聚焦超声介导阳离子脂质体携带HSV-TK增强基因转染及联合更昔洛韦对大鼠胶质瘤C6细胞的影响

Effect of focused ultrasound on enhancing transfection of HSV-TK-loaded cationic liposomes and its influence of combined ganciclovir on glioma C6 cells in rats

目的探讨聚焦超声对载单纯疱疹病毒胸苷激酶(herpes simplex virus thymidine kinase,HSV-TK)基因阳离子脂质体体外基因转染的影响,及其联合更昔洛韦(ganciclovir,GCV)对大鼠脑胶质瘤C6细胞的影响。方法制备含HSV-TK基因质粒,利用阳离子脂质体转染至对数期大鼠胶质瘤C6细胞,并随机分为脂质体组和超声1、2、3、4min组,脂质体组加入含9μg阳离子脂质体-HSV-TK基因质粒的100μL培养液;超声1、2、3、4min组在脂质体组基础上分别给予体外超声(1 MHz,功率3W/cm2,电压150mvPP)辐照1、2、3、4min。5组C6细胞均于37℃、体积分数5%CO2培养箱,含体积分数10%胎牛血清的高糖DMEM完全培养液,继续培养48h。转染48h后,荧光显微镜下观察载HSV-TK基因阳离子脂质体转染效率。将对数生长期C6细胞接种于96孔板,依据转染效率实验结果分为GCV组、超声3min+GCV组,超声3 min+GCV组加入9μg阳离子脂质体-HSV-TK基因质粒的100μL培养液,并超声辐照3min,GCV组转染空白阳离子脂质体;转染24h后,2组均分别加入0.1、1、10、50、100、1 000mg/L GCV作用48h,采用分光光度法检测C6细胞存活率。结果转染48h后,超声1、2、3、4min组C6细胞HSV-TK基因转染效率[(0.14±0.07)%、(1.39±0.11)%、(19.61±4.80)%、(18.81±7.92)%]均高于脂质体组[(0.05±0.01)%](P<0.05),且超声3、4min组转染效率高于超声1、2min组,超声2 min组转染效率高于超声1 min组(P<0.05),超声3 min组与超声4min组转染效率比较差异无统计学意义(P>0.05);随GCV浓度增加,GCV组和超声3min+GCV组C6细胞存活率均逐渐降低;GCV为50、100、1 000mg/L时,超声3min+GCV组C6细胞存活率[(85.88±1.80)%、(75.51±1.43)%、(68.17±5.26)%]均低于GCV组[(95.40±0.85)%、(92.62±4.50)%、(83.66±0.98)%](P<0.05);GCV为0.1、1、10mg/L时,超声3min+GCV组C6细胞存活率[(99.32±3.01)%、(97.01±1.53)%、(94.50±1.15)%]与GCV组[(99.56±5.14)%、(98.60±5.18)%、(97.39±8.06)%]比较差异均无统计学意义(P>0.05)。结论聚焦超声体外辐照可明显提高载HSV-TK基因阳离子脂质体转染效率,增强GCV的抗胶质瘤作用。

Objective To explore the effect of focused ultrasound（FUS）on enhancing the gene transfection of herpes simplex virus thymidine kinase（HSV-TK）-loaded cationic liposomes in vitro,and the influence of ganciclovir（GCV）on glioma C6 cells.Methods Plasmids containing HSV-TK gene were prepared,and transfected into logarithmic phase C6 cells using cationic liposome as vectors.The C6 cells were randomly divided into cationic liposomes group and FUS 1-,2-,3-and 4-min groups.Cationic liposomes group was added 100μL medium containing 9μg HSV-TK-loaded cationic liposomes compound,and FUS 1,2,3 and 4-min groups received FUS irradiation（1 MHz,3 W/cm2,150 mvPP）for 1,2,3 and 4 min respectively in addition to the treatment in cationic liposomes group.C6 cells in 5 groups were cultured in37 ℃,5% CO2 incubator for 5 h,followed by in high-glucose DMEM complete medium containing 10% fetal bovine serum for 48 h.After transfection for 48 h,the transfection efficiency on HSV-TK gene-load cationic liposomes was observed under fluorescence microscope.The C6 cells in logarithmic phase were inoculated in 96-hole plate and divided into GCV group and FUS 3 min ＋ GCV group.FUS 3 min ＋ GCV group was added 100μL culture containing 9μg HSV-TK-loaded cationic liposomes compound and irradiated for 3 min,and GCV group was transfected with blank cationic liposomes.After transfection for 24 h,these two groups were added 0.1,1,10,50,100 and 1 000 mg/L GCV and the survival rates of C6 cells were detected by spectrophotometry after 48 h.Results After transfection for 48 h,the transfection efficiency of HSV-TK gene of C6 cells was significantly higher in FUS 1-,2-,3-and 4-min groups（（0.14±0.07）%,（1.39±0.11）%,（19.61±4.80%,（18.81±7.92）%）than that in cationic liposomes group（（0.05±0.01）%）（P〈0.05）,in FUS 3-and 4-min groups than that in FUS 1-and 2-min groups,and in FUS 2-min group than that in FUS 1-min group（P〈0.05）,and there was no significant difference between FUS 3-min group and 4-min group（P〈0.05）.With the increase of GCV concentration,the survival rate of C6 cells in GCV group and FUS 3 min＋ GCV group decreased gradually.When the concentration of GCV was 50,100 and 1 000 mg/L,the survival rates of C6 cells in FUS 3 min＋ GCV group（（85.88±1.80）%,（75.51±1.43）%,（68.17±5.26）%）were significantly lower than those in GCV group（（95.40±0.85）%,（92.62±4.50）%,（83.66±0.98）%）（P〈0.05）.When the GCV was 0.1,1 and10 mg/L,the survival rates of C6 cells in FUS 3 min ＋ GCV group（（99.32±3.01）%,（97.01±1.53）%,（94.50±1.15）%）showed no significant difference in comparison with those in GCV group（（99.56±5.14）%,（98.60±5.18）%,（97.39±8.06）%）（P〈0.05）.Conclusion FUS can significantly improve the transfection efficiency of HSV-TK-loaded cationic liposomes in vitro and enhance the anti-glioma effect of GCV.