CAJ | 학술논문

目的探讨电穿孔技术介导的重组质粒体内转染兔下颌骨牵引成骨的可行性.方法以新西兰大白兔为实验动物模型,于术后3 d开始下颌骨牵引,每天0.8 mm,连续牵引7 d后,将实验动物分为3组:质粒+电穿孔组(A组),质粒组(B组),生理盐水组+电穿孔组(C组).各组动物分别于注射后3 h及1、3、7、14 d处死,切取牵引区组织0.4 cm×0.4 cm行冰冻切片检查,采用荧光显微镜观察绿色荧光蛋白(GFP)表达以检测外源基因的表达.检测兔血清肝功能指标丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)和肾功能指标尿素氮(BUN)、肌酐(Scr)和心、肝、肾组织学检查.结果 A组转染新西兰大白兔,3 h可观察到GFP的表达,1 d时GFP的表达增强,3 d时GFP的表达最强,其后开始逐渐下降,7 d后GFP的表达减少,14 d仍可观察到微弱GFP的表达.B组的GFP的表达时限与A组相同,但各时相点的GFP的表达强度明显弱于A组,C组在各时间段均未观察到GFP的表达.3组肝、肾功能指标两两比较差异无统计学意义(P>0.05).结论电穿孔技术介导的带有荧光标记的重组质粒体内转染,可在兔下颌骨牵引区组织内表达,电穿孔能明显提高重组质粒的体内转染效率,提示电穿孔技术介导的重组质粒体内转染兔下颌骨牵引成骨的动物模型是可行的,用于体内试验是安全的.
목적 탐토전천공기술개도적중조질립체내전염토하합골견인성골적가행성.방법 이신서란대백토위실험동물모형,우술후3 d개시하합골견인,매천0.8 mm,련속견인7 d후,장실험동물분위3조:질립+전천공조(A조),질립조(B조),생리염수조+전천공조(C조).각조동물분별우주사후3 h급1、3、7、14 d처사,절취견인구조직0.4 cm×0.4 cm행빙동절편검사,채용형광현미경관찰록색형광단백(GFP)표체이검측외원기인적표체.검측토혈청간공능지표병안산전안매(ALT)、천문동안산전안매(AST)화신공능지표뇨소담(BUN)、기항(Scr)화심、간、신조직학검사.결과 A조전염신서란대백토,3 h가관찰도GFP적표체,1 d시GFP적표체증강,3 d시GFP적표체최강,기후개시축점하강,7 d후GFP적표체감소,14 d잉가관찰도미약GFP적표체.B조적GFP적표체시한여A조상동,단각시상점적GFP적표체강도명현약우A조,C조재각시간단균미관찰도GFP적표체.3조간、신공능지표량량비교차이무통계학의의(P>0.05).결론 전천공기술개도적대유형광표기적중조질립체내전염,가재토하합골견인구조직내표체,전천공능명현제고중조질립적체내전염효솔,제시전천공기술개도적중조질립체내전염토하합골견인성골적동물모형시가행적,용우체내시험시안전적.
Objective To evaluate the feasibility of electroporation-mediated transfection of recombinant plasmid to mandibular distraction area of rabbit in vivo. Methods New-Zeland rabbit were employed. The mandible was distracted 3 days after operation at a rate of 0.8 mm per day for 7 days. The rabbits were randomly divided into 3 groups as group A (recombinant plasmid plRES-VEGF165-EGFP), group B(recombinant plasmid plRES-VEGF165-EGFP) and group C(normal saline). The rabbits were sacrified at 3 hours, 1, 3, 7 and 14 d after injection respectively. The tissue at the distraction area was taken out for frozen section. The gene expression was assessed by the detection of expression of green fluorescence protein (GFP) using fluorescence microscope. The liver and kidney function test(ALT, AST, BUN, Scr) and the histological examination of heart, liver and kidney were also performed. Results GFP was seen in the distraction area in group A and group B 3 hours after injection, which increased at the 1st day, reached peak value at the 3rd day, decreased at the 7th day and was very lower at 14th day. The GFP expression was much stronger in group A than in group B. GFP was not expressed in group C. There was no statistical difference in the concentration of ALT,AST,BUN and Set in serum of rabbits among the three groups. Conclusions Electroporation-mediated transfection of recombinant plasmid can be expressed in the distraction area of rabbits, and there was no toxity to the liver and kidney of rabbits. Electroporation could obviously improve transfection efficiency in vivo. It indicates that electroporation-mediated transfection of recombinant plasmid to distraction area tissue of rabbits is feasible.