We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly ...We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.展开更多
A complementary DNA (cDNA) encoding human hepatocyte growth factor was intro-duced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homolo-gous recombination of intracellular plasmid D...A complementary DNA (cDNA) encoding human hepatocyte growth factor was intro-duced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homolo-gous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities to human hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8%±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was de-tected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF, the amount of expression was 76 ng/4.0×105 cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6×109 pfu, 8.6×108 pfu, 8.6×107 pfu, 8.6×106 pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that of the normal skin. The value of HI (hypertrophic index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 109 pfu and 108 pfu. Whereas no therapeutic effects were seen at lower dose (107 pfu and 106 pfu of Ad-HGF) groups. In addition, clusters of hair were ob-served to different extent on healed wound treated with Ad-HGF. Histopathologic examination re-vealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed. In Sirius red-stained sections the amount of type I collagen in the Ad-HGF-treated scars was diminished markedly, compared to that in Ad-GFP group, in which a huge amount of type I collagen was still observed; (iv) immune response against HGF was absent. Antibody against HGF was not detectable by ELISA in serum from rabbit treated with Ad-HGF; (v) no local or systemic side-effects and toxicity associated with the gene transfer were found. These results demonstrated the potential use of treating pathologic scar by Ad-HGF, an alterative strategy of gene therapy for scar in clinical practice.展开更多
Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer Methods A total of 12 female New Zealand rabbits were use...Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer Methods A total of 12 female New Zealand rabbits were used in this study Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad HGF group 1, Ad HGF group 2, Ad HGF group 3, Ad GFP (a reporter gene) group and the solvent group Immediately after surgery, 6×10 7 pfu Ad HGF, 6×10 8 pfu Ad HGF, 6×10 9 pfu of Ad HGF, 6×10 9 pfu of Ad GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad GFP (6×10 9 pfu) into its wounds Ice slides of wounds from this animal were observed under fluorescence microscopy Another additional rabbit was used to evaluate the expression of HGF and TGFβ1 after transferring Ad HGF (6×10 9 pfu) into each of its wound Immunohistochemistry was used for detection Results The effect of HGF on reducing excessive dermal scarring was observed by adenovirus mediated gene transfer Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over expression of TGFβ1, which plays an essential role in the progression of dermal fibrogenesis Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring Conclusion HGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing展开更多
Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adeno- virus with hepat...Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adeno- virus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we estab- lished a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were ran- domly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.展开更多
目的探讨肝细胞生长因子(HGF)对原代培养脊髓神经元的保护作用。方法原代培养脊髓神经元转染不同滴度的绿色荧光蛋白或肝细胞生长因子重组腺病毒(Ad-GFP or Ad-HGF),用流式细胞仪检测转染率,PI-Hoechst双染色观察神经元生长状况。用ELIS...目的探讨肝细胞生长因子(HGF)对原代培养脊髓神经元的保护作用。方法原代培养脊髓神经元转染不同滴度的绿色荧光蛋白或肝细胞生长因子重组腺病毒(Ad-GFP or Ad-HGF),用流式细胞仪检测转染率,PI-Hoechst双染色观察神经元生长状况。用ELISA、中性红、MTT和NSE-ELISA等方法分别检测Ad-HGF转染脊髓神经元后HGF的表达,以及HGF对神经元的保护和迁移作用。结果在转染复数(MO I)为50时,Ad-GFP可高效转染脊髓神经细胞,且细胞生长状态好。转染Ad-HGF后,HGF能有效表达,同时HGF作用后的神经元活性明显高于对照组(P<0.05)。并观察到HGF对脊髓神经元有明显促迁移作用。结论HGF对体外培养的脊髓神经元有保护和迁移作用。展开更多
目的探讨携带肝细胞生长因子基因的重组腺病毒Ad-HGF基因对小鼠慢性阻塞性肺疾病(COPD)模型的治疗作用。方法猪胰弹性蛋白酶(PPE)气管内滴注(4 u/只)复制小鼠COPD模型30只,随机分为模型对照组、Ad-HGF治疗组和Ad-Lac Z治疗组,每组10只...目的探讨携带肝细胞生长因子基因的重组腺病毒Ad-HGF基因对小鼠慢性阻塞性肺疾病(COPD)模型的治疗作用。方法猪胰弹性蛋白酶(PPE)气管内滴注(4 u/只)复制小鼠COPD模型30只,随机分为模型对照组、Ad-HGF治疗组和Ad-Lac Z治疗组,每组10只。造模后3 d Ad-HGF治疗组气管内滴注Ad-HGF(3×1010particle/mouse);Ad-LacZ治疗组及模型对照组气管内分别滴注同剂量的Ad-Lac Z及同体积0.9%氯化钠溶液。每组5只实验动物分别于处理后7 d和14 d处死,光学显微镜下观察肺组织病理形态,测量肺泡平均截线长度(mean linear intercept,Lm);TUNEL法检测细胞凋亡;Ki-67免疫组织化学染色评价内皮细胞再生,并检测血浆HGF浓度。结果处理后7 d及14 d Ad-HGF治疗组血浆HGF浓度均明显高于Ad-LacZ治疗组及模型对照组,肺泡平均截线长度明显小于Ad-LacZ治疗组及模型对照组。TUNEL染色表明Ad-HGF治疗组与Ad-LacZ治疗组相比TUNEL阳性细胞明显减少。免疫组织化学染色显示Ad-HGF治疗组较Ad-LacZ治疗组Ki-67细胞明显增多。结论腺病毒介导的HGF基因可能有部分治疗小鼠COPD的作用。展开更多
基金grants fromthe Chinese Academy of Sciences (No. KJ951-BI608), the National Natural Sciences FOundation ofChina (No. 39625007 and
文摘We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.
基金supported by the State High Technology Development Program(Grant No.2001AA217061).
文摘A complementary DNA (cDNA) encoding human hepatocyte growth factor was intro-duced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homolo-gous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities to human hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8%±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was de-tected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF, the amount of expression was 76 ng/4.0×105 cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6×109 pfu, 8.6×108 pfu, 8.6×107 pfu, 8.6×106 pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that of the normal skin. The value of HI (hypertrophic index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 109 pfu and 108 pfu. Whereas no therapeutic effects were seen at lower dose (107 pfu and 106 pfu of Ad-HGF) groups. In addition, clusters of hair were ob-served to different extent on healed wound treated with Ad-HGF. Histopathologic examination re-vealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed. In Sirius red-stained sections the amount of type I collagen in the Ad-HGF-treated scars was diminished markedly, compared to that in Ad-GFP group, in which a huge amount of type I collagen was still observed; (iv) immune response against HGF was absent. Antibody against HGF was not detectable by ELISA in serum from rabbit treated with Ad-HGF; (v) no local or systemic side-effects and toxicity associated with the gene transfer were found. These results demonstrated the potential use of treating pathologic scar by Ad-HGF, an alterative strategy of gene therapy for scar in clinical practice.
基金theStateHigh TechnologyDepartmentProgram (No 2 0 0 1AA2 170 61)partlybyagrantfromtheBiologicalEngineeringDepartment+1 种基金FarEastAreaofAmericanBaxterHealthcareProductsCo Ltd USA
文摘Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer Methods A total of 12 female New Zealand rabbits were used in this study Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad HGF group 1, Ad HGF group 2, Ad HGF group 3, Ad GFP (a reporter gene) group and the solvent group Immediately after surgery, 6×10 7 pfu Ad HGF, 6×10 8 pfu Ad HGF, 6×10 9 pfu of Ad HGF, 6×10 9 pfu of Ad GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad GFP (6×10 9 pfu) into its wounds Ice slides of wounds from this animal were observed under fluorescence microscopy Another additional rabbit was used to evaluate the expression of HGF and TGFβ1 after transferring Ad HGF (6×10 9 pfu) into each of its wound Immunohistochemistry was used for detection Results The effect of HGF on reducing excessive dermal scarring was observed by adenovirus mediated gene transfer Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over expression of TGFβ1, which plays an essential role in the progression of dermal fibrogenesis Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring Conclusion HGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing
基金National "863" Programme (Grant Nos. 2003AA216080 and 2007AA021007)
文摘Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adeno- virus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we estab- lished a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were ran- domly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.
文摘目的探讨肝细胞生长因子(HGF)对原代培养脊髓神经元的保护作用。方法原代培养脊髓神经元转染不同滴度的绿色荧光蛋白或肝细胞生长因子重组腺病毒(Ad-GFP or Ad-HGF),用流式细胞仪检测转染率,PI-Hoechst双染色观察神经元生长状况。用ELISA、中性红、MTT和NSE-ELISA等方法分别检测Ad-HGF转染脊髓神经元后HGF的表达,以及HGF对神经元的保护和迁移作用。结果在转染复数(MO I)为50时,Ad-GFP可高效转染脊髓神经细胞,且细胞生长状态好。转染Ad-HGF后,HGF能有效表达,同时HGF作用后的神经元活性明显高于对照组(P<0.05)。并观察到HGF对脊髓神经元有明显促迁移作用。结论HGF对体外培养的脊髓神经元有保护和迁移作用。
文摘目的探讨携带肝细胞生长因子基因的重组腺病毒Ad-HGF基因对小鼠慢性阻塞性肺疾病(COPD)模型的治疗作用。方法猪胰弹性蛋白酶(PPE)气管内滴注(4 u/只)复制小鼠COPD模型30只,随机分为模型对照组、Ad-HGF治疗组和Ad-Lac Z治疗组,每组10只。造模后3 d Ad-HGF治疗组气管内滴注Ad-HGF(3×1010particle/mouse);Ad-LacZ治疗组及模型对照组气管内分别滴注同剂量的Ad-Lac Z及同体积0.9%氯化钠溶液。每组5只实验动物分别于处理后7 d和14 d处死,光学显微镜下观察肺组织病理形态,测量肺泡平均截线长度(mean linear intercept,Lm);TUNEL法检测细胞凋亡;Ki-67免疫组织化学染色评价内皮细胞再生,并检测血浆HGF浓度。结果处理后7 d及14 d Ad-HGF治疗组血浆HGF浓度均明显高于Ad-LacZ治疗组及模型对照组,肺泡平均截线长度明显小于Ad-LacZ治疗组及模型对照组。TUNEL染色表明Ad-HGF治疗组与Ad-LacZ治疗组相比TUNEL阳性细胞明显减少。免疫组织化学染色显示Ad-HGF治疗组较Ad-LacZ治疗组Ki-67细胞明显增多。结论腺病毒介导的HGF基因可能有部分治疗小鼠COPD的作用。
