携带BMP-2基因的慢病毒感染大鼠骨髓间质干细胞定向分化

Rat mesenchymal stem cell can be differentiated into osteoblasts by recombinant human BMP2 lentivirus

目的构建含有人骨形态发生蛋白2(human bone morphogenetic protein 2,hBMP2)cDNA的重组慢病毒DNA,在293T细胞中进行慢病毒包装和表达检测。用慢病毒感染大鼠骨髓间质干细胞后,观察其向成骨细胞分化的情况。方法从GenBank中调出hBMP2基因序列,设计带有NheⅠ、AgeⅠ酶切位点引物。从人肝脏组织中抽提总RNA,再反转录成cDNA,扩增出hBMP2 cDNA的全长序列。测序验证后克隆到慢病毒载体pHIV-CS-CDF-CG-PRE上,通过PCR扩增、酶切、测序等方法鉴定重组慢病毒DNA。将该重组慢病毒DNA同辅助质粒一起共感染293T细胞,通过重组而包装成能表达hBMP2基因的有活性的慢病毒。慢病毒感染大鼠骨髓间质干细胞,通过碱性磷酸酶(AP)、钙沉积定量测定、Ⅰ型胶原蛋白表达观察干细胞向成骨细胞转化的情况。结果①扩增出1.2 kb左右的hBMP2全长cDNA与GeneBank中的hBMP2阅读框架序列完全一致;②成功构建和克隆了含hBMP2基因的慢病毒重组DNA(pHIV-hBMP2);③对重组慢病毒DNA进行了成功包装和分析。④大鼠骨髓间质干细胞感染慢病毒后,碱性磷酸酶分泌增加,钙沉积增加,Ⅰ型胶原蛋白表达增加,成功向成骨细胞转化。结论重组慢病毒携带hBMP2基因感染大鼠骨髓间质干细胞可以使其向成骨细胞定向分化,为组织工程研究提供良好的种子细胞。

Purpose To isolate the full-length human BMP2 cDNA and construct the recombinant lentivirus DNA pHIV-CS-CDF-CG-PRE-hBMP, the recombinant lentivirus was packaged and grew in 293T cells DNA. Then to infect the rats BMSCs with the recombinant lentivirus in order to observe the differentiation of the infected cells. Methods The full length hBMP2 gene sequence was searched out from GenBank. Then complify the cDNA by RT-PCR with two primers design by DNAstar. The validated hBMP2 cDNA was cloned and identified. It was inserted into the lentivirus vetor and packaged in 293T cells. The cDNA was expressed when 293T cells were infected with recombinant lentivirus. The differentiation from rMSCs to osteoblasts was investigated by test alkaline phosphatase （AP）, quantitative assay of calcium deposition and the expression of collagen Ⅰ . Results The amplified hBMP2 gene of 1.2 kb was completely in accordance with the sequence of its open reading frame in GeneBank. The full-length cDNA was cloned in lentivirus vector. The recombinant lentivirus-hBMP2 DNA was packaged and expressed in 293T cells successfully. After recombinant lentivirus infected rMSCs, the secretion of AP, calcium deposition and the expression of collagen Ⅰ were all increased in rMSCs. Conclusions The rMSCs infected with the recombinant lentivirus may differentiate to osteoblasts and would provide favourable cell resource for tissue engineering .