论文部分内容阅读
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3.5～4.5kg,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屈趾肌腱分离腱鞘、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5μg/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1,2,3,4d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20μg/L)作用于3种细胞的Ⅰ型胶原含量。结果:①每种细胞培养1d后增长率相近,培养2～4d,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5～10μg/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10～20μg/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的水平可能对肌腱粘连的防止具有重要的作用。
BACKGROUND: Transforming growth factor beta is a cytokine that has multiple biological effects in the repair and renewal of tissue damage. Tenosynoidal fibroblasts and type I collagen play an important role in tendon healing and adhesion formation. OBJECTIVE: To observe the effects of proliferation, collagen production and TGF-β1 on proliferation and collagen production of tendon sheath, tendon adventitia and tendon endothelium in rabbit flexor tendon. Design: Contrast observation experiment. Unit: Department of Traumatic Surgery, Affiliated Hospital of Qingdao University Medical College. MATERIALS: The experiment was performed at the Animal Laboratory of Affiliated Hospital of Qingdao University Medical College from July 2004 to September 2005. Six adult New Zealand white rabbits were selected. The body weight was 3.5-4.5 kg. The experimental animals were provided by Qingdao Experimental Animal Center. Collagenase (sigma), type Ⅰ, Ⅱ, Ⅲ collagen antibody (Sigma), transforming growth factor β1 (Wuhan Boster Biological Company). Methods: According to the method of literature, the tendon of experimental rabbit was separated from the tendon sheath, tendon adventitia and tendon endothelium and cultured. The three kinds of cells were cultured in serum-containing culture medium and transferred to serum-free culture medium, which were divided into Experimental group and control group, experimental group by adding 5μg / L transforming growth factor β1, the control group did not add. MAIN OUTCOME MEASURES: ① Cell proliferation was observed by cell counting method in 1, 2, 3 and 4 days. ② Immunocytochemical staining was used to detect the generation of collagen type Ⅰ, Ⅱ and Ⅲ after 4 days of cell culture. The collagen content of each group was quantified by enzyme-linked immunosorbent assay (ELISA). ③ RT-PCR was used to detect the expression of collagen type Ⅰ gene in two groups of cells. ④ The levels of collagen type Ⅰ (TGF-β1) (0, 5, 10, 15 and 20 μg / L) were measured by enzyme-linked immunosorbent assay in three kinds of cells. Results: ① The growth rate of each cell cultured for 1 day was similar. After cultured for 2 ~ 4 days, the proliferation rate of tendon sheath cell was significantly increased. Compared with the other two cell proliferation rates, the difference was significant (P <0.05). Immunocytochemical staining showed that all the three kinds of cells could produce collagen type I, II, and III. Quantitative analysis of collagen by enzyme linked immunosorbent assay showed that the three kinds of collagen produced by tendon sheath cells in each group were the most, Type Ⅰ collagen content was higher than the control group (P <0.05 ~ 0.01). ③ The expression of type Ⅰ collagen in the cells of experimental group was 1.3 times more than that of the control group (P <0.01). The expression of tendon adventitial cells and tendon endometrial cells was also higher than that of the control group (P <0.05). ④ Collagen production increased significantly when TGF-β1 was treated with 5 ~ 10μg / L TGF-β1, whereas collagen production did not change when TGF-β1 increased to 10 ~ 20μg / L. CONCLUSION: Transforming growth factor β1 can increase the production of collagen and the expression of collagen type Ⅰ in tendon sheath fibroblasts, tendon adventitial cells and tendon intima, and the prevention of tendon adhesion by regulating the level of transforming growth factor β1 after tendon injury Has an important role.