摘要
目的探讨人骨形态发生蛋白-2(BMP-2)基因通过腺病毒转染青山羊脂肪间充质干细胞(ADSCs)的可行性及其向成骨细胞定向分化的能力。方法取麻醉状态下青山羊腹股沟脂肪组织,经体外分离培养获得ADSCs,转染以腺病毒介导的人BMP-2基因。倒置相差显微镜观察细胞形态,MTT法检测ADSCs生长增殖情况,免疫细胞化学法对ADSCs细胞进行碱性磷酸酶(ALP)活性测定,SABC法检测ADSCsⅠ型胶原的表达,通过RT-PCR、Western blot法检测BMP-2基因的表达水平。结果转染人BMP-2基因的ADSCs形态规则;细胞生长速度快,且随培养时间的延长逐渐增多(P<0.05);成骨诱导后,转染组细胞ALP活性和Ⅰ型胶原表达强度明显高于未转染组(P<0.05);转染组ADSCs BMP-2基因m RNA水平和蛋白含量均明显强于未转染组(P<0.05)。结论经腺病毒介导的人BMP-2基因转染的青山羊ADSCs在体外诱导培养中可以向成骨细胞定向分化,并保持较强的增殖能力;转染后细胞BMP-2目的基因表达增高。
Objective To investigate the feasibility of human bone morphogenetic protein-2 （BMP-2） gene transfection into grey goat adipose tissue-derived mesenchymal stern cells （ADSCs） by adenovirus vector, and to explore the directional differentiation potential of ADSCs into osteoblasts. Methods Inguinal fat tissue was excised from anaesthetized grey goat, and then ADSCs were obtained by isolation and culture in vitro. After the human BMP-2 transfection by adenovirus, inverted phase contrast microscope was used to observe the morphology of ADSCs, cell growth was measured by MTT test, alkaline phosphatase （ALP） activity of ADSCs was examined by immunocytochemistry method, expression of collagen I was measured by SABC method, also, expression levels of BMP-2 gene were measured by RT-PCR and Western blot tests. Results After hmuan BMP-2 transfection by adenovirus, the morphology of ADSCs became regular; Cells grew up rapidly with increasing tendency with culture time going by （P 〈0.05）; After osteoinduction, ALP activity and collagen I expression intensity in transfection group showed significant higher than those in control group （P 〈0.05）; The expression levels of mRNA and protein content of BMP-2 in transfection group were significantly higher than those in control group （P 〈0.05）. Conclusion Grey goat＇s ADSCs transfected by adenovirus vector carrying human BMP-2 gene have the potential of directional differentiation into osteoblasts and good proliferation ability, moreover, the expression of BMP-2 gene become increasing after transfection.
出处
《中国骨科临床与基础研究杂志》
2016年第4期221-227,共7页
Chinese Orthopaedic Journal of Clinical and Basic Research
基金
深圳市科创委基金(20150305190447)
关键词
脂肪间充质干细胞
骨形态发生蛋白质类
腺病毒科
转染
细胞
培养的
细胞分化
成骨细胞
Adipose tissue-derived mesenchymal stem cells
Bone morphogenetic proteins
Adenoviridae
Transfection
Cells, cultured
Cell differentiation
Osteoblasts
