Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equi...Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.展开更多
Mesenchymal stem cells(MSCs)are multipotent stem cells and capable of differentiating into multiple cell types including osteoblastic,chondrogenic and adipogenic lineages.We previously identified BMP9 as one of the mo...Mesenchymal stem cells(MSCs)are multipotent stem cells and capable of differentiating into multiple cell types including osteoblastic,chondrogenic and adipogenic lineages.We previously identified BMP9 as one of the most potent BMPs that induce osteoblastic differentiation of MSCs although exact molecular mechanism through which BMP9 regulates osteogenic differentiation remains to be fully understood.Here,we seek to develop a recombinant adenovirus system to optimally silence mouse BMP9 and then characterize the important role of BMP9 in osteogenic differentiation of MSCs.Using two different siRNA bioinformatic prediction programs,we design five siRNAs targeting mouse BMP9(or simB9),which are expressed under the control of the converging H1 and U6 promoters in recombinant adenovirus vectors.We demonstrate that two of the five siRNAs,simB9-4 and simB9-7,exhibit the highest efficiency on silencing exogenous mouse BMP9 in MSCs.Furthermore,simB9-4 and simB9-7 act synergistically in inhibiting BMP9-induced expression of osteogenic markers,matrix mineralization and ectopic bone formation from MSCs.Thus,our findings demonstrate the important role of BMP9 in osteogenic differentiation of MSCs.The characterized simB9 siRNAs may be used as an important tool to investigate the molecular mechanism behind BMP9 osteogenic signaling.Our results also indicate that recombinant adenovirus-mediated expression of siRNAs is efficient and sustained,and thus may be used as an effective delivery vehicle of siRNA therapeutics.展开更多
Background Steroid-induced osteonecrosis of the femoral head （ONFH） is a common clinical disease,with a high disability rate.At present,efficient prevention and treatment of steroid-induced ONFH is still lacking.The...Background Steroid-induced osteonecrosis of the femoral head （ONFH） is a common clinical disease,with a high disability rate.At present,efficient prevention and treatment of steroid-induced ONFH is still lacking.The peroxisome proliferator-activated receptor-γ （PPARγ） is recognized as an important pathogenic gene for the development of steroid-induced ONFH.RNA interference （RNAi） is a tool for functional gene analysis,which has been successfully used to down-regulate the levels of specific target proteins.Therefore,down-regulation of PPARγ expression by RNAi may prevent the incidence of steroid-induced ONFH.Methods According to the principles of siRNA design,three duplex siRNA sequences （971-989,1253-1271 and 1367-1385） derived from the PPARy gene （NM_001082148） were synthesized.These duplexes were annealed,purified and ligated into 1.0-cytomegalovirus （CMV） shuttle vector.The shuttle vector was transfected into HEK293 cells.The HEK293 generated recombinant adenovirus vector carrying PPARγ siRNA sequences was purified and the titer of recombinant adenovirus was determined.Results After the annealing of single-strand DNA oligo encoding short hairpin RNA （shRNA） sequences,products were identified by gel electrophoresis.These products were ligated into the 1.0-CMV shuttle vector and the recombinant shuttle vectors 1.0-CMV-971,1.0-CMV-1253 and 1.0-CMV-1367 were constructed.These sequences of these recombinant vectors were confirmed.We then successfully constructed the recombinant adenovirus vector carrying siRNA targeting PPARγ.After purification,the virus titer was higher than 1010 plaque forming unit （PFU）/ml.Conclusion In this study,three recombinant adenovirus shuttle vectors carrying siRNA targeting PPARγ,including shuttle vectors 1.0-CMV-971,1.0-CMV-1253 and 1.0-CMV-1367,were successfully constructed and high titers of recombinant adenovirus were obtained.展开更多
Background Steroids inhibit osteogenic differentiation and decrease bone formation while concomitantly inducing adipose deposition in osteocytes.This leads to the fatty degeneration and necrosis of bone cells commonly...Background Steroids inhibit osteogenic differentiation and decrease bone formation while concomitantly inducing adipose deposition in osteocytes.This leads to the fatty degeneration and necrosis of bone cells commonly seen in osteonecrosis of the femoral head.The peroxisome proliferator-activated receptor-γ （PPARγ） is an adipogenic transcription factor linked to the development of this disease and responsible for inducing adipogenesis over osteogenesis in bone marrow mesenchymal stem cells （BMSCs）.The aim of this study was to assess whether adipogenic differentiation could be suppressed,and thus osteogenic potential retained,by inhibiting PPARy expression in BMSCs.Methods Cells from the bone marrow of New Zealand rabbits were treated with 10-7 mol/L dexamethasone and infected with one of three small interference RNA （siRNA） adenovirus vectors （S1,S2,and S3） or non-targeting control siRNA （Con） and compared with dexamethasone-treated （model） and untreated （normal） cells.Cells were grown for 21 days and stained with Sudan Ⅲ for adipocyte formation.At various time points,cells were also assessed for changes in PPARγ,osteocalcin （OC）,Runx2,alkaline phosphatase （ALP） activity,and triglyceride （TG） content.Results Dexamethasone-treated model and control groups showed a significant increase in fatty acid-positive staining,which was inhibited in cells treated with PPARγ siRNA-treated,similar to normal untreated cells.All three siRNA groups significantly inhibited PPARγ mRNA and protein,adipocyte number,and TG content compared with the dexamethasonetreated model and control groups,matching that seen in normal cells.OC and Runx2 mRNA and protein,as well as ALP activity,were significantly higher in cells treated with siRNA against PPARγ,similar to that seen in the normal cells.These osteogenic markers were significantly lower in the dexamethasone-treated cell cultures.Conclusions The siRNA adenovirus vector targeting PPARγ can efficiently inhibit steroid-induced adipogenic differentiation in rabbit BMSCs and retain their osteogenic differentiation potential.展开更多
目的:利用低氧干预分离的小鼠神经干细胞,通过构建腺病毒载体将腺病毒5-丝氨酸-苏氨酸蛋白激酶3(Ad5-AKT3)基因和丝氨酸-苏氨酸蛋白激酶3小干扰RNA(AKT3 si RNA)干扰转染至缺血神经干细胞,探索磷酸肌醇三磷酸激酶-丝氨酸-苏氨酸蛋白激酶...目的:利用低氧干预分离的小鼠神经干细胞,通过构建腺病毒载体将腺病毒5-丝氨酸-苏氨酸蛋白激酶3(Ad5-AKT3)基因和丝氨酸-苏氨酸蛋白激酶3小干扰RNA(AKT3 si RNA)干扰转染至缺血神经干细胞,探索磷酸肌醇三磷酸激酶-丝氨酸-苏氨酸蛋白激酶(PI3K-AKT)通路对缺血性神经干细胞的增殖作用。方法:取新生24h昆明小鼠海马,分离培养原代神经干细胞,并采用巢蛋白(Nestin)免疫荧光检测和5-溴脱氧尿嘧啶核苷(Brdu)渗入实验对其进行鉴定;将所培养的传代神经干细胞随机分组:正常组、缺血模型组、低氧组(低氧+缺血模型组)、Lip2000组(低氧+缺血模型组+Lip2000空转染组)、AKT转染组(低氧+缺血模型组+Ad5-AKT3转染组)、AKT干扰组(低氧+缺血模型组+Lip2000+AKT3 si RNA干扰组)。建立脑缺血损伤的体外模型,低氧干预,构建腺病毒Ad5-AKT3载体和AKT3 si RNA后,各组行MTT检测;RT-q PCR检测AKT3 m RNA表达情况;Werstern Blot检测PI3K、AKT3、PCNA。结果:通过免疫荧光检测Nestin鉴定神经干细胞球,在荧光显微镜下观察到绿色明亮且典型的细胞球,球内可见清晰结构;渗入Brdu免疫荧光检测可见整个着色细胞球。MTT OD值和RT-q PCR检测AKT3 m RNA均显示,与正常组相比,模型组和AKT干扰组下降(P<0.05),其余各组均升高(P<0.05),其中低氧组和Lip2000组之间无明显差异(P>0.05)。与模型组相比,除AKT干扰组下降外(P<0.05),其余各组均升高(P<0.05)。Western Blot检测PI3K、AKT3、PCNA蛋白表达与正常组相比,模型组和AKT干扰组表达量减少(P<0.05),低氧组、Lip2000组和AKT转染组均增加(P<0.05),其中AKT转染组差异最明显(P<0.01)。结论:PI3K-AKT信号通路在低氧干预下有效促进神经干细胞的增殖过程。展开更多
目的探讨DNA分化抑制因子(inhibitor of DNA differentiation,Id)Id1/Id3在子宫内膜癌中的表达及对子宫内膜癌细胞增殖、侵袭、迁移及粘附等生物学行为的作用。方法 Western blot方法检测4例子宫内膜癌组织及相应的癌旁组织中的Id1/Id3...目的探讨DNA分化抑制因子(inhibitor of DNA differentiation,Id)Id1/Id3在子宫内膜癌中的表达及对子宫内膜癌细胞增殖、侵袭、迁移及粘附等生物学行为的作用。方法 Western blot方法检测4例子宫内膜癌组织及相应的癌旁组织中的Id1/Id3的表达;将子宫内膜癌RL-952和HEC-1-B两株细胞各分为未处理细胞组、空白病毒组、启动子病毒组和Id1/Id3双敲低组。通过qRT-PCR检测不同腺病毒载体感染细胞后MMP2、CXCR4和P21 mRNA表达量,Western blot检测MMP2、CXCR4、P21蛋白表达情况;通过MTT增殖试验、Transwell侵袭实验、细胞划痕实验和细胞粘附实验检测Id1/Id3表达改变后,对子宫内膜癌细胞增殖、侵袭、移行和粘附能力的影响。结果 Id1/Id3在子宫内膜癌组织中较癌旁组织表达增高(P<0.05);双敲低Id1/Id3,子宫内膜癌细胞中MMP2、CXCR4的mRNA水平和蛋白水平明显降低(P<0.05),P21的mRNA水平和蛋白水平则明显升高(P<0.05),RNAi组与其他3组细胞相比均有显著性差异;Id1/Id3双敲低组子宫内膜癌RL-952和HEC-1-B株细胞增殖能力、侵袭能力、迁移能力和粘附能力均抑制(P<0.05)。结论 Id1/Id3在子宫内膜癌患者组织中高表达,并可通过升调MMP2、CXCR4和降调P21的表达促进子宫内膜癌细胞增殖、侵袭、迁移及粘附作用,靶向Id1/Id3治疗可能成为预防和治疗子宫内膜癌的新方案。展开更多
鸡胚成纤维细胞(CEF)是多种动物及人类病毒的易感细胞;也是研究鸡的基因组功能的理想材料.本试验的目的是研究非复制型腺病毒载体对CEF的转染效率及安全性;并建立在CEF细胞中进行RNAi的技术平台.采用GFP重组非复制型腺病毒载体(Ade-GFP...鸡胚成纤维细胞(CEF)是多种动物及人类病毒的易感细胞;也是研究鸡的基因组功能的理想材料.本试验的目的是研究非复制型腺病毒载体对CEF的转染效率及安全性;并建立在CEF细胞中进行RNAi的技术平台.采用GFP重组非复制型腺病毒载体(Ade-GFP)转染CEF细胞;结果证明0.1-2000 MOI Adv-GFP均可转染CEF细胞并表达GFP;转染后16h开始出现GFP阳性细胞;胞核与胞浆可见明亮的荧光;荧光细胞数量及荧光强度在24～36h达到高峰;以后逐渐衰减;约180h全部消失;转染效率最高为22.3%;形态学观察及流式细胞仪测定结果显示;Adv-GFP转染的CEF细胞未见细胞病变;多数转染组细胞活力不受影响;说明用Adv-GFP携带外源转染CEF细胞是安全可行的;用脂质体转染试剂转染化学合成的GFP-siRNA;成功地干涉了腺病毒载体介导的GFP基因在鸡胚成纤维细胞的表达;干涉效率为85%;证明了鸡胚成纤维细胞中存在RNAi机制;为进一步利用非复制型腺病毒载体递送siRNA在CEF细胞内进行RNAi的研究奠定了基础.展开更多
基金the National Natural Science Foundation of China, No. 30672136
文摘Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.
基金The reported work was supported in part by research grants from the National Institutes of Health(CA226303,DE020140 to TCH and RRR)the U.S.Department of Defense(OR130096 to JMW)+1 种基金the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust(RRR,TCH),the Scoliosis Research Society(TCH and MJL)the National Key Research and Development Program of China(2016YFC1000803 and 2011CB707906 to TCH).
文摘Mesenchymal stem cells(MSCs)are multipotent stem cells and capable of differentiating into multiple cell types including osteoblastic,chondrogenic and adipogenic lineages.We previously identified BMP9 as one of the most potent BMPs that induce osteoblastic differentiation of MSCs although exact molecular mechanism through which BMP9 regulates osteogenic differentiation remains to be fully understood.Here,we seek to develop a recombinant adenovirus system to optimally silence mouse BMP9 and then characterize the important role of BMP9 in osteogenic differentiation of MSCs.Using two different siRNA bioinformatic prediction programs,we design five siRNAs targeting mouse BMP9(or simB9),which are expressed under the control of the converging H1 and U6 promoters in recombinant adenovirus vectors.We demonstrate that two of the five siRNAs,simB9-4 and simB9-7,exhibit the highest efficiency on silencing exogenous mouse BMP9 in MSCs.Furthermore,simB9-4 and simB9-7 act synergistically in inhibiting BMP9-induced expression of osteogenic markers,matrix mineralization and ectopic bone formation from MSCs.Thus,our findings demonstrate the important role of BMP9 in osteogenic differentiation of MSCs.The characterized simB9 siRNAs may be used as an important tool to investigate the molecular mechanism behind BMP9 osteogenic signaling.Our results also indicate that recombinant adenovirus-mediated expression of siRNAs is efficient and sustained,and thus may be used as an effective delivery vehicle of siRNA therapeutics.
文摘Background Steroid-induced osteonecrosis of the femoral head （ONFH） is a common clinical disease,with a high disability rate.At present,efficient prevention and treatment of steroid-induced ONFH is still lacking.The peroxisome proliferator-activated receptor-γ （PPARγ） is recognized as an important pathogenic gene for the development of steroid-induced ONFH.RNA interference （RNAi） is a tool for functional gene analysis,which has been successfully used to down-regulate the levels of specific target proteins.Therefore,down-regulation of PPARγ expression by RNAi may prevent the incidence of steroid-induced ONFH.Methods According to the principles of siRNA design,three duplex siRNA sequences （971-989,1253-1271 and 1367-1385） derived from the PPARy gene （NM_001082148） were synthesized.These duplexes were annealed,purified and ligated into 1.0-cytomegalovirus （CMV） shuttle vector.The shuttle vector was transfected into HEK293 cells.The HEK293 generated recombinant adenovirus vector carrying PPARγ siRNA sequences was purified and the titer of recombinant adenovirus was determined.Results After the annealing of single-strand DNA oligo encoding short hairpin RNA （shRNA） sequences,products were identified by gel electrophoresis.These products were ligated into the 1.0-CMV shuttle vector and the recombinant shuttle vectors 1.0-CMV-971,1.0-CMV-1253 and 1.0-CMV-1367 were constructed.These sequences of these recombinant vectors were confirmed.We then successfully constructed the recombinant adenovirus vector carrying siRNA targeting PPARγ.After purification,the virus titer was higher than 1010 plaque forming unit （PFU）/ml.Conclusion In this study,three recombinant adenovirus shuttle vectors carrying siRNA targeting PPARγ,including shuttle vectors 1.0-CMV-971,1.0-CMV-1253 and 1.0-CMV-1367,were successfully constructed and high titers of recombinant adenovirus were obtained.
基金This study was supported by a grant from the National Natural Science Foundation of China （No. 81071520）.
文摘Background Steroids inhibit osteogenic differentiation and decrease bone formation while concomitantly inducing adipose deposition in osteocytes.This leads to the fatty degeneration and necrosis of bone cells commonly seen in osteonecrosis of the femoral head.The peroxisome proliferator-activated receptor-γ （PPARγ） is an adipogenic transcription factor linked to the development of this disease and responsible for inducing adipogenesis over osteogenesis in bone marrow mesenchymal stem cells （BMSCs）.The aim of this study was to assess whether adipogenic differentiation could be suppressed,and thus osteogenic potential retained,by inhibiting PPARy expression in BMSCs.Methods Cells from the bone marrow of New Zealand rabbits were treated with 10-7 mol/L dexamethasone and infected with one of three small interference RNA （siRNA） adenovirus vectors （S1,S2,and S3） or non-targeting control siRNA （Con） and compared with dexamethasone-treated （model） and untreated （normal） cells.Cells were grown for 21 days and stained with Sudan Ⅲ for adipocyte formation.At various time points,cells were also assessed for changes in PPARγ,osteocalcin （OC）,Runx2,alkaline phosphatase （ALP） activity,and triglyceride （TG） content.Results Dexamethasone-treated model and control groups showed a significant increase in fatty acid-positive staining,which was inhibited in cells treated with PPARγ siRNA-treated,similar to normal untreated cells.All three siRNA groups significantly inhibited PPARγ mRNA and protein,adipocyte number,and TG content compared with the dexamethasonetreated model and control groups,matching that seen in normal cells.OC and Runx2 mRNA and protein,as well as ALP activity,were significantly higher in cells treated with siRNA against PPARγ,similar to that seen in the normal cells.These osteogenic markers were significantly lower in the dexamethasone-treated cell cultures.Conclusions The siRNA adenovirus vector targeting PPARγ can efficiently inhibit steroid-induced adipogenic differentiation in rabbit BMSCs and retain their osteogenic differentiation potential.
文摘目的:利用低氧干预分离的小鼠神经干细胞,通过构建腺病毒载体将腺病毒5-丝氨酸-苏氨酸蛋白激酶3(Ad5-AKT3)基因和丝氨酸-苏氨酸蛋白激酶3小干扰RNA(AKT3 si RNA)干扰转染至缺血神经干细胞,探索磷酸肌醇三磷酸激酶-丝氨酸-苏氨酸蛋白激酶(PI3K-AKT)通路对缺血性神经干细胞的增殖作用。方法:取新生24h昆明小鼠海马,分离培养原代神经干细胞,并采用巢蛋白(Nestin)免疫荧光检测和5-溴脱氧尿嘧啶核苷(Brdu)渗入实验对其进行鉴定;将所培养的传代神经干细胞随机分组:正常组、缺血模型组、低氧组(低氧+缺血模型组)、Lip2000组(低氧+缺血模型组+Lip2000空转染组)、AKT转染组(低氧+缺血模型组+Ad5-AKT3转染组)、AKT干扰组(低氧+缺血模型组+Lip2000+AKT3 si RNA干扰组)。建立脑缺血损伤的体外模型,低氧干预,构建腺病毒Ad5-AKT3载体和AKT3 si RNA后,各组行MTT检测;RT-q PCR检测AKT3 m RNA表达情况;Werstern Blot检测PI3K、AKT3、PCNA。结果:通过免疫荧光检测Nestin鉴定神经干细胞球,在荧光显微镜下观察到绿色明亮且典型的细胞球,球内可见清晰结构;渗入Brdu免疫荧光检测可见整个着色细胞球。MTT OD值和RT-q PCR检测AKT3 m RNA均显示,与正常组相比,模型组和AKT干扰组下降(P<0.05),其余各组均升高(P<0.05),其中低氧组和Lip2000组之间无明显差异(P>0.05)。与模型组相比,除AKT干扰组下降外(P<0.05),其余各组均升高(P<0.05)。Western Blot检测PI3K、AKT3、PCNA蛋白表达与正常组相比,模型组和AKT干扰组表达量减少(P<0.05),低氧组、Lip2000组和AKT转染组均增加(P<0.05),其中AKT转染组差异最明显(P<0.01)。结论:PI3K-AKT信号通路在低氧干预下有效促进神经干细胞的增殖过程。
文摘目的探讨DNA分化抑制因子(inhibitor of DNA differentiation,Id)Id1/Id3在子宫内膜癌中的表达及对子宫内膜癌细胞增殖、侵袭、迁移及粘附等生物学行为的作用。方法 Western blot方法检测4例子宫内膜癌组织及相应的癌旁组织中的Id1/Id3的表达;将子宫内膜癌RL-952和HEC-1-B两株细胞各分为未处理细胞组、空白病毒组、启动子病毒组和Id1/Id3双敲低组。通过qRT-PCR检测不同腺病毒载体感染细胞后MMP2、CXCR4和P21 mRNA表达量,Western blot检测MMP2、CXCR4、P21蛋白表达情况;通过MTT增殖试验、Transwell侵袭实验、细胞划痕实验和细胞粘附实验检测Id1/Id3表达改变后,对子宫内膜癌细胞增殖、侵袭、移行和粘附能力的影响。结果 Id1/Id3在子宫内膜癌组织中较癌旁组织表达增高(P<0.05);双敲低Id1/Id3,子宫内膜癌细胞中MMP2、CXCR4的mRNA水平和蛋白水平明显降低(P<0.05),P21的mRNA水平和蛋白水平则明显升高(P<0.05),RNAi组与其他3组细胞相比均有显著性差异;Id1/Id3双敲低组子宫内膜癌RL-952和HEC-1-B株细胞增殖能力、侵袭能力、迁移能力和粘附能力均抑制(P<0.05)。结论 Id1/Id3在子宫内膜癌患者组织中高表达,并可通过升调MMP2、CXCR4和降调P21的表达促进子宫内膜癌细胞增殖、侵袭、迁移及粘附作用,靶向Id1/Id3治疗可能成为预防和治疗子宫内膜癌的新方案。
文摘鸡胚成纤维细胞(CEF)是多种动物及人类病毒的易感细胞;也是研究鸡的基因组功能的理想材料.本试验的目的是研究非复制型腺病毒载体对CEF的转染效率及安全性;并建立在CEF细胞中进行RNAi的技术平台.采用GFP重组非复制型腺病毒载体(Ade-GFP)转染CEF细胞;结果证明0.1-2000 MOI Adv-GFP均可转染CEF细胞并表达GFP;转染后16h开始出现GFP阳性细胞;胞核与胞浆可见明亮的荧光;荧光细胞数量及荧光强度在24～36h达到高峰;以后逐渐衰减;约180h全部消失;转染效率最高为22.3%;形态学观察及流式细胞仪测定结果显示;Adv-GFP转染的CEF细胞未见细胞病变;多数转染组细胞活力不受影响;说明用Adv-GFP携带外源转染CEF细胞是安全可行的;用脂质体转染试剂转染化学合成的GFP-siRNA;成功地干涉了腺病毒载体介导的GFP基因在鸡胚成纤维细胞的表达;干涉效率为85%;证明了鸡胚成纤维细胞中存在RNAi机制;为进一步利用非复制型腺病毒载体递送siRNA在CEF细胞内进行RNAi的研究奠定了基础.
