Department of Orthopaedics, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China.
Chin Med J (Engl). 2011 Dec;124(23):3976-81.
Most of the basic and clinical studies of osteonecrosis of the femoral head (ONFH) are restricted to bone tissues only, whereas various systems are involved in the onset and development of ONFH, including nervous system. Peptidergic nerve participates in the neuronal regulation of bone metabolism and anabolism, and plays key roles in the growth, repair and reconstruction of bone. Calcitonin gene-related peptide (CGRP), which is secreted by peptidergic nerve, is the main mediator of bone metabolism. It dramatically promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Additionally, it enhances the osteoblast mass and the rate of osteoblast formation, and reduces the bone resorption by acting on osteoblasts and osteoclasts. Hence, we aimed to construct recombinant retrovirus vector pLNCX(2)-hCGRPα and to investigate the proliferation and osteogenic potential of hCGRPα-producing BMSCs (BMSCs/pLNCX(2)-hCGRPα) after virus infection.
The constructed recombinant retrovirus vector pLNCX(2)-hCGRPα was transfected into PT67 packaging cells by lipofectamine 2000. Virus was collected for BMSCs infection. The mRNA and protein expression of hCGRPα was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. The cell proliferation was determined by methyl thiazoleterazolium (MTT) assay. The osteogenic potential of BMSCs was evaluated by alkaline phosphatase (ALP) activity.
Both mRNA and protein expression of hCGRPα was detected in BMSCs/pLNCX(2)-hCGRPα cells. These cells exhibited significantly elevated proliferation and ALP value as compared with control BMSCs (P < 0.05).
BMSCs/pLNCX(2)-hCGRPα cells could stably express hCGRPα and showed promoted proliferation ability and osteogenic potential as compared with control BMSCs.
大多数关于股骨头坏死（ONFH）的基础和临床研究都仅限于骨组织，而ONFH 的发生和发展涉及到多个系统，包括神经系统。肽能神经参与骨代谢和合成的神经元调节，在骨的生长、修复和重建中发挥关键作用。降钙素基因相关肽（CGRP）由肽能神经分泌，是骨代谢的主要介质。它能显著促进骨髓间充质干细胞（BMSCs）的增殖和成骨分化。此外，它通过作用于成骨细胞和破骨细胞来增加成骨细胞的质量和形成速率，并减少骨吸收。因此，我们旨在构建重组逆转录病毒载体 pLNCX(2)-hCGRPα，并研究病毒感染后 hCGRPα 产生的 BMSCs（BMSCs/pLNCX(2)-hCGRPα）的增殖和成骨潜能。
采用脂质体 2000 将构建的重组逆转录病毒载体 pLNCX(2)-hCGRPα转染至 PT67 包装细胞，收集病毒感染 BMSCs。采用逆转录聚合酶链反应（RT-PCR）和 Western blot 分别检测 hCGRPα 的 mRNA 和蛋白表达。采用噻唑蓝（MTT）比色法检测细胞增殖。通过碱性磷酸酶（ALP）活性评估 BMSCs 的成骨潜能。
BMSCs/pLNCX(2)-hCGRPα 细胞中均检测到 hCGRPα 的 mRNA 和蛋白表达。与对照组 BMSCs 相比，这些细胞的增殖和 ALP 值明显升高（P < 0.05）。
BMSCs/pLNCX(2)-hCGRPα 细胞可稳定表达 hCGRPα，与对照组 BMSCs 相比，其增殖能力和成骨潜能增强。
