Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (45): 7370-7374.doi: 10.3969/j.issn.2095-4344.2014.45.028
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Luo Shu-ping1, Du Yu-ting2, Bai Ju2
Online:
2014-11-05
Published:
2014-11-05
About author:
Luo Shu-ping, Nurse in charge, Department of Critical Care Medicine, General Hospital of Lanzhou Military Region, Lanzhou 730050, Gansu Province, China
CLC Number:
Luo Shu-ping, Du Yu-ting, Bai Ju. Coculture of human umbilical vein endothelial cells and bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(45): 7370-7374.
2.1 细胞共培养系统接触模式的选择 2.1.1 非接触共培养/直接接触共培养 非接触共培养是用具有渗透性的物质物理性的将两种细胞分开,或使用条件培养基或通过从其中一种细胞中获得的细胞外基质间接的共培养[10]。直接接触共培养是将两种细胞直接接触,允许两种细胞之间最大限度的相互作用(例如,用可扩散性因子或通过黏附和缝隙连接进行接触)[11]。非接触共培养可以分析和消除两种细胞之间相互污染的危险,其中一种细胞分泌的扩散性因子可以穿过中间的遮挡屏障以诱导细胞分化。陆军等[12]采用具有半透膜的细胞培养池结合6孔板的方式进行非接触共培养诱导,将人脐静脉内皮细胞铺于培养池内,第3代骨髓间充质干细胞以1×105/孔铺在培养池外的6孔板内,两种细胞的初始比例为1∶5,加入含体积分数为10%胎牛血清的低糖DMEM培养液,培养14 d,结果表明在非接触共培养方式下,人脐静脉内皮细胞可成功诱导人骨髓间充质干细胞向内皮细胞分化,表达内皮细胞特有的表面标志CD31和vWF,阳性率均> 99%,并能够在脱细胞牛静脉血管支架上黏附生长。非接触共培养的主要缺点是它们切断了细胞间密切的物理性接触或调节,而这种对于信号转导的限制与体内环境相悖。直接接触共培养可以在二维培养板表面上或球形和三维支架式的共培养体系中培养[13]。Kaigler等[14]研究发现只有当间充质干细胞和内皮细胞直接接触的共培养时碱性磷酸酶活性和骨钙素的分泌才会同时增加。因此,目前的研究大部分都采用的是直接接触共培养系统。彭锦等[15]研究表明人脐静脉内皮细胞可以通过细胞间直接接触诱导共培养的骨髓间充质干细胞开始向内皮分化,其机制可能与细胞间直接接触促进细胞分泌血管内皮生长因子以及细胞融合密切相关。刘春晓等[16]将人骨髓间充质干细胞及人脐静脉内皮细胞按1∶1比例直接共培养,Matrigel实验显示共培养组形成的毛细血管状结构较单一细胞组多,在共培养体系中,未诱导的人骨髓间充质干细胞能促进形成毛细血管状结构,已成骨诱导的人骨髓间充质干细胞成骨矿化能力增强。 2.1.2 二维/三维培养模式 目前,一些用于研究骨髓间充质干细胞和内皮细胞间二维或三维培养基中相互作用的体外共培养模式已建立起来。大部分研究数据显示用传统的二维表面或在三维结构内共培养,骨髓间充质干细胞的成骨和成血管潜能都要比其单独培养时高。二维培养为细胞接触的分子基础提供了详细的信息,也为内皮细胞控制骨髓间充质干细胞分化的细胞机制提供信息。三维培养为细胞间相互作用、功能性骨血管的快速形成、细胞的生存提供了一个生理性优化的环境[17]。当人间充质干细胞与人静脉内皮细胞在悬浮培养基中共培养时,它们能自发的形成三维结构。在这个系统中,内皮细胞受到刺激而形成三维预成血管网架,间充质干细胞中特定的成骨因子(如碱性磷酸酶的活性)表达增强[14,18]。 2.2 基础培养基的选择 在细胞共培养体系中,培养基的选择没有固定的模式。但是在共培养体系中,敏感性较强的细胞类型所占比例一般较重,从而决定了最终培养基的选择[6]。在人静脉内皮细胞和间充质干细胞的研究中,骨血管生成的培养基选择取决于对培养基要求更苛刻内皮细胞[19]。Bidarra等[9]实验结果表明,不同类型细胞在不同培养基中的生长情况表现不同。人静脉内皮细胞适宜的顺序:IMDM>M199=M199+ DMEM>DMEM,间充质干细胞适宜的顺序是:M199+DMEM>DMEM>IMDM=M199,所以得出结论,M199+DMEM的混合基质能为两种细胞提供最适宜的培养环境。也有学者在实验中使用另外4种培养基:促成骨细胞增殖培养基,成骨性培养基,内皮细胞培养基和后两者1∶1的混合物。结果仅在成骨性培养基中观察到有矿化的发生[20]。 2.3 不同细胞比例对间充质干细胞的影响 为了评估骨髓间充质干细胞与人静脉内皮细胞的相对比例对骨髓间充质干细胞行为的影响,一些学者采用了一些对比研究。结果显示骨髓间充质干细胞∶人静脉内皮细胞比例为50%∶50%时,骨髓间充质干细胞的代谢活性增加最多,75%∶25%组骨髓间充质干细胞的增殖率最高。在这个比例下,人静脉内皮细胞的降低最为轻微。25%∶75%和50%∶50%者碱性磷酸酶活性最高[9]。Xue等[21]以1∶5(内皮细胞/间充质干细胞)的比例在骨髓间充质干细胞中加入脐静脉内皮细胞共培养5 d后,两种细胞增殖显著增加,经检测发现两种细胞间存在细胞桥,同时检测到碱性磷酸酶的mRNA表达增强。这种影响比在培养基中加入成骨因子如地塞米松,抗坏血酸和β-甘油磷酸酯要更为显著。Ma等[20]在加入成骨培养基中,碱性磷酸酶活性、矿化程度和CD31+染色在共培养以(内皮细胞/间充质干细胞)50%∶50%的比例时有最高的表达,并得出结论认为50%∶50%的比例是可以获得成骨和成血管分化最佳效果的组合。但目前对细胞比例的选择还没有最后的定论。 2.4 共培养中细胞的数量、形态及分化特征 实验研究证明,共培养中细胞的数量增加明显。Bidarra等[9]将两种细胞分开分析,单独培养的骨髓间充质干细胞数量显著增加,培养14 d以后数量便不再变化;共培养中骨髓间充质干细胞的细胞数量增长一直延续到第21天,而在后两个星期共培养者的增殖率明显高于单独培养者(1.5×103或3×103 /cm2)。研究结果显示,人脐静脉内皮细胞具有刺激骨髓间充质干细胞增殖的潜力。尽管人脐静脉内皮细胞在共培养中的相对比例有所下降,但内皮细胞的增殖时间延长[9]。 细胞的形态变化方面研究发现,单独培养中,前2周内皮细胞随机分布在培养基表面,到第21天,开始出现类似成纤维细胞形状的压力纤维平行伴随于细胞。而共培养中,细胞重排显示出显著不同的形式,其形成了一个管状的细胞网架。到第21天,可以观察到有交错排列的带有密集压力纤维的细胞骨架生成[22]。有研究观察到人脐静脉内皮细胞可沿成骨前体细胞迁移,共培养可刺激趋化因子的释放,而趋化因子又可以反过来促进人脐静脉内皮细胞的迁移[23]。 基因表达方面,共培养骨髓间充质干细胞中碱性磷酸酶和Ⅰ型胶原的基因表达显著高于单独培养[21]。血管内皮生长因子(VEGF165)在共培养的骨髓间充质干细胞中表达明显升高[24]。同时,神经钙黏蛋白的表达增加,伴随着其调节细胞间黏附与信号转导,使得共培养中骨髓间充质干细胞在成骨分化早期数量显著增多[25]。内皮细胞能参与不同水平的诱导成骨:分泌骨形态发生蛋白以诱导成骨,控制调节成骨分化所需的3种转录因子(Dlx 5,cbfa1/runx2,osterix)以及在预成的血管网中产生矿化组织[26]。同时,骨髓间充质干细胞又能促进内皮细胞分泌骨形态发生蛋白2[27]。 2.5 两种类型细胞间的作用机制 共培养环境中,脐静脉内皮细胞与骨髓间充质干细胞之间可以通过以下3种机制进行联系:①通过两个邻近细胞的膜分子间直接接触而相互作用(黏附和紧密连接)。②信号分子通过邻近细胞胞质间的自由扩散而形成的缝隙连接。③靶细胞上的特定受体或从细胞外基质中释放的可扩散性因子旁分泌作用[11]。 2.5.1 细胞间的紧密连接(黏附) Li等[25]研究显示,骨髓间充质干细胞的细胞质中神经钙黏蛋白(N-cadherin)在共培养下主要集中于共培养的骨髓间充质干细胞的细胞膜上,证明了神经钙黏蛋白在共培养中表达增强并且促进了细胞间的黏附。在另一项研究中,血管内皮钙黏蛋白与β-连环蛋白在共培养中的细胞连接区域发生暂时性消失,可扰乱细胞间的黏附以促进人脐静脉内皮细胞的迁移[24]。 2.5.2 缝隙连接 内皮细胞和骨髓间充质干细胞共培养时可以通过细胞质的连接来进行联系和信息交换。在骨髓间充质干细胞中,主要的缝隙连接蛋白是连接蛋白43。这种蛋白和连接蛋白37、连接蛋白40在内皮细胞中也表达[28]。虽然缝隙连接基因表达的确切机制仍不清楚,但其将依赖于:①渗透连接通道的信号类型。②涉及的连接蛋白(connexins)类型,因为它们具有不同的分子渗透率。有实验显示缝隙连接介导的细胞与细胞间反应是干细胞分化中必需的[11]。另外,有研究指出内皮细胞与成骨样细胞间的相互联系依赖于旁分泌因素和在细胞分化中有重要作用的缝隙连接(连接蛋白43) [22]。 2.5.3 旁分泌作用 Wang等[2]研究认为共培养涉及到管状因子如血管内皮生长因子,这可以证实内皮细胞和骨髓间充质干细胞共培养可产生影响骨细胞生长和分化的生长因子。在共培养体系中,血管内皮生长因子为两种细胞的联系起到核心的作用。它由骨形成细胞分泌,如成骨细胞、骨前体细胞或间充质干细胞,能促进内皮细胞增殖、迁移和毛细血管状结构的形成[29]。血管内皮生长因子能延长内皮细胞的生存时间和增殖效力,并能通过激活特定受体以刺激细胞分化和血管生成[8,30]。有研究表明,血管内皮生长因子受体2(KDR)和尿激酶类纤维溶酶原激活剂(uPA)在人脐静脉内皮细胞中的释放增加。同时,共培养增加了骨髓间充质干细胞对血管内皮生长因子165的分泌;血管内皮生长因子继而活化人脐静脉内皮细胞中的尿激酶类纤维溶酶原激活剂,后者可能是和血管内皮钙黏蛋白一起启动了细胞迁移和自组装网络的形成[26]。"
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