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目的:探索小鼠11β-HSD1基因过表达的前成骨细胞系的建立方法。方法:构建过表达小鼠11β-HSD1基因的慢病毒载体,包装、收集、纯化慢病毒,感染小鼠MC3T3-E1前成骨细胞系,用BSD(一种核苷抗生素)筛选出感染成功的细胞,荧光定量PCR及Western blot检测11β-HSD1过表达情况,诱导成骨分化后用茜素红染色法染色矿化结节。结果:成功构建了小鼠11β-HSD1基因过表达慢病毒载体,高效感染MC3T3-E1细胞系。荧光定量PCR及Western blot结果显示,11β-HSD1过表达病毒组细胞11β-HSD1 mRNA水平是空载病毒对照组的17.4倍,蛋白表达量比空载病毒对照组增加。茜素红染色结果表明慢病毒感染的细胞成骨分化良好,细胞正常成骨分化功能未受影响,11β-HSD1过表达细胞成骨分化减少。结论:本研究成功建立了过表达小鼠11β-HSD1的前成骨细胞系,为研究11β-HSD1对成骨细胞的具体作用提供了研究基础。
OBJECTIVE: To explore a method for establishing a pre-osteoblast line that overexpression mouse 11β-HSD1 gene. Methods: The lentiviral vector overexpressing mouse 11β-HSD1 gene was constructed, and the lentivirus was packaged, purified, lentivirus and MC3T3-E1 pre-osteoblasts were infected with BSD (a kind of nucleoside antibiotics) Cell, quantitative PCR and Western blot were used to detect the 11β-HSD1 overexpression. After osteogenic differentiation, mineralized nodules were stained by alizarin red staining. Results: The mouse 11β-HSD1 gene overexpression lentiviral vector was successfully constructed and MC3T3-E1 cells were efficiently infected. Fluorescent quantitative PCR and Western blot results showed that 11β-HSD1 mRNA level in 11β-HSD1 over-expressing virus group was 17.4-fold higher than that in control group without empty vector, and the protein expression level was increased compared with control group. Alizarin red staining results showed that the cells infected by lentivirus had well osteogenic differentiation, normal osteogenic differentiation was not affected, and the osteoblastic differentiation of 11β-HSD1 overexpression decreased. Conclusion: The present study successfully established a pre-osteoblast line that overexpressed mouse 11β-HSD1 and provided the basis for further studies on the specific effects of 11β-HSD1 on osteoblasts.