Sandberg J W, Lau C, Jacomino M, Finegold M, Henning S J
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
Hum Gene Ther. 1994 Mar;5(3):323-9. doi: 10.1089/hum.1994.5.3-323.
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 transfer via the 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 effect in vivo mucus 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) and N-acetyl-cysteine (NAC) versus a 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.
在先前探索肠道上皮作为体细胞基因治疗潜在位点的研究中，我们得出结论，肠道内的黏液对任何通过肠腔途径进行基因转移的尝试都构成了重大障碍。由于作为基因转移合理靶点的上皮干细胞位于肠道隐窝深处，黏液问题更加严重。本研究的目标是开发一些程序，以改善对肠道干细胞的可达性，并在不损伤下层上皮的情况下实现体内黏液清除。初步实验包括评估使用扩张来改善对肠道隐窝的可达性，以及使用黏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）对照溶液进行黏液清除。在麻醉大鼠的3厘米回肠末端节段的两端插入导管。将2毫升试剂注入夹紧的节段中2分钟，然后取出，重复操作。肠腔扩张导致绒毛缩短，绒毛间距变宽，从而改善了对隐窝的可达性。NAC和DTT冲洗都清除了绒毛之间的大量黏液，但未能到达隐窝腔。为了增强黏液从隐窝腔的释放，由于毛果芸香碱的胆碱能特性及其与隐窝杯状细胞上毒蕈碱受体的优先结合，选择了毛果芸香碱。在进行黏液溶解剂或PBS冲洗前30分钟腹腔注射毛果芸香碱，可显著清除隐窝腔内的黏液。如果肠道保持不受干扰，这种效果在3 - 4小时后仍然明显。
