张善强　姚立杰　李宇　邓凤春　金海峰　沈雷

[摘要] 目的 探討神经营养因子-3（NT-3）在低氧环境中对人骨髓间充质干细胞（hBMSC）增殖能力的影响。 方法 在齐齐哈尔医学院分子生物学研究室利用三气培养体系建立低氧体外细胞培养模型，选取2016年5月购买的hBMSC，在低氧环境下，未行任何刺激的hBMSC为低氧对照组；用100 ng/mL人NT-3 重组蛋白刺激的hBMSC为NT-3组；先以MK2206作用30 min，再以NT-3刺激的hBMSC为Akt抑制剂组；正常氧浓度条件下培养的hBMSC为常氧对照组，各组均行成骨诱导实验21 d。分别用噻唑蓝细胞增殖、Western blot、酶联免疫吸附试验（ELISA）等实验检测各组细胞增殖、凋亡，及VEGF和BMP-1等蛋白的表达。 结果 与低氧对照组相比，NT-3组hBMSC增殖OD值（1.438±0.116）明显提高差异有统计学意义（P<0.01），NT-3组VEGF及BMP-1蛋白含量（1.704±0.132）ng/mL；（1.794±0.098）ng/mL较低氧对照组均有增高差异有统计学意义（P<0.01）；相对于NT-3组，Akt抑制剂组hBMSC增殖OD值（0.927±0.103）降低差异有统计学意义（P<0.01），且Akt抑制剂组VEGF和BMP-1蛋白含量（1.428±0.205）ng/mL；（1.157±0.102）ng/mL均低于NT-3组差异有统计学意义（P<0.01）。结论 NT-3可提高hBMSC抗缺氧功能，促进hBMSC在低氧状态下增殖。

[关键词] 神经营养因子-3；骨髓间充质干细胞；增殖； 缺氧

[中图分类号] R392.12 [文献标识码] A [文章编号] 1674-0742（2018）02（c）-0003-05

Neurotrophin-3 Promoting the Proliferation of Human Bone Marrow Mesenchymal Stem Cells in Hypoxic Environment

ZHANG Shan-qiang， YAO Li-jie， LI Yu， DENG Feng-chun， JIN Hai-feng， SHEN Lei

Department of Anatomy， Qiqihar Medical College of Basic Medical College， Qiqihar， Heilongjiang Province， 161006 China

[Abstract] Objective This paper tries to investigate the effect of Neurotrophin-3 （NT-3） on proliferation of human bone marrow mesenchymal stem cell （hBMSC） in hypoxic environment. Methods The hypoxic cell culture model in vitro was established by using three gas culture systems in the molecular biology laboratory of Qiqihar medical college， and the hBMSC purchased in May 2016 were selected. Under the hypoxic environment， the hBMSCs without any stimulation was used as the hypoxic control group； the hBMSCs supplemented with 100 ng/mL human NT-3 recombinant protein composed the NT -3 group； the rBMSCs with MK2206， added to the NT -3 group composed the Akt inhibitor group； the rBMSCs cultured with normal oxygen concentration as the normoxic control group， each group was subjected to osteogenic induction for 21 days. The MTT， Western blot， and ELISA assay was used to detect the cell proliferation， apoptosis， and the expression of VEGF and BMP-1 in each group， respectively. Results The OD value of hBMSC proliferation in NT-3 group was significantly higher than that in hypoxia control group（1.438 ± 0.116），The difference was statisically significant（P<0.01）， and the levels of VEGF and BMP-1 protein in NT-3 group（1.704±0.132）ng/mL；（1.794±0.098）ng/mL were higher than those in hypoxia control group，The difference was statisically significant（P<0.01）. Compared with NT-3 group， OD value of hBMSC proliferation decreased （0.927±0.103） in Akt inhibitor group，The difference was statisically significant（P<0.01）， and the levels of VEGF and BMP-1 in Akt inhibitor group Protein content （1.428±0.205）ng/mL；（1.157±0.102）ng/mL was lower than NT-3 group，The difference was statisically significant（P<0.01）. Conclusion NT-3 can improve the anti hypoxia function of hBMSC and promote the proliferation of hBMSC into osteoblasts under hypoxia.

[Key words] Neurotrophin-3 （NT-3）； Bone marrow mesenchymal stem cell （hBMSC）； Prolifertaion； Hypoxia

目前，来源于骨髓的间充质干细胞（Mesenchymal stem cell， MSC）因具备取材方便、免疫原性低、易在体外培养增殖和多向分化等优势已成为组织工程治疗骨折、骨缺损等骨损伤疾病的首选种子细胞[1]。然而，骨损伤部位的持续缺血缺氧环境会减少MSC的存活时间、抑制MSC的增殖和分化能力，从而降低细胞的组织修复功能[2]。因此，提高MSC抗缺氧能力，将对组织工程利用MSC治疗骨损伤具有重要意义。作为骨组织工程的三个关键因素之一，生物活性因子常被用以参与调节成骨的多种因子表达以及成骨细胞的活性来提高成骨效能。神经营养因子-3（Neurotrophin-3， NT-3）由中枢神经系统和肌肉组织产生，具有促进神经和血管再生的功能[3]。……