus infection. Leukemia K562, MEG01 cell, fluorescence microscopy and flow cytometry. The infection efficiency of adenovirus vector RGD modified on leukemia cells in vitro experiments were divided into Ad.RGD-LIF, Ad.RGD-IL24, Ad.RGD-LIF-IL24 adenovirus vector group and PBS control group, Ad.RGD empty vector group. The experimental group MEG01 cells infected with Western blot detection LIF gene, the expression of IL-24 expression on the proliferation of MEG01 cell by detecting gene LIF and / or IL-24 CCK-8 method, PE-AnexinV/7-AAD staining, flow cytometry analysis with LIF and / or IL-24 gene of adenovirus.PI on apoptosis in MEG01 staining,.Transwell method was used to detect LIF gene changes and / or flow cytometry was used to detect IL-24 of each experimental group. Cell cycle detection of apoptosis related protein Bcl-2.Western blot, effects on the invasion ability of MEG01 cells to Bax, Bad, P53 expression, Real-time PCR detection of cell cycle and invasion related genes P21, E2F1, MMP-2/9 expression. In vivo, the establishment of the nude mouse model of leukemia MEG01 cells in the experimental group, intratumoral injection of adenovirus Ad.RGD-LIF, Ad.RGD-IL24, Ad.RGD-LIF-IL24 treatment, control group were injected with PBS and Ad.RGD virus, once every other day, a total of 5 injections. Through observation and measurement The tumor volume, weight, LIF and / or IL-24 expression inhibits tumor growth in nude mice. Tumor removed, paraffin section, immunohistochemical detection of factor Bcl-2, Bax, Caspase3, P53, E2F1. Results: the expression of adenovirus vector Ad.RGD-LIF was successfully constructed by RGD modified Ad.RGD-IL24. And Ad.RGD-LIF-IL24. modified by RGD infection efficiency on leukemia MEG01 cells increased from 30% to 90%, the infection efficiency of K562 cells increased from 15% to 50%.Western blot confirmed the adenovirus vector carrying LIF and / or IL-24 gene can be successfully expressed in MEG01 cells in vitro, the experimental results show that compared with the PBS group, and the empty virus group Ad.RGD-LIF, Ad.RGD-IL24 and Ad.RGD-LIF-IL24 treatments could significantly inhibit the proliferation of MEG01 cells and induce cell apoptosis, and Ad.RGD-LIF-IL24 genes were more inhibitory effect on cells The cycle; results show that all the experimental groups can produce G2/M phase arrest; invasion experiments show that LIF and / or IL-24 single and double gene expression were able to inhibit MEG01 cell invasion and metastasis, the more significant inhibition and double gene co expression. In vivo, successfully established in nude mice of human leukemia MEG01 cells model, intratumoral injection of adenovirus carrying LIF gene and / or IL-24 after transplantation tumor growth was inhibited, and the double gene co expression inhibition effect was more significant. The results show that the molecular mechanism of co expression of Ad.RGD-LIF-IL24 gene increased the expression of Bax, P53, Caspase3, P21 and down-regulation of Bcl-xl. Bcl-2, E2F1, MMP-2/9, apoptosis, cycle and expression of invasion related genes, and its effect is Ad.RGD-LIF or Ad.RGD-IL24 single genome is more significant. Conclusion: 1, adenovirus vector Ad.RGD-LIF was successfully constructed by RGD modified Ad.R. GD-IL24, Ad.RGD-LIF-IL24.RGD modification can significantly improve the infection efficiency of adenovirus vector.2 of MEG01 and K562 cells, LIF and / or IL-24 single / double gene expression in vitro and in vivo adenovirus, can inhibit leukemia MEG01 cell growth and induce cell apoptosis, and Ad.RGD-LIF-IL24 double gene co expression group with tumor effect of synergism of.3 cancer, adenovirus vector carrying LIF and / or IL-24 single / double gene expression in MEG01 cells was significantly up-regulated the expression of Bax, P53, P21 and down-regulation of Bcl-2 expression of E2F1, apoptosis and cycle related factors, from induced apoptosis and G2/M phase arrest; downregulation of MMP-2/9 inhibits MEG01 cell invasion; and Ad.RGD-LIF-IL24 double gene co expression group more significant role on the apoptosis, cycle related genes, which may be the co expression of tumor suppressor molecules play an important synergistic effect Mechanism.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R733.7;R450

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