电穿孔介导的pIRES-hVEGF165-EGFP转染对牵引成骨过程中早期血管生成的影响

目的 探讨电穿孔介导的基因治疗对下领骨牵引成骨过程中早期血管生成的影响.方法 32只新西兰大白兔随机分为4组:质粒+电穿孔组(A组),质粒组(B组),生理盐水+电穿孔组(C组),空白对照组(D组).各组动物分别于注射后1、3、7、14 d处死,取牵引区组织进行组织学检查、电镜观察、CD34免疫组织化学染色及微血管密度检测.结果 A、B组血管内皮细胞呈增殖活跃状态;C、D组多数血管内皮细胞部分呈现退变及凋亡早期改变.免疫组化染色发现,转染后第1天血管壁内皮细胞浆CD34表达较弱;第3、7、14天,牵引区肉芽组织血管内皮细胞均出现CD34阳性表达.A组CD34阳性表达较B组强,A、B组的CD34表达持续阳性且呈上升趋势;C、D组表达最弱,CD34阳性表达维持在第1天水平上平稳波动.结论 电穿孔介导的pIRES-hVEGF165-EGFP重组质粒体内转染能够促进牵引区早期微血管的生成,使局部血管增生、渗入,增加骨断端的血流量.对调节和促进骨的生长和修复过程具有重要作用.     

电穿孔介导的pIRES-hVEGF165-EGFP转染对牵引成骨过程中早期血管生成的影响

目的 探讨电穿孔介导的基因治疗对下领骨牵引成骨过程中早期血管生成的影响.方法 32只新西兰大白兔随机分为4组:质粒+电穿孔组(A组),质粒组(B组),生理盐水+电穿孔组(C组),空白对照组(D组).各组动物分别于注射后1、3、7、14 d处死,取牵引区组织进行组织学检查、电镜观察、CD34免疫组织化学染色及微血管密度检测.结果 A、B组血管内皮细胞呈增殖活跃状态;C、D组多数血管内皮细胞部分呈现退变及凋亡早期改变.免疫组化染色发现,转染后第1天血管壁内皮细胞浆CD34表达较弱;第3、7、14天,牵引区肉芽组织血管内皮细胞均出现CD34阳性表达.A组CD34阳性表达较B组强,A、B组的CD34表达持续阳性且呈上升趋势;C、D组表达最弱,CD34阳性表达维持在第1天水平上平稳波动.结论 电穿孔介导的pIRES-hVEGF165-EGFP重组质粒体内转染能够促进牵引区早期微血管的生成,使局部血管增生、渗入,增加骨断端的血流量.对调节和促进骨的生长和修复过程具有重要作用.     

电穿孔介导的基因治疗兔下颌骨牵引成骨模型的建立

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.     

电穿孔介导的基因治疗兔下颌骨牵引成骨模型的建立

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.     

电穿孔介导的基因治疗兔下颌骨牵引成骨模型的建立

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.     

电穿孔介导的基因治疗兔下颌骨牵引成骨模型的建立

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.     

电穿孔介导的基因治疗兔下颌骨牵引成骨模型的建立

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.     

电穿孔介导的基因治疗兔下颌骨牵引成骨模型的建立

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.     

电穿孔介导的基因治疗兔下颌骨牵引成骨模型的建立

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.     

电穿孔介导的基因治疗兔下颌骨牵引成骨模型的建立

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.     

Electroporation enhances transfection efficiency in murine cutaneous wounds

Transfection of wounds with DNA-encoding growth factors has the potential to improve healing, but current means of nonviral gene delivery are inefficient. Repeated high doses of DNA, necessary to achieve reliable gene expression, are detrimental to healing. We assessed the ability of in vivo electroporation to enhance gene expression. Full-thickness cutaneous excisional wounds were created on the dorsum of female mice. A luciferase- encoding plasmid driven by a CMV promoter was injected at the wound border. Following plasmid administration, electroporative pulses were applied to injection sites. Pulse parameters were varied over a range of voltage, duration, and number. Animals were euthanized at intervals after transfection and the luciferase activity measured. Application of electric pulses consistently increased luciferase expression. The electroporative effect was most marked at a plasmid dose of 50 micro g, where an approximate tenfold increase was seen. Six 100- micro s-duration pulses of 1750 V/cm were found to be the most effective in increasing luciferase activity. High numbers of pulses tended to be less effective than smaller numbers. This optimal electroporation regimen had no detrimental effect on wound healing. We conclude that electroporation increases the efficiency of transgene expression and may have a role in gene therapy to enhance wound healing.     

Ectopic bone formation by electroporatic transfer of bone morphogenetic protein-4 gene

Orthopedic surgeons have long awaited the clinical application of bone morphogenetic proteins (BMPs) for bone regeneration. However, such possible applications involving proteins or genes transferred with virus vectors have encountered many problems, including high cost, immunological reactions, viral infection, etc. We adopted a new gene transfer system of in vivo electroporation with a plasmid expression vector. A solution of plasmid DNA containing mouse BMP-4 (pMiw-BMP4) was injected into the gastrocnemius of BALB/cA mice, and electric pulses were applied through paired-needle electrodes inserted percutaneously. As a control plasmid, LacZ-containing plasmid (pMiwZ) was transferred by electroporation. A control group in which pMiw-BMP4 was injected and not electroporated was also introduced. In these groups, the gastrocnemius was harvested at 7, 14, 21, and 28 days after electroporation (n = 6 in each). As nonplasmid controls, electroporation with saline injection (n = 6), electroporation without injection (n = 6), and saline injection only (n = 3) were prepared. In these groups, the mice were killed 7 days after experimentation. Ectopic calcification or ossification was examined by histology as well as soft X-ray. In all electroporated groups (pMiwZ, pMiw-BMP4, saline injection, and without injection), dystrophic calcification of muscle bundles and infiltration of mesenchymal cells were observed histologically. Ectopic bone formation was observed only in the pMiw-BMP4 electroporation group. At 7 days after pMiw-BMP4 electroporation, extracellular eosinophilic matrix in a collection of mesenchymal cells was observed. Between 14 and 28 days after electroporation, ectopic bone was observed in 44% of mice, and bone marrow-like cells observed in 22%. The newly formed bone was woven. Injection of pMiw-BMP4 or saline induced neither calcification nor ossification. Our findings indicate that BMP-4 transferred by electroporation can induce in vivo and in situ ectopic bone formation in skeletal muscle.     

Targeting of interstitial cells using a simple gene-transfer strategy.

BACKGROUND: Interstitial fibroblasts are central to the inflammatory response during the progression of tubulointerstitial fibrosis. We examined the efficiency of a new gene transfer method that targets interstitial cells by using parenchymal injection of DNA followed by electroporation. METHODS: Fluoresceinisothiocyanate-labelled oligodeoxynucleotides (FITC-ODNs) or expression vectors were directly injected into the cortex of the kidney, followed by electroporation. RESULTS: Transfection with FITC-ODNs or the EGFP expression vector resulted in efficient transfection in interstitial fibroblasts, but not in tubular epithelial cells or glomerular cells. Transfection efficiency was optimal after using a total of 150 microg of DNA in 1000 microl of PBS, combined with clamping of the renal vessels prior to electroporation. Gene expression peaked at 4 days after transfection and decreased by two orders of magnitude at 6 weeks post-transfection; however, expression recovered to near peak levels after parenchymal or intraperitoneal injection of FR901228, a histone deacetylase inhibitor. CONCLUSION: We demonstrated that direct parenchymal injection of DNA combined with electroporation enables gene transfer into interstitial fibroblasts.     

μ受体在抗神经生长因子抗体减轻大鼠骨癌痛中的作用

目的 评价μ受体在抗神经生长因子抗体(anti-NGF)减轻大鼠骨癌痛中的作用.方法 实验一健康雌性SD大鼠60只,体重200～220 g,随机分为4组(n=15):假手术组(S组)、假手术+anti-NGF组(SN组)、骨癌痛组(P组)和骨癌痛+anti-NGF组(PN组).P组和PN组于左侧胫骨上段骨髓腔内注射10μl Walker256乳腺癌细胞(1×105个)制备骨癌痛模型;S组和SN组于左侧胫骨上段注射PBS 10μl.于肿瘤细胞接种后13 d时,进行鞘内置管.鞘内置管成功后3 d,SN组和PN组鞘内注射anti-NGF 10μg(用生理盐水稀释至10μl),S组和P组鞘内注射生理盐水10μl,2次/d,连续5 d.于肿瘤细胞接种前、肿瘤细胞接种后13、16、18、21 d时测定自发缩足次数(NSF)、热缩足潜伏期(PWL)和机械性痛阈(PWT).肿瘤细胞接种后21 d时,处死大鼠,取L4.5段脊髓背角和背根神经节,测定μ受体及其mRNA的表达.实验二健康雌性SD大鼠30只,体重200～220 g,随机分为2组(n=15):骨癌痛+anti-NGF组(PN组)和骨癌痛+纳洛酮+anti-NGF组(PNN组).于左侧胫骨上段骨髓腔内注射10μlWalker256乳腺癌细胞(1×105个)制备骨癌痛模型.于肿瘤细胞接种后13 d时,进行鞘内置管.鞘内置管成功后3 d,PN组鞘内注射鞘内注入anti-NGF 10μg(生理盐水稀释至25μl);PNN组鞘内注射纳洛酮10μg(生理盐水稀释至25μl),0.5 h后,鞘内注射anti-NGF 10μg(生理盐水稀释至25 μl),2次/d,连续5 d.于肿瘤细胞接种前、肿瘤细胞接种后13、16、18、21 d时测定大鼠NSF、PWL和PWT.结果 实验一与S组比较,SN组NSF、PWL和PWT差异无统计学意义,SN组和PN组μ受体及其mRNA表达差异无统计学意义(P＞0.05),P组和PN组瘤细胞接种后13～21 d时NSF增加,PWL缩短,PWT降低,P组μ受体及其mRNA表达下调(P＜0.05或0.01);与P组比较,PN组肿瘤细胞接种后18～21 d时NSF减少,PWL延长,PWT升高,μ受体及其mRNA表达上调(P＜0.05或0.01).实验二与PN组比较,PNN组肿瘤细胞接种后18～21 d时NSF增加,PWL缩短,PWT降低(P＜0.05或0.01).结论 anti-NGF减轻大鼠骨癌痛与μ受体的激活有关.     

The use of newborn rats and an adenoviral gene delivery vector as a model system for wound-healing research

An attractive experimental method to elucidate the role of growth factors and cytokines in cutaneous wound healing would be to overexpress or "knock out" a molecule using a gene delivery vector and observe the impact on the wound repair process. As a first step toward developing an adenoviral gene delivery procedure to study wound repair, the authors injected beta-galactosidase (beta-gal) adenoviruses either subcutaneously or intradermally into the dorsal skin of 10-day-old postnatal Sprague-Dawley rats. Histological analysis and beta-gal staining were used to determine the expression and localization of the transferred gene. Beta-gal expression was observed as early as day 1 and up to day 7 postintradermal injection and day 9 postsubcutaneous injection, with no obvious inflammatory reaction detected at the injection sites. Furthermore, as expected, greater beta-gal expression was observed in the dermis of intradermally injected rats compared with the dermis of subcutaneously injected rats. Next, the authors sought to determine whether cutaneous wounds would heal before dissipation of the transferred gene. They created incisional and excisional wounds on the backs of similar-age rats. They found that incisional wounds closed by day 5 postwounding, whereas excisional wounds closed by day 14 postwounding. Their study demonstrated that an adenoviral vector delivered a gene efficiently into newborn rat skin and maintained the gene expression for at least as long as it would take for an incisional wound to heal. The combined use of newborn rat wound models and an adenoviral vector may provide a useful in vivo system to define the biological roles of growth factors and cytokines involved in the wound repair process. These discoveries may lead to the development of gene therapy approaches for abnormal wound healing.     

鞘内注射γ-氨基丁酸转运体抑制剂NO-711在骨癌痛大鼠脊髓水平印制磷酸化细胞外信号周节激酶1/2的上调

目的 探讨脊髓水平脊髓细胞外信号调节激酶1/2(extracellular regulated kinase 1/2,ERK1/2)活化在大鼠骨癌痛发生中的作用.方法 实验1:雌性SD大鼠48只,体重160 g～200 g,按随机数字表法分成2组(每组24只),A组(对照组)、B组(模型组).采用胫骨上段骨髓腔接种Wa...     

氯胺酮对新生大鼠海马CREB磷酸化水平的影响

目的 探讨氯胺酮对新生大鼠海马环磷腺苷反应元件结合蛋白(CREB)磷酸化水平的影响.方法 SD大鼠75只,日龄7 d,雌雄不拘,随机分为对照组(C组)、氯胺酮10 mg/kg组(K1组)和氯胺酮20 mg/kg组(K2组),每组25只.K1组和K2组分别皮下注射氯胺酮10、20 mg/kg,C组注射等容量生理盐水,每间隔90 min注射1次,共注射7次.于首次注射后24 h时断头取脑,分离海马,采用TUNEL法检测凋亡神经元,计算凋亡指数;免疫荧光双标法检测p-CREB表达水平;RT-PCR法半定量检测CREB下游基因BDNF mRNA及Bcl-2 mRNA表达水平;于首次注射后6周时采用Morris水迷宫实验测定各组大鼠认知功能.结果 与C组相比,K1组和K2组大鼠海马神经元凋亡指数升高,p-CREB、BDNF mRNA及Bcl-2 mRNA表达下调(P<0.05);与K1组相比,K2组大鼠海马神经元凋亡指数升高,p-CREB、BDNF mRNA及Bcl-2 mRNA表达下调(P<0.05);与C组和K1组相比,K2组逃避潜伏期延长(P<0.05).结论 氯胺酮10、20 mg/kg均可诱导发育期大鼠海马神经元凋亡,而氯胺酮20 mg/kg可导致大鼠发育成熟后认知功能降低,可能与其抑制CREB磷酸化后BDNF及Bcl-2表达下调,导致神经元凋亡,影响大鼠神经系统发育有关.     

Electroporation-mediated transfer of plasmid DNA encoding IL-10 attenuates orthotopic tracheal allograft stenosis in rats

BACKGROUND: Electroporation has been shown to increase the efficacy of intramuscular injection of plasmid DNA, resulting in a higher level of foreign gene expression. Using this technique, we examined the effect of viral IL-10 gene transfer on the prevention of tracheal allograft stenosis in an animal model. METHODS: On the day of tracheal transplantation, recipient Lewis rats were intramuscularly injected with either plasmid pCAGGS-LacZ or plasmid pCAGGS-viral IL-10, followed immediately by electroporation. Tracheas from Brown Norway donors were transplanted into the backs of Lewis recipients, and the histology of the grafts were assessed 2 and 4 weeks after transplantation. RESULTS: The serum level of IL-10 peaked at 2000 pg/ml one day after injection; the level then slowly decreased, but was maintained above 1000 pg/ml until 8 days after injection. At Day 28, the airway lumina of the tracheal allografts were almost completely obliterated by fibroproliferative tissue in the control pCAGGS-LacZ-treated rats. In rats injected once with pCAGGS-viral IL-10, luminal obliteration was significantly decreased compared with the control pCAGGS-LacZ-treated rats (mean luminal opening 46.8% vs 0% p<0.05). The loss of epithelial cells lining the airway was also decreased in the IL-10-treated group (mean epithelial coverage 42% vs 5% p<0.05). Multiple injections with pCAGGS-viral IL-10 did not further improve the histological changes. CONCLUSION: IL-10 gene transfer by intramuscular injection using electroporation attenuated tracheal allograft stenosis associated with mild epithelial injury.