Huang Li, Qiu Ni, Zhang Che, Wei Hong-Yan, Li Ya-Lin, Zhou Hong-Hao, Xiao Zhou-Sheng
Institute of Clinical Pharmacology, Central South University, Changsha 410078, China.
Acta Pharmacol Sin. 2008 May;29(5):580-6. doi: 10.1111/j.1745-7254.2008.00778.x.
To investigate the effect of nitroglycerin (NTG) on cell proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (HBMSC) and its mechanisms.
Primary HBMSC were cultured in osteogenic differentiation medium consisting of phenol red-free alpha-minimum essential media plus 10% fetal bovine serum (dextran-coated charcoal stripped) supplemented with 10 nmol/L dexamethasone, 50 mg/L ascorbic acid, and 10 mmol/L beta-glycerophosphate for inducing osteoblastic differentiation. The cells were treated with NTG (0.1-10 micromol/L) alone or concurrent incubation with different nitric oxide synthase (NOS) inhibitors. Nitric oxide (NO) production was measured by using a commercial NO kit. Cell proliferation was measured by 5-bromodeoxyuridine (BrdU) incorporation. The osteoblastic differentiation of HBMSC culture was evaluated by measuring cellular alkaline phosphatase (ALP) activity and calcium deposition, as well as osteoblastic markers by real-time RT-PCR.
The treatment of HBMSC with NTG (0.1-10 micromol/L) led to a dose-dependent increase of NO production in the conditional medium. The release of NO by NTG resulted in increased cell proliferation and osteoblastic differentiation of HBMSC, as evidenced by the increment of the BrdU incorporation, the induction of ALP activity in the early stage, and the calcium deposition in the latter stage. The increment of NO production was also correlated with the upregulation of osteoblastic markers in HBMSC cultures. However, the stimulatory effect of NTG (10 micromol/L) could not be abolished by either N(G ) -nitro-L-arginine methyl ester, an antagonist of endothelial NOS, or 1400W, a selective blocker of inducible NOS activity.
NTG stimulates cell proliferation and osteoblastic differentiation of HBMSC through a direct release of NO, which is independent on intracellular NOS activity.
研究硝酸甘油（NTG）对人骨髓间充质干细胞（HBMSC）细胞增殖和成骨分化的影响及其机制。
原代HBMSC在无酚红的α-最低必需培养基加10%胎牛血清（葡聚糖包被活性炭处理）组成的成骨分化培养基中培养，添加10 nmol/L地塞米松、50 mg/L抗坏血酸和10 mmol/Lβ-甘油磷酸钠以诱导成骨分化。细胞单独用NTG（0.1 - 10 μmol/L）处理或与不同的一氧化氮合酶（NOS）抑制剂共同孵育。使用商用NO试剂盒测量一氧化氮（NO）生成量。通过5-溴脱氧尿苷（BrdU）掺入法测量细胞增殖。通过测量细胞碱性磷酸酶（ALP）活性和钙沉积以及实时逆转录聚合酶链反应检测成骨标志物来评估HBMSC培养物的成骨分化。
用NTG（0.1 - 10 μmol/L）处理HBMSC导致条件培养基中NO生成量呈剂量依赖性增加。NTG释放的NO导致HBMSC细胞增殖和成骨分化增加，这通过BrdU掺入增加、早期ALP活性诱导以及后期钙沉积得以证明。NO生成量的增加也与HBMSC培养物中成骨标志物的上调相关。然而，内皮型NOS拮抗剂N（G）-硝基-L-精氨酸甲酯或诱导型NOS活性的选择性阻滞剂1400W均不能消除NTG（10 μmol/L）的刺激作用。
NTG通过直接释放NO刺激HBMSC的细胞增殖和成骨分化，这与细胞内NOS活性无关。
