骨形态发生蛋白-7对胚胎小鼠髓核细胞成骨分化和Notch信号表达的影响

Effects of BMP-7 on osteogenic differentiation of prenatal mouse nucleus pulposus cell and Notch signaling pathway

摘要:

目的：探索骨形态发生蛋白-7（bone morphogenetic protein-7，BMP-7）诱导胚胎小鼠椎间盘髓核细胞（nucleus pulposus，NP）的成骨分化及对经典Notch信号通路的调控作用。方法：重组腺病毒BMP-7（recombinant adenovirus-mediated bone mor－phogenetic protein-7，Ad-BMP-7）、绿色荧光蛋白（green fluorescent protein，Ad-GFP）在HEK293细胞中扩增，感染NP细胞。实验分为BMP-7组（n=3）、GFP组（n=3）、空白组（n=3）。RT-PCR检测BMP-7的mRNA水平表达；Real-time PCR检测成骨指标骨桥蛋白（osteopontin，OPN）、骨保护素（osteoprotegerin，OPG）以及经典Notch信号通路分子的mRNA表达水平；Western blot检测成骨转录因子Runx2和OPN的蛋白表达水平；采用碱性磷酸酶（alkaline phosphatase，ALP）读数和ALP染色检测ALP活性。结果：Ad-BMP-7和Ad-GFP在HEK293细胞中高滴度的扩增。NP细胞中BMP-7的基础表达很低，Ad-BMP-7感染NP细胞可高表达BMP-7，并明显提高OPN、OPG的mRNA水平表达（OPN：BMP-7组21.16±0.45，GFP组1.00±0.07，空白组2.84±1.27，F=613.815，P=0.000，BMP-7组与GFP组、空白组比较均P=0.000；OPG：BMP-7组2.05±0.37，GFP组1.00±0.06，空白组1.22±0.36，F=10.046，P=0.012，BMP-7组与GFP组比较P=0.016，与空白组比较P=0.047），成骨转录因子Runx2及OPN的蛋白表达水平也均高于对照组，特异性成骨指标ALP的活性明显增强，ALP染色呈紫蓝色，明显高于对照组（BMP-7组222 676.8±7 643.2，GFP组31 455.5±4 930.6，空白组42 407.3±10 926.8，F=513.417，P=0.000，BMP-7组与GFP组、空白组比较均P=0.000）。Ad-BMP-7感染3 d可促进Notch信号通路Notch-1、Notch-2、Jag-1、Hey-1的mRNA水平表达增高（Notch-1：BMP-7组5.90±0.66，GFP组2.83±0.32，空白组1.00±0.05，F=94.574，P=0.002，BMP-7组与GFP组比较P=0.003，与空白组比较P=0.004；Notch-2：BMP-7组150.35±11.78，GFP组1.94±0.46，空白组1.01±0.19，F=318.962，P=0.001，BMP-7组与GFP组、空白组比较均P=0.000；Jag-1：BMP-7组7.97±0.00，GFP组2.22±0.71，空白组1.00±0.00，F=166.476，P=0.001，BMP-7组与GFP、空白组比较均P=0.000；Hey-1：BMP-7组11.23±0.4，GFP组0.42±0.04，空白组1.00±0.10，F=1 082.054，P=0.000，BMP-7组与GFP组、空白组比较均P=0.000），但第7天回复到基础表达水平，其余信号分子表达无明显差异。结论：腺病毒介导的BMP-7可诱导NP细胞成骨分化，可能与Notch信号通路部分分子的一过性上调相关。

Abstract:

Objective：To investigate the effects of recombinant adenovirus-mediated bone morphogenetic protein-7（Ad-BMP-7） over-expression on osteogenic differentiation of prenatal mouse intervertebral disc nucleus pulposus（NP） cell，and to explore whether BMP-7 regulates classic Notch signal pathway in this process. Methods：Recombinant Ad-BMP-7 and adenovirus-mediated green fluores－cent protein（Ad-GFP） were amplified by HEK293 cell and were used to infect NP cells. The experiment was designed into BMP-7 group（n=3），GFP group（n=3） and blank group（n=3）. Real-time PCR was performed to detect the mRNA expression level of BMP-7，real-time PCR was carried out to detect the mRNA expression level of osteogenesis index osteopontin（OPN），osteoprotegerin（OPG） as well as the classic Notch signal pathway members. Western blot was used to detect the protein expressions level of osteogenesis transcription factor Runx2 and OPN. Alkaline pho-sphatase（ALP） activity was measured by using ALP assay and staining. Results：The high titers Ad-BMP-7 and Ad-GFP were generated by adenovirus amplification in HEK293. The basic mRNA expression of BMP-7 was almost undetectable in NP cell. Compared with those in blank group，Ad-BMP-7 mediated over-expression of BMP-7 significantly increased the mRNA expressions of OPN，OPG and the protein expressions of osteogenesis transcrip－tion factor Runx2，OPN（OPN：F=613.815，P=0.000；BMP-7 group vs. GFP group and blank group，P=0.000；OPG：F=10.046，P=0.012；BMP-7 group vs. GFP group P=0.016；BMP-7 group vs.blank group，P=0.047）. The activity of ALP，a specific indicator for steogenesis in Ad-BMP-7 group was statistically higher than that in blank group（F=513.417，P=0.000；BMP-7 group vs. GFP group and blank group，P=0.000），with enhanced positive purple-blue stained cells. After 3 days of Ad-BMP-7 infection，the mRNA expression of classic Notch signal pathway members Notch-1，Notch-2，Jag-1，Hey-1 significantly increased（Notch-1：F=94.574，P=0.002；BMP-7 group vs. GFP group，P=0.003；BMP-7 group vs. blank group P=0.004）（Notch-2：F=318.962，P=0.001；BMP-7 group vs. GFP group and blank group，P=0.000）（Jag-1：F=166.476，P=0.001，BMP-7 group vs. GFP group and blank group P=0.000）（Hey-1：F=1 082.054，P=0.000；BMP-7 group vs. GFP group and blank group，P=0.000），while reversed to the baseline at day 7. There was no difference in others Notch signal pathway members among treated groups. Conclusion：Adenovirus-mediated BMP-7 over-expression can induce osteogenic dif－ferentiation of NP cell，which partly due to transient up-regulation of some classic Notch signal pathway members.