间充质干细胞注射对大鼠退变椎间盘的作用
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摘要
目的：分离、扩增并鉴定大鼠间充质干细胞，建立并验证大鼠尾椎间盘退变模型，探讨间充质干细胞注射对鼠尾椎间盘退变的作用。
方法：用穿刺抽吸法收集大鼠股骨和胫骨内的骨髓，密度梯度离心法和贴壁法分离并纯化间充质干细胞，进行体外扩增，并用细胞计数法绘制第一代、第三代和第五代细胞的生长曲线。用流式细胞鉴定检测第三代细胞表面标记CD90、CD34和CD45表达情况。对第4代细胞进行成骨和成脂诱导分化，并用茜素红染色鉴定成骨诱导分化结果，以及用油红O染色鉴定成脂诱导分化结果。选取健康3月龄SD大鼠40只，随机分为对照组(8只)、退变组(8只)、培养基注射组(8只)和实验组(16只)，并对后三组的Co7／Co8、Co8／Co9和Co9／Co10椎间盘用细针穿刺纤维环法诱导退变。造模2周后拍摄X光片观察造模效果，并向实验组造模椎间盘内注射间充质干细胞，培养基注射组造模椎间盘内注射等体积干细胞培养基，对照组、退变组不予特殊处理。分别于造模后1周、2周、3周、4周抽取4只实验组大鼠和其他三组大鼠各2只麻醉后拍摄尾椎正位片，测量椎间盘高度，并计算椎间盘高度指数。放射学检查后对抽取的大鼠实施过量麻醉处死，取出Co7／Co8椎间盘和相邻的上下部分椎体，固定、切片，行HE染色和番红-固绿染色评估椎间盘组织学改变。完整取出Co8／Co9和Co9／Co10椎间盘，分别用1，9-二甲胺基甲基蓝结合法和二甲氨基苯甲醛比色法检测Co8／Co9椎间盘内GAG和羟脯氨酸含量；提取出Co9／Co10椎间盘内总RNA后，实时定量PCR检测其中聚集蛋白聚糖mRNA的表达水平。
结果：分离和纯化的间充质干细胞体外扩增迅速，流式细胞鉴定显示细胞表达CD90，不表达CD34和CD45。茜素红染色和油红O染色证明扩增后的细胞仍具有成骨和成脂分化能力。放射学，生物化学和定量PCR检查发现，和对照组相比，退变组和培养基注射组的椎间盘高度、椎间盘内GAG含量、聚集蛋白聚糖mRNA表达水平均有不同程度降低(p＜0．05)，并表现出随时间下降的趋势。而实验组与退变组相比，上述指标均表现出一定的改善趋势，注射4周后GAG含量有统计学差异(p＜0．05)。组织学观察到造模后随时间进展的椎间盘退变过程，实验组与退变组相比，退变程度相对较轻。椎间盘内羟脯氨酸含量，各组间没有明显差异(p＞0．05)。
结论：间充质干细胞分离纯化相对容易、体外扩增迅速，扩增后仍保持其多向分化潜能。细针穿刺法诱导大鼠尾椎间盘退变模型成模时间短，效果可靠，经济便利。向大鼠鼠尾椎间盘退变模型内注射间充质干细胞能延缓椎间盘的退变进程。
Objective:To isolate culture and identify the bone marrow derived mesenchymalstem cells(MSCs).To build and verify the disc degeneration model induced bypercutaneous needle puncture in the rat tail.To explore the effects of injecting MSCs tothe rat model of degenerated intervertebral disc.
Methods:Rat bone marrow was collected by aspiration from the femur and tibia.MSCs were isolated by the gradient isolation of mononuclear cells and cell attachmentto tissue culture plastic.The MSCs were proliferated in vrtro,and the growth curves ofprimary cells,third-generation cells and fifth-generation cells were drawed.The cellsurface markers CD90,CD 45 and CD 34 were checked by flow cytometry.The MSCswere also checked for multi-linage differentiation by adipogenic and osteogeniedifferentiation assays.Forty 3-month-old male Sprague Dawley rats were randomdevided into four groups:8 in normal control group;8 in degeneration group;8 inmedium injection group and experiment group with 16 rats.Degeneration was inducedin degenerative group,medium injection group and experiment group.Theintervertebral discs at regions Co7/Co8,Co8/Co9 and Co9/Co10 were puncturedpercutaneously using a 20-gauge needle with half penetration.2 weeks after induction,MSCs were injected into the inducted discs of all rats in experiment group,and mediumfor MSCs culture was injected into the inducted discs of all rats in medium injectiongroup.At 1,2,3 and 4 weeks after injection,4 rats from experiment group and 2 ratseach from normal control group,degeneration group and medium injection group wereeuthanized with an excess dose of sodium pentobarbital and their spines harvested.
Before harvesting,anteroposterior plain radiographs were taken.Co7/Co8 discs wereisolated with both upper and lower vertebral bodies completely attached for histologicalevaluation by staining with safranin o-fast green and hematoxylin,and hematoxylin andeosin respectively.Co8/Co9 discs were harvested for estimating GAG andhydroxyproline content by using 1-9 dimethylmethylene blue(DMMB)binding assaykit and colorimetric assay kit with chloramine T and dimethylaminobenzaldehyde(DMBA).Co9/Co10 discs were used to extracting total RNAs for quantitative PCR ofaggrecan expression.
Results:The MSCs derived from bone marrow were easy to harvest,isolate and grow.These cells expressed CD90 but were negative for CD34 and CD45.Differentiationassay results demonstrated sufficient differentiation of MSCs in adipogenic andosteogenic lineages.Needle punctures with half penetration caused significant discspace narrowing,progressive histological changes of degeneration and decrease ofGAG content and aggrecan expression after injury.Compared with degeneration group,these indexes of experiment group showed trends of improvement.Significant changein GAG content between degeneration group and experiment group was observed 4weeks after injection.The hydroxyproline content of the discs did not changeappreciably.
Conclusions:The MSCs are easy to harvest,isolate and grow,with minimuminvolvement of in vitro techniques.Tail disc percutaneous needle puncture is a simpleand effective method for inducing disc degeneration.Injection of MSCs can slow theintervertebral disc degeneration process.
方法：用穿刺抽吸法收集大鼠股骨和胫骨内的骨髓，密度梯度离心法和贴壁法分离并纯化间充质干细胞，进行体外扩增，并用细胞计数法绘制第一代、第三代和第五代细胞的生长曲线。用流式细胞鉴定检测第三代细胞表面标记CD90、CD34和CD45表达情况。对第4代细胞进行成骨和成脂诱导分化，并用茜素红染色鉴定成骨诱导分化结果，以及用油红O染色鉴定成脂诱导分化结果。选取健康3月龄SD大鼠40只，随机分为对照组(8只)、退变组(8只)、培养基注射组(8只)和实验组(16只)，并对后三组的Co7／Co8、Co8／Co9和Co9／Co10椎间盘用细针穿刺纤维环法诱导退变。造模2周后拍摄X光片观察造模效果，并向实验组造模椎间盘内注射间充质干细胞，培养基注射组造模椎间盘内注射等体积干细胞培养基，对照组、退变组不予特殊处理。分别于造模后1周、2周、3周、4周抽取4只实验组大鼠和其他三组大鼠各2只麻醉后拍摄尾椎正位片，测量椎间盘高度，并计算椎间盘高度指数。放射学检查后对抽取的大鼠实施过量麻醉处死，取出Co7／Co8椎间盘和相邻的上下部分椎体，固定、切片，行HE染色和番红-固绿染色评估椎间盘组织学改变。完整取出Co8／Co9和Co9／Co10椎间盘，分别用1，9-二甲胺基甲基蓝结合法和二甲氨基苯甲醛比色法检测Co8／Co9椎间盘内GAG和羟脯氨酸含量；提取出Co9／Co10椎间盘内总RNA后，实时定量PCR检测其中聚集蛋白聚糖mRNA的表达水平。
结果：分离和纯化的间充质干细胞体外扩增迅速，流式细胞鉴定显示细胞表达CD90，不表达CD34和CD45。茜素红染色和油红O染色证明扩增后的细胞仍具有成骨和成脂分化能力。放射学，生物化学和定量PCR检查发现，和对照组相比，退变组和培养基注射组的椎间盘高度、椎间盘内GAG含量、聚集蛋白聚糖mRNA表达水平均有不同程度降低(p＜0．05)，并表现出随时间下降的趋势。而实验组与退变组相比，上述指标均表现出一定的改善趋势，注射4周后GAG含量有统计学差异(p＜0．05)。组织学观察到造模后随时间进展的椎间盘退变过程，实验组与退变组相比，退变程度相对较轻。椎间盘内羟脯氨酸含量，各组间没有明显差异(p＞0．05)。
结论：间充质干细胞分离纯化相对容易、体外扩增迅速，扩增后仍保持其多向分化潜能。细针穿刺法诱导大鼠尾椎间盘退变模型成模时间短，效果可靠，经济便利。向大鼠鼠尾椎间盘退变模型内注射间充质干细胞能延缓椎间盘的退变进程。
Objective:To isolate culture and identify the bone marrow derived mesenchymalstem cells(MSCs).To build and verify the disc degeneration model induced bypercutaneous needle puncture in the rat tail.To explore the effects of injecting MSCs tothe rat model of degenerated intervertebral disc.
Methods:Rat bone marrow was collected by aspiration from the femur and tibia.MSCs were isolated by the gradient isolation of mononuclear cells and cell attachmentto tissue culture plastic.The MSCs were proliferated in vrtro,and the growth curves ofprimary cells,third-generation cells and fifth-generation cells were drawed.The cellsurface markers CD90,CD 45 and CD 34 were checked by flow cytometry.The MSCswere also checked for multi-linage differentiation by adipogenic and osteogeniedifferentiation assays.Forty 3-month-old male Sprague Dawley rats were randomdevided into four groups:8 in normal control group;8 in degeneration group;8 inmedium injection group and experiment group with 16 rats.Degeneration was inducedin degenerative group,medium injection group and experiment group.Theintervertebral discs at regions Co7/Co8,Co8/Co9 and Co9/Co10 were puncturedpercutaneously using a 20-gauge needle with half penetration.2 weeks after induction,MSCs were injected into the inducted discs of all rats in experiment group,and mediumfor MSCs culture was injected into the inducted discs of all rats in medium injectiongroup.At 1,2,3 and 4 weeks after injection,4 rats from experiment group and 2 ratseach from normal control group,degeneration group and medium injection group wereeuthanized with an excess dose of sodium pentobarbital and their spines harvested.
Before harvesting,anteroposterior plain radiographs were taken.Co7/Co8 discs wereisolated with both upper and lower vertebral bodies completely attached for histologicalevaluation by staining with safranin o-fast green and hematoxylin,and hematoxylin andeosin respectively.Co8/Co9 discs were harvested for estimating GAG andhydroxyproline content by using 1-9 dimethylmethylene blue(DMMB)binding assaykit and colorimetric assay kit with chloramine T and dimethylaminobenzaldehyde(DMBA).Co9/Co10 discs were used to extracting total RNAs for quantitative PCR ofaggrecan expression.
Results:The MSCs derived from bone marrow were easy to harvest,isolate and grow.These cells expressed CD90 but were negative for CD34 and CD45.Differentiationassay results demonstrated sufficient differentiation of MSCs in adipogenic andosteogenic lineages.Needle punctures with half penetration caused significant discspace narrowing,progressive histological changes of degeneration and decrease ofGAG content and aggrecan expression after injury.Compared with degeneration group,these indexes of experiment group showed trends of improvement.Significant changein GAG content between degeneration group and experiment group was observed 4weeks after injection.The hydroxyproline content of the discs did not changeappreciably.
Conclusions:The MSCs are easy to harvest,isolate and grow,with minimuminvolvement of in vitro techniques.Tail disc percutaneous needle puncture is a simpleand effective method for inducing disc degeneration.Injection of MSCs can slow theintervertebral disc degeneration process.
引文
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