【作者】 孙超；

【导师】 邱勇；

【作者基本信息】 南京医科大学 ， 外科学， 2009， 硕士

【摘要】 青少年特发性脊柱侧凸患者成骨细胞中核心结合因子ɑ1和骨形态发生蛋白-2的表达及意义第一部分青少年成骨细胞的体外分离、培养、扩增及表型鉴定目的建立青少年成骨细胞(osteoblast,OB)体外分离、培养、扩增及表型鉴定的方法,为后续分子生物学检测做准备。方法试验对象为2008年03月至2009年04在我院行后路手术的女性AIS患者26例。根据测量的骨密度值,将AIS患者分为两组:A组15例,为骨量正常患者,年龄12~18岁,平均14.6岁;B组11例,为骨量减低患者,年龄12~18岁,平均14.8岁。所有受试者均需取右侧髂松质骨行植骨融合,在病人知情同意和不影响手术疗效的情况下取少许松质骨标本。胰蛋白酶预消化后植块法分离培养OB,原代培养并传至P2代行细胞记数,同时记录细胞样本各代的增值时间。取P2代细胞通过碱性磷酸酶染色、钙节结染色及RT-PCR检测骨钙素表达行OB的表型鉴定。结果两组胰蛋白酶预消化后植块法分离所得的原代OB均呈贴壁生长。体外培养第5~6天可见少量贴壁细胞,培养第4周见植块周围细胞密集,已呈重叠生长,植块间细胞也接近融合,细胞形态趋向较均匀的短梭形。P2代细胞总数平均可达0.5×107个。两组细胞各代平均增值时间无统计学差异(P>0.05),从P0～P2代平均约需45天。P2代细胞经碱性磷酸酶染色、钙节结染色及RT-PCR检测骨钙素表达鉴定证实获得和培养的细胞为典型的OB。结论1.植块法OB培养能获得大量原代成骨细胞的同时,较好地保持了其生物学特征,是一种较理想的人成骨细胞培养方法。2.两组AIS患者在体外分离、培养、扩增OB时所表现的生长及增殖特性一致。第二部分成骨细胞中核心结合因子ɑ1和骨形态发生蛋白-2的表达与青少年特发性脊柱侧凸患者骨量降低的关系目的从成骨细胞(osteoblast,OB)水平探讨核心结合因子ɑ1(Runx2)、骨形态发生蛋白-2 (bone morphogenetic proteins, BMP-2)与青少年特发性脊柱侧凸(adolescent idiopathic scoliosis,AIS)患者骨量降低的关系。方法试验对象为2008年03月至2009年04在我院行后路手术的女性AIS患者26例。根据测量的骨密度值,将AIS患者分为两组:A组15例,为骨量正常患者,年龄12~18岁,平均14.6岁;B组11例,为骨量减低患者,年龄12~18岁,平均14.8岁。所有AIS患者均采用双能X线吸收测量仪(dual-energy x-ray absorptiometry,DEXA)测量骨密度(bone mineral density,BMD),测量部位包括非优势侧股骨近端及腰椎。所有受试者术中取适量髂前上嵴的松质骨,运用植块法培养成骨细胞。培养至P2代使用RT-PCR和Western blotting检测两组中Runx2和BMP-2的mRNA及蛋白表达的情况。结果B组腰椎(L2~L4)及股骨近端的BMD值明显低于A组。RT-PCR和Western blotting显示:1.两组病例BMP-2的核酸及蛋白的表达量均无显著统计学差异(P>0.05)。2. B组中Runx2的核酸及蛋白水平的表达量均较A组低( P<0.05)。结论AIS骨量降低可能与OB细胞中BMP-2的表达强度无关。Runx2在OB水平核酸及蛋白表达强度的异常可能是导致AIS骨量降低的一个重要分子机制。更多还原

【Abstract】 Expression and significance of transcription factor Runx2 and bone morphogenetic protein-2 (BMP-2) in osteoblasts from patients with adolescent idiopathic scoliosisPartⅠIsolation, cultivation, amplification and phenotype identification of osteoblasts from adolescent subjects in vitro Objective To establish the methods of the isolation,cultivation and amplification of osteoblast (OB) from adolescent subjects in vitro.Methods This study covered 26 female adolescent idiopathic scoliosis (AIS) subjects who underwent spinal surgery from March 2008 to April 2009. According to the status of bone mass, AIS patients were divided into two groups: Group A and Group B. Patients in Group A (15 cases) maintained normal BMD. While patients in Group B (11 cases) suffered from osteopenia. The cancellous bone was harvested from the posterior superior iliac spine of the subjects. The chipped explants were cultured to obtain the osteoblasts. The incremet time of each generation were recorded. The second generation (P2) cells were scraped to count. Formation of calcium nodules,staining of alkaline phosphatase and expression of osteocalcin mRNA detected by RT-PCR were used to confirm the osteoblast phenotype.Results Cell outgrowths appeared after 5-6 days,and then turned to be fusiform in shape and confluent cultures after 4 weeks. 0.5×107 cells could be obtained in each P2 generation culture flask. The positive results of staining of alkaline phosphatase, formation of calcium nodules and expression of osteocalcin confirmed that the P2 cells were typical osteoblasts. There was no statistical difference between the two study groups about increment time of osteoblasts.Conclusion 1. Explant culture system offers copious primary osteoblasts with good cellular characters, it is an ideal culture system for human osteoblasts. 2. Osteoblast from adolescent idiopathic scoliosis in the two study groups has the same capacity of growth and proliferation in vitro. PartⅡThe Association between Runx2, BMP-2 in osteoblasts and the Decreased Bone Mass in Adolescent Idiopathic Scoliosis PatientsObjective To investigate the association between bone morphogenetic proteins-2 (BMP-2), transcription factor Runx2 in osteoblasts and the decreased bone mass of adolescent idiopathic scoliosis patients.Methods This study included 26 female AIS subjects (aged from 12 to 18) who underwent spinal surgery from March 2008 to April 2009. According to the status of bone mass, AIS patients were divided into two groups: Group A and Group B. Patients in Group A (15 cases) had normal BMD. While patients in Group B (11 cases) suffered from osteopenia. The average age was 14.6 years in Group A and 14.8 years in Group B. BMD of the lumbar spine and proximal femur was measured using dual energy X-ray absorptiometry in both groups. The cancellous bone was harvested from the posterior superior iliac spine of the subjects. The chipped explants were cultured to obtain the osteoblasts. Expression of transcription factor Runx2 and bone morphogenetic proteins-2 (BMP-2) of P2 generation osteoblasts from two groups were detected by using RT-PCR and Western blotting.Results BMD of the lumbar spine (L2~L4) and proximal femur in Group B was obviously lower than that in Group A. RT-PCR and Western blotting results showed that:1) Expression of BMP-2 in osteoblasts was comparable between two study group (P>0.05). 2) Expression of Runx2 in Group B were obviously decreased as compared with that in Group A (P<0.05).Conclusion The expression of BMP-2 in OB may not associated with the reduced BMD in AIS, while the abnormal expression of Runx2 may play an important role in the pathogenesis of reduced BMD in AIS.更多还原