【摘要】 目的：比较羧甲基化葡聚糖（carboxymethylated dextran，CMD）与聚乙烯亚胺（polyethyleneimine，PEI）纳米磁颗粒作为基因载体对可溶性血管内皮生长因子受体1（soluble fmslike tyrosine kinase1，sFIt1）胞外第23区sFIt1 (23)基因片段转染的差异。方法：通过磁颗粒载体将构建的真核表达质粒pcDNA 3.1/sFIt1(23)的基因片段转染入人脐静脉血管内皮细胞（HUVEC）中，检测各组的转染效率和对细胞增殖能力及凋亡的影响。实验分为CMD磁颗粒（CMDMAG）组、PEI磁颗粒（PEIMAG）组、单纯PEI组和未转染对照组，用第3～5代处于对数生长期的HUVEC为转染对象。在相同外界磁场条件下分别以N/P(聚合物中的氨基基团与DNA中磷酸基团的摩尔比)为10，15，20，25，30的PEI/DNA/CMDMAG复合物、PEI/DNA/PEIMAG复合物转染HUVEC，并以不加磁场下单纯PEI/DNA复合物转染的和未经转染的HUVEC作为对照。24h后倒置相差荧光显微镜、流式细胞仪检测以上述不同方法转染了增强型绿色荧光蛋白质粒（pcDNA3.1EGFP）的细胞绿色荧光表达百分率以反映目的基因片段sFIt1(23)的转染率；RTPCR法和Western blot法检测转染pcDNA3.1/sFIt1(23)后sFIt1(23) mRNA和蛋白的表达。通过MTT法观测各组细胞的生长情况，计算各组细胞的相对增殖百分率；通过Hoechst染色法观察各组细胞的凋亡百分率，以此对比不同磁颗粒载体转染后对细胞的影响。结果：在N/P为20时，各组GFP的转染率最高，且CMD磁颗粒组各N/P时转染率均高于其他组；转染pcDNA 3.1/sFIt1(23)后24h，CMDMAG组sFIt1(23)的mRNA和蛋白的表达均明显高于其他组；CMD磁颗粒转染组细胞增殖能力与对照组无明显差异，而PEI磁颗粒组、PEI组转染组细胞增殖能力均明显降低；CMD磁颗粒转染组约有5％细胞发生凋亡，PEI磁颗粒组凋亡细胞百分数约为20％～30％，PEI组约为50％。结论：CMD纳米磁颗粒可作为载体实现目的基因片段的高效率转染；在转染率和细胞毒性等方面CMD纳米磁颗粒均优于PEI纳米磁颗粒，可作为sFIt1基因片段转染和眼底新生血管性疾病基因治疗的载体。
【关键词】 眼底新生血管性疾病；可溶性血管内皮生长因子受体1；磁转染；羧甲基化葡聚糖纳米磁颗粒；聚乙烯亚胺纳米磁颗粒
Comparison of sFIt1 receptor gene transfection mediated by two different magnetic nanoparticles
Dan Jiang, Qiang Wu, BeiWen Song, XinHua Du, LiLi Jia
Foundation item: Dengshan Project Foundation of Shanghai(No.064119543)
Department of Ophthalmology, the Sixth Peoples Hospital, Shanghai Jiaotong University, Shanghai 200233, China
AbstractAIM: To compare the 2nd to 3rd transcellular region of sFIt1 receptor gene fragment transfection efficiency mediated by the carboxymethylated dextran (CMD) nanoparticle and polyethyleneimine(PEI) nanoparticle. And to provide a new approach for the gene therapy of neovascularization disease.METHODS: Plasmid pcDNA3.1/sFIt1(23) were constructed and assessed by gel electrophoresis and sequencing. The 3rd to 5th passage HUVECs were divided into four groups: CMDMAG group， PEIMAG group, PEI group and nontransfected control group. Transfections were carried out with PEI/DNA/CMDMAG compound, PEI/DNA/PEIMAG compound at different N/P ratio (10, 15, 20, 25, 30) respectively. HUVECs transfected with the PEI/DNA compound and HUVECs without transfection as control. The plamid pcDNA3.1EGFP which encoding the report gene GFP were also transferred by the ways described above to represent the sFIt1(23) transfection efficiency by fluorescence microscope and flow cytometer at 24 hours. mRNA and protein expressions of sFIt1(23) were detected by RTPCR and Western blotting. The proliferation of HUVEC by MTT was observed and the percentage of apoptosis HUVECs after transfection was calculates by stain Hoechst.RESULTS: The sFIt1(23) gene fragment was same with the sequences in the Gene bank. The peak of GFP transfection efficiency appeared at n=20 in each group and it was much higher in CMDMAG group than the others. Both the mRNA and protein expression of sFIt1(23) were the highest in CMDMAG group. The proliferation in CMDMAG group showed no disparity with control group, but it decreased obviously in PEI group and PEIMAG group. Only 5% cells in CMDMAG group apoptosis, while figures were 20%30% in PEIMAG group and 50% in PEI group.CONCLUSION: CMD nanoparticle surpasses PEI nanoparticle on the transfection efficiency and toxicity, it will be a promising gene vector to mediate the sFIt1(23) and for the gene therapy of neovascularization.
KEYWORDS: neovascularization disease; sFIt1; magnetofection; CMD nanoparticle; PEI nanoparticle
0引言
眼底新生血管的形成是影响糖尿病性视网膜病变、早产儿视网膜病变等多种视网膜疾病预后的重要病理改变，抑制新生血管的生长是治疗这类疾病的关键。除了利用传统的激光光凝和手术治疗之外，人们也做了大量针对血管内皮生长因子（vascular endothelial growth factor, VEGF）及其受体的基因治疗眼底新生血管形成的研究。基因载体是基因治疗的关键环节，非病毒基因载体以其低免疫原性、易于大规模生产和有效的靶向效应展现出极大的优势[1]。磁性纳米磁颗粒是一种新型的非病毒载体，可与聚乙烯亚胺（polyethyleneimine，PEI）结合后在外界磁场的作用下携带目的DNA到达靶细胞或器官，并通过在细胞或组织内释放DNA达到基因转染的目的。本实验采用磁转染方法，比较羧甲基化葡聚糖纳米磁颗粒（carboxymethylated dextrancoated magnetic nanoparticles，CMD）与PEI纳米磁颗粒作为基因载体将转染人可溶性血管内皮生长因子受体1（soluble fmslike tyrosine kinase1，sFIt1）2～3免疫球蛋白样区域转染到对人脐静脉血管内皮细胞（HUVEC）内。以单纯聚乙烯亚胺转染和未转染的HUVEC作为对照，通过对转染率、细胞毒性及凋亡率的检测来比较两种纳米磁颗粒转染能力的差别，为今后眼底新生血管性疾病的基因治疗转染方式的选择提供依据。
1材料和方法
1.1材料
大肠杆菌E.coli DH5α(本实验室保存)，人脐静脉血管内皮细胞株（HUVEC,南京凯基生物公司）；编码增强型绿色荧光蛋白真核表达质粒pcDNA3.1EGFP，pcDNA 3.1载体（Invitrogen公司）；BamH 1酶、Xho 1酶、Sal 1酶、Taq聚合酶、T4DNA连接酶（Promega公司）；质粒DNA抽提试剂盒、胶回收试剂盒、Trizol试剂（Invitrogen公司）；氯仿、异丙醇（上海试一化学试剂公司）；羧甲基化葡聚糖（carboxymethylated dextran，CMD）纳米磁颗粒（4.95×106 g/L）、PEI纳米磁颗粒（4.94×106 g/L）、阳离子聚合物聚乙烯亚胺（polyethyleneimine，PEI）、0.5特斯拉磁板（均由上海交通大学微纳米学院提供）；抗人sFIt1 mAb（CST公司）；二甲基亚砜(DMSO)、四甲基偶氮唑盐(MTT，均购自Aldrich－Sigm a公司)、细胞凋亡－Hoechst染色试剂盒（碧云天生物技术研究所）PCR仪及DNA水平电泳仪（BioRad公司）；凝胶成像系统（上海四星生物技术公司）；高速冷冻离心机（Beckman公司）。
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