课题背景：近年来研究发现，钙通道阻滞剂可以通过调节细胞内外Ca2+离子的浓度来影响细胞外基质的合成和代谢过程，尤其是影响胶原的合成和分泌，临床用于纤维化疾病、硬皮病、增生性瘢痕及术后粘连等细胞外基质代谢异常疾病的预防和治疗获得良好效果。同时在神经再生研究领域，瘢痕对神经功能的影响越来越受到人们的重视，神经卡压综合征、神经损伤后瘢痕形成过程都出现细胞外基质的代谢异常。本实验则从减轻神经瘢痕，改善再生环境角度研究神经再生的抑制因素，希望成为研究神经再生的又一切入点。

应用要点：本实验从减轻神经瘢痕，改善再生环境的角度探讨了神经再生的抑制因素，这是本实验理论上有别于其他观点的地方。另外，实验应用钙通道阻滞剂作用于神经瘢痕，把瘢痕的模型和神经再生的模型交叉应用于神经瘢痕的实验中，为周围神经修复的研究提供了新的途径。

同行评价：该文观察钙通道阻滞剂维拉帕米对体外培养神经瘢痕成纤维细胞增殖及胶原分泌的影响，具有一定的新颖性，得出了较好的结果。

摘要
目的：钙通道阻滞剂可抑制瘢痕成纤维细胞的增殖及胶原蛋白的分泌，减少瘢痕的增生。实验拟进一步观察钙通道阻滞剂对体外培养神经瘢痕成纤维细胞增殖及胶原分泌的影响。
方法：实验于2006-03/06在吉林大学中日联谊医院中心实验室完成。①实验材料：Wistar大鼠10只；盐酸维拉帕米注射液为上海禾丰制药有限公司产品。②实验过程及分组：制作大鼠双侧坐骨神经损伤模型，术后2周取损伤处神经瘢痕，按组织块法培养神经瘢痕成纤维细胞，实验所用细胞为4~8代，分4组，其中3组培养基中维拉帕米药物浓度分别为10，50及100 μmol/L，1组为空白对照组。③实验评估：分别用四甲基偶氮唑盐法观察细胞增殖能力、羟脯氨酸比色法测定细胞上清液中的胶原含量和胞浆中的胶原含量。
结果：①四甲基偶氮唑盐法观察细胞增殖结果：钙通道阻滞剂对神经瘢痕成纤维细胞的生长增殖具有明显抑制作用，且呈现剂量依赖性(P < 0.05)。②羟脯氨酸比色法测定胶原含量：钙通道阻滞剂可使分泌到细胞培养上清中的胶原含量明显减少，以100 μmol/L浓度组作用最为显著(P < 0.05);胞浆中的胶原含量增加，具有明显剂量依赖性，以100μmol/L浓度组作用最为显著（P < 0.05）。
结论：钙通道阻滞剂可以抑制神经瘢痕成纤维细胞的增殖及胶原分泌。
关键词：神经瘢痕；钙通道阻滞药；维拉帕米；成纤维细胞；胶原；组织构建

薛金伟，姜志刚，杨光，李锐，尹维田. 钙通道阻滞剂对体外培养神经瘢痕成纤维细胞增殖及胶原分泌的影响 [J].中国组织工程研究与临床康复，2008，12(7):1239-1242 [www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1239(ps).pdf]

Effects of calcium channel blocking agent on fibroblast proliferation and collagen synthesis in nerve scars cultured in vitro

Abstract

AIM：Calcium channel blocking agent can inhibit the proliferation of scar-derived fibroblasts and synthesis of collagen, and inhibit the formation of hypertrophic scar. This study observed the effect of calcium channel blocking agent on fibroblast proliferation and collagen secretion of nerve scars cultured in vitro.
METHODS: The experiment was performed at the Central Laboratory of China-Japan Union Hospital of Jinlin University from March to June 2006. ①Ten Wistar rats were made into models of bilateral sciatic nerve injury. Two weeks after modeling, injured nerve scars were obtained and fibroblasts were cultivated according to tissue culture method. The cells of the fourth to eighth passage were selected and divided into 4 groups. In 3 Verapamil groups, cells were cultured in culture medium containing 10, 50 and 100 μmol/L Verapamil (Shanghai Harvest Pharmaceutical Co., Ltd.). ②Cell proliferative activity was investigated by MTT method and the collagen content in supernatant fluid of cell and endochylema were examined by hydroxyproline colorimetric analysis.
RESULTS: ①Calcium channel blocking agent could significantly inhibit the growth and proliferation of fibroblasts in a dose dependent manner (P < 0.05). ②Calcium channel blocking agent remarkably decrease collagen content in supernatant of cell culture but increase collagen content in endochylema in a dose dependent manner, especially in 100 μmol/L group (P < 0.05).
CONCLUSION: Calcium channel blocking agent can inhibit fibroblast proliferation and collagen secretion of nerve scars.

Xue JW, Jiang ZG, Yang G, Li R, Yin WT. Effects of calcium channel blocking agent on fibroblast proliferation and collagen synthesis in nerve scars cultured in vitro.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(7):1239-1242(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1239(ps).pdf]