骨形态发生蛋白-2、-3对胚胎小鼠椎间盘细胞的成软骨及成骨作用

Effect of BMP-2 and -3 overexpression on osteogenic and chondrogenic differentiation of prenatal mouse intervertabral disc cells in vitro

目的探索成骨作用的骨形态发生蛋白-2(BMP-2)及抑制成骨作用的骨形态发生蛋白-3(BMP-3)对胚胎小鼠椎间盘细胞的成软骨及成骨作用,为BMPs临床治疗椎间盘相关疾病提供实验依据。方法运用腺病毒介导的BMP-2和BMP-3在胚胎小鼠椎间盘细胞中表达,RT-PCR检测Ⅰ型胶原(ColⅠ)、Ⅱ型胶原(ColⅡ)、蛋白多糖(ACAN)、骨钙素(OC)、骨保护素(OPG)和骨桥蛋白(OPN)成软骨及成骨相关指标mRNA水平的表达,甲苯胺蓝染色检测软骨基质的表达,碱性磷酸酶(ALP)读数及ALP染色检测ALP的活性。结果 BMP-2和BMP-3表达对于椎间盘纤维环(AF)细胞的成软骨和成骨均没有作用。对椎间盘髓核(NP)细胞的软骨相关指标ColⅠ、ColⅡ、ACAN mRNA的表达同样没有影响,但可促进NP细胞的成骨,其中BMP-2和BMP-3均可促进OC mRNA的表达升高,而仅BMP-2可促进其OPG和OPN的mRNA表达升高。甲苯胺蓝染色证明BMP-2和BMP-3对椎间盘细胞软骨基质分泌的影响并不显著。NP细胞在BMP-2和BMP-3腺病毒感染后ALP活性明显增强,在AF细胞中则未观察到这种变化。结论 BMP-2和BMP-3均可促进胚胎小鼠NP细胞的成骨作用,但对AF细胞的成骨及成软骨作用及对NP细胞的成软骨作用没有影响。

Objective To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein （BMP）-2 and -3 overexpressions on chondrogenesis and osteogenesis of prenatal mouse intervertabral disc cells and provide experimental evidences for the application of BMPs in the therapy of disc diseases. Methods The prenatal mouse intervertabral disc cells were infected with a recombinant adenovirus expressing BMP-2 and BMP-3 for 5-7 days, and the expressions of collagen type I （Col I）, collagen type II （Col II）, aggrecan, osteocalcin, osteoprotegerin and osteopontin mRNAs were detected with RT- PCR. The expression of cartilage matrix was evaluated with toluidine blue staining, and alkaline phosphatase （ALP） activity was detected with ALP reading and ALP staining. Results BMP-2 and -3 overexpression did not enhance chondrogenesis and osteogenesis of annulus fibrosus （AF） cells or cause significant increases in the expressions of Col I, Col II or aggrecan mRNA in nucleus pulposus （NP） cells. Adenovirus-mediated overexpression of BMP-2 and BMP-3, however, promoted osteogenesis of NP cells and significantly increased the expression of osteocalcin mRNA; the overexpression of BMP- 2, but not BMP- 3, enhanced the mRNA expressions of osteoprotegerin and osteopontin. Toluidine blue staining demonstrated that BMP- 2 and BMP- 3 overexpression did not obviously affect the secretion of cartilage matrix. In NP cells, BMP-2 and -3 overexpression significantly enhanced ALP activity, which was not observed in AF cells. Conclusion Adenovirus-mediated BMP-2 and -3 overexpression can promote the osteogenic differentiation of NP cells but can not affect osteogenesis of AF cells or chondrogenesis of NP cells.