CAJ | 학술논문

目的探讨超声造影剂声诺维(SonoVue)联合超声辐照介导载单纯疱疹病毒Ⅰ型胸苷激酶基因(hsv1-tk-gfp)质粒转染HXO-44视网膜母细胞的可行性.方法在24孔板中设置6组:空白对照组(只有细胞,第1组);细胞+质粒(第2组);细胞+质粒+超声辐照(第3组);细胞+质粒+超声辐照+声诺维(第4组)(辐照声强:0.5、1.0、1.5 W/cm2,辐照时间:40、60、80 s,造影剂浓度:10%、20%);细胞+质粒+超声辐照+声诺维+昔洛韦(第5组)(辐照方案同第4组,昔洛韦设置不同的浓度:1 mg/L、10 mg/L、100 mg/L、1 g/L);细胞+超声辐照+声诺维(第6组)(辐照方案同第4组);细胞数量为1×105个/孔,质粒用量为1μl每孔.转染48 h后流式细胞仪检测各组绿色荧光蛋白转染率,MTT法观察细胞存活情况.结果辐照声强为1.5 W/cm2、辐照时间为60 s、造影剂浓度为20%的条件下绿色荧光蛋白转染率最高.加入1 g/L的昔洛韦后,辐照声强为0.5、1.0、1.5 W/cm件下,HXO-44视网膜母细胞的存活率分别为(95.29±0.94)%、(92.58±1.29)%、(77.26±16.20)%,细胞存活率差异有统计学意义(F=11.402,P<0.05);辐照声强为1.5 W/cm2时细胞存活率较辐照声强为0.5、1.0 W/cm 2时下降,且差异均有统计学意义(t=12.776、10.856,P<0.01).结论超声造影剂联合一定强度的超声辐照可增强载hsv1-tk-gfp质粒转染HXO-44视网膜母细胞率;hsv1-tk-gfp质粒转染率超过10%的肿瘤细胞在加入前体药物昔洛韦(浓度高于1 g/L)可出现明显的凋亡.
목적탐토초성조영제성낙유(SonoVue)연합초성복조개도재단순포진병독Ⅰ형흉감격매기인(hsv1-tk-gfp)질립전염HXO-44시망막모세포적가행성.방법재24공판중설치6조:공백대조조(지유세포,제1조);세포+질립(제2조);세포+질립+초성복조(제3조);세포+질립+초성복조+성낙유(제4조)(복조성강:0.5、1.0、1.5 W/cm2,복조시간:40、60、80 s,조영제농도:10%、20%);세포+질립+초성복조+성낙유+석락위(제5조)(복조방안동제4조,석락위설치불동적농도:1 mg/L、10 mg/L、100 mg/L、1 g/L);세포+초성복조+성낙유(제6조)(복조방안동제4조);세포수량위1×105개/공,질립용량위1μl매공.전염48 h후류식세포의검측각조록색형광단백전염솔,MTT법관찰세포존활정황.결과복조성강위1.5 W/cm2、복조시간위60 s、조영제농도위20%적조건하록색형광단백전염솔최고.가입1 g/L적석락위후,복조성강위0.5、1.0、1.5 W/cm건하,HXO-44시망막모세포적존활솔분별위(95.29±0.94)%、(92.58±1.29)%、(77.26±16.20)%,세포존활솔차이유통계학의의(F=11.402,P<0.05);복조성강위1.5 W/cm2시세포존활솔교복조성강위0.5、1.0 W/cm 2시하강,차차이균유통계학의의(t=12.776、10.856,P<0.01).결론초성조영제연합일정강도적초성복조가증강재hsv1-tk-gfp질립전염HXO-44시망막모세포솔;hsv1-tk-gfp질립전염솔초과10%적종류세포재가입전체약물석락위(농도고우1 g/L)가출현명현적조망.
@@@@Objective To explore the feasibility of SonoVue combined with ultrasonic irradiation in mediating hsv1-tk-gfp plasmid DNA transfection into HXO-44 retinal matricyte.Methods Six groups were designed in this experiment:(1) only cells ( control group );(2) cells +plasmid;(3) cells +plasmid +ultrasonic irradiation;(4)cells+plasmid+ultrasonic irradiation +SonoVue[with different acoustic intensity (0.5 W/cm 2 ,1.0 W/cm 2 ,1.5 W/cm 2 ), radiating time (40 s,60 s,80 s) and micro bubble concentration (10%,20%)];(5)cells+plasmid+ultrasonic irradiation+SonoVue+drug(GCV)[with the same conditions in the fourth group in addition to different drug concentrations (1 mg/L,10 mg/L,100 mg/L,1 g/L)];cells+ultrasonic irradiation+SonoVue(with the same conditions in the fourth group ).There were 1 μl plasmid and 1 ×10 5 cells in each hole.The transfection efficiency was assessed after 48 h using flow cytometry.And the cell apoptosis was detected by MTT test .Results Under the conditions of 1.5 W/cm 2 acoustic intensity,60 s radiating time and 20% micro bubble concentration, the highest transfection rate was reached.Using 1 g/L GCV,the survival rate of HXO-44 retinal matricyte under the acoustic intensity of 0.5 W/cm2 ,1.0 W/cm2 and 1.5 W/cm2 were (95.29 ±0.94)%,(92.58 ±1.29)%,(77.26 ±16.20)%respectively with significant difference (F=11.402,P<0.05).Under 1.5 W/cm 2 irradiation,cells appeared obvious apoptosis ( t =12.776,10.856, P <0.01).Conclusions Sonovue combined with ultrasonic irradiation can enhance the transfection efficiency of hsv1-tk-gfp plasmid into HXO-44 retinal matricyte. Cells appear obvious apoptosis when the hsv1-tk-gfp plasmid transfection rate above 10% with GVC concentration higher than 1 g/L.