Influence of hypoxia-inducible factor-1 alpha lentiviral vector on osteogenic gene expression of rabbit bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2014.01.010

Abstract

Abstract:

BACKGROUND: Osteogenesis is closely integrated with angiogenesis in bone formation and repair process, and hypoxia-inducible factor 1 alpha (HIF-1α) is considered to be the most important core transcription factor promoting angiogenesis gene regulation, which may promote the formation of blood vessels at hypoxia portion, and thus contribute to bone formation.
OBJECTIVE: To construct the Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) lentiviral eukaryotic expression vectors and to detect their impact on the osteogenic gene expression of rabbit bone marrow mesenchymal stem cells.
METHODS: The Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) lentiviral eukaryotic expression vectors were constructed according to the wild type human HIF-1α gene sequence and the determined restriction sites of human HIF-1α point mutant sequence. Rabbit bone marrow mesenchymal stem cells were transfected with the prepared Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) virus solution.
RESULTS AND CONCLUSION: Immunofluorescence microscopy observations indicated that the cells of Lenti-HIF-1α-IRES-EGFP (wild type) group and Lenti-HIF-1α-IRES-EGFP (point mutant type) group showed no obvious fluorescence on transfected 7 days, and two groups of cells showed a more obvious green fluorescent after transfection 14 days. Quantitative PCR analysis results showed that there were obvious HIF-1α and bonemorphogenetic protein 2 gene expressions on days 7 after transfection and the two genes still showed highly expression levels after transfection 14 days. The two lentiviral eukaryotic expression vectors of Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) could be constructed according to the wild type human HIF-1α gene sequence and the determined restriction sites of human HIF-1α point mutant sequence; HIF-1α gene can promote the osteogenic gene expression of lentivirus-transfected rabbit bone marrow mesenchymal stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: lentivirus, mesenchymal stem cells, bone marrow, bone morphogenetic proteins, viruses, transfection

CLC Number:

R394.2

Zhang Jin-e, Wang Shu-hong, Zhang Xin, Guo Hua-yan, Guo Na, Huang Yuan-liang . Influence of hypoxia-inducible factor-1 alpha lentiviral vector on osteogenic gene expression of rabbit bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(1): 57-62.

Figures/Tables 3

2.1 兔骨髓间充质干细胞培养及慢病毒转染最佳MOI值测定原代细胞培养5 d首次换液时，在倒置相差显微镜下观察，培养皿底部可见少数散在圆形半透明状骨髓间充质干细胞贴壁生长，细胞核略呈卵圆形，周围有大量悬浮细胞为血细胞及造血干细胞。7-10 d再次换液后可见贴壁骨髓间充质干细胞增多并形成散在克隆，细胞胞体膨大，逐渐由圆形向梭形或多边形转化，有时存在大小形态不一的胞浆突起，细胞核较大，位于细胞体一侧，一般含1个核仁，偶见2个。数十个细胞形成集落，初始形成的骨髓基质细胞集落称为成纤维样细胞集落形成单位，悬浮血细胞与造血干细胞随培养时间延长而逐渐坏死，在换液时逐渐被清除。此后，细胞增殖迅速，2周左右融合接近整个培养皿底部，偶见旋涡状排列。传代时，0.25%胰蛋白酶消化后的细胞呈圆形，折光性强且均匀，接种后6 h即开始贴壁并迅速伸展，重新恢复伴有大小不一突起的梭形外观。24 h后绝大多数细胞贴壁生长，呈规则的方向性排列，但无明显集落形成，48 h后贴壁细胞数量增多，形态增大，有些区域见数个细胞形成集落。将生长状态良好的第3代兔骨髓间充质干细胞接种于12孔板中，并按0，30，50，100，200的MOI值梯度用Lenti-GFP病毒进行兔骨髓间充质干细胞慢病毒转染预实验，转染72 h后荧光显微镜观察结果(图2)。图片荧光显示，慢病毒转染兔骨髓间充质干细胞的MOI值选取在30-50为最佳，后续的正式转染实验中即采用MOI=50对兔骨髓间充质干细胞进行慢病毒转染。低氧诱导因子1α基因野生型和点突变型慢病毒转染兔骨髓间充质干细胞：免疫荧光显微镜观察显示，转染7 d后Lenti-HIF1α-IRES-EGFP (野生型)组和Lenti- HIF 1α-IRES- EGFP (点突变型)组细胞均未见明显荧光，转染14 d后两组细胞均呈现较为明显的绿色荧光(图3)。"

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