mTOR蛋白对老龄大鼠肌腱干细胞修复能力影响△

Effect of mTOR protein on repair capacity of tendon stem cells in aged rats

摘要:

[目的] 探讨mTOR 蛋白对老龄大鼠肌腱干细胞肌腱损伤修复能力的影响。[方法] 体外培养青年组和老年组大鼠肌腱干细胞，使用腺病毒转染调控mTOR 表达，Western blot 检测mTOR 和相关蛋白。制作老龄大鼠髌腱损伤模型，分为mTOR表达、mTOR 干扰、GFP（green fluorescent protein 绿色荧光蛋白） 和空白组，在2 周和4 周取大鼠修复肌腱组织，行组织学、组织免疫化学和生物力学检测。[结果] 体外试验方面，Western blot 结果显示，老年组mTOR [(1.8±0.1) vs (0.9±0.1), P＜0.05]、Raptor [(4.1±0.3) vs (2.2±0.3), P＜0.05] 及P-Raptor [(1.8±0.1) vs (0.9±0.1), P＜0.05] 均显著高于青年组；而两组间AKT、P-Akt、S6K、P-S6k 的差异无统计学意义（P＞0.05）。老年大鼠TSCs 体外转染后mTOR、Raptor 及P-Raptor 表达从高到低的顺序：mTOR 的表达组＞GFP 组＞mTOR 干扰组（P＜0.05）。体内试验方面，与术后2 周相比，术后4 周四组HE 组织评分均显著增加（P＜0.05），相应时间点分数从高到低的顺序依次为mTOR 干扰组＞GFP 组＞mTOR 表达组＞空白组，差异均有统计学意义（P＜0.05）。与术后2 周相比，术后4 周，Col1 及SCX 的表达均无显著变化（P＞0.05），相应时间点，四组间Col1、SCX 的表达定量从高到低依次为mTOR 干扰组＞GFP 组＞空白组＞mTOR 表达组，差异均有统计学意义（P＜0.05）。与术后2 周相比，术后4 周四组最大损毁强度均显著增加（P＜0.05），相应时间点，最大损毁强度值从大到小依次为mTOR 干扰组＞GFP 组＞空白组＞mTOR 表达组，差异均有统计学意义（P＜0.05）。[结论] 抑制mTOR 蛋白可加强老龄大鼠肌腱干细胞的修复能力。

Abstract:

[Objective] To investigate the effect of mTOR protein on tendon repair ability of tendon stem cells in aged rats. [Methods]Young and old rat tendon stem cells were cultured in vitro, and mTOR expression was regulated by adenovirus transfection, and mTOR andrelated proteins were detected by Western blot. Aged rat patellar tendon injury models were made, and divided into mTOR expression,mTOR interference, green fluorescent protein (GFP) and blank groups. The repaired tendon tissues of the rats were harvested at 2 and 4weeks for histological, tissue immunochemical and biomechanical assays. [Results] In vitro test, the western blot results showed that theaged rats presented significantly higher level of mTOR [(1.8±0.1) vs (0.9±0.1), P＜0.05], Raptor [(4.1±0.3) vs (2.2±0.3), P＜0.05] and P-Rap-tor [(1.8±0.1) vs (0.9±0.1), P＜0.05] than the young group, despite of insignificant differences in Akt, p-Akt, S6k and P-S6K between the twogroups (P＞0.05). The expression sequence of mTOR, Raptor and P-Raptor after transfection of TSCs in aged rats ranked from high to low:mTOR expression group ＞ GFP Group ＞mTOR interference group (P＜0.05). In vivo test, HE tissue scores significantly increased in all the 4groups 4 weeks after surgery group compared with those 2 weeks after surgery (P＜0.05), which ranked from high to low as the mTOR interfer-ence group ＞GFP Group ＞mTOR expression group ＞ blank control group at time points accordingly with statistically significant differences(P＜0.05). Compared with those 2 weeks after surgery, the Col1 and SCX expressions remained unchanged at 4 weeks after surgery (P＞0.05),which ranked among the 4 groups from high to low as the mTOR interference group ＞GFP Group ＞Blank group ＞mTOR expression group,with statistical significance (P＜0.05). Compared with those 2 weeks after surgery, the maximum failure strength in biomechanical test signifi-cantly increased in 4 weeks after surgery (P＜0.05), which ranked up-down as mTOR interference group ＞GFP Group ＞ blank group ＞mTORexpression group with statistical significances at time points accordingly (P＜0.05). [Conclusion] Inhibition of mTOR protein does enhancethe repair capacity of tendon stem cells in aged rats in this study.