荷载RADA-16/Sox-9的骨髓间充质干细胞在兔退变椎间盘修复中的作用
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摘要
目的探讨荷载RADA-16/Sox-9的骨髓间充质干细胞(mesenchymal stem cells,MSCs)对兔退变椎间盘的修复作用及可能机制。方法40只大白兔随机分为对照组、Sox-9MSCs组、RADA-16MSCs组、RADA-16+Sox-9 MSCs组各10只,4组均采用自行设计的加压装置制备退变椎间盘模型。对照组不作处理,Sox-9 MSCs组、RADA-16 MSCs组、RADA-16+Sox-9MSCs组在造模成功后7d分别向L_(3/4)、L_(4/5)、L_(5/6)椎间隙注射Sox-9转染MSCs、RADA-16转染MSCs、RADA-16+Sox-9转染MSCs溶液各5mL。分别于转染前、转染后3个月行腰椎X线片、MRI检查测量髓核矢状径指数、根袖直径、神经根鞘袖和硬脊膜夹角、病变间隙、临近节段椎间隙高度变化;于转染前、转染后3个月分别处死5只兔,取椎间盘制作病理切片,采用免疫组织化学法检测髓核组织中Ⅱ型胶原表达。结果4组转染前髓核矢状径指数、根袖直径、神经根鞘袖和硬脊膜囊夹角比较差异无统计学意义(P>0.05);转染后3个月,对照组、RADA-16 MSCs组、Sox-9MSCs组、RADA-16+Sox-9MSCs组髓核矢状径指数(0.36±0.13、0.33±0.12、0.20±0.14、0.17±0.11)、神经根鞘袖和硬脊膜夹角[(26.92±1.10)°、(26.23±1.17)°、(25.77±1.17)°、(25.32±1.18)°]依次降低,根袖直径[(0.21±0.05)、(0.29±0.04)、(0.37±0.06)、(0.45±0.05)cm]依次增加(P<0.05);4组转染前病变间隙、临近节段椎间隙高度变化、Ⅱ型胶原表达比较差异均无统计学意义(P>0.05);转染后3个月,对照组、RADA-16 MSCs组、Sox-9 MSCs组、RADA-16+Sox-9MSCs组病变间隙[(26.89±1.11)、(24.77±1.13)、(21.28±1.12)、(16.33±1.13)cm]、临近节段椎间隙高度变化[(1.68±0.12)、(1.50±0.10)、(1.29±0.11)、(1.01±0.10)mm]依次降低,Ⅱ型胶原表达(190.65±1.35、208.80±1.40、221.34±1.56、240.95±1.45)依次增加(P<0.05)。结论荷载RADA-16/Sox-9的MSCs对兔退变椎间盘有较好的修复作用,可能与其能增加髓核组织中Ⅱ型胶原含量有关。
Objective To investigate the role and mechanism of bone marrow derived mesenchymal stem cells(MSCs)loaded with RADA-16/Sox-9in repair of degenerative intervertebral disc in rabbits. Methods The degenerative intervertebral models were prepared in 40 rabbits which were randomly divided into control group,Sox-9 MSCs group,RADA-16 MSCs group and RADA-16+Sox-9M SCs group,with 10 rabbits in each group.On day 7after preparation of the models,Sox-9 MSCs group,RADA-16 MSCs group and RADA-16+Sox-9 MSCs group were injected with 5mL of MSCs solution transfected by Sox-9,RADA-16 and RADA-16+Sox-9into intervertebral disc space of L_(3/4),L_(4/5) and L_(5/6),respectively,while control group received no treatment.The sagittal diameter index of nucleus pulposus,diameter of root sleeve,angle between nerve root sheath sleeve and dural sac,degenerative intervertebral disc space,and height of adjacent intervertebral disc space were detected by the lumbar X-ray and MRI before transfection and 3 months after transfection.Five rabbits in each group were sacrificed before transfection and the other rabbits were sacrificed 3months after transfection.The pathological sections of intervertebral disc were made and the expression of type Ⅱ collagen in nucleus pulposus was detected by immunohistochemistry.Results There were no significant differences in the sagittal diameter index of nucleus pulposus,root sleeve diameter,and angle between nerve root sheath sleeve and dural sac before transfection among four groups(P>0.05).The sagittal diameter index of nucleus pulposus(0.36±0.13,0.33±0.12,0.20±0.14,0.17±0.11),and angle between nerve root sheath sleeve and dural sac((26.92±1.10)°,(26.23±1.17)°,(25.77±1.17)°,(25.32±1.18)°)decreased gradually,and root sleeve diameter((0.21±0.05),(0.29± 0.04),(0.37±0.06),(0.45±0.05)cm)increased gradually 3 months after transfection in control group,RADA-16 MSCs group,Sox-9MSCs group and RADA-16+Sox-9MSCs group in turns(P<0.05).There were no significant differences in the degenerative intervertebral disc space,height of adjacent intervertebral disc space and the expression of type Ⅱcollagen before transfection among four groups(P>0.05).The degenerative intervertebral disc space((26.89±1.11),(24.77±1.13),(21.28±1.12),(16.33±1.13)cm),height of adjacent intervertebral disc space((1.68±0.12),(1.50±0.10),(1.29±0.11),(1.01±0.10)mm)and the expression of typeⅡ collagen(90.65±1.35,208.80±1.40,221.34±1.56,240.95±1.45)increased gradually 3months after transfection in control group,RADA-16 MSCs group,Sox-9MSCs group and RADA-16+Sox-9 MSCs group in turns(P<0.05).Conclusion MSCs loaded with RADA-16/Sox-9is effective in repairing degenerative intervertebral disc in rabbits,probably by increasing type Ⅱcollagen content.
Objective To investigate the role and mechanism of bone marrow derived mesenchymal stem cells(MSCs)loaded with RADA-16/Sox-9in repair of degenerative intervertebral disc in rabbits. Methods The degenerative intervertebral models were prepared in 40 rabbits which were randomly divided into control group,Sox-9 MSCs group,RADA-16 MSCs group and RADA-16+Sox-9M SCs group,with 10 rabbits in each group.On day 7after preparation of the models,Sox-9 MSCs group,RADA-16 MSCs group and RADA-16+Sox-9 MSCs group were injected with 5mL of MSCs solution transfected by Sox-9,RADA-16 and RADA-16+Sox-9into intervertebral disc space of L_(3/4),L_(4/5) and L_(5/6),respectively,while control group received no treatment.The sagittal diameter index of nucleus pulposus,diameter of root sleeve,angle between nerve root sheath sleeve and dural sac,degenerative intervertebral disc space,and height of adjacent intervertebral disc space were detected by the lumbar X-ray and MRI before transfection and 3 months after transfection.Five rabbits in each group were sacrificed before transfection and the other rabbits were sacrificed 3months after transfection.The pathological sections of intervertebral disc were made and the expression of type Ⅱ collagen in nucleus pulposus was detected by immunohistochemistry.Results There were no significant differences in the sagittal diameter index of nucleus pulposus,root sleeve diameter,and angle between nerve root sheath sleeve and dural sac before transfection among four groups(P>0.05).The sagittal diameter index of nucleus pulposus(0.36±0.13,0.33±0.12,0.20±0.14,0.17±0.11),and angle between nerve root sheath sleeve and dural sac((26.92±1.10)°,(26.23±1.17)°,(25.77±1.17)°,(25.32±1.18)°)decreased gradually,and root sleeve diameter((0.21±0.05),(0.29± 0.04),(0.37±0.06),(0.45±0.05)cm)increased gradually 3 months after transfection in control group,RADA-16 MSCs group,Sox-9MSCs group and RADA-16+Sox-9MSCs group in turns(P<0.05).There were no significant differences in the degenerative intervertebral disc space,height of adjacent intervertebral disc space and the expression of type Ⅱcollagen before transfection among four groups(P>0.05).The degenerative intervertebral disc space((26.89±1.11),(24.77±1.13),(21.28±1.12),(16.33±1.13)cm),height of adjacent intervertebral disc space((1.68±0.12),(1.50±0.10),(1.29±0.11),(1.01±0.10)mm)and the expression of typeⅡ collagen(90.65±1.35,208.80±1.40,221.34±1.56,240.95±1.45)increased gradually 3months after transfection in control group,RADA-16 MSCs group,Sox-9MSCs group and RADA-16+Sox-9 MSCs group in turns(P<0.05).Conclusion MSCs loaded with RADA-16/Sox-9is effective in repairing degenerative intervertebral disc in rabbits,probably by increasing type Ⅱcollagen content.
引文
[1]陈明姣,范先群.透明质酸-明胶双网络水凝胶促进骨髓间充质干细胞成骨分化的作用[J].上海交通大学学报(医学版),2018,38(7):722-731.
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[9]张雁儒,张辉,卡卡,等.骨形态发生蛋白质-4转染骨髓间充质干细胞复合双层聚乳酸羟基乙酸共聚物支架修复兔膝关节软骨缺损[J].解剖学杂志,2016,39(6):667-672.
[10]肖仁顺,阮宏,蒋星海,等.含精氨酰-甘氨酰-天冬氨酰环两亲性肽自组装凝胶与骨髓间充质干细胞相容性研究[J].重庆医学,2017,46(35):4912-4916.
[11]于鹏,李明,贾丙申,等.RADA-16自组装多肽的合成表征及负载BMP-2的细胞相容性研究[J].局解手术学杂志,2018,27(7):463-466.
[12]李子建,冯娟.间充质干细胞来源的外泌体的特性及其在疾病治疗中的作用[J].中国生物化学与分子生物学报,2018,34(7):731-739.
[13]徐浩文,孙石磊.骨髓间充质干细胞、过表达Nurr1蛋白的骨髓间充质干细胞与胎脑细胞移植治疗帕金森病大鼠的比较[J].中华实用诊断与治疗杂志,2011,25(10):967-969.
[14]赵正德,陈振银,张慧楠,等.自组装短肽水凝胶支架三维培养环境对骨髓间充质干细胞生物学特性及心肌方向分化的影响[J].中国生物工程杂志,2017,37(11):45-51.
[2]石良晨,秦峰,王岩松.胶质细胞源性神经营养因子修饰骨髓间充质干细胞移植与传统方法治疗脊髓损伤研究进展[J].中华实用诊断与治疗杂志,2015,29(5):423-425.
[3]王小伟,高春生,晏慧超,等.混合共培养条件下骨髓间充质干细胞与脂肪间充质干细胞成骨性能分析[J].实用骨科杂志,2017,23(12):1091-1094.
[4] MOSTAFAVINIA A,DEHDEHI L,GHOREISHI S K,et al.Effect of in vivo,low-level laser therapy on bone marrowderived mesenchymalstemcellsinovariectomy-induced osteoporosis of rats[J].J Photochem Photobiol B,2017,17(5):29-36.
[5]熊晓芊,邵增务,裴洪,等.可控轴向压力致兔腰椎间盘退变模型的建立及评价[J].中国矫形外科杂志,2009,24(19):2077-2080.
[6]杨勇,梁伟,罗卓荆,等.不同Pfirrmann分级椎间盘内髓核细胞生物学特性的比较[J].脊柱外科杂志,2011,9(4):244-248.
[7]贾鹏,赵文国,王勇贵.腰椎滑脱椎弓根螺钉内固定融合术后邻近节段椎间隙变化的相关研究[J].新乡医学院学报,2013,30(8):632-634.
[8]韩祥祯,何惠宇,胡杨,等.人骨形态发生蛋白-2重组慢病毒载体转染羊骨髓间充质干细胞及其成骨调节作用[J].中华实用诊断与治疗杂志,2014,28(6):540-542.
[9]张雁儒,张辉,卡卡,等.骨形态发生蛋白质-4转染骨髓间充质干细胞复合双层聚乳酸羟基乙酸共聚物支架修复兔膝关节软骨缺损[J].解剖学杂志,2016,39(6):667-672.
[10]肖仁顺,阮宏,蒋星海,等.含精氨酰-甘氨酰-天冬氨酰环两亲性肽自组装凝胶与骨髓间充质干细胞相容性研究[J].重庆医学,2017,46(35):4912-4916.
[11]于鹏,李明,贾丙申,等.RADA-16自组装多肽的合成表征及负载BMP-2的细胞相容性研究[J].局解手术学杂志,2018,27(7):463-466.
[12]李子建,冯娟.间充质干细胞来源的外泌体的特性及其在疾病治疗中的作用[J].中国生物化学与分子生物学报,2018,34(7):731-739.
[13]徐浩文,孙石磊.骨髓间充质干细胞、过表达Nurr1蛋白的骨髓间充质干细胞与胎脑细胞移植治疗帕金森病大鼠的比较[J].中华实用诊断与治疗杂志,2011,25(10):967-969.
[14]赵正德,陈振银,张慧楠,等.自组装短肽水凝胶支架三维培养环境对骨髓间充质干细胞生物学特性及心肌方向分化的影响[J].中国生物工程杂志,2017,37(11):45-51.