通过亲和色谱法分离人肺肥大细胞。

The isolation of human lung mast cells by affinity chromatography.

作者信息

van Overveld F J, Terpstra G K, Bruijnzeel P L, Raaijmakers J A, Kreukniet J

机构信息

Department of Pulmonary Disease, State University Hospital, Utrecht, The Netherlands.

出版信息

Scand J Immunol. 1988 Jan;27(1):1-6. doi: 10.1111/j.1365-3083.1988.tb02316.x.

DOI:10.1111/j.1365-3083.1988.tb02316.x

PMID:3340820

Abstract

A method of isolation has been developed to purify mast cells from human lung tissue. The purification steps are: (1) dispersion of human lung tissue in single-cell suspensions by enzymatic digestion, (2) partial purification by counterflow centrifugal elutriation, (3) Percoll gradient centrifugation, and (4) enrichment of the mast cells by affinity chromatography using anti-human IgE-Sepharose. Enzymatic dispersion yielded 0.6 +/- 0.2 x 10(6) mast cells per gram wet tissue with purities of 3.3 +/- 1.0% (mean +/- SEM n = 3). Elutriation and gradient centrifugation yielded 0.36 +/- 0.05 x 10(6) mast cells per gram lung tissue in fractions with purities of 30.8 +/- 10.7%. Enriched mast cell fractions were combined, and disposed of contaminating cells by affinity chromatography, thereby yielding 0.25 +/- 0.03 x 10(6) mast cells per gram lung tissue, and improving the purity to 75.3 +/- 8.3%. The purified mast cells were intact and vitality exceeded 95%. In this way from 1 g wet lung tissue 0.25 +/- 0.03 x 10(6) mast cells may be isolated with a mean recovery of 41.7 +/- 2.4% and a mean purity of 75.3 +/- 8.3%.

摘要

已开发出一种从人肺组织中纯化肥大细胞的分离方法。纯化步骤如下：(1) 通过酶消化将人肺组织分散成单细胞悬液；(2) 通过逆流离心淘析进行部分纯化；(3) 进行Percoll梯度离心；(4) 使用抗人IgE-琼脂糖通过亲和色谱法富集肥大细胞。酶分散法每克湿组织可产生0.6±0.2×10⁶个肥大细胞，纯度为3.3±1.0%（平均值±标准误，n = 3）。淘析和梯度离心法在纯度为30.8±10.7%的组分中每克肺组织可产生0.36±0.05×10⁶个肥大细胞。将富集的肥大细胞组分合并，通过亲和色谱法去除污染细胞，从而每克肺组织产生0.25±0.03×10⁶个肥大细胞，并将纯度提高到75.3±8.3%。纯化的肥大细胞完整无损，活力超过95%。通过这种方法，从1克湿肺组织中可分离出0.25±0.03×10⁶个肥大细胞，平均回收率为41.7±2.4%，平均纯度为75.3±8.3%。