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

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(P0.05);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(P0.05),P-4E/BP1蛋白表达量逐渐减少(P0.05),成骨相关蛋白Runx2、OCN,VEGF表达量逐渐增加(P0.05)。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(P0.05),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(P0.05)。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。

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

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.