目的 探讨不同浓度脂多糖(LPS)对大鼠肠上皮细胞(IEC-6)增殖和白细胞介素(IL)-6、IL-1β及肿瘤坏死因子-α(TNF-α)分泌的影响.方法 体外培养的IEC-6分为正常对照组(0 mg/L,A组)、0.1 mg/L组(B组)、0.5 mg/L组(C组)、1.0 mg/L组(D组)、5.0 mg/L组(E组)和10.0 mg/L组(F组),分别用对应浓度的LPS与细胞共培养3h、5h、7h后,噻唑蓝(MTT)比色法检测细胞增殖率,酶联免疫吸附试验(ELISA)检测细胞培养上清中IL-6、IL-1β及TNF-α的质量浓度.结果 LPS与IEC-6共培养3h、5h、7h时,随着LPS浓度的增加,IEC-6的增殖率逐渐降低.共培养3h和5h时,B组、C组、D组、E组和F组增殖率分别与A组比较,差异均无统计学意义(P均＞0.05);共培养7h时,B组、C组、D组增殖率与A组比较,差异均无统计学意义(P均＞0.05),E组、F组增殖率与A组相比,差异有统计学意义(t=4.216,P=0.014;t=14.991,P=0.000).E组和F组共培养7h时的细胞增殖率较共培养5h时降低,差异均有统计学意义(t=2.576,P=0.033;t2.975,P=0.018);而共培养5h时E组与A组比较,差异无统计学意义(P＞0.05).LPS与IEC-6共培养3h、5h和7h时,B组、C组、D组、E组和F组共培养上清中IL-6水平与A组比较均有升高,差异均有统计学意义(P均＜0.01),其中各时间点E组IL-6水平均最高.E组LPS与IEC-6共培养5h时IL-6水平出现峰值.LPS与IEC-6共培养3h、5h和7h时,B组、C组、D组、E组、F组共培养上清中IL-1β、TNF-α水平与A组比较,差异均无统计学意义(P均＞0.05).结论 在一定浓度、一定培养时间范围内,随LPS浓度的增加,IEC-6增殖率呈下降趋势.0～10.0 mg/L的LPS与IEC-6共培养,可刺激IEC-6分泌IL-6.5.0 mg/L的LPS与IEC-6共培养5h,其培养上清中IL-6水平最高.LPS对IEC-6培养上清中TNF-α和IL-1β水平无影响.5.0 mg/L的LPS与IEC-6共培养5h可制作较好的IEC-6炎性模型.%Objective To investigate the effects of lipopolysaccharides (LPS) in different concentrations on the proliferation and interleukin(IL)-6,IL-1 β and tumor necrosis factor-α(TNF-o) secretion of intestinal epithelial cells (IEC-6) of rats in vitro.Methods IEC-6 of rats were divided into normal group (0 mg/L,group A),0.1 mg/L group (group B),0.5 mg/L group (group C),1.0 mg/L group (group D),5.0 mg/L group (group E) and 10.0 mg/L group(group F).Different groups cells were exposed to LPS with different concentrations for 3 h,5 h and 7 h.Thiazolyl blue(MTT) was performed to investigate the proliferation of IEC-6.The concentrations of IL-6,IL-1 β and TNF-α in culture supernatant were detected by enzyme linked immunosorbent assay(ELISA).Results The proliferation rate of IEC-6 was gradually lower while the concentration of LPS increased.After co-culture with LPS 3h and 5 h,the proliferation rates of group B,group C,group D,group E and group F had no significant difference with those of group A (all P ＞ 0.05);after co-culture with LPS 7 h,the proliferation rates of group B,group C,and group D had no significant difference with those of group A (all P ＞ 0.05);the proliferation rates of group E and group F had significant difference with those of group A(t =4.216,P =0.014;t =14.991,P =0.000).The proliferation rates of group E and group F were lower after co-culture with LPS 5 h than 7 h,and there were significant differences (t =2.576,P =0.033;t =2.975,P =0.018);but there was no significant differences between group E and group A after co-culture with LPS 5 h (P ＞ 0.05).Group B,group C,group D,group E and group F all had a significant higher level of IL-6 than group A after co-culture with LPS 3 h,5 h and 7 h(all P ＜0.01).In addition,group E had the highest level of IL-6 at all time points.And the peak level of IL-6 rose after co-culture with LPS 5 h.The TNF-α level and IL-1 β level of group B,group C,group D,group E and group F all had no significant differences than that of group A after co-culture with LPS 3 h,5 h and 7 h (all P ＞ 0.05).Conclusions In a certain concentration,incubation time range,the proliferation rates of IEC-6 cells were gradually lower while the concentration of LPS increased.Co-cultured IEC-6 cells with LPS(0-10.0 mg/L) can stimulate them secrete to IL-6.The highest level of IL-6 was of group E after 5 h co-culture.LPS had no effects on TNF-α and IL-1 β level of IEC-6 cells cultural supernatant.So 5.0 mg/L concentration of LPS stimulating IEC-6 cells for 5 h can be chosen to build the IEC-6 inflammatory models.
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