Sonovue超声微泡介导DNCREB重组质粒转染人肾癌786—O细胞的实验研究(1)
[摘要] 目的 本研究旨在探讨Sonovue超声微泡介导DNCREB转染对人肾癌786-O细胞增殖、凋亡和周期的影响。方法 构建裸鼠肾癌荷瘤模型，将负显性突变体DNCREB重组质粒结合至Sonovue超声微泡，经尾静脉注射后，在体表瘤块位置给予1 MHz、声强1 W/cm2靶向超声辐照，诱导微泡破裂产生空化效应并释放DNCREB进入靶细胞，观察并记录肿瘤大小变化。使用MTT和流式细胞术分别观察细胞增殖、周期和凋亡情况的变化。 结果 待超声辐照处理后，相比于Vector组和Control组，DNCREB组细胞增殖能力明显受抑制、细胞周期发生S期阻滞、细胞凋亡率显著性增加，同时伴随着细胞周期蛋白cyclin A和凋亡相关蛋白的Bcl-2表达下调。此外，动物实验发现：与Vector组或Control组相比，DNCREB组的瘤块生长能力显著受抑制。 结论 Sonovue超声微泡介导DNCREB重组质粒转染有效地抑制了肾癌细胞的发生发展过程。
[关键词] 超声微泡；DNCREB；肾癌；增殖；周期；凋亡
[中图分类号] R737.1 [文献标识码] A [文章编号] 1673-9701(2018)21-0034-04
[Abstract] Objective To investigate the influence of transfection of DNCREB recombinant plasmid mediated by Sonovue ultrasonic microbubble on the proliferation， cycle and apoptosis of human renal cancer 786-O cell. Methods Tumor bearing model of renal cancer in nude mice was established. Recombinant plasmid of negative dominant mutant of DNCREB was combined with Sonovue ultrasonic microbubble. After caudal vein injection， targeted ultrasonic irradiation of 1MHz and sound intensity 1 W/cm2 was given in the tumor location of body surface to induce the microbubble destruction to generate the cavitation effect and release DNCREB into target cells. The size change of tumor was observed and recorded. Results After the ultrasonic irradiation， compared with Vector group and Control group， cell of DNCREB group showed significantly inhibited ability of proliferation， S arrest of cell cycle and significantly increasing apoptosis rate， along with down-regulated expression of cyclin A and apoptosis-related protein Bcl-2. Moreover， animal experiment showed： compared with Vector group and Control group， the growing ability of tumor in DNCREB group was significantly inhibited. Conclusion Transfection of DNCREB recombinant plasmid mediated by Sonovue ultrasonic microbubble inhibited the occurrence and development process of renal cancer cell effectively.
[Key words] Ultrasonic microbubble； DNCREB； Renal cancer； Proliferation； Cycle； Apoptosis
肾癌源于肾小管上皮细胞恶变，其发病率较高并以2%的速度逐年增长，然而其发病机制目前尚不清楚[1，2]。近年来，原癌基因的异常表达与肾癌之间的相关性研究已成为肿瘤病因学研究的热点。环磷酸腺苷反应元件结合蛋白(cAMP response element binding protein，CREB)作为调控细胞各种行为的转录因子，其Ser-133位点受磷酸化后，能与目的基因的CRE反应元件结合，从而激活靶基因的转录过程。已有文献报道，在多种肿瘤中磷酸化CREB(Phosphated CREB，pCREB)水平呈高表达状态[3，4]。我们的前期研究也发现，肾癌细胞/组织中pCREB水平显著性高于正常肾细胞/组织[5，6]。因此，我们欲通过转染负显性突变CREB(Dominant negative CREB，DNCREB)抑制肾癌细胞的发生发展。本研究旨在研究超声微泡介导DNCREB转染人肾癌786-O细胞后的变化，并深入探讨其机制。
1 材料与方法
1.1 材料来源
人肾癌细胞株786-O购自中国科学院细胞库。RMPI 1640、胎牛血清均购自美国Gbico生物公司；pCREB、CREB、GAPDH兔单克隆抗体及Bcl-2鼠单克隆抗体均购自Cell signaling technology公司；小鼠單克隆Cyclin A、CDK2抗体购自Santa Cruz Biotechnology公司；羊抗兔IgG二抗及羊抗鼠IgG二抗均购自武汉博士德生物公司；细胞周期及凋亡试剂盒均购自杭州联科生物技术股份有限公司。, http://www.100md.com(黄帅帅 王雪 姚许平 翁国斌 任雨)
[关键词] 超声微泡；DNCREB；肾癌；增殖；周期；凋亡
[中图分类号] R737.1 [文献标识码] A [文章编号] 1673-9701(2018)21-0034-04
[Abstract] Objective To investigate the influence of transfection of DNCREB recombinant plasmid mediated by Sonovue ultrasonic microbubble on the proliferation， cycle and apoptosis of human renal cancer 786-O cell. Methods Tumor bearing model of renal cancer in nude mice was established. Recombinant plasmid of negative dominant mutant of DNCREB was combined with Sonovue ultrasonic microbubble. After caudal vein injection， targeted ultrasonic irradiation of 1MHz and sound intensity 1 W/cm2 was given in the tumor location of body surface to induce the microbubble destruction to generate the cavitation effect and release DNCREB into target cells. The size change of tumor was observed and recorded. Results After the ultrasonic irradiation， compared with Vector group and Control group， cell of DNCREB group showed significantly inhibited ability of proliferation， S arrest of cell cycle and significantly increasing apoptosis rate， along with down-regulated expression of cyclin A and apoptosis-related protein Bcl-2. Moreover， animal experiment showed： compared with Vector group and Control group， the growing ability of tumor in DNCREB group was significantly inhibited. Conclusion Transfection of DNCREB recombinant plasmid mediated by Sonovue ultrasonic microbubble inhibited the occurrence and development process of renal cancer cell effectively.
[Key words] Ultrasonic microbubble； DNCREB； Renal cancer； Proliferation； Cycle； Apoptosis
肾癌源于肾小管上皮细胞恶变，其发病率较高并以2%的速度逐年增长，然而其发病机制目前尚不清楚[1，2]。近年来，原癌基因的异常表达与肾癌之间的相关性研究已成为肿瘤病因学研究的热点。环磷酸腺苷反应元件结合蛋白(cAMP response element binding protein，CREB)作为调控细胞各种行为的转录因子，其Ser-133位点受磷酸化后，能与目的基因的CRE反应元件结合，从而激活靶基因的转录过程。已有文献报道，在多种肿瘤中磷酸化CREB(Phosphated CREB，pCREB)水平呈高表达状态[3，4]。我们的前期研究也发现，肾癌细胞/组织中pCREB水平显著性高于正常肾细胞/组织[5，6]。因此，我们欲通过转染负显性突变CREB(Dominant negative CREB，DNCREB)抑制肾癌细胞的发生发展。本研究旨在研究超声微泡介导DNCREB转染人肾癌786-O细胞后的变化，并深入探讨其机制。
1 材料与方法
1.1 材料来源
人肾癌细胞株786-O购自中国科学院细胞库。RMPI 1640、胎牛血清均购自美国Gbico生物公司；pCREB、CREB、GAPDH兔单克隆抗体及Bcl-2鼠单克隆抗体均购自Cell signaling technology公司；小鼠單克隆Cyclin A、CDK2抗体购自Santa Cruz Biotechnology公司；羊抗兔IgG二抗及羊抗鼠IgG二抗均购自武汉博士德生物公司；细胞周期及凋亡试剂盒均购自杭州联科生物技术股份有限公司。, http://www.100md.com(黄帅帅 王雪 姚许平 翁国斌 任雨)