超声微泡介导增强型绿色荧光蛋白基因转染视网膜神经节细胞的体内实验

目的 验证超声微泡介导增强型绿色荧光蛋白(EGFP)基因能否在体内成功转染视网膜神经节细胞(RGCs),是否比传统的转染方式效率高以及这种转染方式是否损伤RGCs.方法 将SD大鼠50只随机分为4组:正常对照组(n=5)、单纯质粒组(n=15)、质粒+超声组(n=15)、超声微泡组(n=15).采用玻璃体腔注射试剂的方法,正常对照组注射5 μl生理盐水；单纯质粒组,注射5μl质粒；质粒+超声组,注射5μl质粒后,立即用0.5 W/cm2超声波辐照大鼠眼球60 s,工作时间控制为1/3(即辐照5s,停10 s,共60 s)；超声微泡组,注射质粒微泡混悬液5 μl后,立即用前述同等能量超声波辐照大鼠眼球.7d后,取大鼠眼球制作视网膜铺片、冰冻纵切片及视网膜EGFPmRNA的RT -PCR.用荧光显微镜观察铺片及切片中EGFP在RGCs的表达情况.用RGCs计数观察RGCs损伤情况[1-2].用RT-PCR对EGFPmRNA进行半定量检测.结果 超声微泡介导EGFP基因转染RGCs的效率明显高于正常对照组、单纯质粒组和质粒+超声组,且对RGCs无明显损伤.结论 在低频超声波的照射下,超声微泡能够在体内安全、有效地介导EGFP基因转染RGCs.

Experiment of ultrasound microbubble mediating gene EGFP transfecting retinal ganglial cells in vivo

Objective To investigate whether ultrasound microbubble could mediate gene EGFP transfecting retinal ganglial cells (RGCs) in vivo,whether this transfection way is more effective than the orthodox way and whether this way could cause damage of RGCs.Methods Fifty SD rats were randomly divided into 4 groups:the normal control group (n =5),plasmid group (n =15),plasmid + ultrasound group (n =15)and ultrasound microbubble group (n =15).The normal control group was injected 5 μL normal saline into vitreous cavity.The plasmid group was injected 5 μL plasmid.The plasmic + ultrasound group was injected 5 μL plasmid,then we exposed rats eyeballs to 0.5 W/cm2 ultrasonic wave immediately for 60 s( the exposure time accounting for 1/3:the exposure time is 5 s,then pause 10 s,total time 60 s.The ultrasound microbubble group was injected 5 μL suspension of plasmid and microvesicle,then we exposed rats eyeballs as the above way immediately.Seven days later,we made stretched preparation and longitudial frozen section of retina,and RT-PCR of EGFP mRNA of retina.Then fluorescence microscope was used to observe stretched preparation and EGFP expression in RGCs.We counted the number of RGCs to observe the damage situation.RGCs EGFP mRNA was detected through RT-PCR semi-quantitatively.Results The efficiency of ultrasound microbubble mediating gene EGFP transfecting RGCs was significantly higher than the normal control group,plasmid group and plasmic + ultrasound group,and this transfection way didn' t cause damage of RGCs.Conclusion By exposing eyeball to ultrasound of low frequency transduction,ultrasound can mediate EGFP gene transfecting RGCs safely and effectively.