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

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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.
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8.	李懷東, 李昕澤. 小干擾 RNA 研究概況. 世界最新醫學信息文摘 (電子版), 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.	朱倫井, 貝朝涌, 段江濤, 等. 脂質體和慢病毒介導 P75 神經生長因子受體及神經生長因子轉染骨髓間充質干細胞的效果比較. 中國組織工程研究, 2019, 23(21): 3302-3308.
11.	陳寧. 雙基因 NGF-IRES-BMP2 真核質粒轉染大鼠 BMSCs 誘導成骨的實驗研究. 桂林: 桂林醫學院, 2015.
12.	粟謀, 貝朝涌, 蔣林彬, 等. pcDNA3-hNGF 真核表達載體的構建及其在大鼠骨髓基質干細胞中的表達. 中國骨質疏松雜志, 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.	張兆弢. 沉默 p75NTR 基因促進舌鱗癌細胞凋亡的分子機制研究. 濟南: 山東大學, 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. 沈夢杰, 楊琨, 劉琪. 骨組織工程研究中 P75 神經營養因子受體的作用及應用價值. 中國組織工程研究, 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, 王銘, 彭稱飛, 等. P75NTR 在兔骨折不愈合局部組織中的表達及意義. 中國骨質疏松雜志, 2017, 23(4): 437-440.
5. 王銘, 彭稱飛, 段江濤, 等. P75NTR 介導細胞凋亡在骨折愈合過程中的研究進展. 世界最新醫學信息文摘 (電子版), 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. 鐘澤祥, 周亦楠, 馮思思, 等. 慢病毒介導小干擾 RNA 干擾促分裂原和應激激活的蛋白激酶 1 對大鼠脊髓損傷的修復作用. 中國修復重建外科雜志, 2018, 32(7): 941-950.
8. 李懷東, 李昕澤. 小干擾 RNA 研究概況. 世界最新醫學信息文摘 (電子版), 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. 朱倫井, 貝朝涌, 段江濤, 等. 脂質體和慢病毒介導 P75 神經生長因子受體及神經生長因子轉染骨髓間充質干細胞的效果比較. 中國組織工程研究, 2019, 23(21): 3302-3308.
11. 陳寧. 雙基因 NGF-IRES-BMP2 真核質粒轉染大鼠 BMSCs 誘導成骨的實驗研究. 桂林: 桂林醫學院, 2015.
12. 粟謀, 貝朝涌, 蔣林彬, 等. pcDNA3-hNGF 真核表達載體的構建及其在大鼠骨髓基質干細胞中的表達. 中國骨質疏松雜志, 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. 張兆弢. 沉默 p75NTR 基因促進舌鱗癌細胞凋亡的分子機制研究. 濟南: 山東大學, 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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