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目的 从正常的人体成骨细胞中克隆IGF 1、PDGF AA和TGFβ1三因子的编码蛋白基因 ,构建上述因子基因的高效表达重组腺病毒 ,旨在为基因治疗研究提供有效的工具。方法 培养正常人体成骨细胞 ,提取细胞总RNA ,RT PCR方法获得IGF 1、PDGF AA和TGFβ1的编码基因。将这些片段克隆到高效表达腺病毒载体Adeno X ,并经过HEK2 93细胞的包装 ,获得成熟的重组腺病毒。Western印迹法检测上述三因子的表达。利用成熟重组腺病毒感染成骨细胞 ,检测细胞增殖和碱性磷酸酶 (ALP)活性的改变。结果 重组T载体、重组腺病毒DNA和成熟的重组腺病毒体中均可得到IGF 1、PDGF AA和TGFβ1基因的PCR片段。Western印迹检测到上述 3种因子蛋白质的表达。当携带上述 3种因子的重组腺病毒感染成骨细胞后 ,细胞增殖明显增强 ,ALP活性明显提高。结论 本实验构建的IGF 1、PDGF AA和TGFβ1因子的高效表达重组腺病毒可以在人体细胞中表达出具有生物活性的蛋白质 ,获得了较好的基因工具。
OBJECTIVE: To clone the gene encoding IGF1, PDGF AA and TGFβ1 from normal human osteoblasts and to construct the recombinant adenovirus with high efficiency of expression of the above factors, so as to provide an effective tool for gene therapy research. Methods Normal human osteoblasts were cultured and total cellular RNA was extracted. The coding genes of IGF1, PDGF AA and TGFβ1 were obtained by RT PCR. These fragments were cloned into adenoviral vector Adeno X and were packaged in HEK2 93 cells to obtain mature recombinant adenovirus. Western blot was used to detect the expression of these three factors. Osteoblasts were infected with mature recombinant adenovirus and the changes of cell proliferation and alkaline phosphatase (ALP) activity were examined. Results PCR fragments of IGF1, PDGF AA and TGFβ1 genes were obtained from recombinant T vector, recombinant adenovirus DNA and mature recombinant adenovirus. Western blot detected the above three factors protein expression. When infected with the above three factors of recombinant adenovirus osteoblasts, cell proliferation was significantly enhanced ALP activity was significantly improved. Conclusion The recombinant adenoviruses expressing IGF1, PDGF AA and TGFβ1 constructed in this study can express bioactive proteins in human cells and obtain better genetic tools.