大鼠枯否细胞的分离、鉴定以及LPS刺激诱导TNF-α的表达

Rat Kupffer cells:Isolation,identif ication and LPSstimulated TNF-α expression and secretion

目的:分离、培养和鉴定枯否细胞,并探讨LPS刺激对细胞肿瘤坏死因子(tumor necrosis factor-α,TNF-α)表达和分泌的影响.方法:采用在体原位灌注、密度梯度离心和早期细胞换液等方法分离纯化大鼠肝枯否细胞(kupffer cell,KC),并采用墨汁吞噬和ED2染色试验对分离培养的细胞进行鉴定.TNF-α表达和分泌采用RT-PCR和酶联免疫技术(enzyme-linked immunosorbent,ELISA)检测.结果:成功分离和纯化大鼠肝KC,并经墨汁吞噬和ED2染色试验鉴定证实;LPS刺激KC细胞内TNF-α mRNA的表达较非刺激细胞(PBS处理细胞)显著升高(1.10±0.02vs0.09±0.01,P<0.001).另外,LPS刺激较非刺激KC培养上清液TNF-α蛋白的水平也显著升高(487.10pg/mL±5.56pg/mLvs39.41pg/mL±15.30pg/mL,P<0.001).结论:原位灌注和密度梯度离心法能有效分离纯化大鼠KC,LPS刺激可诱导其表达和分泌大量TNF-α.

AIM：To isolate,culture and identify rat Kupffer cells,and to investigate the effect of LPS on TNF-α expression and secretion in rat Kupffer cells.METHODS：Rat liver Kupffer cells（KCs） were isolated and purified by means of in situ perfusion,density gradient centrifugation and early medium change.Isolated cells were identified by ink phagocytosis and ED2 staining test.The expression and secretion of TNF-α was detected by RT-PCR and enzyme-linked immunosorbent assay（ELISA）.RESULTS：Rat liver KCs were successfully isolated,purif ied,and conf irmed by ink phagocytosis and ED2 staining test.Compared with non-stimulated KCs,LPS-stimulated cells had a significantly higher level of TNF-α mRNA expression（1.10±0.02 vs 0.09±0.01,P0.001）.TNF-α protein levels in cell supernatants were also significantly increased in LPS-stimulated cells than in non-stimulated cells（487.10 pg/mL±5.56 pg/mL vs 39.41 pg/mL±15.30pg/mL,P0.001）.CONCLUSION：Rat KCs have been successfully isolated and purified by means of in situ perfusion and density gradient centrifugation,and LPS can stimulate the expression and secretion of TNF-α.