Ad-ING4-OSM聯(lián)合放療對喉癌抑瘤增效的體內(nèi)外實驗研究
發(fā)布時間：2018-10-13 20:29
【摘要】：目的：研究腺病毒載體介導(dǎo)的雙基因共表達（Ad-ING4-OSM）聯(lián)合放療在體內(nèi)外對喉癌抑瘤增效的協(xié)同作用及其分子機制。 方法：將基因重組腺病毒載體感染QBI-293A細胞，經(jīng)多輪感染后擴增以備用；利用不同劑量的攜帶有綠色熒光蛋白（GFP）的空腺病毒載體（Ad-GFP）感染喉癌Hep-2細胞以確定最適感染劑量；Annexin-V-PE/7-AAD雙染流式細胞術(shù)（FCM）檢測放療對喉癌Hep-2細胞的促凋亡作用以確定最適放療劑量。在體外實驗中，將Ad-ING4-OSM感染喉癌Hep-2細胞并聯(lián)合放療作用，運用RT-PCR及Westernblot檢測ING4、OSM基因的轉(zhuǎn)錄及翻譯；MTT法和Annexin-V-PE/7-AAD雙染的FCM檢測喉癌Hep-2細胞的生長抑制和凋亡效應(yīng)；PI單染的FCM檢測喉癌Hep-2細胞細胞周期的變化；Hoechst33258染色觀察喉癌Hep-2細胞凋亡核形態(tài)學(xué)變化；半定量RT-PCR檢測分析喉癌Hep-2細胞的細胞周期和細胞凋亡相關(guān)基因P21、P27、P53、Survivin的表達。在體內(nèi)實驗中，建立喉癌Hep-2細胞裸鼠移植瘤動物模型，分組處理，測量瘤體體積、重量并計算抑瘤率，評估Ad-ING4-OSM聯(lián)合放療對人喉癌Hep-2細胞裸鼠移植瘤的抑瘤增效作用；免疫組化檢測細胞周期和凋亡相關(guān)蛋白及腫瘤血管形成相關(guān)因子P21、P53、Bax、Bcl-2、Caspase-3、Cox-2、CD34的表達從而分析可能的相關(guān)分子機制。 結(jié)果：感染QBI-293A細胞后，高效價的基因重組腺病毒載體被成功擴增；腺病毒載體感染喉癌Hep-2細胞的最佳劑量為100MOI（感染復(fù)數(shù)）；聯(lián)合放療選用的最佳放射劑量為6Gy；RT-PCR和Western blot檢測證實腺病毒介導(dǎo)的外源性ING4和OSM基因可以在喉癌Hep-2細胞中穩(wěn)定轉(zhuǎn)錄及翻譯，并且在其它各組中均未發(fā)現(xiàn)內(nèi)源性的ING4和OSM基因。在體外實驗中，Ad-ING4-OSM聯(lián)合放療能明顯抑制喉癌Hep-2細胞的生長、促進其凋亡，其作用顯著優(yōu)于單純放療和Ad-ING4-OSM（P0.05），具有抑瘤增效的協(xié)同作用，并且能更加顯著地引起G1期和G2/M期阻滯、上調(diào)P21、P27、P53并下調(diào)survivin等細胞周期和細胞凋亡相關(guān)基因的表達。在體內(nèi)實驗中，Ad-ING4-OSM聯(lián)合放療能更加顯著抑制喉癌Hep-2細胞裸鼠移植瘤的生長，促進其凋亡，其作用顯著優(yōu)于單純放療和Ad-ING4-OSM（P0.05），具有抑瘤增效的協(xié)同作用；免疫組化檢測結(jié)果表明：Ad-ING4-OSM聯(lián)合放療能更顯著上調(diào)喉癌Hep-2細胞裸鼠移植瘤內(nèi)P21、P53、Bax、Caspase-3和下調(diào)Cox-2、Bcl-2等細胞周期和細胞凋亡相關(guān)蛋白的表達并下調(diào)腫瘤血管形成因子CD34的表達，，且Ad-ING4-OSM聯(lián)合放療作用顯著優(yōu)于單純放療和Ad-ING4-OSM，差異有統(tǒng)計學(xué)意義（P0.05）。 結(jié)論： 1、與單純放療和Ad-ING4-OSM相比，Ad-ING4-OSM聯(lián)合放療在體外能更加顯著地抑制喉癌Hep-2細胞的生長，促進其凋亡，具有抑瘤增效的協(xié)同作用，其作用機制可能與引起G1期和G2/M期阻滯，上調(diào)P21、P27、P53并下調(diào)survivin等細胞周期和細胞凋亡相關(guān)基因表達的作用有關(guān)。 2、與單純放療和Ad-ING4-OSM相比，Ad-ING4-OSM聯(lián)合放療在體內(nèi)能更加顯著地抑制喉癌Hep-2細胞裸鼠移植瘤的生長，具有抑瘤增效的協(xié)同作用，其作用機制可能與上調(diào)喉癌Hep-2細胞裸鼠移植瘤內(nèi)P21、P53、Bax、Caspase-3和下調(diào)Cox-2、Bcl-2等細胞周期和細胞凋亡相關(guān)蛋白的表達并下調(diào)腫瘤血管形成因子CD34的表達有關(guān)。
[Abstract]:Objective: To study the synergistic effect and molecular mechanism of adenovirus vector-mediated co-expression (Ad-ING4-OSM) combined with radiotherapy in laryngeal carcinoma. Methods: The recombinant adenovirus vector (QBI-293A) was infected with recombinant adenovirus vector (QBI-293A), and then amplified for use after infection. The Hep-2 cells were infected with Ad-GFP with different doses of green fluorescent protein (GFP) to determine the optimal infection. Dose; Annexin-V-PE/ 7-AAD Dual-Dye Flow Cytometry (FCM) for Detection of Apoptosis of Hep-2 Cells in Laryngeal Carcinoma by Flow Cytometry (FCM) to Determine Optimal Radiotherapy Dose. In vitro experiments, Ad-ING4-OSM was infected with Hep-2 cells and combined with radiotherapy. RT-PCR and Western blot were used to detect the transcription and translation of ING4 and OSM genes. MTT and Annexin-V-PE/ 7-AAD double-stained FCM were used to detect the growth and apoptosis effects of Hep-2 cells. Morphologic changes of Hep-2 cell apoptosis in laryngeal carcinoma were observed by Hoechst 33258 staining, and semi-quantitative RT-PCR assay was used to analyze the cell cycle and apoptosis-related genes P21, P27, P53 and Survivin of Hep-2 cells in laryngeal carcinoma. To investigate the effect of Ad-ING4-OSM combined radiotherapy on tumor suppressor tumor in nude mice of human laryngeal carcinoma Hep-2 cells. The expressions of P21, P53, Bax, Bcl-2, Caspase-3, Cox-2 and CD34 in cell cycle and apoptosis related proteins and tumor angiogenesis were detected by immunohistochemistry. Results: After QBI-293A cells infected with QBI-293A cells, the recombinant adenovirus vector with high titer was successfully amplified; the optimal dose of adenovirus vector infected with Hep-2 cells was 100MOI (complex number); the best radiation agent selected by combined radiotherapy RT-PCR and Western blot confirmed that adenovirus-mediated exogenous ING4 and OSM genes could be stably transcribed and translated in Hep-2 cells of laryngeal carcinoma, and endogenous ING4 and OSM genes were not found in other groups. In vitro experiments, Ad-ING4-OSM combined radiotherapy could significantly inhibit the growth of Hep-2 cells and promote its apoptosis. The effect of Ad-ING4-OSM was superior to that of radiotherapy alone and Ad-ING4-OSM (P0.05). 1, P27, P53 and down-regulation of cell cycle and apoptosis related to apoptosis In vivo experiments, Ad-ING4-OSM combined radiotherapy could significantly inhibit the growth of Hep-2 cell nude mice transplanted tumor and promote its apoptosis. The effect of Ad-ING4-OSM combined with radiotherapy was superior to that of radiotherapy alone and Ad-ING4-OSM (P0.05). The results showed that Ad-ING4-OSM combined radiotherapy could significantly regulate the expression of P21, P53, Bax, Caspase-3 and down-regulation Cox-2, Bcl-2 and other cell cycle and apoptosis-related proteins in Hep-2 cell nude mice, and down-regulate the expression of tumor angiogenesis factor (CD34), and the combination of Ad-ING4-OSM was superior to radiotherapy alone and Ad-ing. 4-OSM with statistical significance (P 0. 0 Conclusion: 1. Compared with simple radiotherapy and Ad-ING4-OSM, the combination of Ad-ING4-OSM can inhibit the growth of Hep-2 cells in vitro and promote its apoptosis. Up-regulation of P21, P27, P53 and Down-regulation of Cell Cycle and Apoptosis in Vitro Compared with simple radiotherapy and Ad-ING4-OSM, Ad-ING4-OSM combined radiotherapy could significantly inhibit the growth of Hep-2 nude mice xenografts in nude mice. The mechanism of action may be related to the up-regulation of P21, P53, Bax, Cas in nude mice transplanted with Hep-2 cells. pase-3 and down-regulate the expression of cell cycle and apoptosis-related proteins, such as Cox-2 and Bcl-2, and down-regulate tumor blood
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.65
本文編號：2269798
[Abstract]:Objective: To study the synergistic effect and molecular mechanism of adenovirus vector-mediated co-expression (Ad-ING4-OSM) combined with radiotherapy in laryngeal carcinoma. Methods: The recombinant adenovirus vector (QBI-293A) was infected with recombinant adenovirus vector (QBI-293A), and then amplified for use after infection. The Hep-2 cells were infected with Ad-GFP with different doses of green fluorescent protein (GFP) to determine the optimal infection. Dose; Annexin-V-PE/ 7-AAD Dual-Dye Flow Cytometry (FCM) for Detection of Apoptosis of Hep-2 Cells in Laryngeal Carcinoma by Flow Cytometry (FCM) to Determine Optimal Radiotherapy Dose. In vitro experiments, Ad-ING4-OSM was infected with Hep-2 cells and combined with radiotherapy. RT-PCR and Western blot were used to detect the transcription and translation of ING4 and OSM genes. MTT and Annexin-V-PE/ 7-AAD double-stained FCM were used to detect the growth and apoptosis effects of Hep-2 cells. Morphologic changes of Hep-2 cell apoptosis in laryngeal carcinoma were observed by Hoechst 33258 staining, and semi-quantitative RT-PCR assay was used to analyze the cell cycle and apoptosis-related genes P21, P27, P53 and Survivin of Hep-2 cells in laryngeal carcinoma. To investigate the effect of Ad-ING4-OSM combined radiotherapy on tumor suppressor tumor in nude mice of human laryngeal carcinoma Hep-2 cells. The expressions of P21, P53, Bax, Bcl-2, Caspase-3, Cox-2 and CD34 in cell cycle and apoptosis related proteins and tumor angiogenesis were detected by immunohistochemistry. Results: After QBI-293A cells infected with QBI-293A cells, the recombinant adenovirus vector with high titer was successfully amplified; the optimal dose of adenovirus vector infected with Hep-2 cells was 100MOI (complex number); the best radiation agent selected by combined radiotherapy RT-PCR and Western blot confirmed that adenovirus-mediated exogenous ING4 and OSM genes could be stably transcribed and translated in Hep-2 cells of laryngeal carcinoma, and endogenous ING4 and OSM genes were not found in other groups. In vitro experiments, Ad-ING4-OSM combined radiotherapy could significantly inhibit the growth of Hep-2 cells and promote its apoptosis. The effect of Ad-ING4-OSM was superior to that of radiotherapy alone and Ad-ING4-OSM (P0.05). 1, P27, P53 and down-regulation of cell cycle and apoptosis related to apoptosis In vivo experiments, Ad-ING4-OSM combined radiotherapy could significantly inhibit the growth of Hep-2 cell nude mice transplanted tumor and promote its apoptosis. The effect of Ad-ING4-OSM combined with radiotherapy was superior to that of radiotherapy alone and Ad-ING4-OSM (P0.05). The results showed that Ad-ING4-OSM combined radiotherapy could significantly regulate the expression of P21, P53, Bax, Caspase-3 and down-regulation Cox-2, Bcl-2 and other cell cycle and apoptosis-related proteins in Hep-2 cell nude mice, and down-regulate the expression of tumor angiogenesis factor (CD34), and the combination of Ad-ING4-OSM was superior to radiotherapy alone and Ad-ing. 4-OSM with statistical significance (P 0. 0 Conclusion: 1. Compared with simple radiotherapy and Ad-ING4-OSM, the combination of Ad-ING4-OSM can inhibit the growth of Hep-2 cells in vitro and promote its apoptosis. Up-regulation of P21, P27, P53 and Down-regulation of Cell Cycle and Apoptosis in Vitro Compared with simple radiotherapy and Ad-ING4-OSM, Ad-ING4-OSM combined radiotherapy could significantly inhibit the growth of Hep-2 nude mice xenografts in nude mice. The mechanism of action may be related to the up-regulation of P21, P53, Bax, Cas in nude mice transplanted with Hep-2 cells. pase-3 and down-regulate the expression of cell cycle and apoptosis-related proteins, such as Cox-2 and Bcl-2, and down-regulate tumor blood
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.65
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本文編號：2269798
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