中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
CHINESE JOURNAL OF PEDIATRIC SURGERY
2008年
6期
369-372
,共4页
王佚%郭振华%洪思琦%罗庆%金先庆
王佚%郭振華%洪思琦%囉慶%金先慶
왕일%곽진화%홍사기%라경%금선경
造影剂%微气泡%转染
造影劑%微氣泡%轉染
조영제%미기포%전염
Contrast media%Microbubble%Transfection
目的 研究体外超声微泡造影剂促腺病毒转染mdr1基因的效率.方法 扩增表达mdr1基因的重组腺病毒Ad5-mdr1并与微泡造影剂混合;密度梯度离心法分选兔骨髓单个核细胞,与腺病毒微泡造影剂混合,经超声辐照后常规培养;荧光显微镜下观察细胞内荧光强度表达;RT-PCR法检测骨髓单个核细胞基因组中外源性mdr1基因的表达;免疫组织化学法检测转染细胞中mdr1基因的表达产物.结果 ①转染后b(腺病毒转染组)、c(腺病毒转染+超声辐照组)、d(腺病毒微泡造影剂转染组)及e(腺病毒微泡造影剂转染+超声辐照组)组细胞内均有绿色荧光显示,a(常规培养组)组细胞内未见绿色荧光,e组细胞内荧光强度明显高于各对照组;②RT-PCR法显示,除a组外其余4组在157bp处分别观察到特异性mdr1电泳条带,证实外源性mdr1基因通过腺病毒作为载体整合到细胞基因组中;③免疫组织化学法检测e组细胞P-gp表达阳性率(24%)显著高于a、b、c、d(阳性率分别为0.5%、8.5%、10%、11.5%)各组(P<0.05).结论 体外实验证实,超声微泡造影剂能促进以腺病毒为载体的mdr1基因转染和表达.
目的 研究體外超聲微泡造影劑促腺病毒轉染mdr1基因的效率.方法 擴增錶達mdr1基因的重組腺病毒Ad5-mdr1併與微泡造影劑混閤;密度梯度離心法分選兔骨髓單箇覈細胞,與腺病毒微泡造影劑混閤,經超聲輻照後常規培養;熒光顯微鏡下觀察細胞內熒光彊度錶達;RT-PCR法檢測骨髓單箇覈細胞基因組中外源性mdr1基因的錶達;免疫組織化學法檢測轉染細胞中mdr1基因的錶達產物.結果 ①轉染後b(腺病毒轉染組)、c(腺病毒轉染+超聲輻照組)、d(腺病毒微泡造影劑轉染組)及e(腺病毒微泡造影劑轉染+超聲輻照組)組細胞內均有綠色熒光顯示,a(常規培養組)組細胞內未見綠色熒光,e組細胞內熒光彊度明顯高于各對照組;②RT-PCR法顯示,除a組外其餘4組在157bp處分彆觀察到特異性mdr1電泳條帶,證實外源性mdr1基因通過腺病毒作為載體整閤到細胞基因組中;③免疫組織化學法檢測e組細胞P-gp錶達暘性率(24%)顯著高于a、b、c、d(暘性率分彆為0.5%、8.5%、10%、11.5%)各組(P<0.05).結論 體外實驗證實,超聲微泡造影劑能促進以腺病毒為載體的mdr1基因轉染和錶達.
목적 연구체외초성미포조영제촉선병독전염mdr1기인적효솔.방법 확증표체mdr1기인적중조선병독Ad5-mdr1병여미포조영제혼합;밀도제도리심법분선토골수단개핵세포,여선병독미포조영제혼합,경초성복조후상규배양;형광현미경하관찰세포내형광강도표체;RT-PCR법검측골수단개핵세포기인조중외원성mdr1기인적표체;면역조직화학법검측전염세포중mdr1기인적표체산물.결과 ①전염후b(선병독전염조)、c(선병독전염+초성복조조)、d(선병독미포조영제전염조)급e(선병독미포조영제전염+초성복조조)조세포내균유록색형광현시,a(상규배양조)조세포내미견록색형광,e조세포내형광강도명현고우각대조조;②RT-PCR법현시,제a조외기여4조재157bp처분별관찰도특이성mdr1전영조대,증실외원성mdr1기인통과선병독작위재체정합도세포기인조중;③면역조직화학법검측e조세포P-gp표체양성솔(24%)현저고우a、b、c、d(양성솔분별위0.5%、8.5%、10%、11.5%)각조(P<0.05).결론 체외실험증실,초성미포조영제능촉진이선병독위재체적mdr1기인전염화표체.
Objective To research the transfection efficiency of mdr1 gene mediated by adenovirus enhanced by ultrasonic microbubbles in vitro. Methods Recombinant adenovirus Ad5-mdr1, which could express mdr1 gene, was cultured and mixed with ultrasonic microbubbles. Mononuclear cells in bone marrow which contained massive hematopoietic cells of the New Zealand rabbits were gathered and enriched by density gradient centrifugation, and mixed with ultrasonic microbubbles. The mixtures were cultured conventionally after ultrasound wave irradiation. The fluorescence intensity in cells was observed by fluorescence microscope, and the expression of the exogenous mdr1 gene in transferred bone marrow mononuclear cells was detected by RT-PCR, and the P-glycoprotein (expression product of mdr1 gene) in cells was detected by immunohistochemical staining. Results After the transfection, the green fluorescence had been observed in cells of adenovirus transfection group (group B), adenovirus transfection and ultrasonic irradiation group (group C), adenovirus transfection with ultrasonic microbubbles group (group D) and adenovirus transfection with ultrasonic microbubbles and ultrasonic irradiation (group E), while not in adenovirus transfection group (group A). Moreover, the fluorescence intensity in cells of group E was significantly higher than that of the other groups. The results of RT-PCR showed that the specific electrophoresis strips were observed at 157bp in 4 groups except group A, which showed that the mdr1 gene had been transferred into the genome of the cells mediated by adenovirus. The positive rate of P-gp expression in cells of group A, B, C, D and E was 0.5%, 8.5, 10%, 11.5% and 24% respectively, which were detected by immunohistochemical staining. The rate of expression of P-gp in group E was higher than that of the other groups (P<0. 05). ConclusionsThe results demonstrate that the efficiency of gene transfection and expression of mdr1 can be enhanced by adenovirus mediated ultrasonic microbubbles in vitro.
