Improving access to intestinal stem cells as a step toward intestinal gene transfer.
Improving access to intestinal stem cells as a step toward intestinal gene transfer.
复制标题
改善肠道干细胞的获取，作为肠道基因转移的一步。
DOI：
10.1089/hum.1994.5.3-323
复制
发表时间：
1994
影响因子：
4.2
通讯作者：
Henning,SJ
中科院分区：
文献类型：
--
作者：
Sandberg,JW;Lau,C;Jacomino,M;Finegold,M;Henning,SJ
在以前的研究中，探索肠上皮作为体细胞基因治疗的潜在位点，我们得出的结论是，粘膜内衬肠构成了一个显着的障碍，任何尝试在基因transferviolumenal途径。上皮干细胞是基因转移的合理目标，位于肠隐窝深处，这一事实加剧了粘液问题。本研究的目的是开发一种方法，可以改善肠道干细胞的可及性，并且可以在不损伤下层上皮的情况下去除活粘液。最初的实验包括评估使用扩张来改善肠隐窝的可及性，以及使用粘液溶解剂二硫苏糖醇（DTT）和N-乙酰半胱氨酸（NAC）与磷酸盐缓冲盐水（PBS）对照溶液去除粘液。将导管插入麻醉大鼠回肠末端3 cm段的两端。将2毫升试剂滴入夹持的节段中2分钟，取出并重复。管腔扩张导致绒毛缩短，绒毛间距变宽，从而改善隐窝进入。NAC和DTT洗涤均去除了绒毛之间的大量粘液，但未能到达隐窝腔。为了增强粘液从隐窝腔的释放，选择毛果芸香碱，这是由于其胆碱能特性和优先结合隐窝杯状细胞上的毒蕈碱受体。在粘液溶解或PBS洗涤之前30分钟腹膜内给予毛果芸香碱导致粘液显著根除进入隐窝腔。如果肠道保持不受干扰，3-4小时后这种效果仍然明显。
In previous studies exploring the intestinal epithelium as a potential site for somatic gene therapy, we concluded that the mucus lining the intestine constitutes a significant barrier to any attempts at gene transferviathe lumenal route. The mucus problem is aggravated by the fact that the epithelial stem cells, which are the logical target for gene transfer, are located deep in the intestinal crypts. The goals of the current study were to develop procedures that would improve accessibility to the intestinal stem cells and which would effectin vivomucus removal without damaging the underlying epithelium. Initial experiments involved evaluation of the use of distension to improve accessibility to the intestinal crypts and the use of the mucolytic agents dithiothreitol (DTT) andN-acetyl-cysteine (NAC)versusa control solution of phosphate-buffered saline (PBS) for mucus removal. Catheters were inserted in each end of 3-cm terminal ileal segments in anesthetized rats. Two milliliters of agent was instilled into the clamped segment for 2 min, removed, and repeated. Lumenal distension resulted in shortened villi with wider intervillus spacing, thereby improving crypt access. Both NAC and DTT washes removed significant mucus between the villi but failed to reach the crypt lumen. To enhance mucus release from the crypt lumen, pilocarpine was selected due to its cholinergic properties and preferential binding to muscarinic receptors on crypt goblet cells. Pilocarpine given intraperitoneally 30 min prior to the mucolytic or PBS wash resulted in significant eradication of mucus down into the crypt lumen. This effect was still evident 3–4 hr later provided the intestine remained undisturbed.