力生长因子促进骨髓间充质干细胞向成骨细胞的分化

doi:10.3969/j.issn.2095-4344.2016.32.001

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (32): 4717-4724.doi: 10.3969/j.issn.2095-4344.2016.32.001

• 骨髓干细胞 bone marrow stem cells • 下一篇

力生长因子促进骨髓间充质干细胞向成骨细胞的分化

佟雁翔¹，冯卫¹，贾燕飞¹，王彩霞¹，吕慧成¹，吴一民¹，蒋电明²

1内蒙古医科大学第二附属医院，内蒙古自治区呼和浩特市 010059
2重庆医科大学第一附属医院，重庆市 400993

修回日期:2016-05-19 出版日期:2016-08-05 发布日期:2016-08-05
通讯作者: 冯卫，博士，主任医师，内蒙古医科大学第二附属医院，内蒙古自治区呼和浩特市 010059
作者简介:佟雁翔，男，1981年生，河北省人，汉族，2015年重庆医科大学毕业，博士，主治医师，主要从事创伤骨科学、髋部骨折、骨盆环损伤以及骨组织工程研究。

Mechano growth factor promotes the differentiation of bone marrow mesenchymal stem cells into osteoblasts

Tong Yan-xiang¹, Feng Wei¹, Jia Yan-fei¹, Wang Cai-xia¹, Lv Hui-cheng¹, Wu Yi-min¹, Jiang Dian-ming²

1Second Affiliated Hospital, Inner Mongolia Medical University, Hohhot 010059, Inner Mongolia Autonomous Region, China
2the First Affiliated Hospital of Medical University of Chongqing, Chongqing 400993, China

Revised:2016-05-19 Online:2016-08-05 Published:2016-08-05
Contact: Feng Wei, M.D., Chief physician, Second Affiliated Hospital, Inner Mongolia Medical University, Hohhot 010059, Inner Mongolia Autonomous Region, China
About author:Tong Yan-xiang, M.D., Attending physician, Second Affiliated Hospital, Inner Mongolia Medical University, Hohhot 010059, Inner Mongolia Autonomous Region, China

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
力生长因子：来源于胰岛素样生长因子的一个亚型，它存在于受到牵拉刺激的肌肉中，且是非肝脏型的胰岛素样生长因子1。由于其在受到机械刺激时才产生的因子，力生长因子具有使肌肉卫星细胞激活和使肌原细胞生长的功能。同时力生长因子可以修复肌损失、治疗神经损伤并对心肌损伤有预防作用。
反映成骨细胞活动的标志物：①碱性磷酸酶和骨碱性磷酸酶：自1923年Robinson发现碱性磷酸酶以来，碱性磷酸酶已作为评价骨形成和骨转换的重要指标，在骨形成过程中，成骨细胞活动增强，大量分泌骨碱性磷酸酶，骨碱性磷酸酶作为碱性磷酸酶的同功酶，特异性来源于骨组织，能反映成骨细胞活性；②骨钙蛋白：又称骨钙素，是在骨基质矿化过程中由成骨细胞合成的非胶原蛋白，是反映成骨活动和骨转换的重要标志，占骨组织非胶原蛋白10%-20%；③骨形态发生蛋白：主要生物学作用就是诱导间充质细胞分化为成骨细胞，进而产生新骨。

摘要
背景：力生长因子具有激活肌肉卫星细胞和促进肌原细胞生长的作用，同时在维持骨质量和修复骨损伤方面具有作用。
目的：研究力生长因子促进兔骨髓间充质干细胞向成骨细胞分化的作用机制。
方法：采用MTT法检测力生长因子促进兔骨髓间充质干细胞成骨分化的最佳质量浓度与时间。用定量PCR和Western blot检测此质量浓度力生长因子促进兔骨髓间充质干细胞成骨分化后细胞中成骨细胞标志物碱性磷酸酶和骨钙素mRNA和蛋白的表达水平变化，并用Western Blot检测AKT和mTOR磷酸化水平变化。
结果与结论：力生长因子的质量浓度为45 μg/L时作用5 d促进兔骨髓间充质干细胞成骨分化的效果最好。45 μg/L力生长因子促进兔骨髓间充质干细胞成骨分化后细胞中碱性磷酸酶和骨钙素mRNA和蛋白水平在干预后4 h最高，同时AKT和mTOR的磷酸化水平也在干预后4 h时最高。说明力生长因子促进兔骨髓间充质干细胞向成骨细胞的分化的作用机制是通过PI3K/AKT途径实现的，且在45 μg/L干预后4 h时的效果最好。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0002-5751-2803(佟雁翔)

关键词: 干细胞, 骨髓干细胞, 力生长因子, PI3K/AKT通路, 细胞分化, 成骨细胞, mTOR

Abstract:

BACKGROUND: Mechano growth factor has the potential to activate muscle satellite cells and promote myogenic cell growth, and has dual roles in maintaining bone mass and repairing bone defects.
OBJECTIVE: To explore the mechanism underlying osteogenic differentiation of rabbit bone marrow mesenchymal stem cells promoted by the mechano growth factor.
METHODS: The best concentration and time of mechano growth factor to promote osteogenic differentiation of rabbit bone marrow mesenchymal stem cells were detected by MTT. The mRNA and protein expressions of alkaline phosphatase and osteocalcin were detected by qPCR and western blot, respectively. The phosphorylation level of AKT and mTOR were detected by western blot assay.
RESULTS AND CONCLUSION: The best concentration and time of mechano growth factor was 45 μg/L and 5 days for promoting the osteogenic differentiation of rabbit bone marrow mesenchymal stem cells. The expressions of alkaline phosphatase and osteocalcin at mRNA and protein levels were highest after 4-hour intervention with 45 μg/L mechano growth factor, and meanwhile, the phosphorylation levels of mTOR and AKT were also highest. These findings indicate that the mechano growth factor can promote the differentiation of rabbit bone marrow mesenchymal stem cells into osteoblasts via PI3K/AKT pathway, and its best concentration and time are 45 μg/L and 4 hours, respectively.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Cell Differentiation, Alkaline Phosphatase, Tissue Engineering

中图分类号:

R394.2

佟雁翔，冯卫，贾燕飞，王彩霞，吕慧成，吴一民，蒋电明. 力生长因子促进骨髓间充质干细胞向成骨细胞的分化[J]. 中国组织工程研究, 2016, 20(32): 4717-4724.

Tong Yan-xiang, Feng Wei, Jia Yan-fei, Wang Cai-xia, Lv Hui-cheng, Wu Yi-min, Jiang Dian-ming. Mechano growth factor promotes the differentiation of bone marrow mesenchymal stem cells into osteoblasts[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(32): 4717-4724.

图/表（结果） 2

参考文献

[1] Armakolas A, Philippou A, Panteleakou Z, et al. Preferential expression of IGF-1Ec (MGF) transcript in cancerous tissues of human prostate: evidence for a novel and autonomous growth factor activity of MGF E peptide in human prostate cancer cells. Prostate. 2010; 70(11):1233-1242.
[2] Kandalla PK, Goldspink G, Butler-Browne G, et al. Mechano Growth Factor E peptide (MGF-E), derived from an isoform of IGF-1, activates human muscle progenitor cells and induces an increase in their fusion potential at different ages. Mech Ageing Dev. 2011; 132(4):154-162.
[3] Deng M, Zhang B, Wang K, et al. Mechano growth factor E peptide promotes osteoblasts proliferation and bone-defect healing in rabbits. Int Orthop. 2011;35(7): 1099-1106.
[4] Peng Q, Qiu J, Sun J, et al. The nuclear localization of MGF receptor in osteoblasts under mechanical stimulation. Mol Cell Biochem. 2012;369(1-2): 147-156.
[5] Li C, Vu K, Hazelgrove K, et al. Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn's disease and smooth muscle hypertrophy. Am J Physiol Gastrointest Liver Physiol. 2015;309(11):G888-899.
[6] Li H, Lei M, Luo Z, et al. Mechano-growth factor enhances differentiation of bone marrow-derived mesenchymal stem cells. Biotechnol Lett. 2015;37(11): 2341-2348.
[7] Luo Z, Jiang L, Xu Y, et al. Mechano growth factor (MGF) and transforming growth factor (TGF)-β3 functionalized silk scaffolds enhance articular hyaline cartilage regeneration in rabbit model. Biomaterials. 2015;52:463-475.
[8] Goldspink G. Research on mechano growth factor: its potential for optimising physical training as well as misuse in doping. Br J Sports Med. 2005;39(11): 787-788; discussion 787-788.
[9] Gan ZY, Fitter S, Vandyke K, et al. The effect of the dual PI3K and mTOR inhibitor BEZ235 on tumour growth and osteolytic bone disease in multiple myeloma. Eur J Haematol. 2015;94(4):343-354.
[10] Heras-Sandoval D, Pérez-Rojas JM, Hernández-Damián J, et al. The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration. Cell Signal. 2014; 26(12):2694- 2701.
[11] Hsia HE, Kumar R, Luca R, et al. Ubiquitin E3 ligase Nedd4-1 acts as a downstream target of PI3K/PTEN-mTORC1 signaling to promote neurite growth. Proc Natl Acad Sci U S A. 2014;111(36): 13205-13210.
[12] Imanaka M, Iida K, Murawaki A, et al. Growth hormone stimulates mechano growth factor expression and activates myoblast transformation in C2C12 cells. Kobe J Med Sci. 2008;54(1):E46-54.
[13] Deng M, Liu P, Xiao H, et al. Improving the osteogenic efficacy of BMP2 with mechano growth factor by regulating the signaling events in BMP pathway. Cell Tissue Res. 2015;361(3):723-731.
[14] Shang J, Fan X, Liu H. The role of mechano-growth factor E peptide in the regulation of osteosarcoma. Oncol Lett. 2015;10(2):697-704.
[15] Yu C, Sha Y, Guo P, et al. Study on the regulatory effects of mechano growth factor on soft tissue repair. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2015; 32(1):235-239.
[16] Stavropoulou A, Halapas A, Sourla A, et al. IGF-1 expression in infarcted myocardium and MGF E peptide actions in rat cardiomyocytes in vitro. Mol Med. 2009;15(5-6):127-135.
[17] Thevis M, Thomas A, Geyer H, et al. Mass spectrometric characterization of a biotechnologically produced full-length mechano growth factor (MGF) relevant for doping controls. Growth Horm IGF Res. 2014;24(6):276-280.
[18] Yang SY, Goldspink G. Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation. FEBS Lett. 2002;522 (1-3):156-160.
[19] Zhu SB, Zhu J, Zhou ZZ, et al. TGF-β1 induces human aortic vascular smooth muscle cell phenotype switch through PI3K/AKT/ID2 signaling. Am J Transl Res. 2015;7(12):2764-2774.
[20] Tong Y, Feng W, Wu Y, et al. Mechano-growth factor accelerates the proliferation and osteogenic differentiation of rabbit mesenchymal stem cells through the PI3K/AKT pathway. BMC Biochem. 2015; 16:1.
[21] Peña JR, Pinney JR, Ayala P, et al. Localized delivery of mechano-growth factor E-domain peptide via polymeric microstructures improves cardiac function following myocardial infarction. Biomaterials. 2015; 46:26-34.
[22] Luo Q, Wu K, Zhang B, et al. Mechano growth factor E peptide promotes rat bone marrow-derived mesenchymal stem cell migration through CXCR4-ERK1/2. Growth Factors. 2015;33(3): 210-219.
[23] Malo MS. A High Level of Intestinal Alkaline Phosphatase Is Protective Against Type 2 Diabetes Mellitus Irrespective of Obesity. EBioMedicine. 2015; 2(12):2016-2023.
[24] Delli Mauri A, Petrini M, Vitale D, et al. Alkaline phosphatase level in gingival crevicular fluid during treatment with Quad-Helix. J Biol Regul Homeost Agents. 2015;29(4):1017-1023.
[25] Pabis A, Kamerlin SC. Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily. Curr Opin Struct Biol. 2016;37:14-21.
[26] Bobeck EA, Hellestad EM, Helvig CF, et al. Oral antibodies to human intestinal alkaline phosphatase reduce dietary phytate phosphate bioavailability in the presence of dietary 1α-hydroxycholecalciferol. Poult Sci. 2016;95(3):570-580.
[27] Millán JL, Whyte MP. Alkaline Phosphatase and Hypophosphatasia. Calcif Tissue Int. 2016;98(4): 398-416.
[28] Khan AR, Awan FR, Najam SS, et al. Elevated serum level of human alkaline phosphatase in obesity. J Pak Med Assoc. 2015;65(11):1182-1185.
[29] Mruthyunjaya S, Rumma M, Ravibhushan G, et al. c-Jun/AP-1 transcription factor regulates laminin-1-induced neurite outgrowth in human bone marrow mesenchymal stem cells: role of multiple signaling pathways. FEBS Lett. 2011;585(12): 1915-1922.
[30] Olivares-Navarrete R, Hyzy SL, Berg ME, et al. Osteoblast lineage cells can discriminate microscale topographic features on titanium-aluminum-vanadium surfaces. Ann Biomed Eng. 2014;42(12):2551-2561.
[31] Feng HW, Tian YD, Zhang HP, et al. Bone Age and Serum Osteocalcin Levels in Children With Obstructive Sleep Apnea Hypopnea Syndrome Before and After Adenotonsillectomy. Am J Ther. 2015. in press.
[32] Inaba N, Sato T, Yamashita T. Low-Dose Daily Intake of Vitamin K2 (Menaquinone-7) Improves Osteocalcin γ-Carboxylation: A Double-Blind, Randomized Controlled Trials. J Nutr Sci Vitaminol (Tokyo). 2015; 61(6):471-480.
[33] Liu J, Yang J. Uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by preventing the activation of PI3K/Akt in MC3T3-E1 cells. Int J Mol Med. 2016; 37(1):173-181.
[34] Kim KM, Lim S, Moon JH, et al. Lower uncarboxylated osteocalcin and higher sclerostin levels are significantly associated with coronary artery disease. Bone. 2016;83:178-183.
[35] Yun SH, Kim MJ, Choi BH, et al. Low Level of Osteocalcin Is Related With Arterial Stiffness in Korean Adults: An Inverse J-Shaped Relationship. J Clin Endocrinol Metab. 2016;101(1):96-102.
[36] Park JE, Seo YK, Yoon HH, et al. Electromagnetic fields induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation. Neurochem Int. 2013; 62(4):418-424.
[37] Bador KM, Wee LD, Halim SA, et al. Serum osteocalcin in subjects with metabolic syndrome and central obesity. Diabetes Metab Syndr. 2015. in press.
[38] Ho MH, Yao CJ, Liao MH, et al. Chitosan nanofiber scaffold improves bone healing via stimulating trabecular bone production due to upregulation of the Runx2/osteocalcin/alkaline phosphatase signaling pathway. Int J Nanomedicine. 2015;10:5941-5954.
[39] Zwakenberg SR, Gundberg CM, Spijkerman AM, et al. Osteocalcin Is Not Associated with the Risk of Type 2 Diabetes: Findings from the EPIC-NL Study. PLoS One. 2015;10(9):e0138693.
[40] Arya N, Moonarmart W, Cheewamongkolnimit N, et al. Osteocalcin and bone-specific alkaline phosphatase in Asian elephants (Elephas maximus) at different ages. Vet J. 2015;206(2):239-240.
[41] Levinger I, Lin X, Zhang X, et al. The effects of muscle contraction and recombinant osteocalcin on insulin sensitivity ex vivo. Osteoporos Int. 2016;27(2): 653-663.
[42] Ling Y, Gao X, Lin H, et al. A common polymorphism rs1800247 in osteocalcin gene was associated with serum osteocalcin levels, bone mineral density, and fracture: the Shanghai Changfeng Study. Osteoporos Int. 2016;27(2):769-779.
[43] Martins AF, Souza PO, Rege IC, et al. Glucocorticoids, calcitonin, and osteocalcin cannot differentiate between aggressive and nonaggressive central giant cell lesions of the jaws. Oral Surg Oral Med Oral Pathol Oral Radiol. 2015;120(3):386-395.
[44] Peura M, Bizik J, Salmenperä P, et al. Bone marrow mesenchymal stem cells undergo nemosis and induce keratinocyte wound healing utilizing the HGF/c-Met/ PI3K pathway. Wound Repair Regen. 2009;17(4): 569-577.
[45] Wang L, Zhang L, Shen W, et al. High expression of VEGF and PI3K in glioma stem cells provides new criteria for the grading of gliomas. Exp Ther Med. 2016;11(2):571-576.
[46] Abliz A, Deng W, Sun R, et al. Wortmannin, PI3K/Akt signaling pathway inhibitor, attenuates thyroid injury associated with severe acute pancreatitis in rats. Int J Clin Exp Pathol. 2015;8(11):13821-13833.
[47] Robbins HL, Hague A. The PI3K/Akt Pathway in Tumors of Endocrine Tissues. Front Endocrinol (Lausanne). 2016;6:188.
[48] Ma LI, Chang Y, Yu L, et al. Pro-apoptotic and anti-proliferative effects of mitofusin-2 via PI3K/Akt signaling in breast cancer cells. Oncol Lett. 2015; 10(6):3816-3822.
[49] Sledd J, Wu D, Ahrens R, et al. Loss of IL-4Rα-mediated PI3K signaling accelerates the progression of IgE/mast cell-mediated reactions. Immun Inflamm Dis. 2015;3(4):420-430.
[50] Zhao J, Li L, Peng L. MAPK1 up-regulates the expression of MALAT1 to promote the proliferation of cardiomyocytes through PI3K/AKT signaling pathway. Int J Clin Exp Pathol. 2015;8(12):15947-15953.
[51] Li XJ, Luo Q, Sun L, et al. The effects of curcumin on PTEN/PI3K/Akt pathway in Ec109 cells. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2015;31(5):465-468.
[52] Zhang P, Zhang L, Zhu L, et al. The change tendency of PI3K/Akt pathway after spinal cord injury. Am J Transl Res. 2015;7(11):2223-2232.
[53] Lee JJ, Loh K, Yap YS. PI3K/Akt/mTOR inhibitors in breast cancer. Cancer Biol Med. 2015;12(4):342-354.
[54] Chen X, Li YY, Zhang WQ, et al. House dust mite extract induces growth factor expression in nasal mucosa by activating the PI3K/Akt/HIF-1α pathway. Biochem Biophys Res Commun. 2016;469(4): 1055-1061.
[55] Rice GB, Wadhwani NR. PI3K/AKT Pathway and Brain Malformations. Pediatr Neurol Briefs. 2015;29(7):52.
[56] Wang G, Li X, Wang H, et al. Later phase cardioprotection of ischemic post-conditioning against ischemia/reperfusion injury depends on iNOS and PI3K-Akt pathway. Am J Transl Res. 2015;7(12): 2603-2611.
[57] Mayer IA. Clinical Implications of Mutations in the PI3K Pathway in HER2+ Breast Cancer: Prognostic or Predictive? Curr Breast Cancer Rep. 2015;7(4): 210-214.
[58] Sun Y, Tu Y, He LI, et al. High mobility group box 1 regulates tumor metastasis in cutaneous squamous cell carcinoma via the PI3K/AKT and MAPK signaling pathways. Oncol Lett. 2016;11(1):59-62.
[59] Milingos DS, Philippou A, Armakolas A, et al. Insulinlike growth factor-1Ec (MGF) expression in eutopic and ectopic endometrium: characterization of the MGF E-peptide actions in vitro. Mol Med. 2011; 17(1-2):21-28.
[60] Philippou A, Papageorgiou E, Bogdanis G, et al. Expression of IGF-1 isoforms after exercise-induced muscle damage in humans: characterization of the MGF E peptide actions in vitro. In Vivo. 2009;23(4): 567-575.
[61] Qin LL, Li XK, Xu J, et al. Mechano growth factor (MGF) promotes proliferation and inhibits differentiation of porcine satellite cells (PSCs) by down-regulation of key myogenic transcriptional factors. Mol Cell Biochem. 2012;370(1-2):221-230.

引言

材料方法

1.1 设计 随机对照细胞学实验。

1.2 时间及地点 实验于2014年12月在内蒙古医科大学完成。

1.3 材料 健康清洁级新西兰大白兔，体质量1.5- 3.0 kg，购自上海市松联实验动物场，许可证号：SCXK (2007-0016)。

1.4 方法

1.4.1 骨髓间充质干细胞的分离与培养 将新西兰大白兔以氯胺酮(0.5 mg/kg)腹腔注射麻醉，备皮、消毒，取双侧大腿外侧切口，无菌条件下立即离断髋关节和膝关节，摘取股骨，小心剔除股骨上附着的软组织。剪断股骨两端，暴露骨髓腔，用含有双抗(青霉素1×10⁵ U/L、链霉素1×10⁵ U/L)的L-DNEM培养基冲出骨髓于离心管中，吹打制成单细胞悬液。将上述液体离心5 min，1 000 r/min，弃上清。用含体积分数10%的胎牛血清、1×10⁵ U/L青霉素、1×10⁵ U/L链霉素的L-DMEM培养基重悬。将细胞接种于培养瓶中，置于37 ℃、体积分数5%CO₂培养箱中培养。3 d后进行半量换液，5 d后全量换液，以后每3 d换液一次。通过免疫荧光检测CD44和CD34的方法鉴定骨髓间充质干细胞。

1.4.2 MTT检测 收集第4代兔骨髓间充质干细胞作为种子细胞，加于96孔板中，每孔100 μL，接种细胞浓度为1×10⁸ L^-¹左右。加好后加盖，光学显微镜下观察每孔细胞的密度是否均匀，放入培养箱中培养。细胞贴壁且覆盖孔底90%以上时换液一次。加入终质量浓度分别为0，15，30，45，60和75 μg/L力生长因子，分别作用1-10 d，每个质量浓度做5个复孔。每孔中加入10 μL MTT，培养4 h。去上清，每孔加入100 μL DMSO，在振荡器上震荡30 min。紫外分光光度仪检测，酶标仪 570 nm波长测定吸光度值。

1.4.3 力生长因子对兔骨髓间充质干细胞分化的影响 无菌条件下，取兔骨髓间充质干细胞，以DMEM洗涤2遍，1 000 r/min离心5 min，按1×10⁸ L^-¹细胞浓度的接种于25 mL培养瓶内，加入5 mL含体积分数10%胎牛血清的DMEM，在37 ℃、体积分数5% CO₂培养箱中培养，24 h换液，清除悬浮细胞，而后隔日换液，观察细胞生长情况。细胞长满培养瓶底80%后，去培养液，加0.25%胰蛋白酶1 mL，轻轻晃动培养皿，使胰蛋白酶覆盖整个皿底，37 ℃ 消化3-5 min，镜下见细胞皱缩，间距加大，分离为单个小圆细胞时，滴加DMEM培养液中止消化，然后吹打呈细胞悬液，将细胞悬液移入15 mL离心管，1 000 r/min离心10 min，弃上清，沉淀细胞加5 mL含血清DMEM培养液，移入培养瓶在37 ℃、体积分数5% CO₂培养箱中传代培养。传代改用含地塞米松和维生素C的诱导培养基进行细胞培养和扩增，诱导培养基组成：含体积分数10%胎牛血清的DMEM高糖培养基加入地塞米松和维生素C，终末浓度分别为10 mol/L和50 ng/L。加入45 μg/L力生长因子分别培养0，4，8，12，16 h后，收集细胞，提取细胞的RNA。0.8%的琼脂糖电泳，检测RNA的提取情况。在凝胶成像系统中进行观察。

1.4.4 定量RT-PCR分析 将收集到无RNase的无菌离心管中，加入适量的Trizol液充分混匀，室温下静置5 min，加入1/5 Trizol液体积的氯仿混匀，放置冰上5 min，12 000 r/min 4 ℃离心15 min。取上清液，加等体积异丙醇混匀，放于-20 ℃ 冰箱中10 min，之后12 000 r/min 4 ℃离心10 min。弃去上清液，加入300 μL的无RNase体积分数75%冰乙醇，12 000 r/min 4 ℃离心10 min。弃上清液，沉淀干燥5 min，用无RNase的去离子水溶解沉淀。

PrimeScript^RT reagent kit用于SYBR Green的反转录体系：5×PrimeScript Buffer 2.0 μL；PrimeScript RT Enzyme mix Ⅰ 0.5 μL；Oligo dT Primer 0.5 μL；Randon 6 mers 0.5 μL；加无Rnase的dH₂O至10 μL。反转录反应为37 ℃ 15 min，反转录酶的失活反应为 85 ℃ 5 s。放入PCR仪，得到的cDNA，放入-20 ℃保存备用。

SYBR Premix Ex Taq Ⅱ反应体系为：SYBR Premix Ex Taq Ⅱ10 μL，PCR Primer(Forward and Reverse)1.6 μL，ROX Reference Dye or Dye Ⅱ 0.4 μL，cDNA dH₂O 8 μL，20 μL体系。反应条件为：95 ℃变性40 s，退火15 s，72 ℃延伸10 s，40个循环。将cDNA倍比稀释，进行实时定量PCR，反应体系及运行程序同上，检测目的基因及β-actin基因的PCR扩增效率。以2^-^??Ct示其相对表达水平。

1.4.5 Western blot分析 提取蛋白，行SDS-PAGE电泳，将凝胶中的蛋白质转移至硝酸纤维素膜上，5%脱脂奶粉封闭，一抗4 ℃孵育过夜，TBST洗涤，二抗室温振荡孵育1 h，TBST 洗3次，ECL显影，获得蛋白印迹结果。

1.5 主要观察指标 ①兔骨髓间充质干细胞的增殖。②碱性磷酸酶和骨钙素的表达水平。③AKT和mTOR的磷酸化水平。

1.6 统计学分析 采用SPSS 13.0统计软件，实验数据以x(_)±s表示，组间比较用单因素方差分析；当方差不齐时，采用秩和检验，P < 0.05为差异有显著性意义。

讨论

力生长因子是胰岛素样生长因子1基因的一种剪接变异体^[12]。力生长因子能响应力学刺激而表达^[13-14]，现有研究表明其具有促进肌肉肥大^[7^，^14-15]、心肌保护以及神经修复等作用^[16-18]。在实验中发现力生长因子对兔骨髓间充质干细胞的生长增殖有明显促进作用，且当在第5天力生长因子质量浓度达到45 μg/L时对兔骨髓间充质干细胞的作用最为明显。敲除力生长因子基因的小鼠长骨骨化明显延缓，颅盖骨发育缓慢；力生长因子可激活受体，并诱导信号转导，从而调控软骨细胞的增殖和分化。力生长因子促使ERK2磷酸化，促进细胞有丝分裂，同时下调颅骨成骨前体细胞胶原基因表达。力生长因子所诱导的信号途径在膜内骨化过程中调控颅骨细胞的表型，并激活成骨内ERK和p38MAPK信号途径^[5^，⁷^，^19-20]。力生长因子表达是在分化的成骨细胞骨化位点处。变异成纤维生长因子受体2可诱导人颅骨成骨细胞蛋白激酶C激活，该转导途径可提高变异成骨细胞的分化和凋亡。此外力生长因子是一类具有胰岛素样生物活性的多肽物质，可促进成骨细胞有丝分裂，刺激成熟成骨细胞的功能发挥，包括促进骨基质蛋白合成^[6-7^，^21-22]。

碱性磷酸酶和骨的钙化作用密切相关^[23-26]，成骨细胞中的碱性磷酸酶作用产生磷酸^[27-28]，与钙生成磷酸钙沉积于骨中^[29-30]。骨钙素是由成骨合成和分泌的维生素K依赖性钙结合蛋白，是一种非胶原酸性糖蛋白^[31-35]。因为分子中含有依赖维生素K的羧基谷氨酸残基，故又名羧基谷氨酸蛋白。骨钙素分子中的骨钙素是骨钙素与钙离子结合的重要功能基团^[36]。机体骨钙素有2种存在形式即羧化不全骨钙素和羧化骨钙素^[37-40]。骨钙素在骨组织含量较丰富，由分化成熟的成骨细胞合成和分泌^[41-43]，大部分沉积于骨基质，大约有20%释放入血，血清骨钙素和骨组织骨钙素呈正相关性^[44]。为了进一步研究力生长因子对增殖的兔骨髓间充质干细胞向成骨细胞分化情况，实验将兔骨髓间充质干细胞用诱导剂使之诱导成成骨细胞，并检测了成骨细胞标志物碱性磷酸酶和骨钙素mRNA和蛋白表达水平。结果显示碱性磷酸酶和骨钙素无论在mRNA还是在蛋白水平均在干预后4 h表达水平最高，表明力生长因子对增殖的兔骨髓间充质干细胞对其向成骨细胞的转化同样有促进作用。

为进一步研究力生长因子对兔骨髓间充质干细胞向成骨细胞分化的作用机制，实验研究了45 μg/L力生长因子干预兔骨髓间充质干细胞后4 h磷酸化AKT和磷酸化mTOR的表达水平最高。说明力生长因子对兔骨髓间充质干细胞分化的作用主要通过PI3k/AKT的下游途径。PI3K蛋白家族主要参与细胞分化、增殖、凋亡以及葡萄糖转运等多种细胞功能的调节^[45-49]。PI3K激活后，可使第二信使3，4，5-三磷酸磷脂酰肌醇在质膜上产生，进而使其与细胞内的AKT结合，从而使AKT活化^[50-54]。AKT磷酸化TSC1/2，可以使敏感的mTOR复合体(mTORC1)活化^{[45, 55-58]}。这些作用可激活蛋白的翻译，促进细胞的分化^[59-61]。实验发现虽然PI3k/AKT的下游途径可以调控兔骨髓间充质干细胞向成骨细胞的分化，但不是惟一途径，可能与其他的途径有关，比如MAPK途径。

综上，力生长因子促进兔骨髓间充质干细胞向成骨细胞分化的作用机制是通过PI3k/AKT的下游途径实现的，且在干预后4 h时的效果最好。

力生长因子促进骨髓间充质干细胞向成骨细胞的分化

Mechano growth factor promotes the differentiation of bone marrow mesenchymal stem cells into osteoblasts

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表（结果） 2

参考文献

相关文章 15

引言

材料方法

讨论

文章快阅

延伸阅读

编辑推荐

Metrics

本文评价

[1]	蒲锐, 陈子扬, 袁凌燕. 不同细胞来源外泌体保护心脏的特点与效应[J]. 中国组织工程研究, 2021, 25(在线): 1-.
[2]	林清凡, 解一新, 陈婉清, 叶振忠, 陈幼芳. 人胎盘源间充质干细胞条件培养液可上调缺氧状态下BeWo细胞活力和紧密连接因子的表达[J]. 中国组织工程研究, 2021, 25(在线): 4970-4975.
[3]	张秀梅, 翟运开, 赵杰, 赵萌. 类器官模型国内外数据库近10年文献研究热点分析[J]. 中国组织工程研究, 2021, 25(8): 1249-1255.
[4]	王正东, 黄娜, 陈婧娴, 郑作兵, 胡鑫宇, 李梅, 苏晓, 苏学森, 颜南. 丁酸钠抑制氟中毒可诱导小胶质细胞活化及炎症因子表达增多[J]. 中国组织工程研究, 2021, 25(7): 1075-1080.
[5]	汪显耀, 关亚琳, 刘忠山. 提高间充质干细胞治疗难愈性创面的策略[J]. 中国组织工程研究, 2021, 25(7): 1081-1087.
[6]	万然, 史旭, 刘京松, 王岩松. 间充质干细胞分泌组治疗脊髓损伤的研究进展[J]. 中国组织工程研究, 2021, 25(7): 1088-1095.
[7]	廖成成, 安家兴, 谭张雪, 王倩, 刘建国. 口腔鳞状细胞癌干细胞的治疗靶点及应用前景[J]. 中国组织工程研究, 2021, 25(7): 1096-1103.
[8]	谢文佳, 夏天娇, 周卿云, 刘羽佳, 顾小萍. 小胶质细胞介导神经元损伤在神经退行性疾病中的作用[J]. 中国组织工程研究, 2021, 25(7): 1109-1115.
[9]	李珊珊, 郭笑霄, 尤冉, 杨秀芬, 赵露, 陈曦, 王艳玲. 感光细胞替代治疗视网膜变性疾病[J]. 中国组织工程研究, 2021, 25(7): 1116-1121.
[10]	焦慧, 张一宁, 宋雨晴, 林宇, 王秀丽. 乳腺癌类器官研究进展及临床应用前景[J]. 中国组织工程研究, 2021, 25(7): 1122-1128.
[11]	王诗琦, 张金生. 中医药调控缺血缺氧微环境对骨髓间充质干细胞增殖、分化及衰老的影响[J]. 中国组织工程研究, 2021, 25(7): 1129-1134.
[12]	曾燕华, 郝延磊. 许旺细胞体外培养及纯化的系统性综述[J]. 中国组织工程研究, 2021, 25(7): 1135-1141.
[13]	孔德胜, 何晶晶, 冯宝峰, 郭瑞云, Asiamah Ernest Amponsah, 吕飞, 张舒涵, 张晓琳, 马隽, 崔慧先. 间充质干细胞修复大动物模型脊髓损伤疗效评价的Meta分析[J]. 中国组织工程研究, 2021, 25(7): 1142-1148.
[14]	侯婧瑛, 于萌蕾, 郭天柱, 龙会宝, 吴浩. 缺氧预处理激活HIF-1α/MALAT1/VEGFA通路促进骨髓间充质干细胞生存和血管再生[J]. 中国组织工程研究, 2021, 25(7): 985-990.
[15]	史洋洋, 秦英飞, 吴福玲, 何潇, 张雪静. 胎盘间充质干细胞预处理预防小鼠毛细支气管炎[J]. 中国组织工程研究, 2021, 25(7): 991-995.