hBMP7基因修饰的髓核细胞与同种异体椎间盘复合后体内原位移植的实验研究  

Effect of nucleus pulposus cells expressing hBMP7 on the degeneration of canine transplanted intervertebral disc

作　　者：王超锋[1] 张超[1] 徐成[1] 何勍[1] 阮狄克[1] WANG Chao-feng;ZHANG Chao;XU Cheng;HE Qing;RUAN Di-ke(Department of Orthopaedic Surgery,Navy General Hospital,Beijing 100048,China)

机构地区：[1]海军总医院骨科,北京100048

出　　处：《骨科》2018年第4期306-312,共7页ORTHOPAEDICS

基　　金：国家自然科学基金(30730095)

摘　　要：目的探讨用人骨形态发生蛋白7(human bone morphogenetic protein 7,hBMP7)基因修饰的犬髓核细胞与同种异体椎间盘复合后体内原位移植的方法阻止或延缓同种异体椎间盘移植后退变的能力。方法将18个比格犬椎间盘(L_(4-5))置于-196℃冻存液中保存2个月,随机分为A、B、C三组,A组以r AAV2-hBMP7转染犬髓核细胞,将能够表达hBMP7蛋白的髓核细胞悬液(5×10~6个/ml,20μl)注射入复温后的椎间盘中体外培养,B组以相同数量未转染的hBMP7的髓核细胞注入椎间盘后体外培养,C组以20μl DMEM/F12细胞培养液生理盐水注射后体外培养。将培养7d的3组椎间盘分别移植至15只比格犬的L4-5,在术前、术后即刻及术后1、3、6个月通过X线检测移植椎间盘的愈合情况及其高度变化;通过MRIT2像分析椎间盘的退变情况;术后6个月时处死动物取材,分析腰椎屈伸、侧弯及扭转的生物力学变化;组织学染色检测椎间盘的结构及退变情况;PCR法检测3组髓核组织中hBMP7 mRNA的表达。结果X线检测显示3组移植椎间盘高度变化指数(DHVI)差异无统计学意义(P>0.05);MRI检测结果显示:在术后12、24周时,B、C两组移植椎间盘的MRIT2像信号灰度比明显低于A组(P<0.05);生物力学检测结果显示:C组的左右扭转度明显大于A、B组(P<0.05),而A、B两组间的差异无统计学意义(P>0.05),而对屈伸及侧弯活动度检测发现3组间差异无统计学意义;PCR检测结果显示:6个月时A组仍可检测到hBMP mRNA的高表达;组织学检测结果显示:移植后6个月时仍有外源性髓核细胞存活,A组相对于B、C两组髓核结构更加完整,所含细胞外基质更多。结论表达hBMP7的髓核细胞能阻止同种异体椎间盘移植后的退变性。Objective To discuss the effect of nucleus pulposus cells(NPCs)expressing human bone morphogenetic protein 7(hBMP7)on the degeneration of canine transplanted intervertebral disc(IVD).Methods Eighteen intervertebral discs(L4-5)from 18 Beagles were stored in cryopreservation fluid at-196℃for 2 months and divided into 3 groups.The biological tissue engineering IVDs were constructed by injecting the NPCs expressing hBMP7(group A)or non-transfected NPCs(group B)into cryopreserved IVDs,and the biological tissue engineering IVD was transplanted into a beagle dog,and control group(group C)was set for comparison.The healing and height changes of the intervertebral discs were examined before,immediately after the operation,1,3,and 6 months after the operation.The degeneration of the intervertebral disc was analyzed by the MRI T2 image.The animals were sacrificed at 6th month after the operation to analyze the biomechanical changes of the lumbar flexion extension,lateral bending and torsion.The tissue staining was used to detect the structure of the IVD.The expression of hBMP7 mRNA in nucleus pulposus of 3 groups was detected by PCR.Results X-ray examination showed that there was no significant difference in the height change index of IVDs among the three groups(P>0.05).The results of MRI detection revealed that the MRI T2 image grayscale ratio of the transplanted IVDs in groups B and C was significantly lower than that in the group(P<0.05)at 12nd and 24th week after the operation(P<0.05),and the biomechanical test results demonstrated that the left and right twisting degree in the group C was obviously greater than that in the groups A and B(P<0.05),but there was no significant difference between groups A and B(P>0.05).There was no significant difference in the flexion and extension and lateral bending activity among three groups.The results of PCR test indicated that the high expression of hBMP mRNA was still detected in group A at 6th month.The results of histological examination suggested that exogenous nucleus pulmed

关 键 词：人骨形态发生蛋白7 髓核细胞 体外构建 椎间盘移植 基因转染 

分 类 号：R687.3[医药卫生—骨科学]