摘要
目的探讨重组腺病毒(Ad-GFP-NELL1)介导的NELL1因子对小鼠成骨细胞(MC3T3E1)增殖、分化及OCN(骨钙素)和OPN(骨桥蛋白)基因表达的影响。方法免疫荧光染色鉴定重组腺病毒(Ad-GFP-NELL1)转染细胞后绿色荧光蛋白(GFP)及NELL1的表达;不同转染滴度Ad-GFP-NELL1转染MC3T3E1细胞,荧光显微镜下观察转染效果;将MC3T3E1细胞分为对照组,NELL1组(Ad-GFP-NELL1转染)和GFP组(Ad-GFP转染),利用CCK-8试剂盒检测并绘制生长曲线;观察和计量茜素红染色后钙结节数目;定量PCR检测成骨相关基因OCN,OPN表达的改变。结果免疫荧光显示Ad-GFP-NELL1转染大鼠骨髓基质干细胞72h后能同时表达NELL1和GFP;在最佳转染滴度200pfu/cell转染条件下,Ad-GFP-NELL1及Ad-GFP对MC3T3E1细胞增殖没有明显影响(P>0.05)。NELL1组细胞钙结节数目(7±2)比空白对照组(2±1)及Ad-GFP组(2±1)明显增多(P<0.05),OCN(3.7倍),OPN(5.1倍)的表达也明显上升。结论 Ad-GFP-NELL1转染MC3T3E1细胞后能有效促进细胞的成骨分化作用,且对其增殖无明显毒性作用。
Objective To study the effect of recombinant adenovirus-mediated NELL1 on proliferation and differentiation of osteoblasts（MC3T3E1） and expression of osteocalcin（OCN） and osteopontin（OPN） in mice. Methods Expression of recombinant adenovirus-transfected green fluorescence protein（GFP） and NELL1 was identified with immunofluorescence staining. MC3T3E1 transfected with adenovirus-mediated NELL1 were observed under fluorescent microscope and divided into control group, NELL1 group, and GFP group. Growth curve was plotted for MC3T3E1 with a CCK-8 kit. The number of calcium nodules was calculated with alizarin red staining. Expression of osteoblast master genes （OCN and OPN） was detected by real-time PCR. Results The immunofluorescence showed that the bone marrow ma^ix cells expressed both GFP and NELL1 72h after transfected with adenovirus-mediated NELL1. At the best multiplicity of infection （MOI）=200pfu/cell, the adenovims-mediated NELL1 and GFP had no significant effect on the proliferation of MC3T3E1. The number of calcium nodules was significantly greater in NELL1 group than in control group and GFP group（7 ± 2 vs 2 ± 1 and 2 ± 1, P〈0.05）. The expression levels of OCN and OPN were 3.7-fold and 5.1-fold higher in NELL1 group than in control group（P〈0.05）. Conclusion Adenovirus-mediated NELL1 can effectively promote the differentiation of osteoblasts with no significant toxicity to their proliferation.
出处
《军医进修学院学报》
CAS
2012年第5期509-511,共3页
Academic Journal of Pla Postgraduate Medical School
基金
国家自然科学基金项目(30872621
30872633)~~
