Name: isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods
Uploaded: 2012-11-24T14:00:00
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我们非常感谢博士的莱拉Shakeri博士阿雷夫Dournaei的关键铁饼和穆罕默德·礼萨·哈德姆谢里夫先生为他的技术支持。

1。准备的酶液和增殖培养基（PM） <ol><li> 车型胶原酶类型I解：称出I型胶原酶（12毫克/毫升），并溶解在1ml PBS及过滤器，用0.2μm注射器过滤器。然后将其放在15毫升的试管，并保持在-20℃，直到需要。 </li><li> 使分散酶解：称取分散酶（16毫克/毫升），并溶解在1毫升的PBS及过滤器，用0.2μm注射器过滤器。然后将其放在15毫升的试管，并保持在4℃直至需要。 </li><li> <strong...

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该协议描述了hDPSCs牙髓使用两种方法，酶解和牙髓组织的外植体干细胞的直接产物的分离和鉴定。此外， 在这些细胞的体外分化成成牙本质细胞，通过茜素红S的定量测定＆QPCR评估。 现有的协议，用于隔离从人类牙齿的牙髓组织已被用于各种仪器，如钳子（骨镊子）9，摘除手术中，针29，格雷西刮匙30，牙科裂缝毛刺4，等，这些方法是?...

干细胞是细胞克隆细胞具有两个显着的特点，被称为多潜能和自我更新15。在所有的干细胞不同的复制效力，牙齿干细胞的产后干细胞引起的关注，近年来，因为其可访问性，可塑性强，高增殖能力比较与其他成体干细胞16。典型地，类似的间充质干细胞，牙髓干细胞粘附的克隆形成有多个成间充质细胞的分化能力和/或非间充质细胞谱系，无论是在体外和体内的细胞，这些细胞。1-8,17,18稳干细胞鉴定阴性表达造血抗原（ 如 CD45，CD34，CD14），阳性表达的CD90，CD29，CD73，CD105，CD44和STRO-1。19,20 容易获得潜在的疼痛和发病率最低，作为一个有价值的人DPSCS能源间充质干细胞相比普通的来源，如骨髓间充质干细胞21。在一般情况下，稳干细胞已被分离由任何生长的方法， 即 ，从的牙髓组织外植体（DPSC-OG）22-24，和/或酶消化4,6,25（DPSC-ED）的干细胞的迁移。以往的研究表明，隔离方法和培养条件下，可以吸引不同的人群或谱系体外通道26,27。在的箱子恒牙（pDPSCs），Huang 等人透露，酶消化pDPSCs超越全部牙髓之中相比，有较高的增殖潜力，26此外，在箱子乳牙（dDPSCs），它表明一个STRO-1及CD34的标记表示在比较更多的dDPSC-ED与dDPSC-OG。此外，dDPSC-ED显示定义的骨/ odonto介质的高矿化率在27因此，由于出色的潜力DPSCS在regenerative药，将有更多的研究，以更好地了解可能来自不同的隔离方法不同人群的需要。 在这里，它是尝试引入纸浆提取的简单的方法，通过使用一步牙科金刚石盘以方便处理纸浆提取。此外，在申请ED或OG方法的人牙髓源性干细胞的分离，两组间的一般特性及分化能力进行了调查。

<table border="1"><tbody><tr><td>的试剂的名称</td><td>公司</td><td>目录或地段。数</td><td>评论（可选） </td></tr><tr><td>的α-MEM </td><td> GIBCO </td><td> 11900-073 </td><td></td></tr><tr><td> I型胶原酶</td><td> Sigma-Aldrich公司</td><td> C0130-100MG </td><td></td></tr><tr><td>中性蛋白酶</td><td> GIBCO </td><td> 17105-041 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> GIBCO </td><td> 15140-122 </td><td></td></tr><tr><td>两性霉素B </td><td> GIBCO </td><td> 15290-018 </td><td></td></tr><tr><td>定义胎牛血清（FBS） </td><td>胎牛血清</td><td> SH30070.03 </td><td></td></tr><tr><td> L-抗坏血酸-2 - 磷酸</td><td>西格玛</td><td> A8960-5G </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> GIBCO </td><td> 25030-024 </td><td></td></tr><tr><td>地塞米松</td><td>西格玛</td><td> D4902 </td><td></td></tr><tr><td> β-甘油磷酸二钠盐水合物，BioUltra </td><td>西格玛</td><td> G9422-100G </td><td></td></tr><tr><td>磷酸二氢钾</td><td> Sigma-Aldrich公司</td><td> P5655 </td><td></td></tr><tr><td>成骨检测试剂盒</td><td>密理博</td><td> PS01802031 </td><td></td></tr><tr><td>同型对照小鼠IgG2b-PE </td><td> BD Pharmingen公司</td><td> 50808088029 </td><td></td></tr><tr><td> FITC小鼠IgG2b同种型控制</td><td> BD Pharmingen公司</td><td> 559532 </td><td></td></tr><tr><td> FITC小鼠IgG1κ型</td><td> BD Pharmingen公司</td><td> 11471471 </td><td></td></tr><tr><td> FITC / PE的小鼠抗人CD34/CD45 </td><td> BD Pharmingen公司</td><td>341071 </td><td></td></tr><tr><td> PE抗人CD146 </td><td> BD Pharmingen公司</td><td> 550315 </td><td></td></tr><tr><td>单克隆小鼠抗人CD90/FITC </td><td>特嘉</td><td> 00034418 </td><td></td></tr><tr><td> PE的小鼠抗人CD73 </td><td> BD Pharmingen公司</td><td> 550257 </td><td></td></tr><tr><td>抗H CD105/Endoglin PE的</td><td> BD Pharmingen公司</td><td> FAB10971P </td><td></td></tr><tr><td> PE的小鼠抗人CD11b/Mac1 </td><td> BD Pharmingen公司</td><td> 5553888 </td><td></td></tr><tr><td>小鼠抗人CD44 PE </td><td> BD Pharmingen公司</td><td> 555479 </td><td></td></tr><tr><td>磷酸盐缓冲溶液（PBS）中</td><td> GIBCO </td><td> 003000 </td><td></td></tr><tr><td> 70微米的细胞过滤器</td><td>鹰</td><td> 352360 </td><td></td></tr><tr><td&gt; 0.2微米针头式过滤器</td><td>新型Millex-GV </td><td> SLGV033RB </td><td></td></tr><tr><td> 25厘米2的培养瓶中</td><td> Sigma-Aldrich公司</td><td> Z707481 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td> BD FACSCalibur </td><td> BD </td><td> 342975 </td><td></td></tr><tr><td> multiskan微孔板分光光度计</td><td> Thermo Scientific的</td><td> 51119200 </td><td></td></tr><tr><td> Fleurcense显微镜</td><td>奥林巴斯</td><td></td><td></td></tr><tr><td>流动软件版本2.3.1 </td><td></td><td></td><td></td></tr></tbody></table>

isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods

<p class="jove_content"&gt;开发智齿容易访问的源干细胞在成年后可以得到常规正畸治疗。人牙髓干细胞（hDPSCs）具有高增殖能力，多系分化能力比较普通的成体干细胞来源<sup&gt; 1-8</sup因此，hDPSCs可能是自体移植，组织工程和再生医学的很好的候选人。除了这些优点，具有间充质干细胞（MSC）的功能，如immunolodulatory效果，使hDPSCs更有价值，即使在同种异体移植的情况下，<sup&gt; 6,9,10</sup&gt;。因此，使用此的干细胞来源的主要步骤，用于选择最佳的协议，用于隔离hDPSCs从牙髓组织。为了实现这一目标，关键是要研究不同的隔离条件下对不同细胞的行为，如他们共同的表面标记和其分化能力的影响。</p&gt;<p class="jove_content"因此，在这里我们分离的人牙髓组织影响第三磨牙，然后用现有文献的基础上协议，用于隔离hDPSCs，<sup&gt; 11-13</sup&gt;<em&gt; IE浏览器</em&gt;牙髓组织中的酶分解的（DPSC ED）或组织块长出（DPSC OG）。在这方面，我们努力促进的隔离方法，使用牙科金刚石盘。然后，将这些细胞其特征在于基质相关的标志物（CD73，CD90，C​​D105和CD44），造血/内皮标志物（CD34，CD45及CD11b）的，血管周围标记，如CD146和STRO-1。此后，这两个协议进行比较的基础上通过定量聚合酶链反应（QPCR）和茜素红染色成牙本质细胞的分化潜能。 QPCR被用于评估矿化相关基因的表达（碱性磷酸酶ALP，基质细胞外磷酸糖蛋白; MEPE牙本质涎磷蛋白; DSPP）。<sup&gt; 14</sup</p

<ol><li> 酶解（DPSC-ED）是通过以下方式获得的全部牙髓之中这里示出在第10天，15,18（图1）。发起的菌落上形成几乎为3至5天之后，隔离。 </li><li> 超越DPSCS（DPSC-OG），如图2所示。成纤维细胞样细胞开始从牙髓组织迁移到烧瓶平行订购了近5天播种后5天，10日，13日及18。 </li><li> 全部牙髓之中使用这两种方法在通道3的图3中所示，这两种类型的稳干细胞几乎相同的大小...

Watch this Scientific Journal Video about Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods at JoV

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

通过使用所描述的方法分离和鉴定人牙髓干的细胞（hDPSCs）<strong酶解浆（DPSC-ED）</strong&gt;或<strong的直接产物牙髓组织的外植体干细胞（DPSC-OG）。接着</strong&gt;<em在体外</em这两种类型的hDPSCs&gt;比较分化成成牙本质细胞。

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Research

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牙种植体不同材料的口腔生物膜形成

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