目的 探讨超声介导微泡空化效应对鼠肺微血管内皮细胞膜流动性及细胞骨架的影响.方法 6孔板培养鼠肺微血管内皮细胞,每孔加入20μg质粒EGFP及10%微泡后行超声辐照,辐照方式为连续波,探头频率2 MHz,分别以不同照射时间分组:A组30 s,B组60 s,C组90 8,D组120 s,E组180 s(机械指数均为1.0);对B组再以不同机械指数分组:B1组0.75,B2组1.0,133组1.3,134组1.5,135组1.8(照射时间均为60 s).以激光共聚焦显微镜观察细胞膜荧光恢复评价细胞膜流动性,以免疫荧光法观察细胞微管蛋白、微丝蛋白荧光强度变化.结果 胞膜流动性荧光恢复指数分别为:A组0.173±0.013,B组0.250±0.037,C组0.364±0.022,D组0.381±0.019,E组0.395±0.009(B～E组与A组比较,均P＜0.01);B1组0.171±0.017,B2组0.255±0.026.B3组0.378±0.007,B4组0.382±0.009、B5组0.397±0.008(B2～B5组与B1组比较,均P＜0.01).微管蛋白荧光强度分别为:A组159.15±4.79,B组188.23±6.20,C组205.80±4.48,D组208.99±8.34,E组213.70±5.09(B～E组与A组比较,均P＜0.01),B1组176.84±3.10,B2组187.57±14.52,B3组206.41±11.66,B4组220.12±13.39,B5组221.16±12.78(B2～B5组与B1组比较,均P＜0.01);C、D、E组间两两比较及B3、B4、B5组间两两比较差异均无统计学意义(P＞0.05).不同辐照时间及MI时,微丝蛋白荧光强度差异无统计学意义.结论 超声介导微泡空化在一定能量模式下可引起细胞膜流动性及微管蛋白荧光改变,此效应可能是超声及微泡促基因转染机制之一.
目的 探討超聲介導微泡空化效應對鼠肺微血管內皮細胞膜流動性及細胞骨架的影響.方法 6孔闆培養鼠肺微血管內皮細胞,每孔加入20μg質粒EGFP及10%微泡後行超聲輻照,輻照方式為連續波,探頭頻率2 MHz,分彆以不同照射時間分組:A組30 s,B組60 s,C組90 8,D組120 s,E組180 s(機械指數均為1.0);對B組再以不同機械指數分組:B1組0.75,B2組1.0,133組1.3,134組1.5,135組1.8(照射時間均為60 s).以激光共聚焦顯微鏡觀察細胞膜熒光恢複評價細胞膜流動性,以免疫熒光法觀察細胞微管蛋白、微絲蛋白熒光彊度變化.結果 胞膜流動性熒光恢複指數分彆為:A組0.173±0.013,B組0.250±0.037,C組0.364±0.022,D組0.381±0.019,E組0.395±0.009(B～E組與A組比較,均P＜0.01);B1組0.171±0.017,B2組0.255±0.026.B3組0.378±0.007,B4組0.382±0.009、B5組0.397±0.008(B2～B5組與B1組比較,均P＜0.01).微管蛋白熒光彊度分彆為:A組159.15±4.79,B組188.23±6.20,C組205.80±4.48,D組208.99±8.34,E組213.70±5.09(B～E組與A組比較,均P＜0.01),B1組176.84±3.10,B2組187.57±14.52,B3組206.41±11.66,B4組220.12±13.39,B5組221.16±12.78(B2～B5組與B1組比較,均P＜0.01);C、D、E組間兩兩比較及B3、B4、B5組間兩兩比較差異均無統計學意義(P＞0.05).不同輻照時間及MI時,微絲蛋白熒光彊度差異無統計學意義.結論 超聲介導微泡空化在一定能量模式下可引起細胞膜流動性及微管蛋白熒光改變,此效應可能是超聲及微泡促基因轉染機製之一.
목적 탐토초성개도미포공화효응대서폐미혈관내피세포막류동성급세포골가적영향.방법 6공판배양서폐미혈관내피세포,매공가입20μg질립EGFP급10%미포후행초성복조,복조방식위련속파,탐두빈솔2 MHz,분별이불동조사시간분조:A조30 s,B조60 s,C조90 8,D조120 s,E조180 s(궤계지수균위1.0);대B조재이불동궤계지수분조:B1조0.75,B2조1.0,133조1.3,134조1.5,135조1.8(조사시간균위60 s).이격광공취초현미경관찰세포막형광회복평개세포막류동성,이면역형광법관찰세포미관단백、미사단백형광강도변화.결과 포막류동성형광회복지수분별위:A조0.173±0.013,B조0.250±0.037,C조0.364±0.022,D조0.381±0.019,E조0.395±0.009(B～E조여A조비교,균P＜0.01);B1조0.171±0.017,B2조0.255±0.026.B3조0.378±0.007,B4조0.382±0.009、B5조0.397±0.008(B2～B5조여B1조비교,균P＜0.01).미관단백형광강도분별위:A조159.15±4.79,B조188.23±6.20,C조205.80±4.48,D조208.99±8.34,E조213.70±5.09(B～E조여A조비교,균P＜0.01),B1조176.84±3.10,B2조187.57±14.52,B3조206.41±11.66,B4조220.12±13.39,B5조221.16±12.78(B2～B5조여B1조비교,균P＜0.01);C、D、E조간량량비교급B3、B4、B5조간량량비교차이균무통계학의의(P＞0.05).불동복조시간급MI시,미사단백형광강도차이무통계학의의.결론 초성개도미포공화재일정능량모식하가인기세포막류동성급미관단백형광개변,차효응가능시초성급미포촉기인전염궤제지일.
Objective To explore the ultrasound mediated destruction and cavitation of targeted mierobubbles on rat's pulmonary endothelial cell membrane's fluidity and cytoskeletal structure changes.Methods The rat's endothelial cells were cultured in 6-well plates,and were transfected with the EGFP plasmid in the presence of protein microbubbles.During the transfection process,the cells underwent continuous 2 MHz ultrasonic irradiation for 30 to 180 s(group A:30 s:group B:60 s;group C:90 s;group D:120s;group E:180 s)at mechanical Index(MI)1.0,and with different MI from 0.75 to 1.8(group B1:0.75;group B2:1.0;group B3:1.3;group B4:1.5;group 135:1.8)at 60 s exposure time.The changes of endothelial cytoskeletal structure and membrane fluidity were evaluated by immunofluorescence staining after ultrasound exposure.Results The Recovery index of intensity of immunofluorescence,which expressed endothelial membrane fluidity,were 0.173±0.013 for group A,0.250±0.037 for group B,0.364±0.022 for group C,0.381±0.019 for group D,0.395±0.009 for group E(P＜0.01,compared with A),and 0.171±0.017 for group B1,0.255±0.026 for group B2,0.378±0.007 for group 133,0.382±0.009 for group B4,0.397±0.008 for group B5 respectively.The immunofluorescence staining intensity which expressed microtubulin of endothelial cytoskeletal structure was 159.15±4.79 for group A,188.23±6.20 for group B,205.80±4.48 for group C,208.99±8.34 for group D,213.70±5.09 for group E(P＜0.01,compared with group A),and 176.84±3.10 for group B1,187.57±14.52 for group B2,206.41 ±11.66 for group B3,220.12 ±13.39 for group 134,221.16±12.78 for group B5 respectively(P＜0.01,compared with B1),while there was no significant difference among groups of C,D,E and groups of B3,134,B5(P＞0.05).These data showed that the endothelial membrane fluidity and microtubule fluorescence recovery intensity increased remarkably compared with baseline(P＜0.01)within the MI(0.50 to 1.0)and exposure time(30 S to 90 s),however it showed no changes when increase MI to 1.5 and prolonging exposure time to 180 s respectively(P＞0.05).No changes was observed for microfilament fluorescence intensity whatever MI and irradiation time varied.Conclusions The ultrasound mediated destruction and cavitation of targeted microbubbles can increase endothelial membrane fluidity and cytoskeletal structure changes,especially of microtubulin,which contributed much to effective gene transfection of endothelium,but detail mechanism remained further explored.
