小中大
谢谢两位的关注.
dongwp:你好!我看了许多有关你发的胰岛分离的帖子,收获很多.我的这个培养胰腺导管细胞的方案前面取材分离和胰岛分离几乎一样,只是后面培养环节不同.我简单说一下主要步骤,如下:
1. 取材 SD大鼠(8-10wk),称重,麻醉,备皮,无菌条件下剖腹,胆总管逆向插管,灌注V型胶原酶(Sigma)10-12ml.
2. 消化 37℃ 水浴静止消化10分钟,漩涡振荡器振荡2-3次,每次3-5秒.
3. 终止消化, 过40目滤网,细胞悬液离心(1000rpm/min,2min)
4. Ficoll密度梯度离心(2000rpm/min,20min)
5.取下底层吹打,加RPMI1640(10%FBS+1%双抗)接种至25cm2 Costar 培养瓶.37℃,5%co2培养.
主要问题:
1. 由于我们实验室不允许杀动物,怕污染,只好在动物中心买了大鼠直接在那边无菌操作室杀,取了胰腺放含有Hank`s 的50ml离心管,4℃拿回实验室,中间耗时在20-30分钟左右. 然后再水浴消化,是不是这样消化时间过长?
2. Ficoll密度梯度离心后,我原来看到一些资料说胰岛主要集中在上中层,而我要的导管细胞主要在下底层.所以取了下面的和沉淀部分.但参考 Bonner-Weir et al(2000).的一篇文献,意思应该是导管细胞也主要集中于上中层.
After purification on a Ficoll gradient, the top interface (1.062/1.096 densities) was 50–95% islet with varying amounts of duct and degranulated acinar tissue, the middle interface (1.096/1.11 densities) contained 1–15% islets, duct, and degranulated acini, and the pellet was mostly well granulated acinar tissue with less than 1% islets. In the top and middle layers there were sheets of ductal epithelium from larger ducts whereas the clumps of exocrine cells found in all layers consisted of small intercalated ducts continuing into the acini. Tissue from these layers was cultured in 50 ml of CMRL 1066 (5.6 mM glucose) media plus 10% FBS in Falcon nontreated T-75 flasks (#3012 Becton Dickinson) at 37°C, 5% CO2.
这也是我有疑问的一个地方.
望给予指导,谢谢!
