Isolation of intestinal epithelial cells for the study of differential gene expression along the crypt-villus axis.
Isolation of intestinal epithelial cells for the study of differential gene expression along the crypt-villus axis.
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DOI：
10.1152/ajpgi.1991.260.6.g895
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发表时间：
1991-06
期刊：
影响因子：
--
通讯作者：
P. Traber;D. Gumucio;Wei Wang
中科院分区：
文献类型：
--
作者：
P. Traber;D. Gumucio;Wei Wang
从小肠隐窝和绒毛区室中分离肠上皮细胞的方法已被用于肠道功能的研究。然而，使用不同的方法导致了不同的结论，表达基因的定位。因此，我们对膨胀肠囊法和外翻肠囊法两种肠上皮细胞分离方法进行了仔细的比较。分离的细胞部分（膨化囊部分1-10，膨化囊部分1-5）被评估表达两个mrna，它们沿着隐窝-绒毛轴的定位已经被原位杂交阐明：细胞色素P-450IIB1（在绒毛细胞中表达）和隐窝蛋白（在隐窝细胞中表达）。每个细胞群的总RNA或多聚(A)+ RNA的Northern印迹显示，两种方法的第一部分都含有P-450IIB1 mRNA，这表明两种方法都可以分离出源自绒毛的细胞。膨胀囊法仅在细胞组分5 ~ 10中检测到Cryptdin mRNA，膨出囊法未在细胞组分中检测到Cryptdin mRNA。[3H]胸腺嘧啶结合表明，扩张囊法成功地除去了分裂（隐窝）细胞，但仍保留着膨出囊的肠残体。最后，对分离的细胞和肠残体进行光镜和电镜检查，证实在膨大的囊细胞9和10中存在未分化的隐窝细胞，但在外翻的囊残体中仍存在未分化的隐窝细胞。因此，膨胀囊方案是有用的分离细胞从尖端和隐窝室。（摘要删节250字）
Methods for the differential isolation of intestinal epithelial cells from crypt and villus compartments of small intestine have been used in the study of intestinal functions. However, the use of different methods has resulted in discrepant conclusions as to the localization of expressed genes. Therefore, we undertook a careful comparison of two methods of intestinal epithelial cell isolation, the distended intestinal sac method and the everted intestinal sac method. The isolated cell fractions (distended sac fractions 1-10, everted sac fractions 1-5) were evaluated for the expression of two mRNAs whose localization along the crypt-villus axis had been previously elucidated by in situ hybridization: cytochrome P-450IIB1 (expressed in villus cells) and cryptdin (expressed in crypt cells). Northern blots of total or poly(A)+ RNA from each cell population showed that the first fractions from both methods contained P-450IIB1 mRNA, suggesting that both methods allowed the isolation of cells originating from the villus. Cryptdin mRNA was detected only in cell fractions 5-10 using the distended sac method and was not detected in any fractions from the everted sac method. [3H]thymidine incorporation demonstrated that dividing (crypt) cells were successfully removed by the distended sac method, but remained with the everted sac intestinal remnant. Finally, light and electron microscopy of the isolated cells as well as the intestinal remnants confirmed that while undifferentiated crypt cells were present in distended sac cell fractions 9 and 10, they remained with the everted sac remnant. Thus the distended sac protocol was useful for the isolation of cells from tip and crypt compartments.(ABSTRACT TRUNCATED AT 250 WORDS)