Effect of lentivirus-mediated silencing of P75 neurotrophin receptor gene on osteogenic differentiation of bone marrow mesenchymal stem cells in rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Ning ¹ , CHEN Junyi ¹ , CHEN Ximiao ² , ZHU Lunjing ¹ , DUAN Jiangtao ¹ , WANG Ye ² ,  BEI Chaoyong ¹

1. Department of Trauma and Limb Surgery, Affiliated Hospital of Guilin Medical College, Guilin Guangxi, 541000, P.R.China;
2. Guilin Medical College, Guilin Guangxi, 541000, P.R.China;

Corresponding author：

BEI Chaoyong, Email: beichy2008@126.com

Keywords：

P75 neurotrophin receptor; small interfering RNA; lentivirus; gene silencing; bone marrow mesenchymal stem cells; osteogenic differentiation; rat

DOI：

10.7507/1002-1892.201912086

Video：

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Objective To investigate the effect of small interfering RNA (siRNA) lentivirus-mediated silencing of P75 neurotrophin receptor (P75NTR) gene on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats.Methods Three lentivirus-mediated P75NTR gene siRNA sequences (P75NTR-siRNA-1, 2, 3) and negative control (NC)-siRNA were designed and transfected into the 3rd generation Sprague Dawley (SD) rat BMSCs. The cells morphological changes were observed under an inverted microscope, and the expressions of P75NTR gene and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. Then the best silencing P75NTR-siRNA for subsequent osteogenic differentiation experiments was screened out. The 3rd generation SD rat BMSCs were randomly divided into experimental group, negative control group, and blank control group (normal BMSCs). The BMSCs of negative control group and experimental group were transfected with NC-siRNA and the selected P75NTR-siRNA lentiviral vector, respectively. The cells of each group were cultured by osteogenic induction. The expressions of osteogenic related proteins [osteocalcin (OCN) and Runx related transcription factor 2 (Runx2)] were detected by Western blot; the collagen type Ⅰ expression was observed by immunohistochemical staining; the osteogenesis of BMSCs was observed by alkaline phosphatase (ALP) detection and alizarin red staining.Results After lentivirus-mediated P75NTR transfected into BMSCs, the expressions of P75NTR mRNA and protein significantly reduced (P<0.05), and the best silencing P75NTR-siRNA was P75NTR-siRNA-3. After P75NTR gene was silenced, MTT test showed that the cell proliferation in the experimental group was significantly faster than those in the two control groups (P<0.05). After osteogenic induction, the relative expressions of OCN and Runx2 proteins, collagen type Ⅰ expression, and ALP activity were significantly higher in the experimental group than in the two control groups, the differences were significant (P<0.05). With the prolongation of osteogenic induction, the mineralized nodules in the experimental group gradually increased.Conclusion Silencing the P75NTR gene with siRNA lentivirus can promote the osteogenic differentiation of rat BMSCs and provide a new idea for the treatment of bone defects.

Citation： WANG Ning, CHEN Junyi, CHEN Ximiao, ZHU Lunjing, DUAN Jiangtao, WANG Ye, BEI Chaoyong. Effect of lentivirus-mediated silencing of P75 neurotrophin receptor gene on osteogenic differentiation of bone marrow mesenchymal stem cells in rats. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(8): 1052-1058. doi: 10.7507/1002-1892.201912086 Copy

1.	沈梦杰, 杨琨, 刘琪. 骨组织工程研究中 P75 神经营养因子受体的作用及应用价值. 中国组织工程研究, 2018, 22(36): 5852-5857.
2.	Edalat H, Hajebrahimi Z, Movahedin M, et al. p75NTR suppression in rat bone marrow stromal stem cells significantly reduced their rate of apoptosis during neural differentiation. Neurosci Lett, 2011, 498(1): 15-19.
3.	Ko KI, Coimbra LS, Tian C, et al. Diabetes reduces mesenchymal stem cells in fracture healing through a TNFα-mediated mechanism. Diabetologia, 2015, 58(3): 633-642.
4.	李家勇, 王铭, 彭称飞, 等. P75NTR 在兔骨折不愈合局部组织中的表达及意义. 中国骨质疏松杂志, 2017, 23(4): 437-440.
5.	王铭, 彭称飞, 段江涛, 等. P75NTR 介导细胞凋亡在骨折愈合过程中的研究进展. 世界最新医学信息文摘 (电子版), 2018, 18(6): 81-83.
6.	Karnes JM, Daffner SD, Watkins CM. Multiple roles of tumor necrosis factor-alpha in fracture healing. Bone, 2015, 78: 87-93.
7.	钟泽祥, 周亦楠, 冯思思, 等. 慢病毒介导小干扰 RNA 干扰促分裂原和应激激活的蛋白激酶 1 对大鼠脊髓损伤的修复作用. 中国修复重建外科杂志, 2018, 32(7): 941-950.
8.	李怀东, 李昕泽. 小干扰 RNA 研究概况. 世界最新医学信息文摘 (电子版), 2019, 19(35): 116-117.
9.	Liu X, Hu G. In vivo calpain knockdown using delivery of siRNA. Methods Mol Biol, 2019, 1915: 219-232.
10.	朱伦井, 贝朝涌, 段江涛, 等. 脂质体和慢病毒介导 P75 神经生长因子受体及神经生长因子转染骨髓间充质干细胞的效果比较. 中国组织工程研究, 2019, 23(21): 3302-3308.
11.	陈宁. 双基因 NGF-IRES-BMP2 真核质粒转染大鼠 BMSCs 诱导成骨的实验研究. 桂林: 桂林医学院, 2015.
12.	粟谋, 贝朝涌, 蒋林彬, 等. pcDNA3-hNGF 真核表达载体的构建及其在大鼠骨髓基质干细胞中的表达. 中国骨质疏松杂志, 2016, 22(1): 41-44.
13.	Mesentier-Louro LA, Rosso P, Carito V, et al. Nerve growth factor role on retinal ganglion cell survival and axon regrowth: effects of ocular administration in experimental model of optic nerve injury. Mol Neurobiol, 2019, 56(2): 1056-1069.
14.	Fahnestock M, Shekari A. ProNGF and neurodegeneration in Alzheimer’s disease. Front Neurosci, 2019, 13: 129.
15.	张兆弢. 沉默 p75NTR 基因促进舌鳞癌细胞凋亡的分子机制研究. 济南: 山东大学, 2013.
16.	Zanin JP, Montroull LE, Volosin M, et al. The p75 neurotrophin receptor facilitates TrkB signaling and function in rat hippocampal neurons. Front Cell Neurosci, 2019, 13: 485.

1. 沈梦杰, 杨琨, 刘琪. 骨组织工程研究中 P75 神经营养因子受体的作用及应用价值. 中国组织工程研究, 2018, 22(36): 5852-5857.
2. Edalat H, Hajebrahimi Z, Movahedin M, et al. p75NTR suppression in rat bone marrow stromal stem cells significantly reduced their rate of apoptosis during neural differentiation. Neurosci Lett, 2011, 498(1): 15-19.
3. Ko KI, Coimbra LS, Tian C, et al. Diabetes reduces mesenchymal stem cells in fracture healing through a TNFα-mediated mechanism. Diabetologia, 2015, 58(3): 633-642.
4. 李家勇, 王铭, 彭称飞, 等. P75NTR 在兔骨折不愈合局部组织中的表达及意义. 中国骨质疏松杂志, 2017, 23(4): 437-440.
5. 王铭, 彭称飞, 段江涛, 等. P75NTR 介导细胞凋亡在骨折愈合过程中的研究进展. 世界最新医学信息文摘 (电子版), 2018, 18(6): 81-83.
6. Karnes JM, Daffner SD, Watkins CM. Multiple roles of tumor necrosis factor-alpha in fracture healing. Bone, 2015, 78: 87-93.
7. 钟泽祥, 周亦楠, 冯思思, 等. 慢病毒介导小干扰 RNA 干扰促分裂原和应激激活的蛋白激酶 1 对大鼠脊髓损伤的修复作用. 中国修复重建外科杂志, 2018, 32(7): 941-950.
8. 李怀东, 李昕泽. 小干扰 RNA 研究概况. 世界最新医学信息文摘 (电子版), 2019, 19(35): 116-117.
9. Liu X, Hu G. In vivo calpain knockdown using delivery of siRNA. Methods Mol Biol, 2019, 1915: 219-232.
10. 朱伦井, 贝朝涌, 段江涛, 等. 脂质体和慢病毒介导 P75 神经生长因子受体及神经生长因子转染骨髓间充质干细胞的效果比较. 中国组织工程研究, 2019, 23(21): 3302-3308.
11. 陈宁. 双基因 NGF-IRES-BMP2 真核质粒转染大鼠 BMSCs 诱导成骨的实验研究. 桂林: 桂林医学院, 2015.
12. 粟谋, 贝朝涌, 蒋林彬, 等. pcDNA3-hNGF 真核表达载体的构建及其在大鼠骨髓基质干细胞中的表达. 中国骨质疏松杂志, 2016, 22(1): 41-44.
13. Mesentier-Louro LA, Rosso P, Carito V, et al. Nerve growth factor role on retinal ganglion cell survival and axon regrowth: effects of ocular administration in experimental model of optic nerve injury. Mol Neurobiol, 2019, 56(2): 1056-1069.
14. Fahnestock M, Shekari A. ProNGF and neurodegeneration in Alzheimer’s disease. Front Neurosci, 2019, 13: 129.
15. 张兆弢. 沉默 p75NTR 基因促进舌鳞癌细胞凋亡的分子机制研究. 济南: 山东大学, 2013.
16. Zanin JP, Montroull LE, Volosin M, et al. The p75 neurotrophin receptor facilitates TrkB signaling and function in rat hippocampal neurons. Front Cell Neurosci, 2019, 13: 485.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of lentivirus-mediated silencing of P75 neurotrophin receptor gene on osteogenic differentiation of bone marrow mesenchymal stem cells in rats

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