细胞系（株）的鉴定方法

<p class="MsoNormal"><strong><span style="background-color: rgb(255, 204, 0);"><span style="font-family: 宋体; font-size: 9pt;">(<font face="宋体">一</font><font face="Calibri">)</font><font face="宋体">形态学鉴定</font></span></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="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">光学显微镜检查</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"><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;">(1)</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">培养瓶</font><font face="Calibri">(</font><font face="宋体">皿</font><font face="Calibri">)</font><font face="宋体">或培养板直接置于倒置显微镜下观察，上皮细胞呈多边形，边界清晰，大小规则，核呈圆形，核质比例小，细胞间排列整齐，为单层培养物，无重叠生长，是正常上皮组织来源；</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;">成纤维细胞呈长梭形，核小而圆，细胞排列规则、整齐，为单层培养物，无重叠生长，是正常间质组织来源。肿瘤组织来源的贴壁细胞，其单层培养物呈多层重叠生长。</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></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;">(2)</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">细胞染色检查：将无菌小玻片</font><font face="Calibri">(O</font><font face="宋体">．</font><font face="Calibri">5cm</font><font face="宋体">&times;</font><font face="Calibri">2cm)</font><font face="宋体">于细胞传代接种前放人培养瓶皿内，接种细胞悬液后培养，在玻片上制备培养的单层细胞，然后将载有细胞的玻片取出，用磷酸盐缓冲液</font><font face="Calibri">(phosphate-buffeer saline</font><font face="宋体">，</font><font face="Calibri">PBS)</font><font face="宋体">轻轻漂洗后，将玻片放在普通玻片上，空气中自然干燥。</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;"><font face="宋体">经</font>PBS<font face="宋体">／甲醇</font><font face="Calibri">(1</font><font face="宋体">：</font><font face="Calibri">1)</font><font face="宋体">固定液同定</font><font face="Calibri">15min</font><font face="宋体">，弃固定液，加吉姆萨</font><font face="Calibri">(Giemsa)</font><font face="宋体">染色液染色</font><font face="Calibri">10</font><font face="宋体">～</font><font face="Calibri">15min</font><font face="宋体">，用清水漂洗干净，置于空气中干燥，用二甲苯透明，用光学树脂胶片封片后观察。上皮细胞和成纤维细胞的形态与直接镜检相同，细胞核染成紫红或蓝紫色，胞质染成粉红色。</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> <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></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">电子显微镜观察通常用透射电镜检查细胞的超微结构。取对数生长期的细胞，用胰蛋白酶消化，加培养液悬浮消化的细胞，用</font><font face="Calibri">1000r</font><font face="宋体">／</font><font face="Calibri">min</font><font face="宋体">离心</font><font face="Calibri">5min</font><font face="宋体">。</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;">收集离心管底部细胞，经戊二醛固定，包埋，超薄切片；也可采用细胞原位包埋的方法，将细胞培养于电镜用特殊膜上，进行固定，包埋，该法不破坏细胞问排列关系。在电镜下可见到上皮细胞的张力原纤维和桥粒等，腺细胞可见胞质中的分泌颗粒，这对确定培养细胞系组织来源有重要意义。</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></p> <p class="MsoNormal"><span style="background-color: rgb(255, 204, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">(<font face="宋体">二</font><font face="Calibri">)</font><font face="宋体">细胞核型分析</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;"><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;">细胞染色体核型分析能简易、有效地确定细胞种来源和鉴定正常或恶性的性质，以及检查细胞有无交叉污染等。细胞染色体核型分析包括制备中期细胞分裂象样本，将分散的单个染色体进行计数和排列分组分析。</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></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">制备细胞分裂样本取对数生长期的细胞悬液，加入</font><font face="Calibri">1</font><font face="宋体">&times;</font><font face="Calibri">1 0-5 mol</font><font face="宋体">／</font><font face="Calibri">L</font><font face="宋体">秋水仙素</font><font face="Calibri">(</font><font face="宋体">终浓度为</font><font face="Calibri">1</font><font face="宋体">&times;</font><font face="Calibri">10-7mol</font><font face="宋体">／</font><font face="Calibri">L)</font><font face="宋体">到养液中，处理</font><font face="Calibri">6h</font><font face="宋体">后，轻轻吸出培养液，加</font><font face="Calibri">0</font><font face="宋体">．</font><font face="Calibri">25</font><font face="宋体">％胰蛋白酶消化细胞或手振摇培养瓶使细胞脱落，收集中期分裂象细胞，</font><font face="Calibri">37</font><font face="宋体">℃静置</font><font face="Calibri">20min</font><font face="宋体">后加入冰醋酸一甲醇</font><font face="Calibri">(1</font><font face="宋体">：</font><font face="Calibri">1)1ml</font><font face="宋体">，固定细胞</font><font face="Calibri">1</font><font face="宋体">～</font><font face="Calibri">20min</font><font face="宋体">，再次以</font><font face="Calibri">1000r</font><font face="宋体">／</font><font face="Calibri">min</font><font face="宋体">离心：</font><font face="Calibri">10min</font><font face="宋体">，去上清液，加入新固定液，吹打混匀，第</font><font face="Calibri">3</font><font face="宋体">次经</font><font face="Calibri">1000r</font><font face="宋体">／</font><font face="Calibri">mln</font><font face="宋体">离心</font><font face="Calibri">10min</font><font face="宋体">，收集分裂象细胞。</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></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">制备染色体玻片将沉集的细胞加入</font><font face="Calibri">O</font><font face="宋体">．</font><font face="Calibri">2ml</font><font face="宋体">醋酸甲醇分散细胞，取一滴细胞悬液加到冰冷的玻片上，同时用口吹散细胞液滴，使细胞均匀分散于玻片上。同时可多制备几张染色体玻片，待干燥后染色、镜检。</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></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">染色体计数及核型分析取染色体玻片经吉萨姆染色后镜检。在显微镜下可观察到多个中期分裂象细胞染色体，计数</font><font face="Calibri">50</font><font face="宋体">～</font><font face="Calibri">100</font><font face="宋体">个细胞染色体数目，并计数出细胞染色体的分布，细胞群体中分布</font><font face="Calibri">zui</font><font face="宋体">多的染色体数目是染色体数。人正常细胞有</font><font face="Calibri">46</font><font face="宋体">条染色体，为</font><font face="Calibri">23</font><font face="宋体">对二倍体。</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;"><font face="宋体">染色体分组排列，每组染色体无增加或减少，无异常染色体。小鼠染色体为</font>40<font face="宋体">条，不仅数目与人染色体不同，而且以顶端着丝点为主。人的染色体则为中央着丝点，数目与着丝点位置可作为细胞系是人或鼠来源的依据。</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>