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

Osteopractic total flavone promoting rat extra-articular tendon-bone healing through mTOR pathway

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。

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.