胰岛素样生长因子-1与血小板源生长因子-AA以及肿瘤生长因子β1基因克隆及其在腺病毒载体中的表达

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HEK293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。

Cloning and expression of insulin-like growth factor-1 platelet-derived growth factor-AA,and tumor growth factor beta1 genes in adenovirus vector

OBJECTIVE: To establish recombinant adenovirus highly expressing genes of insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-AA (PDGF-AA), and tumor growth factor beta1 (TGF beta 1) to be used in gene therapy. METHODS: Normal human osteoblasts were obtained from cancellous bone of lilium left from bone grafting and then cultured. Total RAN was extracted and IGF-1, PDGF-AA, and TGF beta 1 cDNAs was obtained by RT-PCR. Vector p-Shuttle containing these cNDAs was cloned into adenovirus and then the recombinant adenovirus was transfected into HEK293 cells. Recombinant adenovirus containing reporter gene LacZ was used in control group. Western blotting was used to detect the expression of these growth factors. Human osteoblasts were cultured and transfected with recombinant adenovirus. MTT method was used to detect the proliferation of the cells. Paranitrophenol method was used to examine the activity of alkaline phosphatase (ALP) in osteoblasts. RESULTS: Expression of IGF-1, PDGF-AA, and TGF beta 1 cDNA and expression of IGF-1, PDGF-AA, and TGF beta 1 proteins were found in HEK293 cells transfected with the recombinant adenovirus and not in the control HEK293 cells. The proliferation and ALP activity of osteoblasts transfected with the recombinant virus were significantly increased in comparison with those of the control osteoblasts (all P < 0.010). Immunohistochemical staining showed significant brown particles in the osteoblasts transfected with the recombinant virus and none in the control osteoblasts. CONCLUSION: The recombinant adenovirus thus constructed expresses the proteins of several growth factors with bioactivity in human cells and can be used as a satisfactory gene tool.