中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (2): 262-266.doi: 10.3969/j.issn.1673-8225.2010.02.017

• 组织构建与中医药 tissue construction and traditional Chinese medicine • 上一篇    下一篇

杜仲对大鼠骨髓基质细胞骨桥蛋白和骨保护素表达的影响
水提液与醇提液有区别吗？

张艳红1，谢焕松1，夏树林2，赵  春2，谭湘陵2   

  1. 1南通大学生命科学学院细胞生物学教研室，江苏南通市  226007；  2南通大学生物工程研究所，江苏省南通市  226007
  • 出版日期:2010-01-08 发布日期:2010-01-08
  • 通讯作者: 谭湘陵，硕士，教授，南通大学生物工程研究所，江苏省南通市 226007 tanxl@ntu.edu.cn
  • 作者简介:张艳红，女，1963年生，汉族，江苏省通州市人，1984年南京师范大学毕业，副教授，主要从事细胞生物学领域的相关研究。 ntuzyh@126.com
  • 基金资助:

    无锡市社会发展基金(CSE00723)。

Effect of eucommia bark on osteopontin and osteoprotegerin expressions in rat bone marrow stem cells: Is there any difference between water extract and methanol extract?

Zhang Yan-hong1, Xie Huan-song1, Xia Shu-lin2, Zhao Chun2, Tan Xiang-ling2   

  1. 1 Department of Cell Biology, Life School of Nantong University, Nantong  226007, Jiangsu Province, China; 2 Institute of Bioengineering, Nantong University, Nantong  226007, Jiangsu Province, China
  • Online:2010-01-08 Published:2010-01-08
  • Contact: Tan Xiang-ling, Master, Professor, Institute of Bioengineering, Nantong University, Nantong 226007, Jiangsu Province, China tanxl@ntu.edu.cn
  • About author:Zhang Yan-hong, Associate professor, Department of Cell Biology, Life School of Nantong University, Nantong 226007, Jiangsu Province, China ntuzyh@126.com
  • Supported by:

    the Social Development Foundation of Wuxi, No. CSE00713*

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摘要：

背景：以往实验证实杜仲能促进骨髓基质细胞向成骨细胞分化，但对促进成骨分化的分子机制报道很少。
目的：观察中药杜仲水/醇提取物对大鼠骨髓基质细胞骨桥蛋白和骨保护素表达的影响
方法：盐杜仲2 g，分别加蒸馏水或甲醇至15 mL，室温振荡1 h，静置过夜后，15 000 r/min离心10 min，弃沉淀。水浸上清不经处理即得水提取物；甲醇浸上清经真空浓缩，再加水至15 mL溶解剩余物后，得醇提取物水、醇提取物，用0.22 μm滤膜过滤后于-20 ℃保存。取2月龄SD 大鼠6 只，体外培养至第3代SD大鼠骨髓基质细胞进行杜仲水/醇提取物诱导，诱导物分为1×10-2，1×10-3，1×10-4和1×10-5 4个稀释度；阴性对照组加入PBS；诱导培养6 d。以免疫细胞化学法测定诱导6 d的骨髓基质细胞骨桥蛋白和骨保护素表达。应用反转录-聚合酶链反应方法测定1×10-3，1×10-4稀释度诱导3 d的骨髓基质细胞骨桥蛋白和骨保护素的基因表达。
结果与结论：细胞免疫化学显示，杜仲水/醇提取物对大鼠骨髓基质细胞骨桥蛋白的表达均有显著的刺激作用，以 1×10-4的稀释度作用最强，且醇提取物的作用大于水提取物；骨保护素的表达却无显著变化。反转录-聚合酶链反应显示，水提取物对诱导3 d后大鼠骨髓基质细胞骨桥蛋白的基因表达量显著高于醇提取物，骨保护素未检测出表达。证实杜仲水/醇提取物诱导骨髓基质细胞成骨分化与刺激骨桥蛋白表达相关，与骨保护素无关；水提取物促进骨髓基质细胞骨桥蛋白表达早于醇提取物。

关键词: 杜仲, 骨髓基质细胞, 骨桥蛋白, 骨保护素

Abstract:

BACKGROUND: Previous studies demonstrated that eucommia bark can promote bone marrow stem cells (BMSCs) differentiated into osteoblasts, but relative mechanism is poorly understood. 
OBJECTIVE: To investigate the effects of eucommia bark water/methanol extracts on expressions of osteopontin (OPN) and osteoprotegerin (OPG) in rat BMSCs.
METHODS: Totally 2 g eucommia bark powder were added into water or methanol to 15 mL and oscillated for 1 hour at room temperature. After soaked overnight, both extracts were centrifuged at 15 000 r/min for 10 minutes. Water extract was obtained from supernatant in water soaked powder. In methanol soaked powder, methanol extracts was obtained by concentrated supernatant in vacuo and resolved using 15 mL water. Water and methanol extracts were then filtered by 0.22 μm membrane, and conserved at -20 ℃. Six SD rats, aged 2 months, were selected, and the 3rd passage of BMSCs were induced by water or methanol extracts with dilution of 1×10-2, 1×10-3, 1×10-4 and 1×10-5, respectively. PBS was added in the negative control group. All cells were cultured for 6 days. Expressions of OPN and OPG was measured by immunocytochemistry at 6 days with induction. The expression of OPN and OPG induced by water and methanol at 1×10-3 and 1×10-4 dilution was detected by RT-PCR.
RESULTS AND CONCLUSION: Immunocytochemistrical results indicated that both water and methanol extracts of eucommia bark simulated OPN and OPG expression, in particular with dilution of 1×10-4. The methanol extracts had a stronger effect than water extract, but the expression of OPG did not change obviously. RT-PCR demonstrated that at the 3rd day of inducement, the level of OPN expression induced by water extract was higher than that of methanol extract, and no OPG expression was detected. Osteogenic differentiation of rat MSCs induced by eucommia bark water/methanol extracts relates to stimulating expression of OPN, which has no correlation to OPG. OPN expression induced by water extract is early than that of methanol extract. 

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