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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: Large doses of nasal spray or intramuscular calcitonin can effectively prevent the loss of spinal bone in postmenopausal women. However, calcitonin takes a long period of repeated medication, expensive, and weak antigenicity, limiting the long-term use. Gene therapy can provide more effective and economical treatment for osteoporosis, while reducing the side effects of drugs. Objective: To observe the expression of human calcitonin gene in myoblasts and analyze the effect of recombinant human calcitonin on osteoblasts in vitro. Design: Gene as the experimental object, comparative observation experiment. Unit: Institute of Radiation Medicine, Fudan University. Materials: The experiment was performed at Institute of Radiation Medicine, Fudan University from December 2005 to June 2006. Selection of healthy SD fetus 10, provided by the Fudan University Institute of Radiation Medicine. Human calcitonin monoclonal antibody was purchased from Santa Cruz Biotechnology Corporation in the United States. L6 myoblasts from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences Institute of Biochemistry and Cell Biology. Methods: L6 myoblast medium was added with pcDNA3.0-hCT liposome transfection mixture (transfected group) and empty vector pcDNA3.0 liposome mixture (control group) were cultured. The protein expression of the target gene was identified by ELISA, Western Blot and immunohistochemistry. 1 × 10-14, 1 × 10-13 and 1 × 10-12 mol / L recombinant human calcitonin and MEM were respectively added into the osteoblast medium. MAIN OUTCOME MEASURES: The changes of rat osteoblast proliferation and differentiation were observed by MTT assay and alkaline phosphatase activity assay. Results: The results of ELISA showed that calcitonin was detected in cell culture medium. Western blot and immunohistochemistry confirmed that human calcitonin was stably expressed in myoblasts after transfection. When the concentration of recombinant human calcitonin was 1 × 10-14 and 1 × 10-13 mol / L, the activity of osteoblast and the activity of alkaline phosphatase of osteoblasts increased compared with the control group, but the difference was not significant (P> 0.05). When the concentration was increased to 1 × 10-12 mol / L, the level was significantly higher than that of the control group (P <0.05). Conclusion: By means of gene transfection, myoblasts can stably synthesize and secrete human calcitonin. Recombinant human calcitonin can promote bone formation.