细胞核与线粒体的分级分离

<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;">细胞内不同结构的比重和巨细都不相同，在同一离心场内的沉降速度也不相同，依据这一原理，常用不同转速的离心法，将细胞内各种组分分级别离出来。</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;">别离细胞器最常用的办法是将安排制成匀浆，在均匀的悬浮介质中用差速离心法进行别离，其过程包含安排细胞匀浆、分级别离和剖析三步，这种办法已成为研讨亚细胞成分的化学组成、理化特性及其功用的主要手法。</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">匀浆（</font>Homogenization<font face="宋体">）低温条件下，将安排放在匀浆器中，参加等渗匀浆介质（即</font><font face="Calibri">0.25mol</font><font face="宋体">／</font><font face="Calibri">L</font><font face="宋体">蔗糖一</font><font face="Calibri">0.003mol</font><font face="宋体">／</font><font face="Calibri">L</font><font face="宋体">氯化钙）进行破碎细胞使之成为各种细胞器及其包含物的匀浆。</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">分级别离（</font>Fractionation<font face="宋体">）由低速到高速离心逐步沉降。先用低速使较大的颗粒沉积，再用较高的转速，将浮在上清液中的颗粒沉积下来，从而使各种细胞结构，如细胞核、线粒体等得以别离。</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;">因为样品中各种巨细和密度不同的颗粒在离心开始时均匀分布在整个离心管中，所以每级离心得到的第一次沈淀必定不是纯的最重的颗粒，须经重复悬浮和离心加以纯化。</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">剖析</font> &nbsp;<font face="宋体">分级别离得到的组分，可用细胞化学和生化办法进行形态和功用判定。</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="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">细胞核的别离提取</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;">（一）操作过程</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;">1<font face="宋体">．用颈椎脱位的办法处死小白鼠后，敏捷剖开腹部取出肝脏，剪成小块（去除结缔安排）尽快置于盛有</font><font face="Calibri">0.9</font><font face="宋体">％</font><font face="Calibri">NaCl</font><font face="宋体">的烧杯中，重复洗涤，尽量除掉血污，用滤纸吸去外表的液体。</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;">2<font face="宋体">．将湿重约</font><font face="Calibri">1g</font><font face="宋体">的肝安排放在小平皿中，用量筒量取</font><font face="Calibri">8ml</font><font face="宋体">预冷的</font><font face="Calibri">0.25mol</font><font face="宋体">／</font><font face="Calibri">L</font><font face="宋体">蔗糖一</font><font face="Calibri">0.003mol</font><font face="宋体">／</font><font face="Calibri">L</font><font face="宋体">氯化钙溶液，先加少量该溶液于平皿中，尽量剪碎肝安排后，再悉数参加。</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;">3<font face="宋体">．剪碎的肝安排倒入匀浆管中，使匀浆器下端浸入盛有冰块的器皿中，左手持之，右手将匀浆捣杆笔直插入管中，上下转动研磨</font><font face="Calibri">3</font><font face="宋体">～</font><font face="Calibri">5</font><font face="宋体">次，用</font><font face="Calibri">3</font><font face="宋体">层纱布过滤匀浆液于离心管中，然后制备一张涂片①，做好符号，天然枯燥。</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;">4<font face="宋体">．将装有滤液的离心管配平后，放入一般离心机，以</font><font face="Calibri">2500rpm</font><font face="宋体">，离心</font><font face="Calibri">15</font><font face="宋体">分钟；（</font><font face="Calibri">1</font><font face="宋体">）慢慢取上清液，移入高速离心管中，保存于有冰块的烧杯中，待别离线粒体用；（</font><font face="Calibri">2</font><font face="宋体">）同时涂一张上清液片②做好符号，天然枯燥；（</font><font face="Calibri">3</font><font face="宋体">）余下的沉积物进行下一过程。</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;">5<font face="宋体">．用</font><font face="Calibri">6ml0.25mol/L</font><font face="宋体">蔗糖一</font><font face="Calibri">0.003mol</font><font face="宋体">／</font><font face="Calibri">L</font><font face="宋体">氯化钙溶液悬浮沉积物，以</font><font face="Calibri">2500rpm</font><font face="宋体">离心</font><font face="Calibri">15</font><font face="宋体">分钟弃上清，将残留液体用吸管吹打成悬液，滴一滴于洁净的载玻片上，涂片③，天然枯燥。</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;">6<font face="宋体">．将①、②、③涂片用</font><font face="Calibri">l</font><font face="宋体">％甲苯胺兰染色后盖片即可调查。</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;">（二）成果</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;">别离于高倍镜下调查三张涂片，描述镜下所见。</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="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">高速离心别离提取线粒体</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;">（一）操作过程</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;">1<font face="宋体">．将装有上清液的高速离心管，从装有冰块的烧杯中取出，配平后，以</font><font face="Calibri">17000rpm</font><font face="宋体">离心</font><font face="Calibri">20</font><font face="宋体">分钟，弃上清，留取沉积物。</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;">2<font face="宋体">．参加</font><font face="Calibri">0</font><font face="宋体">．</font><font face="Calibri">25mol</font><font face="宋体">／</font><font face="Calibri">L</font><font face="宋体">蔗糖一</font><font face="Calibri">0</font><font face="宋体">．</font><font face="Calibri">003mol</font><font face="宋体">／</font><font face="Calibri">L</font><font face="宋体">氯化钙液</font><font face="Calibri">lml</font><font face="宋体">，用吸管吹打成悬液，以</font><font face="Calibri">17000rpm</font><font face="宋体">离心</font><font face="Calibri">20</font><font face="宋体">分钟，将上清吸入另一试管中，留取沉积物，参加</font><font face="Calibri">0.1ml 0</font><font face="宋体">．</font><font face="Calibri">25mol</font><font face="宋体">／</font><font face="Calibri">L</font><font face="宋体">蔗糖一</font><font face="Calibri">0.003mol</font><font face="宋体">／</font><font face="Calibri">L</font><font face="宋体">氯化钙溶液混匀成悬液（可用牙签）。</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;">3<font face="宋体">．取上清液和沉积物悬液，别离滴一滴于洁净载玻片上（别离符号④、⑤涂片），各滴一滴</font><font face="Calibri">0.02</font><font face="宋体">％詹纳斯绿</font><font face="Calibri">B</font><font face="宋体">染液盖上盖片染</font><font face="Calibri">20</font><font face="宋体">分钟。</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;">（二）成果</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">油镜下调查，颗粒状的线粒体被詹纳斯绿</font>B<font face="宋体">染成蓝绿色。</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>