建立pEGFP—N1-TGF-β1重组质粒和脂质体介导转染原代培养新生猪AEC-Ⅱ方法

Construction of pEGFP-N1-TGF-β1 recombinant plasmid and transfected it into primary cultured AEC-Ⅱ of neonatal piglet using lipofectamine 2000

目的构建pEGFP—N1—TGF-β1重组质粒，利用脂质体介导方法将重组质粒转染入原代培养的新生猪肺泡Ⅱ型上皮细胞（AEC-Ⅱ）中，为构建实验性干预急性肺损伤的真核表达载体及转染原代培养AEC-Ⅱ提供方法学基础。方法应用含人TGF—β1CDS的质粒，设计PCR引物扩增出其CDS片段，将其与空质粒pEGFP—N1用Xhol和EcoRI双酶切，T4DNA Ligase连接双酶切产物，将连接产物转化入DH5α中，培养16h后抽提质粒，PCR法鉴定和测序证实TGF—β1 CDS序列正确。采用Lipofectamine 2000脂质体介导转染，将新生猪AEC—Ⅱ原代培养48h至细胞融合80％-90％，换为无血清培养液；将质粒DNA（pEGFP—N1-TGF—β1重组质粒）和Lipofectamine 2000加至DMEM培养液中混匀，置于培养箱中培养4—6h，更换为完全培养液；转染24—48h后，荧光显微镜下观察细胞EGFP表达水平。结果PCR法及测序鉴定证实TGF—β1 CDS序列正确，重组质粒构建成功，采用Nanovue超微量分光光度计测浓度质粒浓度为195．5μg/mL，转染后48h在荧光显微镜下可见细胞有绿色荧光表达，证实已将其成功转染入原代培养的AEC—Ⅱ中。结论成功构建了pEGFP-N1—TGF-β1真核表达载体，并首次采用脂质体介导方法将重组质粒转染入原代培养的新生猪AEC—Ⅱ中，为开展其他实验性干预ALI的基因转染并进一步研究其修复作用及转归提供方法学基础。

Objective To construct pEGFP-N1-TGF-β1 recombinant plasmid and transfect it into primary cultured neonatal piglet type Ⅱ alveolar epithelium cell （ AEC-Ⅱ） by using lipofectamine 2000, in order to provide basis of methodology for producing recombinant plasmids for transplantation of transfected AEC-Ⅱ into ALI/ARDS animal model lungs. Methods PCR primers were designed to amplify the human TGF-β1 cDNA fragment from plasmid. XhoI and EcoRI were used for double digesting the empty plasmid pEGFP-N1 and cDNA fragment of human TGF-β1. Then the products of double enzyme digestion by using T4 DNA ligase were connected and transformed into DH5α and cultured over night for 16 hours. The structure of recombinant plasmid was identified by using PCR and base sequencing to verify the correctness of pEGFP-N1-TGF-β1 recombinant plasmid. It was then transfected into primarily cultured AEC-Ⅱ by lipofectamine2000 mediated transfection and cultured for another 48 hour. Plasmid DNA （ pEGFP-N1-TGF-β1 recombinant plasmid） and lipofectamine 2000 were added into serum-free DMEM respectively, then DNA suspension and Lipofectamine 2000 suspension were blended together and added into cells. After 24 -48 hours later, the expression level of enhanced green fluorescent protein （EGFP） was evaluated under fluorescence microscope. Results The structure of vector was verified as pEGFP-N1-TGF-β1 recombinant plasmid by using PCR and base sequencing.Green fluorescence found in some cells showed that the pEGFP-N1-TGF-β1 recombinant plasmids had been successfully transfected into primary cultured AEC-Ⅱ, however, the transfection efficiency still need to be further improved such as repeating the transfection procedure once again or using adenovirus mediated transfection method to improve the efficiceny and to transplant the cells into animal lungs eventually. Conclusions pEGFP-N1-TGF-β1 recombinant plasmid was successfully constructed and, for the first time, transfected into primarily cuhured AEC-Ⅱ of newborn piglets. This established method should be useful for investigation of therapeutic effect and outcomes of lung with experimental acute lung injury.