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
전화생장인자β1대기건건초、건외막화건내막세포증식화효원산생적영향
Effects of transforming growth factor beta-1 on proliferation and collagen production of tendon sheath fibroblasts, epitenon tenocytes and endotenon tenocytes
  • 万方数据
    中国组织工程研究与临床康复 중국조직공정연구여림상강복
    JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
    2007年 41期 8404-8407 ,共4页

    李海燕%姚光慧%夏长所

    리해연%요광혜%하장소

    肌腱%转化生长因子β1%胶原组织 肌腱%轉化生長因子β1%膠原組織
    기건%전화생장인자β1%효원조직
    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.
    배경:전화생장인자β시일충재조직손상적수복화경신중구유다충생물학효응적세포인자;건초성섬유세포화Ⅰ형효원재기건유합화점련형성과정중기착중요적작용.목적:관찰토굴지기건건초、건외막화건내막세포증식、효원산생화전화생장인자β1대세포적증식화효원산생적영향.설계:대비관찰실험.단위:청도대학의학원부속의원창상외과.재료:실험우2004-07/2005-09재청도대학의학원부속의원동물실험실완성,선용6지성년신서란대백토,자웅불구,체질량3.5～4.5 kg,유청도시실험동물중심제공.효원매(sigma), Ⅰ、Ⅱ、Ⅲ형효원항체(Sigma),전화생장인자β1(무한박사덕생물공사).방법:안문헌방법진행장실험토계지기건분리건초、건외막화건내막세포병배양,장3충세포채용함혈청배양액배양후,전이도불함혈청적배양액중,분위실험조급대조조,실험조매공가입5 μg/L전화생장인자β1,대조조불여첨가.주요관찰지표:①용세포계수법관찰배양1,2,3,4 d각조세포증식정황.②채용면역세포화학염색검측세포배양4d후Ⅰ、Ⅱ화Ⅲ형효원적산생;통과매련면역흡부시험법정량검측2조세포적각형효원함량.③채용RT-PCR정량검측2조세포Ⅰ형효원기인적표체.④매련면역흡부시험법측정불동제량적전화생장인자β1(0,5,10,15화20 μg/L)작용우3충세포적Ⅰ형효원함량.결과:①매충세포배양1 d후증장솔상근,배양2～4 d,건초세포증식솔현저증고.여기타량충세포증식솔비교,차이유현저성의의(P＜0.05).②면역세포화학염색현시3충세포균가이산생Ⅰ、Ⅱ、Ⅲ형효원;매련면역흡부시험법정량측정효원결과현시:각조건초세포산생적3충효원량최다,차실험조각충세포Ⅰ형효원함량고우대조조(P＜0.05～0.01).③실험조건초세포Ⅰ형효원기인표체비대조조증가1.3배,차이유통계학의의(P＜0.01),건외막세포화건내막세포표체야고우대조조(P＜0.05).④효원산생량재5～10 μg/L전화생장인자β1작용시명현증가,이전화생장인자β1증가도10～20 μg/L시효원산생량무명현개변.결론:전화생장인자β1가증가건초성섬유세포、건외막세포화건내막세포효원적산생화Ⅰ형효원적기인표체,재기건손상후조절전화생장인자β1적수평가능대기건첩련적방지구유중요적작용.
    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 antibody 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:Immunocytochemical 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 the 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 collagen Ⅰ gene. In addition, it is important for regulating level of TGF-β1 after tendon injury to prevent adhesion of tendon.
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