摘要
目的应用原核细胞基因工程方法生产出有活性的骨形态发生蛋白-4(bonemorphoge-neticprotein-4,BMP-4),观察其对骨髓基质干细胞生物学行为的影响。方法利用RT-PCR技术,从成熟的人胎盘组织中扩增出长0.34kb编码人BMP-4成熟肽的基因序列,装入表达载体pET-22b(+),转化大肠杆菌BL-21菌株,并诱导目的蛋白表达,SDS-PAGE检测表达蛋白。获得的蛋白制品经小鼠异位成骨测活证实后,诱导培养的兔骨髓基质干细胞,观测细胞形态变化、碱性磷酸酶和骨钙素的含量。结果大肠杆菌中目的蛋白表达量达菌体蛋白的15%,该蛋白可诱导骨髓基质干细胞向成骨细胞分化,形成钙结节,碱性磷酸酶和骨钙素的含量也明显增加。结论大肠杆菌可表达出有活性的BMP-4,该蛋白可诱导骨髓基质干细胞向成骨细胞分化。
Objective To produce bioactive human bone morphogenetic protein-4 by Escherichia coli genetic engineering and investigate the effect of the product, recombinant BMP-4, on the bone marrow stem cells. Methods cDNA of human morphogenetic protein-4 mature peptide was obtained by RT PCR from tissue of human placenta. The gene was constructed in the pET-22b(+) expression vector and expressed in Escherichia coli BL-21 after transformation and induction by IPTG. The harvested protein was proved to be bioactive by inducing ectopic bone information in mouse thigh. The protein was applied to induce the cultured bone marrow stem cell. Shape change of the cell, ability of ALP (alkaline phosphatase) and concentration of OC (osteocalcin) were investigated. Results SDS-PAGE revealed a new protein band that located in position of 14×103 after 4 hours induction, the new protein made 15% of total bacteria protein, the rhBMP-4 could induce the cultured bone marrow stem cell of New Zealand rabbit to differentiate into osteoblasts and form calcified node. The ability of ALP and concentration of OC of tested group increased significantly than that of the control group. Conclusion The bioactive rhBMP-4 can be produced by Escherichia coli genic engineering, this protein can induce bone marrow stem cells to differentiate into osteoblast cells.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2002年第11期685-689,共5页
Chinese Journal of Orthopaedics
