| 骨碎补总黄酮通过激活mTOR信号通路促进大鼠腱骨愈合的实验研究 |
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投稿时间：2017-04-17
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| 作者 | Author | 单位 | Address | E-Mail |
| 张新涛 |
ZHANG Xin-tao |
北京大学深圳医院运动医学与康复医学科, 广东 深圳 518036 |
Department of Sports medicine and rehabilitation, Shenzhen Hospital of Peking University, Shenzhen 518036, Guangdong, China |
zhangxintao@sina.com |
| 江华基 |
JIANG Hua-ji |
南方医科大学第三附属医院骨科, 广东 广州 510630 |
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| 梁祖儒 |
LIANG Zu-ru |
北京大学深圳医院运动医学与康复医学科, 广东 深圳 518036 |
Department of Sports medicine and rehabilitation, Shenzhen Hospital of Peking University, Shenzhen 518036, Guangdong, China |
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| 贺飞林 |
HE Fei-lin |
北京大学深圳医院运动医学与康复医学科, 广东 深圳 518036 |
Department of Sports medicine and rehabilitation, Shenzhen Hospital of Peking University, Shenzhen 518036, Guangdong, China |
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| 廖小卿 |
LIAO Xiao-qing |
北京大学深圳医院运动医学与康复医学科, 广东 深圳 518036 |
Department of Sports medicine and rehabilitation, Shenzhen Hospital of Peking University, Shenzhen 518036, Guangdong, China |
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| 任玉香 |
REN Yu-xiang |
北京大学深圳医院运动医学与康复医学科, 广东 深圳 518036 |
Department of Sports medicine and rehabilitation, Shenzhen Hospital of Peking University, Shenzhen 518036, Guangdong, China |
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| 张文涛 |
ZHANG Wen-tao |
北京大学深圳医院运动医学与康复医学科, 广东 深圳 518036 |
Department of Sports medicine and rehabilitation, Shenzhen Hospital of Peking University, Shenzhen 518036, Guangdong, China |
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| 期刊信息：《中国骨伤》2018年，第31卷，第3期，第248-253页 |
| DOI：10.3969/j.issn.1003-0034.2018.03.011 |
| 基金项目:广东省中医药局科研课题（编号：20131027） |
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中文摘要:
目的：探讨骨碎补总黄酮对大鼠腱骨愈合的作用及相关分子机制。
方法：选取10只8周龄雄性SD大鼠（体重180~220 g），培养原代跟腱干细胞，取第3代跟腱干细胞用于实验。向大鼠跟腱干细胞中加入不同浓度（0、0.1、1、10 ng/ml）骨碎补总黄酮处理，14 d后分别检测ALP、Runx2、OCN、VEGF、P-S6和P-4E/BP1表达的变化。选取40只8周龄的雄性SD大鼠（体重180~220 g）行大鼠关节外骨隧道-游离肌腱移植造模，术后随机分为实验组和对照组，实验组灌胃骨碎补总黄酮（100 mg·kg-1·d-1），对照组灌胃等量的生理盐水。分别于术后3、6周取材进行生物力学检测腱骨愈合强度，组织学切片观察腱骨愈合情况及新生血管数目。
结果：不同浓度的骨碎补总黄酮处理后，ALP染色及活性指标检测提示0 ng/ml组（0.55±0.11）、0.1 ng/ml组（0.87±0.08）、1 ng/ml组（1.18±0.11）、10 ng/ml组（1.48±0.10），各组比较差异有统计学意义（P<0.05）；体外培养14 d后提取细胞蛋白免疫印迹实验提示，随着骨碎补总黄酮浓度的增加，mTOR下游标记蛋白P-S6蛋白表达量逐渐增加（P<0.05），P-4E/BP1蛋白表达量逐渐减少（P<0.05），成骨相关蛋白Runx2、OCN，VEGF表达量逐渐增加（P<0.05）。生物力学检测显示术后3周时，对照组（0.51±0.02）N/mm和实验组（0.78±0.05）N/mm力学强度比较差异无统计学意义（P>0.05），而6周时实验组（1.36±0.09）N/mm的力学强度明显高于对照组（1.01±0.08）N/mm（P<0.05）。组织学结果显示实验组3周和6周时腱骨界面细胞成熟程度更高，Sharpey纤维生长更加密集，间质钙化程度更高，新骨和血管发生明显增多。
结论：骨碎补总黄酮可以通过激活mTOR信号通路，促进肌腱干细胞的成骨分化，在活体可以加快腱骨愈合速度，提高腱骨愈合质量。 |
| 【关键词】骨碎补 黄酮 大鼠 跟腱 |
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| Osteopractic total flavone promoting rat extra-articular tendon-bone healing through mTOR pathway |
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ABSTRACT
Objective: To explore function and related molecular mechanism of osteopractic total flavone (OTF) on tendon healing in rats.
Methods: Ten male rats aged for 8 weeks were collected and weighted from 180 to 220 g. Tendon stem cells were cultivated,the third tendon stem cells were used for experiment. OTP treated with 0,0.1,1,10 ng/ml were added into tendon stem cells,and expression change of ALP,Runx2,OCN,VEGF,P-S6,P-4E/BP1 were detected after 14 days. Forty male rats aged for 8 weeks (weighted 180 to 220 g) were established extra-articular tendon-bone transplanting healing model,and divided into experimental group and control group. Experimental group were treated with OTF(100 mg·kg-1·d-1),while control group was treated by normal saline with the same volume. Tendon-bone healing degree were detected by biomechanical testing at 3 and 6 weeks after surgery,histological detection were applied to detect tendon-bone healing and number of new vessles.
Results: After treated by OTP,ALP staining and active index detection showed there were statistical differences among 0,0.1,1,10 ng/ml group. After 14 days' cultivation,western blotting results showed mTOR downstream marker protein P-S6 protein expression were gradually increased with increase of density of OTP,expression of P-4E/BP1 was reduced,while expression of Runx2,OCN,VEGF were increased. Biological detection results showed that there was no significant difference in mechanical strength between experimental group(0.78±0.05) N/mm and control group (0.51±0.02) N/mm at 3 weeks after surgery,while mechanical strength in experimental group (1.36±0.09) N/mm was higher than control group (1.01±0.08) N/mm at 6 weeks after surgery. Histological results showed maturity of tendon-bone surface cell were higher at 3 and 6 weeks in experimental group,sharpey fiber growth more density,calcification extent of mesenchyme was high,and new bone,vessels were increased.
Conclusion: OTF could promote osteogenic differentiation of tendon stem cells through mTOR signaling in vitro,and stimulate tendon-bone healing in bone tunnel and enhance connection quality between tendon and bone. |
| KEY WORDS Drynaria fortunei Flavone Rats Achilles tendon |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 张新涛,江华基,梁祖儒,贺飞林,廖小卿,任玉香,张文涛.骨碎补总黄酮通过激活mTOR信号通路促进大鼠腱骨愈合的实验研究[J].中国骨伤,2018,31(3):248~253 |
| 英文格式: | ZHANG Xin-tao,JIANG Hua-ji,LIANG Zu-ru,HE Fei-lin,LIAO Xiao-qing,REN Yu-xiang,ZHANG Wen-tao.Osteopractic total flavone promoting rat extra-articular tendon-bone healing through mTOR pathway[J].zhongguo gu shang / China J Orthop Trauma ,2018,31(3):248~253 |
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