CAJ | 학술논문

背景:目前常用的动物肌腱研究模型(比如兔,罗曼鸡等)的培养方法存在周期长,细胞获取量少等不足,往往成为制约肌腱组织工程实验研究进程的瓶颈.目的:希望建立SD大鼠鼠尾肌腱细胞培养流程,以期用较短时间获取大量种子细胞,为更高的工程化肌腱构造研究创造模型构建条件.设计、时间及地点:对比观察,于2006-02/11在四川大学生物治疗国家重点实验室干细胞与组织工程研究室完成.材料:7～10dSD大鼠2只用于获取鼠尾腱细胞;第4代人包皮成纤维细胞由四川大学华西医院干细胞与组织工程研究室提供.方法:选用SD乳鼠,无菌条件下抽取尾腱,体积分数为10%的小牛血清+DF培养基悬浮组织块培养法获得鼠尾肌腱原代细胞,将第3代细胞与人包皮成纤维细胞为对照,行Ⅰ,Ⅲ型胶原免疫细胞化学染色,染色结果用image-pro plus 5.02行吸光度测量,并做统计分析.主要观察指标:SD大鼠尾腱细胞免疫细胞化学染色结果及染色吸光度测量结果.结果:第2代SD大鼠尾腱细胞Ⅰ型胶原染色呈阳性,Ⅲ型胶原染色呈阴性;人成纤维细胞Ⅰ、Ⅲ型胶原免疫组织化学皆呈阳性.尾腱细胞和人成纤维细胞Ⅰ型胶原表达吸光度值均明显高于Ⅲ型胶原(P＜0.05).两种细胞Ⅲ型胶原表达吸光度值与空白对照之间差异无显著性意义(P＞0.05).结论:SD乳鼠尾腱源细胞符合肌腱细胞的生物学特性.采用组织块悬浮培养可以在短期内大量获取原代或传代尾腱细胞.
배경:목전상용적동물기건연구모형(비여토,라만계등)적배양방법존재주기장,세포획취량소등불족,왕왕성위제약기건조직공정실험연구진정적병경.목적:희망건립SD대서서미기건세포배양류정,이기용교단시간획취대량충자세포,위경고적공정화기건구조연구창조모형구건조건.설계、시간급지점:대비관찰,우2006-02/11재사천대학생물치료국가중점실험실간세포여조직공정연구실완성.재료:7～10dSD대서2지용우획취서미건세포;제4대인포피성섬유세포유사천대학화서의원간세포여조직공정연구실제공.방법:선용SD유서,무균조건하추취미건,체적분수위10%적소우혈청+DF배양기현부조직괴배양법획득서미기건원대세포,장제3대세포여인포피성섬유세포위대조,행Ⅰ,Ⅲ형효원면역세포화학염색,염색결과용image-pro plus 5.02행흡광도측량,병주통계분석.주요관찰지표:SD대서미건세포면역세포화학염색결과급염색흡광도측량결과.결과:제2대SD대서미건세포Ⅰ형효원염색정양성,Ⅲ형효원염색정음성;인성섬유세포Ⅰ、Ⅲ형효원면역조직화학개정양성.미건세포화인성섬유세포Ⅰ형효원표체흡광도치균명현고우Ⅲ형효원(P＜0.05).량충세포Ⅲ형효원표체흡광도치여공백대조지간차이무현저성의의(P＞0.05).결론:SD유서미건원세포부합기건세포적생물학특성.채용조직괴현부배양가이재단기내대량획취원대혹전대미건세포.
BACKGROUND:The low output of seed cells and long cycle of traditional ceils culture methods in tendon animal models(rabbits and chicken) restrict the researches of tendon tissue engineering study.OBJECTIVE:To establish an ideal culture protocol of tail tendon in SD rats,to get more seed cells within less time for subsequent engineered tendon construction research.DESIGN,TIME AND SETTING:Controlled observation was performed in the National Key Laboratory of Biotherapy,Department of Stem Cells and Tissue Engineering,Sichuan University between February and November in 2006.MATERIALS:Rat tail tendon cells were harvested from 2 SD rats,aged 7-10 days;human prepuce fibroblasts were offered by National Key Laboratory of Biotherapy,Department of Stem Cells and Tissue Engineering,Sichuan University.METHODS:Tail tendon of SD rats was draw off and cut into pieces,which were then cultured in 10% fetal bovine serum+DFculture medium for getting primary tendoncyte by using suspension tissue culture method. The third generation cells were processed into immuocytochemistry stain with collagen type Ⅰ and Ⅲ,while human prepuce fibroblasts served as controls.Absorbance of stain result was measured by image-pro plus 5.02 for statistical analysis.MAIN OUTCOME MEASURES:Immuocytochemistry stain and absorbance measurement of SD rat tail tendon cells.RESULTS:The second generation of SD rat tail tendon cells were positive for type Ⅰ collagen stain,and negative for type Ⅲ collagen stain;human fibroblast were positive for both Ⅰ and Ⅲ collagen. In the rat tail tendon cells and human flbroblasts,the absorbance value of type Ⅰ collagen expression was dramatically higher than of type Ⅲ collagen(P＜0.05). There was no significant differences addressing the absorbance of type Ⅲ collagen expression between type Ⅰ and Ⅲ collagen of SD rat tail tendon cells and blank control group (P＞0.05).CONCLUSION:Cells cultured from SD rat tail tendon have biological characteristic of tendon cells. Tissue piece suspensionculture can obtain a quantity of primary or subcuitured cells of rat tail tendon within a short time.