超声造影剂促进野生型p53基因转染抑制大鼠卵巢癌生长的实验研究

目的 探讨超声辐照造影剂微泡促进野生型p53基因转染进而抑制大鼠卵巢癌生长的有效性。方法构建野生型p53真核质粒表达载体pcDNA3．1＋／p53，将40只卵巢癌荷瘤大鼠分成4组。第1组瘤体内注入造影剂微泡和野生型p53质粒混合物，并用超声辐照大鼠腹部瘤区；第2组注入裸质粒后予以超声辐照瘤体；第3组注射超声造影剂和基因混合物后无超声辐照；第4组仅注入裸质粒而无超声辐照。每组进行基因转染每周1次，共进行8次。2月后用逆转录-聚合酶链式反应（RT-PCR）检测野生型p53 mRNA在卵巢癌组织中的表达，Western Blot检测野生型p53基因蛋白表达。结果成功构建野生型p53真核质粒表达载体pcDNA3．1＋／p53，埋线法建立卵巢癌动物模型；用超声波辐照微泡促进基因转染后见p53基因转录及表达。第1组p53 mRNA的表达明显增强，高于其他3组，约是第2组的2．65倍，第4组的5．95倍。第2组高于未行超声辐照组，是第4组的2．23倍。第3、4组基因表达差异无统计学意义；Western Blot分析示p53基因蛋白表达的差异模式基本同mRNA，即第1组p53 mRNA的表达增强，高于其他3组，约是第2组的1．75倍，第4组的3．13倍；第2组高于未行超声辐照组，是第4组的1．79倍；第3、4组基因表达差异无统计学意义。结论瘤体内注射造影剂微泡和野生型p53质粒混合物后行超声辐照，可明显增加质粒基因在癌瘤组织中的导人及表达，超声联合造影剂微泡可作为卵巢癌基因治疗中促进基因转移的辅助方法。

Experimental gene therapy study on rat ovarian cancer with naked human wild type p53 plasmid delivered by echo contrast agent mediated by ultrasound

Objective To determine whether ultrasound mediated microbubble destruction could increase naked human wild type p53 plasmid delivery to ovarian cancer in rats by intratumoral injection. Methods Forty rats with ovarian cancer successfully induced by chemical intoxicant were divided into four groups.The wild type p53 gene was cloned into a high expressing efficiency eukaryotic plasmid pcDNA 3.1+ and the mixture of naked human wild type p53 plasmid was carefully injected directly into the center of ovarian neoplasms with or without echo contrast agent, and it was exposed to ultrasound for twenty minutes once a week.Two months after p53 gene transfection, semiquantitative RT-PCR was used to detect wild type p53 mRNA expression and western blot was to evaluate p53 protein expression level. Results Recombinated eukaryotic expression plasmid DNA encoding pcDNA 3.1+ /p53 was successfully constructed and confirmed by sequence analyzing and endonuclease cutting. The ovarian cancer model of rat was obtained by exposure to intoxicant. Expression of human wild type p53 mRNA was significantly higher in rats transfected with wild type p53 plasmid by means of ultrasound and contrast agent than others(P< 0.05 ),and it was about 5.95 fold increase compared with naked plasmid alone.Compared with the rats without ultrasound irradiation, transgene expression in rats with plasmid DNA using ultrasound was enhanced up to 2.23 fold(P< 0.05 ); and the pattern of p53 protein expression was observed the same as the RT-PCR results. Conclusions Transfection of naked plasmid DNA directly injected into tumors using ultrasound and echo contrast agent is an effective and safe technique for gene therapy compared with viral vector system, and it can be applied for ovarian cancer treatment as a gene delivering method.