转化生长因子β1对肌腱腱鞘、腱外膜和腱内膜细胞增殖和胶原产生的影响

转化生长因子β1对肌腱腱鞘、腱外膜和腱内膜细胞增殖和胶
原产生的影响
李海燕;姚光慧;夏长所
【期刊名称】《中国组织工程研究》
【年(卷),期】2007(011)041
【摘要】BACKGROUND:Transforming growth factor beta-1(TGF-β1)is a cytokine having variously biological effects in repair and renew of tissue injuries; meanwhile, tendon sheath fibroblasts and collagen Ⅰ play important roles in healing and desmoplasia of tendon.OBJECIVE:To study the effects of TGF-β1 on the proliferation and collagen production of tendon sheath fibroblasts.epitenon tenocytes and endotenon tenocytes in the three cell types of rabbit fexor tendon.DESIGN:Contrast observation study.SETTING:Department of Trauma Surgery,Affiliated Hospital of Medical College,Qingdao University.MATERIALS:The experiment was carried out in the Animal Laboratory,Affiliated Hospital of Medical College,Qingdao University from July 2004 to September 2005.A total of 6 adult New Zealand rabbits,of either gender,weighing 3.5-4.5 kg,were selected from Qingdao Experimental Animal Center.Collagenase was provided by Sigma Company;collagen Ⅰ,Ⅱand Ⅲ an tibody by Sigma Company;TGF-β1 by Wuhan Boster Biology Company.METHODS: Three cell lines of tendon sheath,epitenon and endotenon were isolated from rabbit flexor tendon and cultured in serum culture media and then in serum-free
culture media.In addition,the cells in the experimental group were added with 5 μg/L TGF-β1 in each well,but they were not added with any additive in the control group.MAIN OUTCOME MEASURES:①Proliferation in the two groups was measured with cytometry at 1,2,3 and 4 days after culture.②Preduction of collagens Ⅰ,Ⅱ and Ⅲ was measured with immunohistochemical staining at 4 days after culture.③Collagen contents of the three types were measured with enzyme linked immunosorbent assay(ELISA)in the two groups;expressJon of collagen Ⅰ gene was detected with reverse transcription polymerase chain reaction(RT-PCR).④Contents of collagen Ⅰ induced by TGF-β1 in various dosages of 0,5.10,15 and 20 μg/L were detected with ELISA
technique.RESULTS:①Proliferated rates were similar in the two groups at 1 day after culture;however,proliferated rate of tendon sheath fibroblasts was rapidly increased, and there was significant difference as compared with that of epitenontenocytes and endotenon tenocytes(P＜
0.05).②Expressions of collagens Ⅰ, Ⅱ and Ⅲ:Immunocytoch emical stain demonstrated that three kinds of cells could produce collagens Ⅰ, Ⅱ and Ⅲ;while ELISA indicated that the contents of collagens in three types produced by tendon sheath fibroblasts were the most;in addition,content of collage Ⅰ was higher in th e experimental group than that in the control group(P＜0.05-0.01).③Expression of collage Ⅰ gene of tendon sheath fibroblasts was increased as 1.3 times in the experimental group as that in the control group and there was signiflcant difierence(P＜
0.01);meanwhile,expressions in epitenon tenocytes and endotenon
tenocytes were also higher in the experimental group than those in the control group(P＜0.05).④TGF-β1 in the dosage of 5-10 μg/L had obvious effects on increasing production of collagen;however,production of collagen was not obviously changed when it was affeCted by TGF-β1 in the dosage of 10-20 μg/L.CONCLUSION: TGF-β1 can increase the production of collagen in tendon sheath fibroblasts,epitenon tenocytes and endotenon tenocytes and the expression of colla gen Ⅰ gene. In addition, it is important for regulating level of TGF-β1 after tendon injury to prevent adhesion of tendon.%背景:转化生长因子β是一种在组织损伤的修复和更新中具有多种生物学效应的细胞因子;腱鞘成纤维细胞和Ⅰ型胶原在肌腱愈合和粘连形成过程中起着重要的作用.目的:观察兔屈趾肌腱腱鞘、腱外膜和腱内膜细胞增殖、胶原产生和转化生长因子β1对细胞的增殖和胶原产生的影响.设计:对比观察实验.单位:青岛大学医学院附属医院创伤外科.材料:实验于2004-07/2005-09在青岛大学医学院附属医院动物实验室完成,选用6只成年新西兰大白兔,雌雄不拘,体质量
3.5～
4.5 kg,由青岛市实验动物中心提供.胶原酶(sigma), Ⅰ、Ⅱ、Ⅲ型胶原抗体(Sigma),转化生长因子β1(武汉博士德生物公司).方法:按文献方法进行将实验兔届趾肌腱分离腱鞘、腱外膜和腱内膜细胞并培养,将3种细胞采用含血清培养液培养后,转移到不含血清的培养液中,分为实验组及对照组,实验组每孔加入5 μg/L转化生长因子β1,对照组不予添加.主要观察指标:①用细胞计数法观察培养1,2,3,4 d各组细胞增殖情况.②采用免疫细胞化学染色检测细胞培养4d后Ⅰ、Ⅱ和Ⅲ型胶原的产生;通过酶联免疫吸附试验法定量检测2组细胞的各型胶原含量.③采用RT-PCR 定量检测2组细胞Ⅰ型胶原基因的表达.④酶联免疫吸附试验法测定不同剂量的转化生长因子β1(0,5,10,15和20 μg/L)作用于3种细胞的Ⅰ型胶原含量.结果:①每种细胞培养1 d后增长率相近,培养2～4 d,腱鞘细胞增殖率显著增高.与其他两种
细胞增殖率比较,差异有显著性意义(P＜0.05).②免疫细胞化学染色显示3种细胞均可以产生Ⅰ、Ⅱ、Ⅲ型胶原;酶联免疫吸附试验法定量测定胶原结果显示:各组腱鞘细胞产生的3种胶原量最多,且实验组各种细胞Ⅰ型胶原含量高于对照组(P＜
0.05～0.01).③实验组腱鞘细胞Ⅰ型胶原基因表达比对照组增加1.3倍,差异有统计学意义(P＜0.01),腱外膜细胞和腱内膜细胞表达也高于对照组(P＜0.05).④胶原产生量在5～10 μg/L转化生长因子β1作用时明显增加,而转化生长因子β1增加到10～20 μg/L时胶原产生量无明显改变.结论:转化生长因子β1可增加腱鞘成纤维细胞、腱外膜细胞和腱内膜细胞胶原的产生和Ⅰ型胶原的基因表达,在肌腱损伤后调节转化生长因子β1的水平可能对肌腱帖连的防止具有重要的作用.
【总页数】4页(P8404-8407)
【作者】李海燕;姚光慧;夏长所
【作者单位】青岛大学医学院附属医院创伤骨科,山东省青岛市,266003;青岛大学医学院附属医院创伤骨科,山东省青岛市,266003;青岛大学医学院附属医院创伤骨科,山东省青岛市,266003
【正文语种】中文
【中图分类】R329
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