骨髓间充质干细胞修复椎动脉型颈椎病的血管损伤

doi:10.3969/j.issn.2095-4344.2014.01.005

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (1): 27-32.doi: 10.3969/j.issn.2095-4344.2014.01.005

• 骨髓干细胞 bone marrow stem cells • 上一篇下一篇

骨髓间充质干细胞修复椎动脉型颈椎病的血管损伤

张夏梦¹，寿折星²

1辽宁医学院附属第一医院中医科，辽宁省锦州市 121000
2华中科技大学同济医学院协和医院中医科，湖北省武汉市 430022

修回日期:2013-10-12 出版日期:2014-01-01 发布日期:2014-01-01
通讯作者: 寿折星，博士，主治医师，华中科技大学同济医学院协和医院中医科，湖北省武汉市430022
作者简介:张夏梦，女，1986年生，湖北省赤壁市人，汉族，硕士，辽宁医学院在读硕士，主要从事中医骨伤方向的研究。

Bone marrow mesenchymal stem cells repair vascular injury after cervical spondylotic vertebral arteriopathy

Zhang Xia-meng¹, Shou Zhe-xing²

1Department of Traditional Chinese Medicine, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, Liaoning Province, China
2Department of Traditional Chinese Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China

Revised:2013-10-12 Online:2014-01-01 Published:2014-01-01
Contact: Shou Zhe-xing, M.D., Attending physician, Department of Traditional Chinese Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China
About author:Zhang Xia-meng, Studying for master’s degree, Department of Traditional Chinese Medicine, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, Liaoning Province, China

摘要/Abstract

摘要：

背景：炎症刺激是椎动脉型颈椎病发病的重要因素，骨髓间充质干细胞具有很强的免疫调节作用，能有效抑制炎症，具有治疗椎动脉型颈椎病的潜能。
目的：探讨椎动脉型颈椎病血管损伤机制及骨髓间充质干细胞的治疗作用。
方法：40只日本大耳兔随机分为4组：空白组不作任何处理；模型组、丹参酮组、干细胞组采用硬化剂注射法制作椎动脉型颈椎病模型。造模24 h后，空白组，模型组不进行干预，丹参酮组和干细胞组经耳缘静脉注射丹参酮ⅡA磺酸钠溶液10 mL和骨髓间充质干细胞混悬液10 mL，干预2周后取材。
结果与结论：与模型组相比，干细胞组管壁中膜平滑肌细胞肥大增生明显受到抑制，血管内皮皱折较匀称；干细胞组与丹参酮组比较无明显差别。与模型组相比，干细胞组椎动脉血管cathepsin B、白细胞介素6、肿瘤坏死因子α表达显著下降，差异有显著性意义(P < 0.05)；模型组与丹参酮组比较差异无显著性意义(P > 0.05)。结果可见炎症反应可能是椎动脉血管受到损伤的机制之一；骨髓间充质干细胞能有效抑制椎动脉血管炎症反应，修复受损椎动脉血管。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

关键词: 干细胞, 骨髓干细胞, 骨髓间充质干细胞, 椎动脉型颈椎病, cathepsin B, 白细胞介素6, 肿瘤坏死因子α

Abstract:

BACKGROUND: Inflammation is an important factor in cervical spondylotic vertebral arteriopathy, bone marrow mesenchymal stem cells have the potential to treat cervical spondylotic vertebral arteriopathy because of the immunomodulatory effects to inhibit inflammation.
OBJECTIVE: To investigate the injury mechanism of vascular injury in the model of cervical spondylotic vertebral arteriopathy and to study the therapeutic effects of bone marrow mesenchymal stem cells on it.
METHODS: Forty Japanese big ear rabbits were randomly divided into four groups: control group, model group, tanshinone group, and stem cell group. After modeling, the control and model groups were not given intervention, while the tanshinone and stem cell groups were injected with tanshinone II A sodium sulfonate solution (10 mL) and bone marrow mesenchymal stem cell suspension (10 mL) along the ear vein, respectively. After 2 weeks, the routine pathological examination was done to observe the vascular morphological changes, immunofluorescence staining was done to observe the cathepsin B expression in the vertebral artery, and ELISA was used to detect the expression of tumor necrosis factor-α and interleukin-6 in the vertebral artery.
RESULTS AND CONCLUSION: Compared with the model group, the arterial smooth muscle cell hypertrophy and hyperplasia was obviously restrained in stem cell group, and vascular endothelial fold was in symmetry, while no significant difference was found between stem cell group and tanshinone group. Compared with the model group, expressions of cathepsin B, interleukin 6 and tumor necrosis factor-α expression were reduced significantly in the stem cell group (P < 0.05), while there was no difference between the model and tanshinone groups (P > 0.05). Inflammatory reaction may be one of mechanisms for vertebral artery damage, and bone marrow mesenchymal stem cells can effectively inhibit inflammation of the vertebral artery and improve vascular remodeling.

全文链接：

Key words: bone marrow, mesenchymal stem cells, cervical vertebrae, interleukin-6, tumor necrosis factor-alpha, cathepsin B

中图分类号:

R394.2

张夏梦，寿折星 . 骨髓间充质干细胞修复椎动脉型颈椎病的血管损伤[J]. 中国组织工程研究, 2014, 18(1): 27-32.

Zhang Xia-meng, Shou Zhe-xing. Bone marrow mesenchymal stem cells repair vascular injury after cervical spondylotic vertebral arteriopathy[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(1): 27-32.

图/表（结果） 4

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引言

材料方法

设计：随机对照动物实验。

时间及地点：实验于2012年11月至2013年6月在华中科技大学同济医学院附属协和医院中心实验室完成。

材料：

实验动物及分组：清洁级日本大耳兔40只，6月龄，体质量2.5-3.0 kg，雌雄各半，购于同济医学院动物实验中心(合格证号：0129836)。随机分为空白组，模型组，干细胞组，丹参酮组，每组10只。

实验药物、试剂及仪器：消痔灵注射液[吉林省集安益盛药业股份有限公司生产(国药准字Z22026175)，批号：1203452]。丹参酮ⅡA磺酸钠溶液(上海第一生化药业有限公司，批号：081002)。cathepsin B、白细胞介素6、肿瘤坏死因子α试剂盒(武汉博士德生物)。HMIAS-2000型高清晰度彩色医学图文分析系统(武汉千屏影像技术有限责任公司)。

实验方法：

造模方法：空白组不作任何处理；模型组、丹参酮组、干细胞组参照文献[21]，采用硬化剂注射法制作椎动脉型颈椎病模型。先用脱毛剂脱掉日本大耳兔左侧自枕后及颈部的体毛，在左侧C₃-C₅椎体处消毒，抽取消痔灵10 mL注射于兔左侧C₃-C₅颈椎横突侧面，进针以感觉到横突边缘骨头为准，回抽针栓无血，进而推注消痔灵，并于第2周，第4周各重复1次。

干细胞分离、培养、鉴定：取4周龄SD雄性日本大耳兔，体质量3 kg，颈椎脱臼法处死，浸泡于体积分数为75%乙醇5 min，取四肢股骨和胫骨，剔除骨表面的软组织，体积分数为75%乙醇浸泡后取出，用无菌磷酸盐缓冲液浸泡清洗，剪断两端骨头，显露骨髓腔，放置于含有体积分数为20%胎牛血清的低糖DMEM完全培养基的无菌培养皿中，以5 mL注射器吸取培养基冲洗骨髓腔，由一端骨髓腔冲出骨髓，重复3次，再反方向冲出骨髓，重复3次，直到骨髓腔冲洗液变清亮后为止。收集骨髓悬液于15 mL离心管中，以1 000×g/min室温离心5 min，离心后去上清，用 6 mL完全培养基重悬，轻轻吹打制成单细胞悬液，转移至25 cm²培养瓶中，轻轻摇匀，置于37 ℃体积分数为5%CO₂饱和湿度的培养箱中。48 h后换新鲜培养基以去除未贴壁的细胞，以后每隔3 d换1次液，直到贴壁细胞达到80%融合，以0.25%胰蛋白酶消化，以后每次重复相同的步骤，第3代细胞用于做后续实验。

取第3代骨髓间充质干细胞以0.25%胰蛋白酶消化，以1 000×g/min室温离心5 min，离心后去上清，以PBS制成单细胞悬液，每管100 μL，分别加入荧光标记的CD29、CD90、CD45、CD11b，4 ℃避光孵育30 min，加入PBS清洗，离心后弃上清，除去未结合的荧光抗体，加1 mL磷酸盐缓冲液悬浮上机检测。

干预方法：所有动物均正常喂养，造模24 h后，空白组、模型组不作特殊治疗，丹参酮组给予10 mL丹参酮ⅡA磺酸钠溶液(含丹参酮ⅡA磺酸钠5 mg)经耳缘静脉注射， 1次/d，共14 d；干细胞组给予1×10⁷的骨髓间充质干细胞混悬液10 mL经耳缘静脉注射1次。

检测方法：

病理检查：给药14 d后，空气栓塞法处死4组动物。选取椎动脉第Ⅱ段注射部位靠近头部一侧同一部位做成石蜡切片。40 g/L的多聚甲醛固定24 h后行常规石蜡包埋、切片，切片厚度为5 μm，送病理科进行常规病理检查。部分标本包埋后做免疫荧光染色检测，部分标本直接冰冻后做ELISA检测。

免疫荧光染色：①切片脱蜡：依次将切片放入二甲苯Ⅰ-二甲苯Ⅱ-无水乙醇Ⅰ-无水乙醇Ⅱ-体积分数为95%乙醇-体积分数为90%乙醇-体积分数为80%乙醇-体积分数为70%乙醇，然后蒸馏水浸泡2 min。②抗原修复：采用微波进行抗原修复，将脱蜡水化后的组织切片置于烧杯(或修复盒)中的不锈钢(或耐高温塑料)切片架上，加适量的修复液(0.01 mol/L枸橼酸缓冲液，pH 6.0)于烧杯中，液面要浸过切片组织一定高度，微波炉可先用中高档加热使液体沸腾，当加热至沸腾时调到中档，此时开始计时，修复时间为10-15 min，此过程中勿使组织干片(修复液要足量)。到时间后将烧杯从微波炉中取出，放入冷水中冷却降温，当修复液降至室温后取出玻片，用磷酸盐缓冲液(pH 7.4)冲洗3遍，每次3 min。③血清封闭：用吸水纸擦干玻片，免疫组化笔在组织周围画圈，滴加稀释好的正常山羊血清，室温封闭30 min，以减少非特异性染色。④加一抗：甩去多余液体，不洗，然后滴加稀释好的一抗，如果做阴性对照实验，就在对照组的切片上滴加磷酸盐缓冲液。加完一抗后于4 ℃湿盒中孵育过夜。⑤加荧光二抗：PBST冲洗切片3次，每次3 min，吸水纸擦干切片后滴加稀释好的荧光二抗，湿盒中20-37 ℃孵育1 h，PBST冲洗切片4次，每次3 min。⑥复染核：滴加DAPI避光孵育5 min，对标本进行染核，PBST 5 min×4次洗去多余的DAPI。用吸水纸擦干切片上的液体，用含抗荧光淬灭剂的封片液封固，然后在荧光显微镜下观察采集图像。⑦图像分析：IPP 6.0对采集图像进行荧光特异性表达的分析，计算荧光面积百分比。

ELISA检测白细胞介素6、肿瘤坏死因子α在椎动脉血管表达：操作步骤按照说明书说明进行操作。终止反应后用酶标仪在450 nm处测吸光度值(A值)，以标准品吸光度值为横坐标，标本吸光度值为纵坐标绘制标准曲线，所有样品孔值均减除空白孔值后计算含量。

主要观察指标：①病理检查观察椎动脉血管形态。②免疫荧光染色检测椎动脉血管cathepsin B表达。③ELISA检测椎动脉血管白细胞介素6、肿瘤坏死因子α的表达。

统计学分析：采用SPSS 12.0统计软件进行统计分析，多组间比较采用SNK-q检验，P < 0.05为差异有显著性意义。

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讨论

颈椎病是指颈椎间盘组织退行性改变及椎间结构继发性改变，刺激或压迫神经根、脊髓、椎动脉、交感神经等周围组织，出现相应的各种症状和(或)体征^[22-24]。椎动脉型颈椎病是由Bayye和Lieon于1926年率先提出，究其原因为椎基底动脉供血不足引起一系列临床症状。

椎动脉型颈椎病病理机制非常复杂，临床上往往出现影像学结果和临床症状不一致的病例。许多学者从解剖学、病理学、神经及临床多方面研究^[25-30]，对其发病病理机制的认识渐趋统一。炎症刺激被认为是颈椎病急性发作的重要因素^[2]。既往以硬化剂注射法复制椎动脉型颈椎病模型^[21]，硬化剂能凝固兔颈背部伸肌群，造成颈椎动力性及静力性平衡失调引起颈椎退行性变。同时硬化剂凝固肌肉后形成无菌性炎症^[21]，本实验也证实兔颈部肌肉组织出现炎症、水肿、瘢痕、机化，椎动脉血管出现增殖，血管增厚，血管内皮皱折不均匀，血管α-平滑肌肌动蛋白及基质金属蛋白酶2呈强阳性表达。

白细胞介素6和肿瘤坏死因子α被认为是介导基质金属蛋白酶2活化和促进其表达的炎症因子^[31-32]。因而猜测动物模型椎动脉血管白细胞介素6和肿瘤坏死因子α表达呈阳性。实验证实空白组椎动脉血管白细胞介素6和肿瘤坏死因子α表达较少；与空白组相比，模型组椎动脉血管白细胞介素6和肿瘤坏死因子α表达显著增加，差异有显著性意义 (P < 0.05)。炎症因子刺激动脉血管可引起血管收缩、血管内皮损伤^[3-4]。

cathepsin B以酶原形式储存在溶酶体中，胞浆中约含10%，激活后的组织蛋白酶B具有广谱的蛋白水解酶活性。直接降解或激活纤溶酶原激活剂及基质金属蛋白酶等而间接降解许多细胞外基质成分，从而参与血管壁重构^[33-38]。有研究表明，炎症因子白细胞介素6和肿瘤坏死因子α均能活化cathepsin B^[39-40]。本实验证实空白组椎动脉血管cathepsin B表达较少；与空白组相比，模型组椎动脉血管cathepsin B显著增加，差异有显著性意义(P < 0.05)。说明椎动脉型颈椎病动物模型椎动脉血管因炎症刺激受到损害。

如何抑制炎症反应并修复受损的血管呢？骨髓间充质干细胞作为一种新的治疗方法能治疗炎症介导的多种疾病。动物实验证实骨髓间充质干细胞具有免疫赦免特性，可在同种异基因或异种间进行移植，而很少发生移植排斥反应^[41]，说明骨髓间充质干细胞具有异基因移植物耐受和免疫调节作用。

CD4⁺CD25⁺调节性T细胞(Regulatory T cell，Treg细胞)为一类具有免疫抑制作用的T细胞，以“主动”方式参与免疫耐受的调控^[42-46]。间充质干细胞能上调Treg细胞水平，且该作用呈剂量依赖性，Treg细胞在自身免疫性疾病中能抑制炎症因子肿瘤坏死因子α、白细胞介素6表达^[47-48]。因此猜想，经静脉注射骨髓间充质干细胞上调Treg细胞水平，可以抑制椎动脉肿瘤坏死因子α、白细胞介素6产生。实验证实，与模型组相比，干细胞组椎动脉血管肿瘤坏死因子α、白细胞介素6表达降低，差异有显著性意义(P < 0.05)。干细胞组与丹参酮组比较无明显差别(P > 0.05)。说明干细胞能抑制椎动脉血管炎症反应。

免疫荧光染色检测显示：与模型组相比，干细胞组椎动脉血管cathepsin B表达明显减少，差异有显著性意义 (P < 0.05)；丹参酮为临床常用的治疗椎动脉型颈椎病的中药单体，它能抑制血管炎症反应^[49-50]，所以本实验以丹参酮作为对照。干细胞组与丹参酮组比较差异无显著性意义(P > 0.05)。说明治疗组椎动脉血管炎症受到抑制后，使血管蛋白酶活性受到抑制，从而减轻血管损伤。

总的来说，椎动脉血管炎症可能是血管损伤的发病机制之一，骨髓间充质干细胞作为一种新的治疗椎动脉型颈椎病的方法，可能通过抑制炎症和细胞分化作用修复椎动脉血管，其具体作用机制尚需进一步研究。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
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骨髓间充质干细胞修复椎动脉型颈椎病的血管损伤

Bone marrow mesenchymal stem cells repair vascular injury after cervical spondylotic vertebral arteriopathy

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