中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2008年
4期
337-342
,共6页
李东侃%王传富%宋跃%吕振华
李東侃%王傳富%宋躍%呂振華
리동간%왕전부%송약%려진화
神经生长因子%转染%内皮%角膜%内皮细胞%再生%细胞,培养的
神經生長因子%轉染%內皮%角膜%內皮細胞%再生%細胞,培養的
신경생장인자%전염%내피%각막%내피세포%재생%세포,배양적
Nerve growth factor%Transfection%Endothelium,corneal%Endothelial cells%Regeneration%Cells,cultured
目的 探讨人β神经生长因子真核表达载体(pcDNA4-13-NGF)转染体外培养猫角膜内皮细胞并促进细胞分裂再生的机制,为将该基因应用于促进人角膜内皮细胞再生的研究打下基础.方法 为实验研究.通过EffecteneTM脂质体介导将自行构建并经测序证实的人pcDNA4-B-NGF转染到体外培养的猫角膜内皮细胞中.采用分组对照的方法研究转染前后猫角膜内皮细胞分裂再生能力.转基因后48 h通过逆转录聚合酶链反应(RT-PCR)和免疫组织化学染色方法分别在mRNA和蛋白水平检测人β神经生长因子(β-NGF)的表达.转基因后96 h采用细胞四甲基偶氮唑盐染色(MTT)测量吸光度值、细胞有丝分裂指数、流式细胞仪检测G1期细胞比例及细胞损伤后愈合面积测量等方法检测目的 基因对猫角膜内皮细胞增殖活性的作用.结果 EffecteneTM脂质体可有效介导重组真核表达载体peDNA4-β-NGF转染到经改良方法体外培养的猫角膜内皮细胞中,转染效率为11.3%,并促进该细胞表达β-NGF.正常对照组β-NGF/β-actin比值结果为3.14,加脂质体组为3.23,pcDNA4质粒转染组为3.21,pcDNA4-β-NGF重组质粒转染组为4.53.培养液、正常对照组、加脂质体组、pcDNA4质粒转染组及pcDNA4-β-NGF重组质粒转染组平均A值分别为0.178±0.007、0.482±0.033、0.488±0.017、0.520±0.021及0.623±0.041.正常对照组、加脂质体组、pcDNA4质粒转染组及pcDNA4-β-NGF重组质粒转染组细胞有丝分裂指数分别为3.3%、3.0%、3.1%及7.7%.正常对照组、加脂质体组、pcDNA4质粒转染组及pcDNA4-β-NGF重组质粒转染组G1期细胞比例分别为68.1%、51.6%、60.4%及87.9%.结论 人β-NGF基因可通过EffecteneTM脂质体介导有效转染到体外培养的猫角膜内皮细胞中,并促进细胞分裂再生,为进一步将此基因转入人角膜内皮细胞的研究提供实验经验.
目的 探討人β神經生長因子真覈錶達載體(pcDNA4-13-NGF)轉染體外培養貓角膜內皮細胞併促進細胞分裂再生的機製,為將該基因應用于促進人角膜內皮細胞再生的研究打下基礎.方法 為實驗研究.通過EffecteneTM脂質體介導將自行構建併經測序證實的人pcDNA4-B-NGF轉染到體外培養的貓角膜內皮細胞中.採用分組對照的方法研究轉染前後貓角膜內皮細胞分裂再生能力.轉基因後48 h通過逆轉錄聚閤酶鏈反應(RT-PCR)和免疫組織化學染色方法分彆在mRNA和蛋白水平檢測人β神經生長因子(β-NGF)的錶達.轉基因後96 h採用細胞四甲基偶氮唑鹽染色(MTT)測量吸光度值、細胞有絲分裂指數、流式細胞儀檢測G1期細胞比例及細胞損傷後愈閤麵積測量等方法檢測目的 基因對貓角膜內皮細胞增殖活性的作用.結果 EffecteneTM脂質體可有效介導重組真覈錶達載體peDNA4-β-NGF轉染到經改良方法體外培養的貓角膜內皮細胞中,轉染效率為11.3%,併促進該細胞錶達β-NGF.正常對照組β-NGF/β-actin比值結果為3.14,加脂質體組為3.23,pcDNA4質粒轉染組為3.21,pcDNA4-β-NGF重組質粒轉染組為4.53.培養液、正常對照組、加脂質體組、pcDNA4質粒轉染組及pcDNA4-β-NGF重組質粒轉染組平均A值分彆為0.178±0.007、0.482±0.033、0.488±0.017、0.520±0.021及0.623±0.041.正常對照組、加脂質體組、pcDNA4質粒轉染組及pcDNA4-β-NGF重組質粒轉染組細胞有絲分裂指數分彆為3.3%、3.0%、3.1%及7.7%.正常對照組、加脂質體組、pcDNA4質粒轉染組及pcDNA4-β-NGF重組質粒轉染組G1期細胞比例分彆為68.1%、51.6%、60.4%及87.9%.結論 人β-NGF基因可通過EffecteneTM脂質體介導有效轉染到體外培養的貓角膜內皮細胞中,併促進細胞分裂再生,為進一步將此基因轉入人角膜內皮細胞的研究提供實驗經驗.
목적 탐토인β신경생장인자진핵표체재체(pcDNA4-13-NGF)전염체외배양묘각막내피세포병촉진세포분렬재생적궤제,위장해기인응용우촉진인각막내피세포재생적연구타하기출.방법 위실험연구.통과EffecteneTM지질체개도장자행구건병경측서증실적인pcDNA4-B-NGF전염도체외배양적묘각막내피세포중.채용분조대조적방법연구전염전후묘각막내피세포분렬재생능력.전기인후48 h통과역전록취합매련반응(RT-PCR)화면역조직화학염색방법분별재mRNA화단백수평검측인β신경생장인자(β-NGF)적표체.전기인후96 h채용세포사갑기우담서염염색(MTT)측량흡광도치、세포유사분렬지수、류식세포의검측G1기세포비례급세포손상후유합면적측량등방법검측목적 기인대묘각막내피세포증식활성적작용.결과 EffecteneTM지질체가유효개도중조진핵표체재체peDNA4-β-NGF전염도경개량방법체외배양적묘각막내피세포중,전염효솔위11.3%,병촉진해세포표체β-NGF.정상대조조β-NGF/β-actin비치결과위3.14,가지질체조위3.23,pcDNA4질립전염조위3.21,pcDNA4-β-NGF중조질립전염조위4.53.배양액、정상대조조、가지질체조、pcDNA4질립전염조급pcDNA4-β-NGF중조질립전염조평균A치분별위0.178±0.007、0.482±0.033、0.488±0.017、0.520±0.021급0.623±0.041.정상대조조、가지질체조、pcDNA4질립전염조급pcDNA4-β-NGF중조질립전염조세포유사분렬지수분별위3.3%、3.0%、3.1%급7.7%.정상대조조、가지질체조、pcDNA4질립전염조급pcDNA4-β-NGF중조질립전염조G1기세포비례분별위68.1%、51.6%、60.4%급87.9%.결론 인β-NGF기인가통과EffecteneTM지질체개도유효전염도체외배양적묘각막내피세포중,병촉진세포분렬재생,위진일보장차기인전입인각막내피세포적연구제공실험경험.
Objective To explore the mechanisms of proliferation and regeneration effects of a human nerve growth factor(p-NGF)expression vector(pcDNA4-β-NGF)on the transfected cat corneal endothelial cells in vitro.To provide a new method for long term cultivation of human corneal endothelial cells in vitro and to establish theoretical basis of gene therapy for corneal endothelial defects.Methods It was a experimental study.The human pcDNA4-B-NGF expression vector was constructed and transfected into cultured cat corneal endothelial cells bv EffecteneTM lipofectine transfection technique.The expression of the reporter gene pcDNA4-β-LacZ expression Was used to determine the transfection efficiency 48 hours after the transfection．RT-PCR and immunohistochemistry techniques were used to check the transient expression status at mRNA and protein levels in cat comeal endothelial cells.Mitotic index and methyl thiazolyl tetrazolium(MTT) value were measured and cell numbers at different stages of cell cycles were deterd by flow cytometer 96 hours after transfection.An in vitro quantitative cat corneal endothelial cell traumatic model was established which was used for observing the effect of human B-NGF expression product on the DNA synthesis of cat endothelial cells and heMing process of traumatized endothelial cells.Results Ahuman nerve growth factor(B-NGF)expression vector(pcDNA4-13-NGF)was successfully constructed and confirmed by sequence analysis.Single layered pure cat corneal endothelial cells were obtained by a modified sliced tissue culture technique and confirmed by morphological analysis,neurone specific enolase immunohistoehemistry study and transmission electronic microscope.EffecteneTM lipefectine mediated transfection efficiency of pcDNA4-β-NGF into cat Corueal endothelial cells in vitro was 11.3%.The human β-NGF could be highly expressed in the transfected comeal endothelial cells at mRNA and protein levels.Mitotic index.MTT valae and G1 stage cell numbers,as well as traumatically defected endothelial cells numbers during the healing process of human β-NGF transfected comeal endothelial cells were statistically differed from the pre-transfected cells and control groups.Conclusions EffeeteneTM lipefectine transfection technique could be effectively used for transfecting pcDNA4-B-NGF into cat comeal enl cells in vitro with good efficacy and the gene could stably express to improve the proliferation and regeneration of the cat comeal endothelial cells.This method could be managed as an experimental basis to be applied in the experimental study for transfecting the human B-NGF gene into human corneal endothelial cells.Therefore a new method for resolving the problem of impossible regeneration of corneal endothelial cells could become possible.
