細胞的型態鑒定與核型分析

<p class="MsoNormal"><strong><span style="background-color: rgb(255, 204, 0);"><span style="font-family: 宋體; font-size: 9pt;">(<font face="宋體">一</font><font face="Calibri">)</font><font face="宋體">形態學鑒定</font></span></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="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">光學顯微鏡檢查</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;">(1) </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">培養瓶</font><font face="Calibri">(</font><font face="宋體">皿</font><font face="Calibri">)</font><font face="宋體">或培養板直接置于倒置顯微鏡下觀察，上皮細胞呈多邊形，邊界清晰，大小規則，核呈圓形，核質比例小，細胞間排列整齊，為單層培養物，無重疊生長，是正常上皮組織來源；</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;">成纖維細胞呈長梭形，核小而圓，細胞排列規則、整齊，為單層培養物，無重疊生長，是正常間質組織來源。腫瘤組織來源的貼壁細胞，其單層培養物呈多層重疊生長。</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></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;">(2) </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">細胞染色檢查：將無菌小玻片</font><font face="Calibri">(O</font><font face="宋體">．</font><font face="Calibri">5cm</font><font face="宋體">&times;</font><font face="Calibri">2cm)</font><font face="宋體">于細胞傳代接種前放人培養瓶皿內，接種細胞懸液后培養，在玻片上制備培養的單層細胞，然后將載有細胞的玻片取出，用磷酸鹽緩沖液</font><font face="Calibri">(phosphate-buffeer saline</font><font face="宋體">，</font><font face="Calibri">PBS)</font><font face="宋體">輕輕漂洗后，將玻片放在普通玻片上，空氣中自然干燥。</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">經</font>PBS<font face="宋體">／甲醇</font><font face="Calibri">(1</font><font face="宋體">：</font><font face="Calibri">1)</font><font face="宋體">固定液同定</font><font face="Calibri">15min</font><font face="宋體">，棄固定液，加吉姆薩</font><font face="Calibri">(Giemsa)</font><font face="宋體">染色液染色</font><font face="Calibri">10</font><font face="宋體">～</font><font face="Calibri">15min</font><font face="宋體">，用清水漂洗干凈，置于空氣中干燥，用二甲苯透明，用光學樹脂膠片封片后觀察。上皮細胞和成纖維細胞的形態與直接鏡檢相同，細胞核染成紫紅或藍紫色，胞質染成粉紅色。</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></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">電子顯微鏡觀察通常用透射電鏡檢查細胞的超微結構。取對數生長期的細胞，用胰蛋白酶消化，加培養液懸浮消化的細胞，用</font><font face="Calibri">1000r</font><font face="宋體">／</font><font face="Calibri">min</font><font face="宋體">離心</font><font face="Calibri">5min</font><font face="宋體">。</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;">收集離心管底部細胞，經戊二醛固定，包埋，超薄切片；也可采用細胞原位包埋的方法，將細胞培養于電鏡用特殊膜上，進行固定，包埋，該法不破壞細胞問排列關系。在電鏡下可見到上皮細胞的張力原纖維和橋粒等，腺細胞可見胞質中的分泌顆粒，這對確定培養細胞系組織來源有重要意義。</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></p> <p class="MsoNormal"><span style="background-color: rgb(255, 204, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">(<font face="宋體">二</font><font face="Calibri">)</font><font face="宋體">細胞核型分析</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;"><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;">細胞染色體核型分析能簡易、有效地確定細胞種來源和鑒定正常或惡性的性質，以及檢查細胞有無交叉污染等。細胞染色體核型分析包括制備中期細胞分裂象樣本，將分散的單個染色體進行計數和排列分組分析。</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></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">制備細胞分裂樣本取對數生長期的細胞懸液，加入</font><font face="Calibri">1</font><font face="宋體">&times;</font><font face="Calibri">1 0-5 mol</font><font face="宋體">／</font><font face="Calibri">L</font><font face="宋體">秋水仙素</font><font face="Calibri">(</font><font face="宋體">終濃度為</font><font face="Calibri">1</font><font face="宋體">&times;</font><font face="Calibri">10-7mol</font><font face="宋體">／</font><font face="Calibri">L)</font><font face="宋體">到養液中，處理</font><font face="Calibri">6h</font><font face="宋體">后，輕輕吸出培養液，加</font><font face="Calibri">0</font><font face="宋體">．</font><font face="Calibri">25</font><font face="宋體">％胰蛋白酶消化細胞或手振搖培養瓶使細胞脫落，收集中期分裂象細胞，</font><font face="Calibri">37</font><font face="宋體">℃靜置</font><font face="Calibri">20min</font><font face="宋體">后加入冰醋酸一甲醇</font><font face="Calibri">(1</font><font face="宋體">：</font><font face="Calibri">1)1ml</font><font face="宋體">，固定細胞</font><font face="Calibri">1</font><font face="宋體">～</font><font face="Calibri">20min</font><font face="宋體">，再次以</font><font face="Calibri">1000r</font><font face="宋體">／</font><font face="Calibri">min</font><font face="宋體">離心：</font><font face="Calibri">10min</font><font face="宋體">，去上清液，加入新固定液，吹打混勻，第</font><font face="Calibri">3</font><font face="宋體">次經</font><font face="Calibri">1000r</font><font face="宋體">／</font><font face="Calibri">mln</font><font face="宋體">離心</font><font face="Calibri">10min</font><font face="宋體">，收集分裂象細胞。</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></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">制備染色體玻片將沉集的細胞加入</font><font face="Calibri">O</font><font face="宋體">．</font><font face="Calibri">2ml</font><font face="宋體">醋酸甲醇分散細胞，取一滴細胞懸液加到冰冷的玻片上，同時用口吹散細胞液滴，使細胞均勻分散于玻片上。同時可多制備幾張染色體玻片，待干燥后染色、鏡檢。</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></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">染色體計數及核型分析取染色體玻片經吉薩姆染色后鏡檢。在顯微鏡下可觀察到多個中期分裂象細胞染色體，計數</font><font face="Calibri">50</font><font face="宋體">～</font><font face="Calibri">100</font><font face="宋體">個細胞染色體數目，并計數出細胞染色體的分布，細胞群體中分布</font><font face="Calibri">zui</font><font face="宋體">多的染色體數目是染色體數。人正常細胞有</font><font face="Calibri">46</font><font face="宋體">條染色體，為</font><font face="Calibri">23</font><font face="宋體">對二倍體。</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">染色體分組排列，每組染色體無增加或減少，無異常染色體。小鼠染色體為</font>40<font face="宋體">條，不僅數目與人染色體不同，而且以頂端著絲點為主。人的染色體則為中央著絲點，數目與著絲點位置可作為細胞系是人或鼠來源的依據。</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>