大鼠γ-干扰素基因转染大鼠肺泡巨噬细胞和气道上皮细胞的实验研究  

Transfection of recombined rat IFN-γ into rat alveolar macrophage and airway epithelial cells in vitro

作　　者：黄桂君[1] 农江[2] 马壮[1] 钱桂生[1] 李淑萍[1] 陈维中[1] 

机构地区：[1]第三军医大学附属新桥医院全军呼吸内科研究所,重庆400037 [2]解放军第303医院病理科,南宁530021

出　　处：《第三军医大学学报》2002年第1期73-75,共3页Journal of Third Military Medical University

基　　金：全军"十五"攻关课题面上项目 (0 1MB0 0 1)

摘　　要：目的　构建重组大鼠γ 干扰素真核表达载体 ,探讨其转染大鼠肺泡巨噬细胞 (AM)和气道上皮细胞的可行性及表达水平。方法　采用RT PCR技术克隆了大鼠γ 干扰素 ,并将其定向克隆到逆转录病毒载体pLXSN上 ,构成真核表达重组载体。将重组载体转染培养的AM和气道上皮细胞。用PCR法鉴定重组载体整合在细胞基因组DNA中。并检测细胞培养上清中γ 干扰素浓度和活性。结果　DNA测序证明 ,构建的重组载体中γ 干扰素的基因方向正确 ,DNA序列与GeneBank中大鼠的γ 干扰素cDNA序列相同。转染后AM和气道上皮细胞基因组DNA的PCR产物电泳可见转染的重组载体DNA片段条带。pLXSN IFN载体组的AM和气道上皮细胞培养上清中大鼠γ 干扰素浓度和活性显著高于pLXSN空载体组 (P <0 .0 1)。结论　本研究构建的大鼠γ 干扰素重组表达载体可成功地转染大鼠AM和气道上皮细胞 ,整合入基因组DNA ,并分泌有活性的γ 干扰素 。Objective To recombine IFN γ expression vector and study its expression in alveolar macrophage (AM) and airway epithelial cells of Wistar rats after transfection in vitro . Methods IFN γ gene was cloned from Wistar rats with RT PCR, and then inserted into pLXSN vector with DNA sequencing. The vector was transfected into AM and airway epithelial cells in cell culture by using cationic liposomes. The integration of pLXSN IFN and pLXSN with the genomic DNA of the transfected cells was detected with PCR. And the expression level and activity of IFN γ in the supernatant were investigated with ELISA and L929 cytopathogenicity inhibition assay. Results DNA sequencing proved that the sequence of the cloned IFN γgene was identical to its counterpart in GenBank and the recombinant pLXSN IFN expression vector was successfully constructed. A PCR product of 620 bp in the cells with pLXSN IFN transfection and one of 140 bp in pLXSN transfected cells were demonstrated in agrose gel with eletrophoresis. The levels and activity of IFN γ in the culture supernatant after pLXSN IFN transfection were markedly higher than those of the cells transfected with pLXSN ( P <0.01). Conclusion The recombinant IFN γ gene expression vector is successfully transfected into AM and airway epithelial cells in vitro . It can integrate with the genomic DNA of the cells, and express active IFN γ, which is useful for further investigation in vivo .

关 键 词：Γ-干扰素 基因转染 肺泡巨噬细胞 气道上皮细胞 大鼠 

分 类 号：R563[医药卫生—呼吸系统] R978.7[医药卫生—内科学]