[6-磷酸甘露糖对屈肌腱细胞转化生长因子β及转化生长因子β受体表达的影响]

Yang Ruixiang, Xia Changsuo, Wang Xiuying, Sun Kang, Yang Xuanying, Tian Shaoqi, Hong Guangxiang

Department of Orthopaedics, the Affiliated Hospital of Medical College, Qingdao University, Qingdao Shandong, 266003, PR China.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 Jan;24(1):64-8.

OBJECTIVE

By culturing tendon sheath fibroblasts, epitenon tenocytes and endotenon tenocytes of rabbits' tendon in vitro, to study the effects of mannose-6-phosphate on transforming growth factor beta (TGF-beta) peptide and receptor expression, and to provide the experimental basis for preventing the tendon healing adhesion by mannose-6-phosphate.

METHODS

Eight adult New Zealand white rabbits, regardless of their gender and weighing 4.0-4.5 kg, were selected. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendon and cultured separately. All 3 cells were divided into 2 groups at random after cells were adjusted to a concentration of 4 x 10(4) per well and 1 x 10(4)/mL. The first was the control group without supplementation. The experimental group was supplemented with mannose-6-phosphate. The expressions of TGF-beta and TGF-beta receptor were quantified with enzyme-linked immunosorbent assay. The expression of TGF-beta1 mRNA was also assessed with in situ hybridization and the expression of TGF-beta1 was assessed with immunohistochemistry.

RESULTS

The expressions of TGF-beta and TGF-beta receptor in experimental group were significantly lower than that in control group (P < 0.05). The expression levels of TGF-beta1 and TGF-beta2 decreased in descending order of tendon sheath fibroblasts (36.1%, 37.9%), epitenon tenocytes (31.0%, 32.1%), and endotenon tenocytes (31.2%, 27.0%). The expression levels of TGF-beta3 decreased in descending order of endotenon tenocytes (42.5%), tendon sheath fibroblasts (41.2%), and epitenon tenocytes (33.3%). The expression levels of TGF-beta receptor 1 and TGF-beta receptor 2 decreased in descending order of epitenon tenocytes (29.9%, 26.2%), endotenon tenocytes (27.8%, 23.5%), and tendon sheath fibroblasts (23.1%, 20.0%). The expression levels of TGF-beta receptor 3 decreased in descending order of endotenon tenocytes (26.1%), epitenon tenocytes (19.2%), and tendon sheath fibroblasts (15.8%). In experimental group, the positive expression of TGF-beta1 mRNA and the expression level of intracellular TGF-beta1 mRNA in all 3 tendon cells were significantly lower than those in the control group (P < 0.05). Immunohistochemical staining showed the expressions of TGF-beta1 in all 3 tendon cells were significantly lower in the experimental group than in the control group.

CONCLUSION

Mannose-6-phosphate can significantly decrease the expressions of TGF-beta peptide, TGF-beta receptor, and TGF-beta1 mRNA. Modulation of mannose-6-phosphate levels may provide a mean of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.

目的

通过体外培养兔肌腱的腱鞘成纤维细胞、腱外膜成纤维细胞和腱内膜成纤维细胞，研究6-磷酸甘露糖对转化生长因子β（TGF-β）肽和受体表达的影响，为6-磷酸甘露糖预防肌腱愈合粘连提供实验依据。

方法

选取8只成年新西兰白兔，雌雄不限，体重4.0-4.5 kg。从兔屈肌腱中分离腱鞘成纤维细胞、腱外膜成纤维细胞和腱内膜成纤维细胞并分别培养。将3种细胞调整至浓度为每孔4×10⁴个和1×10⁴/mL后随机分为2组。第一组为不添加任何物质的对照组。实验组添加6-磷酸甘露糖。采用酶联免疫吸附测定法定量检测TGF-β和TGF-β受体的表达。采用原位杂交法评估TGF-β1 mRNA的表达，采用免疫组织化学法评估TGF-β1的表达。

结果

实验组中TGF-β和TGF-β受体的表达明显低于对照组（P<0.05）。TGF-β1和TGF-β2的表达水平按腱鞘成纤维细胞（36.1%，37.9%）、腱外膜成纤维细胞（31.0%，32.1%）和腱内膜成纤维细胞（31.2%，27.0%）的顺序依次降低。TGF-β3的表达水平按腱内膜成纤维细胞（42.5%）、腱鞘成纤维细胞（41.2%）和腱外膜成纤维细胞（33.3%）的顺序依次降低。TGF-β受体1和TGF-β受体2的表达水平按腱外膜成纤维细胞（29.9%，26.2%）、腱内膜成纤维细胞（27.8%，23.5%）和腱鞘成纤维细胞（23.1%，20.0%）的顺序依次降低。TGF-β受体3的表达水平按腱内膜成纤维细胞（26.1%）、腱外膜成纤维细胞（19.2%）和腱鞘成纤维细胞（15.8%）的顺序依次降低。在实验组中，所有3种肌腱细胞中TGF-β1 mRNA的阳性表达及细胞内TGF-β1 mRNA的表达水平均明显低于对照组（P<0.05）。免疫组织化学染色显示，实验组中所有3种肌腱细胞中TGF-β1的表达均明显低于对照组。