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中华老年骨科与康复电子杂志

中华老年骨科与康复电子杂志 ›› 2019, Vol. 05 ›› Issue (01) : 25 -32. doi: 10.3877/cma.j.issn.2096-0263.2019.01.006

所属专题： 文献；

基础研究

雷帕霉素调节自噬抑制凋亡促进骨折愈合的机制研究
殷照阳1, 霍永峰1, 刘新晖2, 殷建2,†()   
  1. 1. 222000　连云港市第一人民医院骨科
    2. 211100　南京医科大学附属江宁医院骨科
  • 收稿日期:2018-10-20 出版日期:2019-02-05
  • 通信作者: 殷建
  • 基金资助:
    南京医科大学科技发展基金(2016NJMU156); 连云港市卫计委面上项目(201703)

Rapamycin promotes fracture healing by regulating autophagy and inhibition of apoptosis

Zhaoyang Yin1, Yongfeng Huo1, Xinhui Liu2, Jian Yin2,†()   

  1. 1. Department of Orthopaedics, Lianyungang First People's Hospital, Lianyungang 222000
    2. Department of Orthopaedics, the Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing 211100, China
  • Received:2018-10-20 Published:2019-02-05
  • Corresponding author: Jian Yin
  • About author:
    Corresponding author: Yin Jian, Email:
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殷照阳, 霍永峰, 刘新晖, 殷建. 雷帕霉素调节自噬抑制凋亡促进骨折愈合的机制研究[J/OL]. 中华老年骨科与康复电子杂志, 2019, 05(01): 25-32.

Zhaoyang Yin, Yongfeng Huo, Xinhui Liu, Jian Yin. Rapamycin promotes fracture healing by regulating autophagy and inhibition of apoptosis[J/OL]. Chinese Journal of Geriatric Orthopaedics and Rehabilitation(Electronic Edition), 2019, 05(01): 25-32.

目的

探讨雷帕霉素调节细胞自噬抑制凋亡促进骨折愈合的机制。

方法

建立SD大鼠右侧股骨干骨折模型，随机分为两组：空白对照组和雷帕霉素组（1 mg/kg/d）。空白对照组无特殊处理，雷帕霉素组在术后12 h、24 h，3 d和7 d腹腔注射1 mg/kg的雷帕霉素。分别在造模后2 w，4 w和6 w应用X线和Micro CT评估骨组织愈合情况，免疫荧光检测LC3-Ⅱ，免疫组化检测Beclin-1的表达水平，western blot检测Bax、VEGF及p-mTOR，HE染色计数骨折后骨组织中成骨细胞数量来评估成骨活性。

结果

与对照组相比，雷帕霉素组骨折端骨组织中p-mTOR及p-mTOR/mTOR比值降低[2 w（t=6.703，P=0.004；t=16.346，P=0.000）、4 w（t=16.407，P=0.000；t=16.738，P=0.000）、6 w（t=6.101，P=0.001；t=6.104，P=0.001）]；自噬蛋白LC3-Ⅱ和Beclin-1表达明显增加[2 w（t=9.509，P=0.000；t=9.962，P=0.000）、4 w（t=3.868，P=0.008；t=24.490，P=0.000）、6 w（t=5.071，P=0.002；t=12.454，P=0.001）]；抗凋亡蛋白Bcl-2表达增加[2 w（t=-9.099，P=0.000）、4 w（t=-8.128，P=0.000）、6 w（t=-9.497，P=0.000）]；促凋亡蛋白Bax表达降低[2 w（t=-4.263，P=0.005）、4 w（t=-2.936，P=0.026）和6 w（t=-8.343，P=0.000）]；VEGF表达增加[2 w（t=-5.754，P=0.001）、4 w（t=-5.077，P=0.002）和6 w（t=-12.200，P=0.000）]，差异均具有统计学意义。X线结果显示雷帕霉素组术后2 w、4 w和6 w Garrett评分高于对照组（t=4.371，P=0.005；t=5.166，P=0.002；t=4.243，P=0.005），micro-CT结果显示雷帕霉素组术后2 w、4 w和6 w骨密度评分高于对照组（t=8.765，P=0.000；t=25.649，P=0.000；t=11.199，P=0.000），差异具有统计学意义。

结论

雷帕霉素通过诱导细胞自噬，抑制细胞凋亡促进骨折愈合。

关键词: 雷帕霉素, 自噬, 凋亡, 骨折愈合
Objective

To investigate the changes in cell autophagy and the molecularmechanism of rapamycin affecting the fracture healing.

Methods

Sprague-Dawley (SD) rats were used to establish the right femoral shaft fracture models. After model establishment, SD rats were divided into two groups, the control group and the rapamycin group (1 mg/kg/d). There was no special treatment in the blank control group. The rapamycin group was intraperitoneally injected with 1 mg/kg rapamycin at 12 h, 24 h, 3 d and 7 d after surgery. X-ray and Micro CT were used to evaluate the healing of bone tissue at 2 w, 4 w and 6 w after model establishment. Immunofluorescence was used to detect LC3-Ⅱ. Immunohistochemistry was used to detect the expression level of Beclin-1. Western blot was used to detect Bax and VEGF. p-mTOR, hematoxylin and eosin staining (HE staining) to evaluate theosteoblastic activity through count of osteoblast in bone tissue at the end of fracture.

Results

Compared with the control group, the ratio of p-mTOR and p-mTOR/mTOR in the fractured bone tissue of the Papamycin group were decreased [2 w (t=6.703, P=0.004; t=16.346, P=0.000), 4 w (t=16.407, P=0.000; t=16.738, P=0.000) and 6 w (t=6.101, P=0.001, t=6.104, P=0.001)]. The expression of autophagy proteins LC3-II and Beclin-1 were significant increased[2 w (P=0.000, t=9.509; P=0.000, t=9.962), 4 w (t=3.868, P=0.008, t=24.490, P=0.000,) and 6 w(t=5.071, P=0.002, t=12.454, P=0.001,)]. Expression of anti-apoptotic protein Bcl-2 were increased [2 w (t=-9.099, P=0.000), 4 w (t=-8.128, P=0.000, And 6 w (t=-9.497, P=0.000)]. Pro-apoptotic protein Bax expression were decreased [2 w (t=-4.263, P=0.005), 4 w (t=-2.936, P=0.026) and 6 w (t=-8.343, P=0.000)]. VEGF expression were increased, 2 w [(t=-5.754, P=0.001), 4 w (t=-5.077, P=0.002) and 6 w (t=-12.200, P=0.000)], all the differences were statistically significant. X-ray results showed that Garrett scores were significantly higher in the rapamycin group than in the control group at 2 w, 4 w and 6 w after surgery (t=4.371, P=0.005, t=5.166, P=0.002, t=4.243, P=0.005), micro-CT results showed that the bone density scores at 2 w, 4 w and 6 w after rapamycin group were significantly higher than those in the control group (t=8.765, P=0.000, t=25.649, P=0.000, t=11.199, P=0.000), the difference was statistically significant.

Conclusion

Rapamycin promotes fracture healing by inducing autophagy and inhibiting apoptosis.

Key words: Sirolimus, Autophagy, Apoptosis, Fracture healing
图1~6 免疫荧光检测骨折愈合过程中LC3-Ⅱ蛋白的表达。图1~5　空白对照组造模术后12 h、24 h、3 d和7 d骨折区骨痂LC3免疫荧光（200×）；图6 LC3-Ⅱ阳性点计数直方图
图7~20 X线及micro-CT检测骨折愈合情况。图7~12　对照组及雷帕霉素组术后2 w、4 w和6 w X线检查；图13~18　两组术后2 w、4 w和6 w micro-CT检查；图19　两组术后2 w、4 w和6 w时X线Garrett评分；图20　两组术后2 w、4 w和6 w骨密度评分
图21~31 HE组织学染色检测两组骨折愈合过程。图21~26　对照组及雷帕霉素组术后2 w、4 w和6 w HE染色（400×）；图27 Western blotting检测两组术后2 w、4 w和6 w Bax、Bcl-2和VEGF蛋白的表达；图28 VEGF蛋白表达定量分析；图29 Bax蛋白表达定量分析；图30 Bcl-2蛋白表达定量分析；图31 Bax/Bcl-2比值分析
图32~45 免疫荧光和免疫组化分别检测LC3-Ⅱ和Beclin-1的表达水平。图32~37　免疫荧光法检测对照组及雷帕霉素组术后2 w、4 w和6 w LC3-Ⅱ蛋白的表达水平；图38~43　免疫组化法检测两组术后2 w、4 w和6 w Beclin-1蛋白的表达水平；图44　两组术后2 w、4 w和6 w LC3-Ⅱ蛋白阳性点定量分析；图45　两组术后2 w、4 w和6 w Beclin-1蛋白阳性点定量分析
图46~48 Western blotting检测mTOR通路蛋白的表达水平。图46 Western blotting法检测对照组及雷帕霉素组术后2 w、4 w和6 w mTOR和p-mTOR蛋白的表达水平；图47 p-mTOR蛋白表达定量分析；图48 p-mTOR/mTOR比值分析
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