Department of Orthopaedic Surgery, The Second Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang Province 325000, People's Republic of China.
J Cell Biochem. 2013 Aug;114(8):1720-8. doi: 10.1002/jcb.24512.
A range of biological and molecular effects caused by nicotine are considered to effect bone metabolism. Vitamin C functions as a biological antioxidant. This study was to evaluate the in vitro effects of nicotine on human bone marrow stromal cells and whether Vitamin C supplementation show the antagonism action to high concentration nicotine. We used CCK-8, alkaline phosphatase (ALP) activity assay, Von Kossa staining, real-time polymerase chain reaction and Western Blot to evaluate the proliferation and osteogenic differentiation. The results indicated that the proliferation of BMSCs increased at the concentration of 50, 100 ng/ml, got inhibited at 1,000 ng/ml. When Vitamin C was added, the OD for proliferation increased. For ALP staining, we found that BMSCs treated with 50 and 100 ng/ml nicotine showed a higher activity compared with the control, and decreased at the 1,000 ng/ml. Bone morphogenetic protein-2 (BMP-2) expression and the calcium depositions decreased at 100 and 1,000 ng/ml nicotine, while the addition of Vitamin C reversed the down regulation. By real-time PCR, we detected that the mRNA expression of collagen type I (COL-I) and ALP were also increased in 50 and 100 ng/ml nicotine groups (P < 0.05), while reduced at 1,000 ng/ml (P < 0.05). When it came to osteocalcin (OCN), the changes were similar. Taken all together, it is found that nicotine has a two-phase effect on human BMSCs, showing that low level of nicotine could promote the proliferation and osteogenic differentiation while the high level display the opposite effect. Vitamin C could antagonize the inhibitory effect of higher concentration of nicotine partly.
尼古丁引起的一系列生物学和分子效应被认为会影响骨代谢。维生素 C 作为一种生物抗氧化剂。本研究旨在评估尼古丁对人骨髓基质细胞的体外影响，以及维生素 C 补充是否对高浓度尼古丁表现出拮抗作用。我们使用 CCK-8、碱性磷酸酶（ALP）活性测定、Von Kossa 染色、实时聚合酶链反应和 Western Blot 来评估增殖和成骨分化。结果表明，BMSCs 的增殖在 50、100ng/ml 的浓度下增加，在 1000ng/ml 时受到抑制。当添加维生素 C 时，增殖的 OD 值增加。对于 ALP 染色，我们发现，与对照组相比，50 和 100ng/ml 尼古丁处理的 BMSCs 表现出更高的活性，而在 1000ng/ml 时则降低。骨形态发生蛋白-2（BMP-2）表达和钙沉积在 100 和 1000ng/ml 尼古丁时降低，而添加维生素 C 则逆转了下调。通过实时 PCR，我们还检测到 50 和 100ng/ml 尼古丁组中胶原 I（COL-I）和 ALP 的 mRNA 表达也增加（P<0.05），而在 1000ng/ml 时降低（P<0.05）。对于骨钙素（OCN），变化也是相似的。总的来说，发现尼古丁对人 BMSCs 有两相作用，表现为低水平的尼古丁可以促进增殖和成骨分化，而高水平则显示相反的效果。维生素 C 可以部分拮抗更高浓度尼古丁的抑制作用。
