骨髓间充质干细胞的旁分泌能影响成骨细胞生物学功能

doi:10.3969/j.issn.2095-4344.2014.10.001

摘要/Abstract

摘要：

背景：有研究表明骨髓间充质干细胞移植可促进骨缺损的修复，但受损组织的缺血、缺氧和炎症反应限制了其临床应用。
目的：观察骨髓间充质干细胞旁分泌作用对成骨细胞MG63增殖、迁徙、分化功能的影响。
方法：Ficoll-Paque密度梯度离心法分离骨髓间充质干细胞，制备骨髓间充质干细胞条件培养基培养成骨细胞MG63。CCK-8法检测MG63细胞增殖能力变化；细胞划痕法检测MG63细胞迁徙能力变化；微板检测MG63细胞合成碱性磷酸酶能力的变化；Real-time PCR法检测碱性磷酸酶、Ⅰ型胶原及骨钙素基因表达量的变化；茜素红染色检测其矿化能力的变化。
结果与结论：骨髓间充质干细胞表型为CD44、CD73、CD90表达强阳性，CD34表达阴性。与普通培养基(DMEM)相比，在骨髓间充质干细胞条件培养基作用下，MG63细胞的增殖速度明显加快；细胞划痕结果示其迁徙能力明显提高；诱导第4，7天后碱性磷酸酶基因表达量及蛋白合成量明显增多(P < 0.01)；Ⅰ型胶原及骨钙素基因表达量在诱导第4天后差异无显著性意义，诱导第7天后较对照组明显升高(P < 0.05)；茜素红染色示骨髓间充质干细胞条件培养基诱导MG63细胞21 d后钙结节形成增多，矿化沉积作用增强。结果证实，骨髓间充质干细胞的旁分泌物质可以显著促进成骨细胞的增殖、迁徙、分化和矿化能力。

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, 旁分泌, 成骨细胞, 增殖, 分化, 矿化

Abstract:

BACKGROUND: Some studies have shown that bone marrow mesenchymal stem cells have a certain role in the repair of bone defects, but the clinical application is limited because of ischemia, hypoxia and inflammation of injured tissues.
OBJECTIVE: To investigate the paracrine effects of bone marrow mesenchymal stem cells on osteoblast biological function.
METHODS: Bone marrow mesenchymal stem cells were isolated using standard Ficoll-Paquedensity gradient centrifugation. Mesenchymal stem cell conditioned medium was prepared to cultivate osteoblasts, MG63. Proliferation of MG63 cells was analyzed by cell counting kit-8. Migration of MG63 cells was analyzed by cell scratch method. Alkaline phosphatase activity of MG63 cells was analyzed by microplate test kit. Real-time PCR was performed to evaluate osteoblast differentiation markers, alkaline phosphatase, collagen type I and osteocalcin. Alizarin red staining was performed to evaluate osteoblast mineralization.
RESULTS AND CONCLUSION: The cells were strongly positive for CD44, CD73 and CD90, but negative for CD34. MG63 cells cultured in the conditioned medium showed better proliferation and migration than those cultured in the Dulbecco’s modified Eagle’s medium. The activity and mRNA expression of alkaline phosphatase were much higher after induction of 4, 7 days (P < 0.01). There was no significant difference in expression of collagen type I and osteocalcin after induction of 4 days, but they were significantly higher than those in the control group after induction of 7 days (P < 0.05). Alizarin red staining showed that the number of calcium nodules was increased and the mineral apposition was enhanced after induction of 21 days with the conditioned medium. These findings suggest that the paracrine substance of bone marrow mesenchymal stem cells can significantly promote osteoblast proliferation, migration, differentiation and mineralization.

全文链接：

Key words: bone marrow, mesenchymal stem cells, culture media, conditioned, osteoblasts, cell proliferation, cell differentiation, cell migration assays

中图分类号:

R394.2

李成，周海斌. 骨髓间充质干细胞的旁分泌能影响成骨细胞生物学功能[J]. 中国组织工程研究, 2014, 18(10): 1477-1483.

Li Cheng, Zhou Hai-bin. Paracrine effects of bone marrow mesenchymal stem cells on biological function of osteoblasts [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(10): 1477-1483.

图/表（结果） 4

2.1 骨髓间充质干细胞及MG63细胞形态观察 骨髓间充质干细胞多为长梭形的纤维状，少量为多突起的星形样细胞。细胞折光度好，核仁明显，培养至7 d左右，可见大量的细胞集落形成，细胞形态均一。MG63细胞呈成纤维细胞形贴壁生长，外观呈短梭形、三角形、多边形和鳞片形，折光性强，胞核呈满圆形，位于细胞中央，核质比较大(图1)。

2.2 骨髓间充质干细胞免疫表型分析 培养至第3代的骨髓间充质干细胞表型检测结果显示CD44、CD73、CD90表达强阳性，CD34表达阴性(图1)。

2.3 骨髓间充质干细胞条件培养基对MG63增殖作用的影响 骨髓间充质干细胞的条件培养基培养的MG63细胞，CCK-8法检测结果示其在第1，3，5天的增殖速度明显快于对照组(P < 0.01)，提示骨髓间充质干细胞所分泌物质具有促进成骨细胞生长的作用(图2A)。

2.4 骨髓间充质干细胞条件培养基对MG63细胞迁徙能力的影响 见图3。相比于对照组，骨髓间充质干细胞条件培养基中的MG63细胞于12 h呈现出一定迁徙能力(图3B和F)；24 h后MG63细胞的迁徙速度明显加快(图3C和G)；48 h后实验组的MG63细胞基本合拢，但DMEM培养基中MG63细胞仍存有一定间隙(图3D和H)。提示骨髓间充质干细胞条件培养基可以明显提高MG63细胞的迁徙能力。

2.5 骨髓间充质干细胞条件培养基对MG63细胞分化及矿化能力的影响 分别于DEME培养基和骨髓间充质干细胞条件培养基条件下诱导成骨细胞。碱性磷酸酶活性检测结果显示，在第4，7天时，骨髓间充质干细胞条件培养基诱导组的碱性磷酸酶活性明显高于对照组(P < 0.01，图2B)。Real-time PCR结果显示，骨髓间充质干细胞条件培养基诱导组的碱性磷酸酶mRNA表达量也较对照组明显升高(P < 0.01)，与碱性磷酸酶活性检测结果一致(图2C)；在第4天时两组的Ⅰ型胶原mRNA表达量差异无显著性意义(P > 0.05)，但于第7天时骨髓间充质干细胞条件培养基组Ⅰ型胶原表达量升高(P < 0.05)；骨钙素mRNA表达量在第4天时有轻微升高但差异无显著性意义(P > 0.05，图2D)，而至第7天时则明显升高(P < 0.01，图2E)。21 d后行茜素红染色DMEM对照组可见少量红色钙化物沉积(图4A)，而骨髓间充质干细胞条件培养基诱导组可见深红色钙化结节，细胞间布满红色颗粒，提示钙化基质沉积完全(图4B)。IPP软件分析显示诱导组累积吸光度值明显高于对照组(P < 0.01，图4C)。

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引言

材料方法

设计：细胞学体外观察实验。

时间及地点：实验于2013年1至10月在苏州大学唐仲英血液研究所实验室完成。

材料：

实验细胞：经患者授权同意，抽取苏州大学附属第二医院骨折患者骨髓分离培养骨髓间充质干细胞，血液检测肝炎病毒、HIV、梅毒、衣原体等病原学均为阴性。MG63细胞株由苏州大学骨科研究所惠赠。

实验方法：

骨髓间充质干细胞的分离与培养：无菌条件下行骨髓穿刺，用肝素化注射器收集骨髓，每份10 mL。等量的1×PBS稀释后沿管壁将骨髓液缓慢加入装有Ficoll-Paque分离液的离心管中，2 000 r/min、离心20 min，吸取界面白膜层细胞，加入培养基，吹打混匀，1 000 r/min、离心 5 min，共洗涤2次。所得单核细胞按1×10⁹ L^-¹细胞浓度接种于10 cm培养皿中。置于含体积分数为5% CO₂的37 ℃温箱中培养。3 d后换液，去除非贴壁细胞，以后每隔2 d换液1次。细胞培养基为：DMEM-LG、体积分数为10%胎牛血清、100 U青霉素和链霉素。细胞达到80%融合时，用0.25%胰酶/0.02% EDTA消化2 min，按1∶3传代扩增。

骨髓间充质干细胞表型的流式细胞学分析：当第3代细胞长至80%融合时，胰酶消化收集骨髓间充质干细胞，1×PBS洗涤后细胞计数，按2×10⁵每管的浓度将细胞加入流式管中，离心后用100 µL的1%牛血清白蛋白(1×PBS配制)重悬，分别加入鼠抗人直标PE或FITC单抗CD34、CD44、CD73、CD90各20 µL，室温避光反应30-60 min，设立阴性对照，1×PBS洗涤3次，并悬浮在450 µL的1×PBS中，流式细胞仪进行检测。

骨髓间充质干细胞条件培养基制备：复苏第3代骨髓间充质干细胞于10 cm²细胞培养皿，待细胞融合度达90%(约2×10⁶)时，细胞换液并弃掉。加入10 mL新鲜DMEM培养基，置于37.5 ℃、体积分数为5%CO₂的饱和湿度的培养箱中，培养24 h后收集骨髓间充质干细胞的培养上清，用0.22 μm滤膜过滤上清，分装并冻存于-80 ℃冰箱备用。此培养基定义为100%浓度的骨髓间充质干细胞的条件培养基，实验组采用50%骨髓间充质干细胞条件培养基+50% DMEM培养基。

CCK-8法检测MG63细胞增殖活性：用DMEM-LG(含体积分数为10%胎牛血清)制备MG63细胞悬液，按3×10³/孔接种于96孔板，每孔加培养基100 µL，培养12 h后移去培养液，分别加入DMEM、骨髓间充质干细胞条件培养基，每组设6个复孔。分别在第1，3，5天后，每孔加入CCK-8 10 µL，37 ℃反应2 h，吸取黄色上清液于酶标仪450 nm波长处读取吸光度值。

细胞迁徙实验：用DMEM-LG(含体积分数为10%胎牛血清)制备MG63细胞悬液，按1×10⁵/孔接种于6孔板培养皿，每孔加培养基2 mL，培养12 h后移去培养液，用1 mL枪尖于每孔中央纵行划痕。对照组加入DMEM-LG(含体积分数为10%胎牛血清)培养，实验组加入骨髓间充质干细胞条件培养基培养，每组设2个复孔，分别于0，12，24和48 h后用相差显微镜拍照，拍照时每孔至少应包括5个不同视野，分析细胞迁徙程度。

碱性磷酸酶活性检测：用DMEM-LG(含体积分数为10%胎牛血清)制备MG63细胞悬液，细胞以1×10⁵/孔接种于6孔板培养皿，培养12 h后移去培养液。对照组加入DMEM-LG(含体积分数为10%胎牛血清)培养，实验组加入骨髓间充质干细胞条件培养基培养，收集第4，7天的细胞，用1% Triton裂解细胞，2 000 r/min离心5 min，取上清。按试剂盒使用说明处理收集的样本，用酶联免疫检测仪在520 nm波长下测定其吸光度值，BCA法计算出细胞内总蛋白质量浓度，最后按公式计算单位蛋白质量浓度的碱性磷酸酶活性。

特异性基因的Real-time PCR检测：通过Primer Premier 5 软件设计引物。对照组加入DMEM-LG(含体积分数为10%胎牛血清)培养，实验组加入骨髓间充质干细胞条件培养基培养，收集第4，7天的细胞。采用TRIZOL一步法提取MG63细胞总RNA，取2 μg总RNA，用反转录试剂盒合成cDNA；取1 μL cDNA行Real-time PCR扩增碱性磷酸酶、Ⅰ型胶原及骨钙素基因，以β-actin基因为内参引物。反应体系为 25 μL，双蒸水 10.7 μL，2×mix 12.5 μL，cDNA 模板1 μL，上游引物0.4 μL，下游引物0.4 μL。反应参数：总循环数：34；预变性：95 ℃ 5 min；变性：95 ℃ 35 s；退火60 ℃ 35 s；延伸：72 ℃ 30 s；最后延伸72 ℃ 10 min。荧光定量PCR仪上机检测(7500，ABI)，ABI系统软件分析基因表达量变化。

矿化结节染色：用DMEM-LG(含体积分数为10%胎牛血清)制备MG63细胞悬液，细胞以1×10⁵/孔接种于6孔板培养皿，培养12 h后移去培养液。对照组加入DMEM-LG(含体积分数为10%胎牛血清)培养，实验组加入骨髓间充质干细胞条件培养基培养，21 d后进行茜素红染色。使用PBS洗涤细胞2次后，经40 g/L多聚甲醛固定10 min，再用去离子水冲洗3次；加入Tris-HCl (pH 8.3)配置的0.1%茜素红，在37 ℃环境下孵育30 min，蒸馏水冲洗，置于光学显微镜下观察。随机选取5个视野照相，采用Image Pro Plus 6.0版专业图像分析软件对图像进行分析。

主要观察指标：观察矿化沉积及钙化结节(骨化作用沉积过程中无机质)形成情况。

统计学分析：采用SPSS 17.0统计软件包进行分析。实验数据以x(_)±s形式表示，组间比较采用成组t 检验，P < 0.05为差异有显著性意义。

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讨论

骨髓间充质干细胞因具有来源广、易分离，低免疫原性和多向分化潜能等诸多优势，广泛应用于骨组织工程，并且在修复骨质和软骨缺损方面显示出良好的应用前景^[9-12] 。然而有研究显示将间充质干细胞移植到组织损伤部位后，由于持续的缺血、缺氧和炎症反应，致使间充质干细胞增殖速度减慢，直接分化效率低下，从而使间充质干细胞移植的临床应用受到限制^[13-16] 。
随着研究的深入，间充质干细胞的旁分泌作用逐渐引起广泛的关注。研究表明骨髓间充质干细胞的旁分泌物质对多种实验性器官和组织创伤模型，如爆发性肝功能衰竭、心肌梗死、急性肾小管损伤和皮肤创伤等，具有较好的促进创伤愈合和功能恢复的治疗效果^[17-24] 。其作用机制主要是通过抑制细胞死亡，减轻炎症反应，刺激细胞内源性再生的作用，促进细胞分化、增殖而实现对受损组织的修复。Parekkadan等^[25]还对骨髓间充质干细胞条件培养基进行蛋白组学分析，检测到69种蛋白，其中大部分是细胞因子、生长因子和免疫活性分子。其中一些分子已经证明具有抗凋亡和刺激成骨细胞增殖分化作用，例如白细胞介素6能够促进成骨细胞分化，增加碱性磷酸酶和骨钙素的表达；胰岛素样生长因子1、碱性成纤维细胞生长因子能刺激成骨细胞增殖和基质合成；血管内皮生长因子能促进成骨细胞增殖、迁移，增强甲状旁腺素刺激的cAMP反应，增加碱性磷酸酶合成；转化生长因子β能够增加成骨前体细胞的增殖、分化能力，促进骨基质蛋白如骨桥蛋白和Ⅰ型胶原的合成^[26-37] 。
成骨细胞增殖、分化及基质的矿化在新骨形成中起着关键作用。碱性磷酸酶是成骨细胞分化的早期标志物，分化早期开始出现，基质成熟期达到高峰；Ⅰ型胶原从增殖期开始表达，基质合成期达到高峰；骨钙素是成骨细胞分化后期和矿化期表达的主要蛋白，反映成骨细胞的分化和成熟，一般将碱性磷酸酶、Ⅰ型胶原、骨桥蛋白作为成骨细胞功能状态的评价指标^[38-41] 。本研究结果表明，骨髓间充质干细胞条件培养基明显促进成骨细胞MG63的增殖和迁徙，有助于成骨细胞从增殖相到分化相的转变；在成骨细胞分化早期即显著增强碱性磷酸酶活性及表达量，随着细胞外基质的成熟，Ⅰ型胶原、骨钙素的基因表达量也较对照组明显增多；在细胞外基质矿化阶段，骨髓间充质干细胞条件培养基诱导的成骨细胞钙结节形成更完整，矿化沉积作用更显著。总之，骨髓间充质干细胞条件培养基旁分泌作用显著促进成骨细胞增殖与迁徙，同时促进成骨细胞基质成熟与矿化，从多个阶段加强了成骨细胞的成骨能力。
骨缺损修复过程受多种体内环境因素的影响，其中包括多种生长因子对细胞分化、发育的调节起协同或制约作用。与体内复杂的内环境相比，单一的生长因子促进组织生长相对不足，多因子协同作用较单个因子更有效^[42-44] 。本研究证实，骨髓间充质干细胞的旁分泌物质可以显著促进成骨细胞的增殖、迁徙、分化和矿化能力，有望使骨髓间充质干细胞旁分泌作用在骨缺损的临床治疗中发挥更重要的作用。

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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