牙周膜细胞和牙髓细胞的培养方法

<font>牙髓细胞的培养</font>

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<font><span><span>1、<span> </span> </span> </span> <span>培养用液：</span> </font>

<font><span>PBSA</span> <span>；</span> </font>

<font><span>胶原酶：</span> <span>I</span> <span>型，用</span> <span>D</span> <span>－</span> <span>Hank’s</span> <span>液配制，</span> <span>625U/ml</span> <span>；</span> </font>

<font><span>培养液：</span> <span>DMEM</span> <span>＋</span> <span>10</span> <span>％</span> <span>FBS</span> <span>；</span> </font>

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<font><span><span>2、<span> </span> </span> </span> <span>Protocol</span> <span>：</span> </font>

<font><span><span>u<span> </span> </span> </span> <span>取拔除的正畸牙或阻生牙（年龄小者较佳），尽量完整的取出牙髓，置于培养皿中，用</span> <span>PBS</span> <span>冲洗；</span> </font>

<font><span><span>u<span> </span> </span> </span> <span>将牙髓放入离心管，加入胶原酶</span> <span>2</span> <span>－</span> <span>3ml/</span> <span>牙，</span> <chmetcnv><span>37</span> <span>℃</span> </chmetcnv> <span>，</span> <span>2</span> <span>－</span> <span>3</span> <span>小时（<b><i>原则是将牙髓消化彻底</i> </b> ）；</span> </font>

<font><span><span>u<span> </span> </span> </span> <span>加入等量培养液，吹打至单细胞，离心，重悬，接种，牙</span> <span>/6well</span> <span>；</span> </font>

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<font> </font>

<font><span>牙周细胞的培养</span> </font>

<font> </font>

<font><span><span>1、<span> </span> </span> </span> <span>培养用液：</span> </font>

<font><span>PBSA</span> <span>；</span> </font>

<font><span>胶原酶：</span> <span>I</span> <span>型，用</span> <span>D</span> <span>－</span> <span>Hank’s</span> <span>液配制，</span> <span>625U/ml</span> <span>；</span> </font>

<font><span>培养液：</span> <span>DMEM</span> <span>＋</span> <span>10</span> <span>％</span> <span>FBS</span> <span>；</span> </font>

<font> </font>

<font><span><span>2、<span> </span> </span> </span> <span>Protocol</span> <span>：</span> </font>

<font><span><span>u<span> </span> </span> </span> <span>取拔除的正畸牙或阻生牙（年龄小者较佳），置于培养皿中，用手术刀仔细刮除附着在牙根上的牙龈组织，然后用</span> <span>PBS</span> <span>反复冲洗，以去除残余组织以及牙根表面的血细胞；</span> </font>

<font><span><span>u<span> </span> </span> </span> <span>将牙放入离心管，加入胶原酶</span> <span>2</span> <span>－</span> <span>3ml/</span> <span>牙，</span> <chmetcnv><span>37</span> <span>℃</span> </chmetcnv> <span>，</span> <span>1</span> <span>小时；</span> </font>

<font><span><span>u<span> </span> </span> </span> <span>加入等量培养液，仔细吹打牙根表面，收集细胞，离心，重悬，接种，牙</span> <span>/6well</span> <span>；</span> </font>

<font><b><span>~undefined</span> </b> <b><span>培养获得的细胞是牙周膜细胞和成牙骨质细胞的混合体</span> <span>!</span></b></font>

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<font><b><span>PS</span> </b> <b><span>：</span> </b></font>

<font><span><span>u<span> </span> </span> </span> <span>年龄对细胞产量以及活力影响较大，最好选用年龄在</span> <span>10</span> <span>－</span> <span>14</span> <span>岁之间的正畸牙或阻生牙；</span> </font>

<font><span><span>u<span> </span> </span> </span> <span>如果选用大鼠牙齿作为培养目标，培养液应稍作调整，基本原则是抑制细胞的衰老，具体做法是：适当降低血清浓度（<i>选用进口血清最佳</i> ），如果降低血清浓度后，细胞增殖减慢，可加入促进细胞增殖的因子（例如</span> <i><span>BPE 25ug/ml</span> </i> <i><span>等</span> </i> <span>）。</span> </font>