目的:对比观察VEGF165b重组蛋白(recombinant human VEGF165b protein,rhVEGF165b)与贝伐珠单抗对人胃癌裸鼠移植瘤生长的影响及作用机制.方法:用人胃癌细胞BGC-823接种于裸鼠皮下,建立裸鼠移植瘤模型,随机分为3组,每组21只:rhVEGF165b...目的:对比观察VEGF165b重组蛋白(recombinant human VEGF165b protein,rhVEGF165b)与贝伐珠单抗对人胃癌裸鼠移植瘤生长的影响及作用机制.方法:用人胃癌细胞BGC-823接种于裸鼠皮下,建立裸鼠移植瘤模型,随机分为3组,每组21只:rhVEGF165b组(腹腔注射rhVEGF165b 10?g/kg)、贝伐珠单抗组(腹腔注射贝伐珠单抗5 mg/kg)、对照组(腹腔注射生理盐水10 mL/kg),分别于1、2、3 wk测量裸鼠移植瘤体积及瘤质量,免疫组织化学方法检测肿瘤组织中CD34表达(以阳性细胞数计算肿瘤微血管密度),TUNEL方法检测肿瘤组织细胞凋亡.结果:贝伐珠单抗组移植瘤体积(cm3)及瘤质量(g)在第1、2、3周均小于对照组(1 wk:0.453±0.119 vs 0.637±0.084,0.32±0.097vs 0.46±0.093;2 wk:1.691±0.381 vs 2.238±0.29,1.168±0.524 vs 2.013±0.833;3 wk:1.709±0.474 vs 4.872±0.594,1.747±0.557 vs3.463±0.986,均P<0.05),而rhVEGF165b组仅在第1、2周较对照组小(1 wk:0.546±0.132 vs0.637±0.084,1.894±0.599 vs 0.46±0.093;2wk:1.894±0.599 vs 2.238±0.29,1.537±0.568vs 2.013±0.833,均P<0.05),第3周其体积及瘤质量大于贝伐珠单抗组(3 wk:3.843±1.339 vs1.709±0.474,3.066±1.281 vs 1.747±0.557,P<0.05),与对照组无明显差异.rhVEGF165b组、贝伐珠单抗组CD34的表达水平在第1、2、3周均低于对照组(P<0.05,P<0.01),凋亡指数高于对照组(P<0.05,P<0.01);与贝伐珠单抗组相比,第1周rhVEGF165b组CD34的表达水平更低(P<0.05),但第2、3周却高于贝伐珠单抗组(P<0.01);rhVEGF165b组的凋亡指数在第1周明显高于贝伐珠单抗组(P<0.01),而第2、3周却低于贝伐珠单抗组(P<0.05).结论:rhVEGF165b、贝伐珠单抗对人胃癌移植瘤的生长有明显抑制作用,rhVEGF165b早期抑制血管生成、促进细胞凋亡表现更为明显.展开更多
Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells (MSCs) with human vascular endothelial growth factor 165 (hVEGF165) gene and human bon...Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells (MSCs) with human vascular endothelial growth factor 165 (hVEGF165) gene and human bone morphogenetic protein 2 (hBMP2) gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein (copGFP). The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 (Lv-VEGF) or hBMP2 (Lv-BMP), respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF (BMP+VEGF group), or each alone (BMP group and VEGF group), or with no virus (Control group). The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay (ELISA). Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP+VEGF group. No significant difference of BMP2 expression was detected between BMP+VEGF and BMP groups (P>0.05). Similarly, there was no significant difference of VEGF165 expression between BMP+VEGF and VEGF groups (P>0.05). Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.展开更多
背景:目前自体脂肪移植已广泛运用于美容整形和修复创伤导致的软组织缺损的修复,有研究表明,移植后1年移植脂肪存活率为20%-80%,因此,在移植后的早期及时、充分的血供建立,对于移植脂肪的存活是非常重要的。目的:观察血管内皮生长因子16...背景:目前自体脂肪移植已广泛运用于美容整形和修复创伤导致的软组织缺损的修复,有研究表明,移植后1年移植脂肪存活率为20%-80%,因此,在移植后的早期及时、充分的血供建立,对于移植脂肪的存活是非常重要的。目的:观察血管内皮生长因子165转染人脂肪间充质干细胞的增殖情况。方法:体外传代培养人脂肪间充质干细胞,将重组血管内皮生长因子165基因入腺病毒液和空病毒液转染至脂肪间充质干细胞内,分别设为实验组和对照组,另设正常培养的细胞为空白组。结果与结论:RT-PCR,Western blot,MTT检测显示,与对照组和空白组相比,实验组血管内皮生长因子165 m RNA和蛋白的表达及细胞增殖均增高(P<0.05)。结果证实,腺病毒承载的血管内皮生长因子165基因转染脂肪间充质干细胞后不仅可以持续的表达目的蛋白,同时也能显著促进脂肪间充质干细胞的增殖。展开更多
文摘目的:对比观察VEGF165b重组蛋白(recombinant human VEGF165b protein,rhVEGF165b)与贝伐珠单抗对人胃癌裸鼠移植瘤生长的影响及作用机制.方法:用人胃癌细胞BGC-823接种于裸鼠皮下,建立裸鼠移植瘤模型,随机分为3组,每组21只:rhVEGF165b组(腹腔注射rhVEGF165b 10?g/kg)、贝伐珠单抗组(腹腔注射贝伐珠单抗5 mg/kg)、对照组(腹腔注射生理盐水10 mL/kg),分别于1、2、3 wk测量裸鼠移植瘤体积及瘤质量,免疫组织化学方法检测肿瘤组织中CD34表达(以阳性细胞数计算肿瘤微血管密度),TUNEL方法检测肿瘤组织细胞凋亡.结果:贝伐珠单抗组移植瘤体积(cm3)及瘤质量(g)在第1、2、3周均小于对照组(1 wk:0.453±0.119 vs 0.637±0.084,0.32±0.097vs 0.46±0.093;2 wk:1.691±0.381 vs 2.238±0.29,1.168±0.524 vs 2.013±0.833;3 wk:1.709±0.474 vs 4.872±0.594,1.747±0.557 vs3.463±0.986,均P<0.05),而rhVEGF165b组仅在第1、2周较对照组小(1 wk:0.546±0.132 vs0.637±0.084,1.894±0.599 vs 0.46±0.093;2wk:1.894±0.599 vs 2.238±0.29,1.537±0.568vs 2.013±0.833,均P<0.05),第3周其体积及瘤质量大于贝伐珠单抗组(3 wk:3.843±1.339 vs1.709±0.474,3.066±1.281 vs 1.747±0.557,P<0.05),与对照组无明显差异.rhVEGF165b组、贝伐珠单抗组CD34的表达水平在第1、2、3周均低于对照组(P<0.05,P<0.01),凋亡指数高于对照组(P<0.05,P<0.01);与贝伐珠单抗组相比,第1周rhVEGF165b组CD34的表达水平更低(P<0.05),但第2、3周却高于贝伐珠单抗组(P<0.01);rhVEGF165b组的凋亡指数在第1周明显高于贝伐珠单抗组(P<0.01),而第2、3周却低于贝伐珠单抗组(P<0.05).结论:rhVEGF165b、贝伐珠单抗对人胃癌移植瘤的生长有明显抑制作用,rhVEGF165b早期抑制血管生成、促进细胞凋亡表现更为明显.
基金Supported by Key Program of Shanghai Science and Technology Committee (054119520)
文摘Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells (MSCs) with human vascular endothelial growth factor 165 (hVEGF165) gene and human bone morphogenetic protein 2 (hBMP2) gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein (copGFP). The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 (Lv-VEGF) or hBMP2 (Lv-BMP), respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF (BMP+VEGF group), or each alone (BMP group and VEGF group), or with no virus (Control group). The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay (ELISA). Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP+VEGF group. No significant difference of BMP2 expression was detected between BMP+VEGF and BMP groups (P>0.05). Similarly, there was no significant difference of VEGF165 expression between BMP+VEGF and VEGF groups (P>0.05). Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.
文摘背景:目前自体脂肪移植已广泛运用于美容整形和修复创伤导致的软组织缺损的修复,有研究表明,移植后1年移植脂肪存活率为20%-80%,因此,在移植后的早期及时、充分的血供建立,对于移植脂肪的存活是非常重要的。目的:观察血管内皮生长因子165转染人脂肪间充质干细胞的增殖情况。方法:体外传代培养人脂肪间充质干细胞,将重组血管内皮生长因子165基因入腺病毒液和空病毒液转染至脂肪间充质干细胞内,分别设为实验组和对照组,另设正常培养的细胞为空白组。结果与结论:RT-PCR,Western blot,MTT检测显示,与对照组和空白组相比,实验组血管内皮生长因子165 m RNA和蛋白的表达及细胞增殖均增高(P<0.05)。结果证实,腺病毒承载的血管内皮生长因子165基因转染脂肪间充质干细胞后不仅可以持续的表达目的蛋白,同时也能显著促进脂肪间充质干细胞的增殖。
