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目的:克隆大鼠转化生长因子β3(TGFβ3)基因并研究其转染肝星状细胞(HSC)后的表达情况。方法:采用逆转录—聚合酶链式应(RT-PCR)从大鼠成骨细胞中扩增出特异性片段,经酶切鉴定证实为大鼠TGFβ3cDNA,将此片段插入pcDNA3.1(+)表达载体,构建得到pcDNA3.1(+)-TGFβ3重组质粒。应用阳离子脂质体介导的基因转移技术将TGFβ3表达载体导入大鼠HSC,24 h、48 h、72 h后用荧光定量PCR法进行瞬时表达的检测。结果:重组真核表达载体pcDNA3.1(+)-TGFβ3经限制性内切酶NheI、XbaI酶切,电泳后显示的TGFβ3目的片段和5.4 kb的pcDNA3.1(+)载体片段,测序证实酶切片段与GenBank中登记的TGFβ3全长序列相同,证实pcDNA3.1(+)-TGFβ3真核表达载体构建成功,转染48 h后,肝星状细胞的表达明显增高(P<0.05)。结论:重组TGFβ3真核表达载体构建正确,并在转染大鼠HSC48 h后获得高效表达,为转基因治疗肝纤维化疾病提供理论依据。
OBJECTIVE: To clone the gene of transforming growth factor β3 (TGFβ3) in rat and study the expression of transforming growth factor β3 (TGFβ3) in rat hepatic stellate cells (HSC). Methods: Specific fragments were amplified from rat osteoblasts by reverse transcription-polymerase chain reaction (RT-PCR) and identified as TGFβ3 cDNA by restriction enzyme digestion. The fragment was inserted into pcDNA3.1 (+) Expression vector, construct pcDNA3.1 (+) - TGFβ3 recombinant plasmid. The TGFβ3 expression vector was introduced into rat HSC by cationic liposome-mediated gene transfer technique and detected by fluorescence quantitative PCR at 24 h, 48 h and 72 h. Results: The recombinant eukaryotic expression vector pcDNA3.1 (+) - TGFβ3 was digested by restriction endonuclease NheI and XbaI, and the target fragment of TGFβ3 and 5.4 kb pcDNA3.1 (+ The full-length cDNA sequence of TGFβ3 in GenBank was identical with that in pcDNA3.1 (+) - TGFβ3. The expression of pcDNA3.1 (+) - TGFβ3 was successfully established and the expression of hepatic stellate cells was significantly increased 48 h after transfection (P <0.05). CONCLUSION: The eukaryotic expression vector of recombinant TGFβ3 is constructed correctly, and is highly expressed 48 h after HSC transfection in rats, providing a theoretical basis for transgenic treatment of liver fibrosis.