CN107354126A - Ncam沉默在促进atdc5细胞的软骨细胞肥大分化中的应用 - Google Patents
Ncam沉默在促进atdc5细胞的软骨细胞肥大分化中的应用 Download PDFInfo
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Abstract
本发明提供了NCAM缺失在促进BMSCs细胞向肥大软骨细胞分化中的应用。通过在ATDC5细胞中转染干扰NCAM表达质粒siRNA的方法以下调内源性NCAM的表达，并将其分别进行软骨细胞分化。
Description
技术领域
本发明涉及软骨细胞培养技术，具体涉及NCAM沉默在促进ATDC5细胞的软骨细胞肥大分化中的应用。
背景技术
关节软骨主要是由软骨细胞和细胞外基质组成的无血管组织，主要依靠关节的运动和挤压吸收营养物质，所以软骨损伤后自我修复能力比较弱，其损伤后的修复移植一直以来是医学界的难题之一。近年来，随着组织工程技术的发展，自体软骨移植在软骨损伤的临床治疗在国内外都有应用。临床研究对患者组织取材有限，并且也因为软骨细胞是终末分化细胞，体外增殖能力有限且易去分化，所以对体外软骨细胞的培养和扩增成为目前要解决的关键。由于软骨细胞属于终末分化细胞，自身的修复能力有限，破坏后很难再生，目前尚缺乏满意的治疗措施。虽然可以采用自体移植的方法治疗软骨损伤，但由于软骨细胞的取材困难，来源有限等缺点，大规模应用受到制约。骨髓间充质干细胞(bone marrowstem cells,BMSCs)是一种多向分化潜能的成体干细胞，具有来源丰富、取材方便、增殖快、定向分化能力强等优点。在体外特定的条件下，具有向软骨、成骨、脂肪、肌腱、韧带等分化的能力。因此，BMSC因其显著优点逐渐成为软骨组织修复构建的最佳种子细胞来源之一。
软骨发生的早期，骨髓间充质干细胞聚集(Condensation)，密集的细胞表达TGF-β、N-CAM等多种因子，并启动软骨细胞分化(Chondrocyte differentiation)，随着间充质细胞向软骨细胞分化，开始分泌富含Ⅱ型胶原蛋白(ColⅡ)和聚集蛋白聚糖(Aggrecan)的细胞外基质。软骨细胞在凝聚的中心将停止分裂起始终末分化，形成肥大的软骨细胞 (Hypertrophic chondrocyte)，进入肥大成熟阶段，X型胶原蛋白(ColX)、RunX2及碱性磷酸酶(ALP)等表达增强，钙累积增多，细胞增大并产生矿化的细胞外基质，肥大的软骨细胞继续钙化将形成软骨下骨细胞。综上，BMSC向软骨细胞分化并形成软骨的过程分为凝结(condensation)、软骨细胞(chondrocyte)和软骨细胞肥大(chondrocyte hypertrophy)三个阶段。
神经细胞粘附分子(neural cell adhesion molecule,NCAM；即CD56)属于免疫球蛋白超家族中的一员。NCAM具有三个不同的亚型，即NCAM120、NCAM140、NCAM180，都是以它们表面的分子大小来命名的。这三个亚型都有一个共同的细胞外的区域，但是NCAM120无胞质区及跨膜区，通过糖基磷脂酰肌醇(GPI)直接与膜相连，易于在膜表面运动，且只锚定于膜上的脂筏区域。NCAM140和NCAM180都有跨膜区域和不同长度的胞质尾，两者通过跨膜区与胞内细胞骨架连接便于信号转导，但是NCAM140相对于NCAM180来说，缺乏268个氨基酸序列。NCAM在神经细胞中起着非常重要的作用，包括：神经退化、迁移、分化、增殖、细胞粘附、信号传导以及学习和记忆的调节。
发明内容
为了解决现有技术的不足，本发明提供了NCAM沉默在促进ATDC5细胞的软骨细胞肥大分化中的应用。
本发明的技术方案是：NCAM沉默在促进ATDC5细胞的软骨细胞肥大分化中的应用。
本发明的进一步改进包括：
通过在ATDC5细胞中转染干扰NCAM表达质粒siRNA的方法以下调内源性NCAM的表达，并将其分别进行软骨细胞分化。
NCAM沉默促进ATDC5细胞钙沉积。
附图说明
图1：NCAM缺失的BMSCs的建立；
图2A：NCAM缺失促进BMSCs的软骨细胞肥大标志物COL10a的表达；
图2B：NCAM缺失促进BMSCs的软骨细胞肥大标志物RUNX2的表达；
图2C：NCAM缺失促进BMSCs的软骨细胞肥大标志物SOX9的表达；
图2D：NCAM缺失促进BMSCs的软骨细胞肥大标志物COL2a的表达；
图3A：NCAM缺失促进BMSCs的软骨细胞肥大标志物RunX2蛋白质水平的表达；
图3B：NCAM缺失促进BMSCs的软骨细胞肥大标志物ColX蛋白质水平的表达；
图4A：NCAM沉默促进ATDC5细胞的软骨细胞肥大中NCAM的表达量；
图4B：NCAM沉默促进ATDC5细胞的软骨细胞肥大中RunX2的表达量；
图4C：NCAM沉默促进ATDC5细胞的软骨细胞肥大中钙沉积的含量。
具体实施方式
下面结合附图对本发明做详细说明。
材料与方法：
材料与试剂
C57/BL6小鼠原代野生型(NCAM+/+)和NCAM敲除(NCAM-/-)的BMSC由本实验室分离培养，ATDC5细胞购自日本细胞库RIKEN Cell Bank；NCAM siRNA质粒由本实验室构建；DMEM低糖、DMEM/F12培养基购自HyClone，胎牛血清(FBS)购自Gibco，0.25％胰蛋白酶(含EDTA)、青霉素-链霉素、细胞裂解液、蛋白酶抑制剂、磷酸酶抑制剂、蛋白上样缓冲液购自上海碧云天生物技术有限公司，Bradford购自BIO-RAD公司；逆转录试剂盒、real-time PCR试剂盒购自南京爱必梦生物材料有限公司；ITS诱导液、茜素红、阿尔新蓝购自Sigma公司；Trizol试剂和Lipofectamine 2000购自Invitrogen公司；Runx2和β-actin抗体购自于CST公司，COLX抗体购自Abcam公司，NCAM抗体购自上海生工生物工程有限公司。
BMSC的培养与软骨诱导分化
BMSC用含10％FBS、100U/mL青霉素/链霉素的DMEM低糖培养基，置于37℃、5％CO2饱和湿度的细胞培养箱培养。
取生长状态良好第三代BMSC接种于12孔板，待细胞贴壁生长24h后，去除原有的培养液，加入预先配制好的软骨细胞分化培养液(FBS 10％、10ng/mL TGF-β1、50nM抗坏血酸、0.1μM地塞米松、10ng/mL bFGF)，每隔两天换液1次。
ATDC5细胞的培养与软骨细胞诱导分化
ATDC5细胞用含5％FBS、100U/mL青霉素/链霉素的DMEM/F12培养基，置于37℃、5％CO2饱和湿度的细胞培养箱培养。
取生长状态良好的ATDC5细胞接种于12孔板，待细胞贴壁生长24h后，去除原有的培养液，加入预先配制好的含ITS的软骨诱导分化培养液(FBS 5％、10μg/mL人胰岛素、5.5μg/mL人转铁蛋白、5ng/mL亚硒酸钠)，每隔两天换液1次。
Western blot分析
用RIPA裂解液裂解细胞，提取总蛋白，并用Bradford测定总蛋白的浓度。采用聚丙烯酰胺凝胶电泳(SDS-PAGE)分离，转膜，用5％的脱脂牛奶封闭1h，一抗4℃孵育过夜，TBST漂洗3次后，二抗室温孵育40min，洗膜后用ECL发光液以及全自动曝光仪曝光。用ImageJ软件对蛋白条带进行扫描，定量分析相应蛋白的表达变化情况。
实时荧光定量PCR分析
采用Trizol试剂，按照试剂盒的要求提取细胞总RNA。根据反转录试剂盒说明书逆转录合成cDNA的第一条链后，以此为模版，进行实时荧光定量PCR(real-time PCR)检测目的基因的表达变化，引物序列如表1所示。PCR的反应程序：95℃预变性30s，95℃变性5s，64℃退火延伸30s，共40个循环。基因的相对表达量用法计算，采用小鼠GAPDH作为内对照。
表1引物序列
免疫荧光
将细胞加入到铺有细胞玻片12孔板中，待细胞爬片后进行相应处理。4％多聚甲醛固定细胞30min，PBS漂洗3次，每次5min。0.1％Triton X-100室温透化10min，PBS漂洗3次，每次5min。用3％BSA室温封闭1h，PBS漂洗3次，每次5min。加入一抗4℃孵育过夜，PBS漂洗3次，每次5min。然后加入FITC标记的二抗，孵育2h，DAPI复染5min，PBS漂洗3次，每次5min，90％甘油封片，激光共聚焦观察拍照。
统计学分析
实验数据统计分析采用Graphpad Prism 5统计软件进行统计分析，结果以均数±标准差来表示，并用单因素方差分析进行处理，组间进行t检验，以P<0.05认为差异具有统计学意义。
结果：
从正常的和NCAM缺失的C57/BL6小鼠中提取BMSCs，将其培养至P3后，提取细胞的总蛋白质，Western blot检测发现NCAM已经完全被敲除(图1)。
图1：取细胞培养板中生长状态良好的正常BMSCs，加入细胞裂解液，提取总蛋白质，用anti-NCAM做Western blot检测NCAM的变化情况，并以β-actin作为内参。
NCAM缺失促进BMSCs的软骨细胞肥大标志物mRNA的表达
Real-time PCR的结果显示，将BMSCs进行软骨细胞分化后，NCAM缺失的BMSCs与正常的BMSCs相比，软骨细胞肥大标志物RunX2和COLXmRNA的表达量明显上调；而软骨细胞标志物COL2a和Sox9mRNA的表达量几乎无显著变化(图2)。
图2A至图2D可以看出：将正常的BMSCs和NCAM缺失的BMSCs分别进行软骨细胞分化0d，1d，3d后，提取细胞的总RNA，并进行反转录PCR后，将cDNA加入到相应的反应体系中，进行实时荧光定量PCR(Real-time PCR)检测RunX2和COLX，COL2a和Sox9的表达情况，并以GAPDH作为内对照。其中n＝4，其中，*P<0.05,**P<0.01的结果为NCAM缺失的BMSCSs和正常的BMSCSs相比较。
NCAM缺失促进BMSCs的软骨细胞肥大标志物蛋白质水平的表达
Western blot的结果显示，与正常的BMSCs相比，NCAM缺失能明显的促进软骨细胞肥大标志物RunX2蛋白质的表达。另外，细胞免疫荧光的结果也证明，与正常的BMSCs相比，软骨细胞肥大标志物ColX的表达量在NCAM缺失的BMSCs中同样具有明显增强的作用(图3)。
图3A将正常的BMSCSs和NCAM缺失的BMSCs分别进行软骨细胞分化0d，1d，3d后，加入一定体积的细胞裂解液，提取总蛋白质，用anti-RunX2做Western blot检测RunX2的变化情况，并以β-actin作为内参。图3B同样的，将正常的BMSCSs和NCAM缺失的BMSCs铺到带有爬片的12孔板中，培养24h后更换为软骨细胞分化培养液，分化1d后，做细胞免疫荧光检测ColX的变化情况。
NCAM缺失促进BMSCs钙结节的形成
为了更好的说明NCAM缺失后能促进BMSCs的软骨细胞肥大，将正常的BMSCSs和NCAM缺失的BMSCs进行分别进行软骨细胞分化0d，1d，3d后进行茜素红染色和阿尔新蓝染色。结果发现，与正常的BMSCs相比，NCAM缺失后，茜素红染色明显的加深，能显著地促进钙的累积；而阿尔新蓝染色没有明显的差异，说明NCAM对蛋白聚糖的产生几乎没有太大的影响。
将生长状态良好的正常BMSCs和NCAM缺失的BMSCs铺至12孔板中，培养24h后更换为软骨细胞分化的培养液，进行软骨细胞分化0d，1d，3d后，分别用茜素红染色(A)和阿尔新蓝染色(B)来检测钙结节含量和蛋白聚糖表达量的变化情况。
NCAM沉默促进ATDC5细胞的软骨细胞肥大
为了进一步的证明NCAM在软骨细胞分化中的作用，我们选用软骨前体细胞(即ATDC5细胞)进行软骨细胞分化的研究。通过在ATDC5细胞中转染干扰NCAM表达质粒siRNA的方法来下调内源性NCAM的表达，并将其分别进行软骨细胞分化。结果发现，与空质粒的siRNA转染的ATDC5细胞相比，在转染NCAM siRNA的ATDC5细胞中，NCAM的表达量明显下调(图4A)，软骨细胞肥大标志物RunX2表达量明显上调(图4B)；与此类似的，茜素红染色后，钙沉积的含量也具有明显的增加(图4C)。
在ATDC5细胞中转染pSilencer-4.1-based的质粒作为阴性对照组，转染si-NCAM作为实验组。正如图4A所示，在转染siRNA后，提取生长状态良好的细胞，加入细胞裂解液，提取总蛋白质，用anti-NCAM做Western blot检测NCAM的表达情况，并以β-actin作为内参。图4B：将转染的ATDC5细胞进行软骨细胞分化0d和3d后，用anti-RunX2做Western blot检测RunX2的变化情况，并以β-actin作为内参。将转染的ATDC5细胞铺至12孔板中，培养24h后更换为ATDC5细胞的软骨细胞分化的培养液，进行软骨细胞分化0d和3d后，分别用茜素红染色来检测钙沉积含量的变化情况。图4C：茜素红染色后，用十六烷基吡啶溶解，测定其OD值，从而定量的测定钙结节含量的变化情况。其中n＝4，其中***P<0.001的结果为si-NCAM与si-ctl相比较。
小结：
1.在BMSC中，NCAM敲除后，软骨肥大标志物(hypertrophy markers)RunX2和COLX的表达明显上调；NCAM敲除能显著地促进软骨细胞肥大(chondrocyte hypertrophy)阶段中钙的沉积。
2.在ATDC-5细胞中，利用RNAi将NCAM沉默后，软骨细胞肥大标志物RunX2的表达明显上调，钙沉积的也明显增加，与BMSC中的结果一致。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (3)
1.NCAM沉默在促进ATDC5细胞的软骨细胞肥大分化中的应用。
2.根据权利要求1所述的应用，其特征在于，通过在ATDC5细胞中转染干扰NCAM表达质粒siRNA的方法以下调内源性NCAM的表达，并将其分别进行软骨细胞分化。
3.根据权利要求1所述的应用，其特征在于，NCAM沉默促进ATDC5细胞钙沉积。
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