CAJ | 학술논문

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丟失.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、Westem 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:EUSA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1×10-14,1×10-13 mol/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1×10-12 mol/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.
배경:대제량다차분비혹자기주강개소능유효지예방절경후부녀척주골적주실.단시강개소수요장시기반복용약、개격앙귀、차유약적항원성,한제료장기사용.기인치료가이위골질소송증제공경가유효、경제적치료방안,동시감소약물적부작용.목적:관찰인강개소기인재성기세포중적표체,분석중조인강개소대체외대서성골세포적영향.설계:이기인위실험대상,대비관찰실험.단위:복단대학방사의학연구소.재료:실험우2005-12/2006-06재복단대학방사의학연구소완성.선용건강SD태서10지,유복단대학방사의학연구소제공.인강개소단극륭항체구우미국santa Cruz생물기술공사.L6성기세포유중국과학원상해생명과학연구원생물화학여세포생물학연구소제공.방법:재L6성기세포배양기중분별가입pcDNA3.0-hCT지질체전염혼합물(전염조)화공재체pcDNA3.0지질체혼합물(대조조)진행배양.채용ELISA법、Westem Blot화면역조직화학감정목적기인적단백표체.재성골세포배양기중분별가입1×10-14,1×10-13,1×10-12mol/L중조인강개소화MEM.주요관찰지표:이용MTT화검측감성린산매활성적방법관찰대서성골세포증식화분화적변화.결과:EUSA법가재세포배양액중검측도강개소단백;Western blot급면역조화균증실인강개소재전염후적성기세포중획득은정표체.당중조인강개소적농도위1×10-14,1×10-13 mol/L시성골세포활성화성골세포감성린산매활성교대조조증고,단차이무현저성의의(P＞0.05);농도승고위1×10-12 mol/L시,명현고우대조조(P＜0.05).결론:차조기인전염적방법,성기세포가이은정합성、분비인강개소.중조인강개소유촉진골형성적작용.
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 osteoblast cells.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 were observed by MTT and alkaline phosphatase (ALP).RESULTS: Human calcitonin was found by ELISA in the supematant 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 1×10-12 mol/L than the control group (P＜0.05).CONCLUSION:The stable synthesis and secretion of biologically active human calcitonin can be achieved in myoblasts by gene transfection.Recombinant human calcitonin can enhance proliferation and differentiation of osteoblasts.