骨发育相关基因在SEDT中差异表达的研究  

Differential expression of bone development-related gene in SEDT

作　　者：胡玉娟[1] 熊丰[1] 朱高慧[1] 罗庆[1] 朱岷[1] 

机构地区：[1]重庆医科大学附属儿童医院内分泌科儿童发育疾病研究省部共建教育部重点实验室儿科学重庆市重点实验室重庆市(儿童发育重大疾病诊治与预防)国际科技合作基地,重庆400014

出　　处：《重庆医科大学学报》2016年第2期118-123,共6页Journal of Chongqing Medical University

基　　金：重庆市自然科学基金计划重点资助项目(编号:CSTS;2007BA5028)

摘　　要：目的:分别靶向增强和干扰人软骨细胞(human chondrocytes-articular,HC-a)X-连锁迟发性脊椎骨骺发育不良(Xlinked spondyloepiphyseal dysplasia tarda,SEDL)基因,骨再生PCR芯片检测细胞内骨发育相关基因表达变化,观察干扰后基因表达谱的变化。方法:分别转入前期构建的靶向增强SEDL的腺病毒质粒p Ad5-SEDL和靶向干扰SEDL的慢病毒质粒LVsi SEDL到293T细胞包装成重组腺病毒p Ad5-SEDL和重组慢病毒LV-si SEDL,扩增重组腺病毒,收集病毒液,检测病毒滴度。将重组腺病毒、重组慢病毒以最适感染复数(multiplicity of infection,MOI)分别感染HC-a,实验分4组:增强组(HC-a/p Ad5-SEDL,重组腺病毒p Ad5-SEDL感染HC-a增强SEDL基因表达)、增强阴性对照组(HC-a/空载体(p Ad5),空白腺病毒p Ad5感染HC-a作阴性对照)、干扰组(HC-a/LV-si SEDL,重组慢病毒LV-si SEDL感染HC-a干扰SEDL基因表达)、干扰阴性对照组(HC-a/空载体(LV),空白慢病毒LV感染HC-a作阴性对照),通过QPCR检测HC-a中SEDL基因表达、Western blot检测HC-a中sedlin蛋白表达,验证是否成功干扰或增强SEDL基因表达,收集4组细胞m RNA进行骨再生PCR芯片(含84个基因)检测。结果:(1)收获高滴度重组腺病毒p Ad5-SEDL,病毒滴度为2.6×1011 pfu/ml。并使重组腺病毒p Ad5-SEDL和重组慢病毒LV-si SEDL成功感染HC-a。(2)HC-a SEDL基因表达量中增强组(2.14±0.36)较增强阴性对照组(1.21±0.25)增高(P=0.022),干扰组(0.86±0.11)较干扰阴性对照组(1.34±0.06)降低(P=0.003);sedlin蛋白表达量中,增强组(0.56±0.04)较增强阴性对照组(0.37±0.05)增高(P=0.006),干扰组(0.13±0.02)较干扰阴性对照组(0.36±0.04)降低(P=0.001)。(3)基因芯片结果:HC-a内SEDL基因干扰后,芯片结果显示:基因上调21个,下调5个。HC-a内SEDL基因增强后,骨再生PCR芯片结果显示:基因上调27个,下调12个。干扰组的差异表达基因中,在增强组中呈反向变化,且与软骨发育相关的基因有5个:COL2A、BMP3、Objective：To detect the varied expressions of bone development-related genes by PCR microarray in enhanced and interfered human chondrocytes-articular（HC-a） X-linked spondyloepiphyseal dysplasia tarda（SEDL） gene expression groups,respectively. Methods：The vectors which targeting enhanced SEDL adenovirus vector p Ad5-SEDL and interfered SEDL lentivirus plasmid LV-si SEDL were constructed previously. 293 T cells were transformed with p Ad5-SEDL and LV-si SEDL respectively,and packaged recombinant adenovirus p Ad5-SEDL and lentivirus LV-si SEDL were amplified by HEK293 cells. The virus was collected and the virus titers were detected. The recombinant adenovirus and lentivirus infected HC-a on optimal multiplicity of infection（MOI）;the experiment was divided into four groups：enhanced group（HC- a / p Ad5- SEDL,p Ad5- SEDL infected HC-a）,reinforced the negative control group（HC-a/empty vector（p Ad5）,p Ad5 infected HC-a）,interference group（HC-a/LV-si SEDL,LV-si SEDL infected HC-a）,interference group negative control group（HC-a/empty vector（LV）,LV infected HC-a）. SEDL gene expression was detected by q PCR;sedlin protein was detected by Western blot to verify whether the SEDL gene expression was interfered or enhanced successfully. Then cellular mRNA of four groups was collected and detected osteogenesis PCR array. Results：①High titer recombinant adenovirus p Ad5-SEDL was harvested and the viral titer was 2.6×10^11pfu/ml. Both of recombinant adenovirus p Ad5-SEDL and lentivirus LV-si SEDL infected HC-a successfully. ② SEDL gene expression was higher in enhanced group（2.14 ±0.36） than in negative control group（1.21 ±0.25）（P =0.022）;SEDL gene expression was lower in interference group（0. 86 ± 0. 11） than in interference group negative control group（1.34±0.06）（P=0.003）. Sedlin protein expression was increased in enhanced group（0.56±0.04）than in negative control group（0.37±0.05）（P=0.006）;Sedlin protein expression was lower in interferen

关 键 词：X-连锁迟发性脊椎骨骺发育不良 基因芯片 基因重组病毒 

分 类 号：R725.8[医药卫生—儿科]