重组成肌细胞合成人降钙素促进大鼠成骨细胞增殖和分化(英文)

Recombinant human calcitonin in myoblasts promotes the proliferation and differentiation of rat osteoblasts

背景:大剂量多次喷鼻或者肌注降钙素能有效地预防绝经后妇女脊柱骨的丢失。但是降钙素需要长时期反复用药、价格昂贵、且有弱的抗原性,限制了长期使用。基因治疗可以为骨质疏松症提供更加有效、经济的治疗方案,同时减少药物的副作用。目的:观察人降钙素基因在成肌细胞中的表达,分析重组人降钙素对体外大鼠成骨细胞的影响。设计:以基因为实验对象,对比观察实验。单位:复旦大学放射医学研究所。材料:实验于2005-12/2006-06在复旦大学放射医学研究所完成。选用健康SD胎鼠10只,由复旦大学放射医学研究所提供。人降钙素单克隆抗体购于美国Santa Cruz生物技术公司。L6成肌细胞由中国科学院上海生命科学研究院生物化学与细胞生物学研究所提供。方法:在L6成肌细胞培养基中分别加入pcDNA3.0-hCT脂质体转染混合物(转染组)和空载体pcDNA3.0脂质体混合物(对照组)进行培养。采用ELISA法、Western Blot和免疫组织化学鉴定目的基因的蛋白表达。在成骨细胞培养基中分别加入1×10-14,1×10-13,1×10-12mol/L重组人降钙素和MEM。主要观察指标:利用MTT和检测碱性磷酸酶活性的方法观察大鼠成骨细胞增殖和分化的变化。结果:ELISA法可在细胞培养液中检测到降钙素蛋白;Western blot及免疫组化均证实人降钙素在转染后的成肌细胞中获得稳定表达。当重组人降钙素的浓度为1×10-14,1×10-13mol/L时成骨细胞活性和成骨细胞碱性磷酸酶活性较对照组增高,但差异无显著性意义(P>0.05);浓度升高为1×10-12mol/L时,明显高于对照组(P<0.05)。结论:借助基因转染的方法,成肌细胞可以稳定合成、分泌人降钙素。重组人降钙素有促进骨形成的作用。

BACKGROUND： Repeated injections or nasal spray of large doses of calcitonin can effectively prevent postmenopausal osteoporosis. Calcitonin should be taken for a long time. But the use of calcitonin is limited by the need for repeated protein administration, costly production methods and antigenicity. Gene therapy can provide effective economic therapeutic regimen for osteoporosis, and reduce side effect of drugs. OBJECTIVE： To describe the expression of human calcitonin produced in myoblasts and determine the effects of the recombinant protein on murine osteobiast ceUs. DESIGN ： A gene-based controlled observational experiment SETTING： Institute of Radiation Medicine, Fudan University MATERIALS： The experiment was carried out at the Institute of Radiation Medicine, Fudan University from December 2005 to June 2006. Ten healthy SD fetal rats were selected from Institute of Radiation Medicine, Fudan University. Human calcitoninsas monoclonal antibody was purchased from American Santa Cruz Biotechnology Company. L6 myoblast line was provided by Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. METHODS： The pcDNA3.0-hCT liposome transfection mixture （transfection group） and empty vector pcDNA3.0 liposome mixture （control group） were added in the L6 myoblast medium, respectively. The expression and secretion of human calcitonin by myoblast cells were confirmed by enzyme-linked immunosorbent assay （ELISA）, Western blot analysis and immunohistochemical analysis. 1 ×10^-14, 1 ×10^-13, 1 ×10^-12 mol/L recombinant human calcitonin and MEM were respectively added in myoblast medium. MAIN OUTCOME MEASURES： The proliferation and differentiation of rat myoblasts ware observed by MTT and alkaline phosphatase （ALP）. RESULTS： Human calcitonin was found by ELISA in the supernatant of cell culture. Western blot and immunohistochemical analysis verified that human calcitonin could be expressed stably in myoblasts after transfection. Osteoblast proliferation and ALP activity were higher when recombinant human calcitonin was 1 ×10^-14 and 1 ×10^-13 mol/L than the control group （P 〉 0.05）. It was significantly higher when the concentration was 1 ×10^-2 mol/L than the control group （P〈 0.05）.