Ad-ING4-OSM联合放疗对喉癌抑瘤增效的体内外实验研究
发布时间：2018-10-13 20:29
【摘要】：目的：研究腺病毒载体介导的双基因共表达（Ad-ING4-OSM）联合放疗在体内外对喉癌抑瘤增效的协同作用及其分子机制。 方法：将基因重组腺病毒载体感染QBI-293A细胞，经多轮感染后扩增以备用；利用不同剂量的携带有绿色荧光蛋白（GFP）的空腺病毒载体（Ad-GFP）感染喉癌Hep-2细胞以确定最适感染剂量；Annexin-V-PE/7-AAD双染流式细胞术（FCM）检测放疗对喉癌Hep-2细胞的促凋亡作用以确定最适放疗剂量。在体外实验中，将Ad-ING4-OSM感染喉癌Hep-2细胞并联合放疗作用，运用RT-PCR及Westernblot检测ING4、OSM基因的转录及翻译；MTT法和Annexin-V-PE/7-AAD双染的FCM检测喉癌Hep-2细胞的生长抑制和凋亡效应；PI单染的FCM检测喉癌Hep-2细胞细胞周期的变化；Hoechst33258染色观察喉癌Hep-2细胞凋亡核形态学变化；半定量RT-PCR检测分析喉癌Hep-2细胞的细胞周期和细胞凋亡相关基因P21、P27、P53、Survivin的表达。在体内实验中，建立喉癌Hep-2细胞裸鼠移植瘤动物模型，分组处理，测量瘤体体积、重量并计算抑瘤率，评估Ad-ING4-OSM联合放疗对人喉癌Hep-2细胞裸鼠移植瘤的抑瘤增效作用；免疫组化检测细胞周期和凋亡相关蛋白及肿瘤血管形成相关因子P21、P53、Bax、Bcl-2、Caspase-3、Cox-2、CD34的表达从而分析可能的相关分子机制。 结果：感染QBI-293A细胞后，高效价的基因重组腺病毒载体被成功扩增；腺病毒载体感染喉癌Hep-2细胞的最佳剂量为100MOI（感染复数）；联合放疗选用的最佳放射剂量为6Gy；RT-PCR和Western blot检测证实腺病毒介导的外源性ING4和OSM基因可以在喉癌Hep-2细胞中稳定转录及翻译，并且在其它各组中均未发现内源性的ING4和OSM基因。在体外实验中，Ad-ING4-OSM联合放疗能明显抑制喉癌Hep-2细胞的生长、促进其凋亡，其作用显著优于单纯放疗和Ad-ING4-OSM（P0.05），具有抑瘤增效的协同作用，并且能更加显著地引起G1期和G2/M期阻滞、上调P21、P27、P53并下调survivin等细胞周期和细胞凋亡相关基因的表达。在体内实验中，Ad-ING4-OSM联合放疗能更加显著抑制喉癌Hep-2细胞裸鼠移植瘤的生长，促进其凋亡，其作用显著优于单纯放疗和Ad-ING4-OSM（P0.05），具有抑瘤增效的协同作用；免疫组化检测结果表明：Ad-ING4-OSM联合放疗能更显著上调喉癌Hep-2细胞裸鼠移植瘤内P21、P53、Bax、Caspase-3和下调Cox-2、Bcl-2等细胞周期和细胞凋亡相关蛋白的表达并下调肿瘤血管形成因子CD34的表达，，且Ad-ING4-OSM联合放疗作用显著优于单纯放疗和Ad-ING4-OSM，差异有统计学意义（P0.05）。 结论： 1、与单纯放疗和Ad-ING4-OSM相比，Ad-ING4-OSM联合放疗在体外能更加显著地抑制喉癌Hep-2细胞的生长，促进其凋亡，具有抑瘤增效的协同作用，其作用机制可能与引起G1期和G2/M期阻滞，上调P21、P27、P53并下调survivin等细胞周期和细胞凋亡相关基因表达的作用有关。 2、与单纯放疗和Ad-ING4-OSM相比，Ad-ING4-OSM联合放疗在体内能更加显著地抑制喉癌Hep-2细胞裸鼠移植瘤的生长，具有抑瘤增效的协同作用，其作用机制可能与上调喉癌Hep-2细胞裸鼠移植瘤内P21、P53、Bax、Caspase-3和下调Cox-2、Bcl-2等细胞周期和细胞凋亡相关蛋白的表达并下调肿瘤血管形成因子CD34的表达有关。
[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
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.65
本文编号：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
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.65
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本文编号：2269798
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