应用要点：①为了突出研究胰岛素样生长因子Ⅰ对肌腱细胞体外培养条件下增殖的影响，并初步探讨其量效关系，作者采用罗曼鸡的肌腱细胞完成实验，干扰因素少，操作较简便，效果较稳定。②实验结果发现胰岛素样生长因子Ⅰ在10 μg/L浓度时效价比最高，而在超过10 μg/L时，细胞增殖并非呈上升趋势，而有下降趋势。

偏倚或不足：一方面，罗曼鸡肌腱与人体肌腱存在一定差别，所以试验结果的临床意义受一定限制。另外，虽然对胰岛素样生长因子Ⅰ作用的量效关系作了初步探讨，但是由于经费和时间等条件的限制，试验的大环境是在传代为第2代的肌腱细胞，这一代细胞本身增殖活力较强，外界因素的干扰作用就相对较弱，所以具体的量效关系指标可能需要进一步推敲不能盲目定论。

相关链接：目前，关于组织工程人工肌腱的研究主要包括以下几个方面的内容：①细胞支架的研究。②种子细胞的研究。③生长因子对肌腱细胞增殖的研究。④力学刺激对肌腱细胞作用的研究。

摘要
目的：胰岛素样生长因子Ⅰ是一种潜在的促有丝分裂剂，对肌腱细胞有促进分裂增殖的作用。实验将不同剂量胰岛素样生长因子Ⅰ作用于第2代肌腱细胞，进一步验证其对细胞增殖的影响并探讨其量效关系。
方法：实验于2004-11/2005-04在天津医院实验室完成。①实验材料：天津医院实验室提供的精选法国罗曼鸡受精鸡蛋200只，在孵育19 d时，随机取出10只鸡胚肌腱。②实验过程及分组：分离培养肌腱细胞，观察肌腱细胞形态及生长规律。取第2代肌腱细胞接种于六孔板，分别给予不同浓度的胎牛血清，观察血清浓度对肌腱贴壁及生长的影响。取第2代肌腱细胞接种于96孔板，分为7组。前5组分别加入含不同剂量胰岛素样生长因子Ⅰ（1，5，10，50，200 μg/L）的体积分数为0.02的胎牛血清培养液；第6组加入体积分数为0.05的胎牛血清培养液做为阳性对照，第７组加入体积分数为0.02的胎牛血清培养液做为阴性对照。③实验评估：采用四甲基偶氮唑盐法及瑞士－姬姆萨染色观察不同剂量的胰岛素样生长因子Ⅰ对细胞增殖的影响。
结果：①肌腱细胞贴壁后生长很快，原代细胞1周左右即可传代，增殖速度与营养液中胎牛血清浓度呈正相关。②肌腱细胞增殖速度随胰岛素样生长因子Ⅰ剂量加大而有增加趋势。第２天、第4天，胰岛素样生长因子Ⅰ1 μg/L组、5 μg/L组和阴性对照组之间差异无显著性(P > 0.05)；胰岛素样生长因子Ⅰ10 μg/L组、50 μg/L组和200 μg/L组之间差异无显著性(P > 0.05)。②第2天，阳性对照组增殖高于胰岛素样生长因子Ⅰ1 μg/L组与5 μg/L组，低于10 μg/L组、50 μg/L组和200 μg/L组，差异有显著性(P < 0.000或< 0.006)；第4天，阳性对照组增殖高于胰岛素样生长因子Ⅰ各剂量组和阴性对照组，差异有显著性(P < 0.000或< 0.006)。③第2天、第4天，胰岛素样生长因子Ⅰ1μg/L组、5μg/L组和阴性对照组低于10μg/L组，50μg/L组，200μg/L组，差异有显著性(P < 0.000)；阳性对照组增殖高于阴性对照组，统计分析差异有显著性(P < 0.000)。
结论：胰岛素样生长因子Ⅰ对肌腱细胞增殖的促进作用，随胰岛素样生长因子Ⅰ浓度增高而有增高趋势，10 μg/L为其较适合浓度，同时发现培养血清浓度对肌腱细胞有其特殊作用，浓度越高，肌腱细胞贴壁越快，并对增殖有明显促进作用。
关键词：肌腱/细胞；胰岛素样生长因子Ⅰ；组织构建

邱南海，夏英鹏，李明新，李瑞华，阚世廉.胰岛素样生长因子Ⅰ对组织工程肌腱细胞增殖影响的量效关系[J].中国组织工程研究与临床康复，2008，12(2):221-226 [www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-221(ps).pdf]

Dose-effect relationship of insulin-like growth factor Ⅰand the proliferation of tenocytes in tissue engineering

Abstract

AIM：Insulin-like growth factor Ⅰ(IGF-Ⅰ) is a potent mitogen and powerful stimulator for the division and growth of tenocytes. In this study, we cultured the tenocytes of the second generation with IGF-Ⅰat different doses to verify the effects of IGF-Ⅰon cell proliferation and the dose effect.
METHODS: The experiment was performed in the laboratory of Tianjin Hospital from November 2004 to April 2005. ①200 France Lohmann fertilized eggs were provided by the laboratory of Tianjin Hospital. On day 19, the tendons of 10 embryo chickens were obtained. ②Tenocytes were isolated and cultured. The cell morphous and growth rules were observed. The second generation cells were seeded onto 6-well culture plate and added with fetal calf serum (FCS) of different concentrations. The effects of serum concentration on cell attachment and growth were observed. The second generation tenocytes were seeded onto 96-well culture plate and divided into 7 groups. The former 5 groups were cultured in 0.02 volume fraction FCS culture medium containing IGF-Ⅰat doses of 1, 5, 10, 50, 200μg/L; the sixth group was cultured in 0.05 volume fraction FCS culture medium as positive control, and the seventh group was cultured in 0.02 volume fraction FCS culture medium as negative control. ③The effects of IGF-Ⅰon cell proliferation were observed with MTT test and Swiss-gimmsa taint test.
RESULTS: ①The tenocytes grew very fast after attaching to the wall. The primary cells started to passage in about 1 week. The proliferative rate of tenocytes was positively correlated with the concentration of FCS in culture medium. ②The proliferative rate of tenocytes was accelerated with the increase in IGF-Ⅰdose. There was no difference among the 1μg/L and 5μg/L IGF-Ⅰgroups and negative control group on days 2 and 4 (P > 0.05); There was no difference among the 10μg/L, 50μg/L and 200μg/L IGF-Ⅰgroups (P > 0.05). ③On the second day, the proliferative rate of tenocytes in the positive control group was significantly higher than that in 1μg/L and 5μg/L IGF-Ⅰgroups but lower than in 10μg/L, 50μg/L and 200μg/L IGF-Ⅰgroups (P < 0.000 or < 0.006). On the fourth day, the proliferative rate of tenocytes in the positive control group was significantly higher than that in each IGF-Ⅰgroup and negative control group (P < 0.000 or < 0.006). ④On days 2 and 4, the proliferative rate of tenocytes in 1μg/L and 5μg/L IGF-Ⅰgroups was significantly lower than that in 10μg/L, 50μg/L and 200μg/L IGF-Ⅰgroups (P < 0.000); the positive control group was remarkably higher than the negative control group (P < 0.000).
CONCLUSION: IGF-Ⅰpromotes the proliferation of tenocytes in a dose-dependent manner. 10μg/L is found to be the most effective concentration. Meanwhile, the proliferative rate of tenocytes is accelerated when the cells are cultured in higher concentration of FCS.

Qiu NH, Xia YP, Li MX, Li RH, Kan SL.Dose-effect relationship of insulin-like growth factor Ⅰand the proliferation of tenocytes in tissue engineering.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(2):221-226(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-221(ps).pdf]