详解常用细胞凋亡检测方法汇总！

<p class="MsoNormal"><span style="background-color: rgb(204, 255, 255);"><strong><span style="font-family: 宋体; font-size: 9pt;">细胞凋亡的形态学检测</span></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">光学显微镜和倒置显微镜</span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">（</font>1<font face="宋体">）</font></span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">未染色细胞：凋亡细胞的体积变小、变形，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体，贴壁细胞出现皱缩、变圆、脱落。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">（</font>2<font face="宋体">）</font></span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">染色细胞：常用姬姆萨染色、瑞氏染色等</font><font face="Calibri">.</font><font face="宋体">凋亡细胞的染色质浓缩、边缘化，核膜裂解、染色质分割成块状和凋亡小体等典型的凋亡形态。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">磷脂酰丝氨酸外翻分析（</font>Annexin V<font face="宋体">法）</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">磷脂酰丝氨酸（</font>Phosphatidylserine<font face="宋体">， </font><font face="Calibri">PS</font><font face="宋体">）正常位于细胞膜的内侧，但在细胞凋亡的早期，</font><font face="Calibri">PS</font><font face="宋体">可从细胞膜的内侧翻转到细胞膜的表面，暴露在细胞外环境中。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">Annexin-V<font face="宋体">是一种分子量为</font><font face="Calibri">35~36KD</font><font face="宋体">的</font><font face="Calibri">Ca2+</font><font face="宋体">依赖性磷脂结合蛋白，能与</font><font face="Calibri">PS</font><font face="宋体">高亲和力特异性结合。将</font><font face="Calibri">Annexin-V</font><font face="宋体">进行荧光素（</font><font face="Calibri">FITC</font><font face="宋体">、</font><font face="Calibri">PE</font><font face="宋体">）或</font><font face="Calibri">biotin</font><font face="宋体">标记，以标记了的</font><font face="Calibri">Annexin-V</font><font face="宋体">作为荧光探针，利用流式细胞仪或荧光显微镜可检测细胞凋亡的发生。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="font-family: 宋体; font-size: 9pt;">线粒体膜势能的检测<o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">线粒体在细胞凋亡的过程中起着枢纽作用，多种细胞凋亡刺激因子均可诱导不同的细胞发生凋亡，而线粒体跨膜电位</font>DYmt<font face="宋体">的下降，被认为是细胞凋亡级联反应过 程中最早发生的事件，它发生在细胞核凋亡特征（染色质浓缩、</font><font face="Calibri">DNA</font><font face="宋体">断裂）出现之前，一旦线粒体</font><font face="Calibri">DYmt</font><font face="宋体">崩溃，则细胞凋亡不可逆转。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">DNA<font face="宋体">片断化检测</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">细胞凋亡时主要的生化特征是其染色质发生浓缩，</font> <font face="宋体">染色质</font>DNA<font face="宋体">在核小体单位之间的连接处断裂， 形成</font><font face="Calibri">50~300kbp</font><font face="宋体">长的</font><font face="Calibri">DNA</font><font face="宋体">大片段，或</font><font face="Calibri">180~200bp</font><font face="宋体">整数倍的寡核苷酸片段， 在凝胶电泳上表现为梯形电泳图谱（</font><font face="Calibri">DNAladder</font><font face="宋体">）。细胞经处理后。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">TUNEL<font face="宋体">法</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">细胞凋亡中，染色体</font>DNA<font face="宋体">双链断裂或单链断裂而产生大量的粘性</font><font face="Calibri">3'-OH</font><font face="宋体">末端，可在脱氧核糖核苷酸末端转移酶（</font><font face="Calibri">TdT</font><font face="宋体">）的作用下，将脱氧核糖核苷酸和荧光 素、过氧化物酶、碱性磷酸酶或生物素形成的衍生物标记到</font><font face="Calibri">DNA</font><font face="宋体">的</font><font face="Calibri">3'-</font><font face="宋体">末端，从而可进行凋亡细胞的检测，这类方法称为脱氧核糖核苷酸末端转移酶介导的缺口末端标记法。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">凋亡相关蛋白</font>TFAR19<font face="宋体">蛋白的表达和细胞定位分析</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">TFAR19<font face="宋体">（</font><font face="Calibri">PDCD5</font><font face="宋体">）是由本研究室在国际上首先报导的一个拥有自己知识产权的人类新基因，前期的功能研究表明，它是促进细胞凋亡的增强剂。利用荧光 素（</font><font face="Calibri">FITC</font><font face="宋体">）标记的</font><font face="Calibri">TFAR19</font><font face="宋体">单克隆抗体为探针，对细胞凋亡过程中</font><font face="Calibri">TFAR19</font><font face="宋体">蛋白的表达水平及定位研究发现，凋亡早期</font><font face="Calibri">TFAR19</font><font face="宋体">表达水平增高并出 现快速核转位现象。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">&nbsp;</span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p>&nbsp;</p>