细胞因子的应用及培养、冻存和复苏

<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;">大多数细胞因子是免疫应答的产物，在动物和人体内可发挥免疫学效应，终会通过上调免疫细胞对抗原物质的免疫应答，介导炎症反应而清除抗原物质。</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;">抗原刺激和病原微生物感染均可诱导体内产生细胞因子，因此细胞因子与疾病的发生、发展有着密切的关系，它们可参与疾病的发生、发展。</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;">另一方面，细胞株的培养、冻存和复苏分为以下三点：</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;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="background-color: rgb(255, 255, 153);"><span style="color: rgb(255, 0, 0);"><span style="font-family: 宋体; font-size: 9pt;">细胞株的培养、冻存和复苏</span></span></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;">1<font face="宋体">）</font></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;"><font face="宋体">细胞株的培养和传代 培养： 用于检测细胞因子的细胞株通常培养于含</font><font face="Calibri">10</font><font face="宋体">％小牛血清和抗生素的培养液中，培养细胞因子依赖细胞株还有加入适量细胞因子。</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">在</font>37<font face="宋体">℃ </font><font face="Calibri">5</font><font face="宋体">％ </font><font face="Calibri">CO2</font><font face="宋体">的饱和水汽二氧化碳培养箱中培养，</font><font face="Calibri">3</font><font face="宋体">～</font><font face="Calibri">4</font><font face="宋体">天传代一次。 </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, 102, 0);"><span style="font-family: 宋体; font-size: 9pt;">★</span></span><span style="color: rgb(255, 255, 153);"><span style="font-family: 宋体; font-size: 9pt;"> </span></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;">悬浮生长细胞的传代： 直接直接吸去或离心后吸去培养上清液，用新鲜培养液悬浮细胞，<font face="Calibri">1</font><font face="宋体">：</font><font face="Calibri">3</font><font face="宋体">或</font><font face="Calibri">1</font><font face="宋体">：</font><font face="Calibri">5</font><font face="宋体">稀释细胞后，分瓶继续培养。 </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, 102, 0);"><span style="font-family: 宋体; font-size: 9pt;">★</span></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;"> 帖壁生长细胞的传代： 吸去培养液，用<font face="Calibri">PBS</font><font face="宋体">洗涤细胞一次，加入消化液，在</font><font face="Calibri">37</font><font face="宋体">℃中消化约</font><font face="Calibri">3</font><font face="宋体">～</font><font face="Calibri">10</font><font face="宋体">分钟，待细胞开始脱落时，倒去消化液，加入新鲜培养液，悬浮和吹打分散细胞。也可以待细胞完全消化脱落，</font><font face="Calibri">500</font><font face="宋体">&times;</font><font face="Calibri">g</font><font face="宋体">离心</font><font face="Calibri">5</font><font face="宋体">分钟，去除消化液，用新鲜培养液悬浮细胞。</font><font face="Calibri">1</font><font face="宋体">：</font><font face="Calibri">3</font><font face="宋体">或</font><font face="Calibri">1</font><font face="宋体">：</font><font face="Calibri">5</font><font face="宋体">稀释细胞后，分瓶继续培养。</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;">2<font face="宋体">）</font></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;"><font face="宋体">细胞株的冻存： </font><font face="Calibri">1</font><font face="宋体">． 取培养</font><font face="Calibri">2</font><font face="宋体">～</font><font face="Calibri">3</font><font face="宋体">天生长旺盛的细胞，用细胞培养液将细胞配成</font><font face="Calibri">2</font><font face="宋体">&times;</font><font face="Calibri">106</font><font face="宋体">～</font><font face="Calibri">2</font><font face="宋体">&times;</font><font face="Calibri">107/ml</font><font face="宋体">。 在</font><font face="Calibri">1ml</font><font face="宋体">细胞冻存管中加入</font><font face="Calibri">0.5ml</font><font face="宋体">细胞悬液，</font><font face="Calibri">0.4ml</font><font face="宋体">小牛血清和</font><font face="Calibri">0.1ml</font><font face="宋体">二甲基亚砜（或甘油），混匀后密封。置</font><font face="Calibri">4</font><font face="宋体">℃ </font><font face="Calibri">1</font><font face="宋体">小时，－</font><font face="Calibri">20</font><font face="宋体">℃ </font><font face="Calibri">2</font><font face="宋体">小时，然后直接放入液氮中或置液氮蒸汽上过夜后浸入液氮中。</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="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">3<font face="宋体">）</font></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;"><font face="宋体">细胞株的复苏： 复苏时，从液氮中取出冻存管，立即置</font><font face="Calibri">37</font><font face="宋体">℃水浴中融化细胞。将细胞直接加入</font><font face="Calibri">10ml</font><font face="宋体">含</font><font face="Calibri">15</font><font face="宋体">％小牛血清的</font><font face="Calibri">RPMI-1640</font><font face="宋体">培养液中，置</font><font face="Calibri">37</font><font face="宋体">℃ </font><font face="Calibri">5</font><font face="宋体">％</font><font face="Calibri">CO2</font><font face="宋体">饱和水汽二氧化碳培养箱中培养。悬浮细胞待细胞全部沉降后小心吸去上清液，更换新鲜的上述培养液，继续培养；帖壁细胞则培养</font><font face="Calibri">2</font><font face="宋体">～</font><font face="Calibri">3</font><font face="宋体">小时，待细胞帖壁后，倒去培养液，更换新鲜的上述培养液，继续培养。也可以在加入新鲜培养液以后，</font><font face="Calibri">500</font><font face="宋体">&times;</font><font face="Calibri">g</font><font face="宋体">离心</font><font face="Calibri">5</font><font face="宋体">分钟，去上清液，加入新鲜培养液，继续培养。&nbsp;</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>