目的:水泡口炎病毒(Vesicular Stomatitis Virus,VSV)基质蛋白(Matrix protein, M 蛋白)具有诱导肿瘤细胞凋亡的作用,本研究探讨水泡口炎病毒基质蛋白对癌性腹水形成的抑制和治疗作用。方法:采用旋转蒸发仪法制备纳米脂质体,检测其体外转染效率;采用脂质体转染技术将已构建的水泡口炎病毒基质蛋白(VSVM)重组真核表达质粒pcDNA31M 转入MethA肿瘤细胞,转染后6小时将细胞接种于小鼠腹腔,观察小鼠腹水的形成情况;腹水治疗组,则先将MethA肿瘤细胞接种于小鼠腹腔,将小鼠随机分成4组,于接种后第二天分别用脂质体包裹的pcDNA31M、pcDNA31空载体、单纯脂质体及生理盐水治疗,观察腹水的形成情况。结果:自制的DOTAP:DOPE脂质体与Sigma公司购买的Lipofectamine 2000的转染效率相似,pcDNA31M明显抑制MethA腹水的形成,对已经形成的腹水也有明显的治疗作用,与对照组比较有统计学意义(Plt;005),同时明显延长了小鼠的存活期。结论: VSVM蛋白真核表达质粒pcDNA31M对小鼠腹水的形成有抑制作用,能延长小鼠的存活期,对于恶性腹水的治疗具有一定的意义,值得进一步研究。
Objective To investigate the feasibility of intramuscular gene therapy for acute arterial ischemic diseases by use of plasmid pcDNA3-VEGF121 and to evaluate therapeutic efficiency of vascular endothelial growth factor(VEGF) by different routes of administration. Methods Fifty New Zealand White rabbits were randomly assigned to either gelation sponge carryingpcDNA3-VEGF121 (n=18), intramuscular injectionpcDNA3-VEGF121 (n=18), or pcDNA3 (as control group,n=14). After ligation of the external iliac artery and complete excision of the femoral artery, 500 μg of the plasmid pcDNA 3-VEGF121 were transfected into the muscles of the ischemic limb by gelation sponge carrying or direct intramuscular-injection. Immediately after gene transfection, blood flow of the internal iliac artery were measured. VEGF121gene expression was detected by RT-PCR after 2 days, 1 week, 2 weeks, 3 weeks and 4 weeks of transfection. After 30 days, blood flow of the internal iliac artery, angiographic score and histologicalvessels of ischemic hindlimbs were measured respectively. Results In the two VEGF-treated groups, VEGF121 mRNA expressed in the transfected ischemic muscles after 2 days and lasted 2 weeks. Immediately after gene transfection, blood flow of the internal iliac artery had no significant difference between three groups. After 30 days, blood flow of the internal iliac artery, angiographicscore and capillary density were significantly greater in both VEGF-treated groups than in control group. Complexity of vascular branching and vessel density of gelation sponge-VEGF treated limbs were significantly greater when comparedwith the intramuscular-injection limbs. Conclusion These findings suggest the feasibility of employing gene therapy of pcDNA3-VEGF121could augmentcollatal development and tissue perfusion in an animal model of hindlimb ischemia, andgelation sponge carrying VEGF gene may respect a potential therapy methods.
目的:探讨PLGA材料构建的纳米粒载体导入表皮生长因子受体(EGFR)反义寡核苷酸在头颈鳞癌基因治疗中的可行性,为头颈肿瘤基因治疗中载体的选择提供一个新的研究思路。方法:以PLGA为材料,采用油包水双乳化溶剂蒸发法制备载EGFR正义、反义寡核苷酸纳米颗粒;纳米颗粒转染SCCⅦ细胞株;MTT法了解纳米颗粒对细胞的毒性;通过实时荧光定量PCR检测转染后EGFR基因mRNA表达水平。结果:获得了制备载寡核苷酸PLGA纳米颗粒工艺流程,PLGA纳米颗粒平均粒径116nm±7.57nm。纳米颗粒体外转染SCCⅦ细胞,MTT结果显示纳米颗粒对细胞生长无明显抑制效应,同时具有明显抑制EGFR基因mRNA表达效应。结论:PLGA纳米颗粒可以有效地载入反义寡核苷酸,达到抑制靶基因的效果,同时无明显的细胞毒性。