嗜酸乳杆菌S-层蛋白对肠道细胞黏附及巨噬细胞增殖的影响

Effects of Lactobacillus acidophilus S-layer Protein on Adhesion to Intestinal Cells and Macrophage Proliferation

摘要:

比较去除S-层蛋白前后嗜酸乳杆菌对肠道细胞黏附及菌体自我凝集率的变化，并探究S-层蛋白对巨噬细胞增殖及溶酶体分泌的影响。采用4M氯化锂提取嗜酸乳杆菌ATCC 4356的表层蛋白，经透析及微孔过滤等步骤得到S-层蛋白，SDS-PAGE确定其分子量。利用酶标仪测定嗜酸乳杆菌在37 ℃孵育过程中(0 h~5.5 h)自我凝集率的变化情况，光学显微镜观察去除S-层蛋白前后嗜酸乳杆菌对结肠癌细胞HT-29的黏附情况变化。将S-层蛋白与巨噬细胞RAW264.7孵育2 h后，MTS法测定细胞增殖情况，并采用溶酶体荧光探针研究溶酶体分泌量的变化。结果表明：所提蛋白为嗜酸乳杆菌S-层蛋白(分子量M≈46ku)。嗜酸乳杆菌自我凝集率随孵育时间的延长而呈现出上升趋势，去除S-层蛋白后，菌体自我凝集率降低，嗜酸乳杆菌对HT-29细胞的黏附量也明显减少，且S-层蛋白能促进巨噬细胞增殖及其溶酶体分泌。

Abstract:

To investigate the autoaggregation and adherence rate to HT-29 cells of Lactobacillus acidophilus ATCC4356 with or without S-layer protein as well as the influence of S-layer protein on the proliferation and lysosomal secretion of macrophages, S-layer protein of L. acidophilus ATCC 4356 was extracted by 4 M lithium chloride, followed by further purification using centrifugation, dialysis, and microfiltration. The molecular weight was then confirmed by SDS-PAGE. Autoaggregation rate of L. acidophilus at 37°C was measured by using a microplate reader during the incubation (0 to 5.5 h). Adhesion of L. acidophilus ATCC4356 to HT-29 cells was observed by light microscopy. After incubation of S-layer proteins with RAW 264.7 macrophages for 2 h, proliferation and changes in lysosomal secretion were evaluated by MTS and Lyso-Tracker Red, respectively. The results showed that the protein extracted was S-layer protein from L. acidophilus ATCC 4356 with a molecular weight of about 46 ku. L. acidophilus ATCC4356 autoaggregation rate and adhesion to HT-29 cells were reduced when the S-layer protein was removed. L. acidophilus ATCC 4356 autoaggregation rate increased as the incubation time was extended. S-layer protein could also promote macrophage proliferation and lysosomal secretion.