詳解常用細胞凋亡檢測方法匯總！

<p class="MsoNormal"><span style="background-color: rgb(204, 255, 255);"><strong><span style="font-family: 宋體; font-size: 9pt;">細胞凋亡的形態(tài)學檢測</span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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;">光學顯微鏡和倒置顯微鏡</span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">（</font>1<font face="宋體">）</font></span></strong><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">未染色細胞：凋亡細胞的體積變小、變形，細胞膜完整但出現(xiàn)發(fā)泡現(xiàn)象，細胞凋亡晚期可見凋亡小體，貼壁細胞出現(xiàn)皺縮、變圓、脫落。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">（</font>2<font face="宋體">）</font></span></strong><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">染色細胞：常用姬姆薩染色、瑞氏染色等</font><font face="Calibri">.</font><font face="宋體">凋亡細胞的染色質濃縮、邊緣化，核膜裂解、染色質分割成塊狀和凋亡小體等典型的凋亡形態(tài)。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">磷脂酰絲氨酸外翻分析（</font>Annexin V<font face="宋體">法）</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">磷脂酰絲氨酸（</font>Phosphatidylserine<font face="宋體">， </font><font face="Calibri">PS</font><font face="宋體">）正常位于細胞膜的內側，但在細胞凋亡的早期，</font><font face="Calibri">PS</font><font face="宋體">可從細胞膜的內側翻轉到細胞膜的表面，暴露在細胞外環(huán)境中。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">是一種分子量為</font><font face="Calibri">35~36KD</font><font face="宋體">的</font><font face="Calibri">Ca2+</font><font face="宋體">依賴性磷脂結合蛋白，能與</font><font face="Calibri">PS</font><font face="宋體">高親和力特異性結合。將</font><font face="Calibri">Annexin-V</font><font face="宋體">進行熒光素（</font><font face="Calibri">FITC</font><font face="宋體">、</font><font face="Calibri">PE</font><font face="宋體">）或</font><font face="Calibri">biotin</font><font face="宋體">標記，以標記了的</font><font face="Calibri">Annexin-V</font><font face="宋體">作為熒光探針，利用流式細胞儀或熒光顯微鏡可檢測細胞凋亡的發(fā)生。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">線粒體膜勢能的檢測<o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">線粒體在細胞凋亡的過程中起著樞紐作用，多種細胞凋亡刺激因子均可誘導不同的細胞發(fā)生凋亡，而線粒體跨膜電位</font>DYmt<font face="宋體">的下降，被認為是細胞凋亡級聯(lián)反應過 程中最早發(fā)生的事件，它發(fā)生在細胞核凋亡特征（染色質濃縮、</font><font face="Calibri">DNA</font><font face="宋體">斷裂）出現(xiàn)之前，一旦線粒體</font><font face="Calibri">DYmt</font><font face="宋體">崩潰，則細胞凋亡不可逆轉。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">片斷化檢測</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">細胞凋亡時主要的生化特征是其染色質發(fā)生濃縮，</font> <font face="宋體">染色質</font>DNA<font face="宋體">在核小體單位之間的連接處斷裂， 形成</font><font face="Calibri">50~300kbp</font><font face="宋體">長的</font><font face="Calibri">DNA</font><font face="宋體">大片段，或</font><font face="Calibri">180~200bp</font><font face="宋體">整數倍的寡核苷酸片段， 在凝膠電泳上表現(xiàn)為梯形電泳圖譜（</font><font face="Calibri">DNAladder</font><font face="宋體">）。細胞經處理后。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">法</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">細胞凋亡中，染色體</font>DNA<font face="宋體">雙鏈斷裂或單鏈斷裂而產生大量的粘性</font><font face="Calibri">3'-OH</font><font face="宋體">末端，可在脫氧核糖核苷酸末端轉移酶（</font><font face="Calibri">TdT</font><font face="宋體">）的作用下，將脫氧核糖核苷酸和熒光 素、過氧化物酶、堿性磷酸酶或生物素形成的衍生物標記到</font><font face="Calibri">DNA</font><font face="宋體">的</font><font face="Calibri">3'-</font><font face="宋體">末端，從而可進行凋亡細胞的檢測，這類方法稱為脫氧核糖核苷酸末端轉移酶介導的缺口末端標記法。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">凋亡相關蛋白</font>TFAR19<font face="宋體">蛋白的表達和細胞定位分析</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">（</font><font face="Calibri">PDCD5</font><font face="宋體">）是由本研究室在國際上首先報導的一個擁有自己知識產權的人類新基因，前期的功能研究表明，它是促進細胞凋亡的增強劑。利用熒光 素（</font><font face="Calibri">FITC</font><font face="宋體">）標記的</font><font face="Calibri">TFAR19</font><font face="宋體">單克隆抗體為探針，對細胞凋亡過程中</font><font face="Calibri">TFAR19</font><font face="宋體">蛋白的表達水平及定位研究發(fā)現(xiàn)，凋亡早期</font><font face="Calibri">TFAR19</font><font face="宋體">表達水平增高并出 現(xiàn)快速核轉位現(xiàn)象。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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:宋體;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>