細胞因子的應(yīng)用及培養(yǎng)、凍存和復(fù)蘇

<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;">大多數(shù)細胞因子是免疫應(yīng)答的產(chǎn)物，在動物和人體內(nèi)可發(fā)揮免疫學(xué)效應(yīng)，終會通過上調(diào)免疫細胞對抗原物質(zhì)的免疫應(yīng)答，介導(dǎo)炎癥反應(yīng)而清除抗原物質(zhì)。</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;">抗原刺激和病原微生物感染均可誘導(dǎo)體內(nèi)產(chǎn)生細胞因子，因此細胞因子與疾病的發(fā)生、發(fā)展有著密切的關(guān)系，它們可參與疾病的發(fā)生、發(fā)展。</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;">另一方面，細胞株的培養(yǎng)、凍存和復(fù)蘇分為以下三點：</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;"><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: 宋體; font-size: 9pt;">細胞株的培養(yǎng)、凍存和復(fù)蘇</span></span></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="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">1<font face="宋體">）</font></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;"><font face="宋體">細胞株的培養(yǎng)和傳代 培養(yǎng)： 用于檢測細胞因子的細胞株通常培養(yǎng)于含</font><font face="Calibri">10</font><font face="宋體">％小牛血清和抗生素的培養(yǎng)液中，培養(yǎng)細胞因子依賴細胞株還有加入適量細胞因子。</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">在</font>37<font face="宋體">℃ </font><font face="Calibri">5</font><font face="宋體">％ </font><font face="Calibri">CO2</font><font face="宋體">的飽和水汽二氧化碳培養(yǎng)箱中培養(yǎng)，</font><font face="Calibri">3</font><font face="宋體">～</font><font face="Calibri">4</font><font face="宋體">天傳代一次。 </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="color: rgb(255, 102, 0);"><span style="font-family: 宋體; font-size: 9pt;">★</span></span><span style="color: rgb(255, 255, 153);"><span style="font-family: 宋體; font-size: 9pt;"> </span></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;">懸浮生長細胞的傳代： 直接直接吸去或離心后吸去培養(yǎng)上清液，用新鮮培養(yǎng)液懸浮細胞，<font face="Calibri">1</font><font face="宋體">：</font><font face="Calibri">3</font><font face="宋體">或</font><font face="Calibri">1</font><font face="宋體">：</font><font face="Calibri">5</font><font face="宋體">稀釋細胞后，分瓶繼續(xù)培養(yǎng)。 </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="color: rgb(255, 102, 0);"><span style="font-family: 宋體; font-size: 9pt;">★</span></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;"> 帖壁生長細胞的傳代： 吸去培養(yǎng)液，用<font face="Calibri">PBS</font><font face="宋體">洗滌細胞一次，加入消化液，在</font><font face="Calibri">37</font><font face="宋體">℃中消化約</font><font face="Calibri">3</font><font face="宋體">～</font><font face="Calibri">10</font><font face="宋體">分鐘，待細胞開始脫落時，倒去消化液，加入新鮮培養(yǎng)液，懸浮和吹打分散細胞。也可以待細胞完全消化脫落，</font><font face="Calibri">500</font><font face="宋體">&times;</font><font face="Calibri">g</font><font face="宋體">離心</font><font face="Calibri">5</font><font face="宋體">分鐘，去除消化液，用新鮮培養(yǎng)液懸浮細胞。</font><font face="Calibri">1</font><font face="宋體">：</font><font face="Calibri">3</font><font face="宋體">或</font><font face="Calibri">1</font><font face="宋體">：</font><font face="Calibri">5</font><font face="宋體">稀釋細胞后，分瓶繼續(xù)培養(yǎng)。</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="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">2<font face="宋體">）</font></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;"><font face="宋體">細胞株的凍存： </font><font face="Calibri">1</font><font face="宋體">． 取培養(yǎng)</font><font face="Calibri">2</font><font face="宋體">～</font><font face="Calibri">3</font><font face="宋體">天生長旺盛的細胞，用細胞培養(yǎng)液將細胞配成</font><font face="Calibri">2</font><font face="宋體">&times;</font><font face="Calibri">106</font><font face="宋體">～</font><font face="Calibri">2</font><font face="宋體">&times;</font><font face="Calibri">107/ml</font><font face="宋體">。 在</font><font face="Calibri">1ml</font><font face="宋體">細胞凍存管中加入</font><font face="Calibri">0.5ml</font><font face="宋體">細胞懸液，</font><font face="Calibri">0.4ml</font><font face="宋體">小牛血清和</font><font face="Calibri">0.1ml</font><font face="宋體">二甲基亞砜（或甘油），混勻后密封。置</font><font face="Calibri">4</font><font face="宋體">℃ </font><font face="Calibri">1</font><font face="宋體">小時，－</font><font face="Calibri">20</font><font face="宋體">℃ </font><font face="Calibri">2</font><font face="宋體">小時，然后直接放入液氮中或置液氮蒸汽上過夜后浸入液氮中。</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="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">3<font face="宋體">）</font></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;"><font face="宋體">細胞株的復(fù)蘇： 復(fù)蘇時，從液氮中取出凍存管，立即置</font><font face="Calibri">37</font><font face="宋體">℃水浴中融化細胞。將細胞直接加入</font><font face="Calibri">10ml</font><font face="宋體">含</font><font face="Calibri">15</font><font face="宋體">％小牛血清的</font><font face="Calibri">RPMI-1640</font><font face="宋體">培養(yǎng)液中，置</font><font face="Calibri">37</font><font face="宋體">℃ </font><font face="Calibri">5</font><font face="宋體">％</font><font face="Calibri">CO2</font><font face="宋體">飽和水汽二氧化碳培養(yǎng)箱中培養(yǎng)。懸浮細胞待細胞全部沉降后小心吸去上清液，更換新鮮的上述培養(yǎng)液，繼續(xù)培養(yǎng)；帖壁細胞則培養(yǎng)</font><font face="Calibri">2</font><font face="宋體">～</font><font face="Calibri">3</font><font face="宋體">小時，待細胞帖壁后，倒去培養(yǎng)液，更換新鮮的上述培養(yǎng)液，繼續(xù)培養(yǎng)。也可以在加入新鮮培養(yǎng)液以后，</font><font face="Calibri">500</font><font face="宋體">&times;</font><font face="Calibri">g</font><font face="宋體">離心</font><font face="Calibri">5</font><font face="宋體">分鐘，去上清液，加入新鮮培養(yǎng)液，繼續(xù)培養(yǎng)。&nbsp;</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>