超声靶向微泡破坏联合聚乙烯亚胺增强裸鼠移植瘤基因输送的初步研究

目的 探讨超声靶向微泡破坏(UTMD)联合聚乙烯亚胺(PEI)增强裸鼠Hela细胞(人宫颈癌)移植瘤基因输送的可行性和应用价值.方法 分别将2种质粒DNA红色荧光蛋白质粒(RFP)和荧光素酶质粒(pCMV-LUC)]与PEI以不同氮/磷酸盐比(N/P比)混合,利用凝胶阻滞实验对PEI/DNA复合物进行分析.经荷瘤裸鼠尾静脉分别注入PBS、质粒、质粒+Sono Vue微泡、PEI/DNA复合物+Sono Vue微泡,仅对一侧肿瘤行超声辐照(3 MHz、2 W/cm2、2 min、20%占空比),另一侧肿瘤作为对照,并对该转染方法 的靶向性进行分析.超声辐照3 d后处死动物,行RFP表达观察、荧光素酶活性检测和组织学检查.结果 琼脂糖凝胶电泳显示PEI可有效地缩合质粒DNA.与裸质粒组比较,UTMD(超声辐照+Sono Vue微泡)能明显提高RFP转染率.与非辐照对照侧比较,UTMD的运用使RFP表达明显增强,荧光素酶活性增加了14倍(P＜0.01).UTMD联合PEI可显著增强基因转染,受辐照移植瘤的荧光素酶活性增加了10倍(P＜0.01);与非联合PEI时比较,荧光素酶表达增加了111倍(P＜0.01).无论有否超声照射,裸鼠其他器官组织中均无明显的基因表达(P＞0.05),且未观察到明显的组织损伤.结论 UTMD联合PEI可显著增强报告基因在肿瘤组织的靶向传输和转染,是一种很有前景、新型而安全的体内基因输送方法.

The initial study of ultrasound-targeted microbubble destruction enhanced gene delivery in tumor xenografts accompanied with polyethylenimine

Objective To determine whether it could enhance gene delivery and tumor transfection in vivo by combination of ultrasound-targeted microbubble destruction(UTMD)with polyethylenimine(PEI)in tumor xenografts.Methods Two different reporter plasmidlueiferase(pCMV-LUC)and red fluorescent protein(RFP)]were incubated with PEI to prepare cationic compound(PEI/DNA)in various nitrogen:phosphate ratios(N/P ratios,nmol of nitrogen in the PEI/nmol of phosphate in DNA).Formation of PEI/DNA complexes were confirmed by the gel retardation assay.Human cervical carcinoma(Hela)tumors were planted subcutaneously in both flanks of female nude mice.Tumor-bearing mice were administered by tail vein with PBS,plasmid,plasmid and Sono Vue microbubble,PEI/DNA and Sono Vue microbubble.One tumor was exposed to ultrasound irradiation (3 MHz,2 W/cm2,2 min exposure,duty cycle 20%),while the other served as control.The feasibility of targeted delivery and tissue specificity facilitated by UTMD and PEI was investigated.The mice were sacrificed 3 days after ultrasound exposure.Tissue specimens were viewed with microscopy to determine the presence of RFP expression.The efficiencies of luciferase transgene expression were determined.Histology analysis was detected.Results Electrophoresis experiment revealed that PEI was mixed with plasmid to condense DNA efficiently.The application of UTMD significantly increases the tissue transfection in vivo compared to plasmid alone.RFP expression was present in all sections of tumors that received ultrasound exposure but not in control tumors.Results of luciferase activity showed that the expression of luciferase was to be 14 times greater in ultrasound-exposed tumors(P＜0.01).More importantly,the increase in transgene expresgion was related to UTMD with the presence of PEI dramatically.At least 10-fold increase of luciferase gene transfer was obtained in irradiated tumors compared to non-irradiated controls(P＜0.01),111-fold increase compared to UTMD alone(P＜0.01).There was not significantly gene expression in other organs or tissues regardless of US exposure(P＞0.05).No tissue damage was seen histologically.Conclusions The combination of UTMD with PEI can enhance targeted delivery and expression of reporter gene to tumors at intravenous administration.It is a promising new and safe method for gene delivery in vivo.