脂多糖对人腹膜间皮细胞葡萄糖转运蛋白-1表达的影响

Effects of Lipopolysaccharide on Glucose Transporter-1 Expression in Cultured Human Peritoneal Mesothelial Cell

目的:研究脂多糖(lipopolysaccharide,LPS)对人腹膜间皮细胞(HPMC)葡萄糖转运蛋白1(GLUT-1)表达的影响。方法:胰蛋白酶消化法进行HPMC的原代培养及传代;免疫组化法(ABC)用于GLUT-1的鉴定和半定量分析;采用逆转录多聚酶链反应(RT-PCR)检测GLUT-1的mRNA表达;己糖激酶法用于葡萄糖浓度的测定,计算出培养液内葡萄糖的减少量作为细胞对糖的净利用。结果:(1)体外培养的HPMC细胞角蛋白用间接免疫荧光染色为绿色荧光,此为HPMC的特征;(2)LPS可以使正常糖浓度下(0.1%)HPMCGLUT-1的蛋白和mRNA的表达增加(P<0.05),呈时间和浓度依赖,并伴有细胞对葡萄糖净利用增加,其中作用24h后,浓度为100μg/ml的LPS使GLUT-1的蛋白和mRNA表达最大(P<0.01),而100μg/ml组LPS作用24h后,GLUT-1的蛋白和mRNA的表达最大(P<0.01),作用36h有所下降;(3)高糖条件下(4.25%),LPS可以使GLUT-1的蛋白和mRNA表达增加,与高糖组和正常对照相比有统计学差异(P<0.05)。结论:LPS可以使HPMCGLUT-1表达和葡萄糖摄取增加,这种作用在高糖条件下更为显著。这为研究和防治腹膜炎时腹透失超滤提供了线索。

Objective：To study the effect of lipopolysaccharide（LPS） on glucose transporter- 1 （GLUT- 1） expression in cultured human peritoneal mesothdial cell（HPMC）. Methods： Enzymatic disaggregation was used for primary culture and passage of HPMC. Cytokeratin was determined by indirect immunofluorescence for HPMC identification. Immunohistochemistry （ABC） was used for GLUT- 1 identification and semi - quantitative analysis. The expression of GLUT- 1 mRNA was evaluated by reverse transcription polymerase chain reaction（RT- PCR） technology. Hexokinase assay was used for measurement of concentration of glucose in cell culture fluid, and then net glucose utilization was calculated. Results： （1）The result of indirect immunofluorescence showed that the antibody to cytokeratin of cultured HPMC dyeing presented green fluorescence and this is the character of HPMC. （2）LPS can stimiulate HPMC to increase the expression of GLUT- 1 protein and mRNA and net glucose utilization significantly in normal glucose concentration（0.1% ） in a concentration - and time - dependent manner（ P 〈 0.05）. At concentration of 100 μg/mL for 24 h, LPS made the expression of GLUT- 1 protein and mRNA increase to the most in different concentrations and periods（ P 〈 0.01 ）. （3）At high concentration of glucose（4.25 % ）, LPS can increase the expression of GLUT - 1 protein and mRNA significantly compared to the high glucose group and the control group（P〈0.05）. Conclusion： LPS can increase the GLUT- 1 expression and net glucose utilization of HPMC, which displayed more significantly in high concentration glucose. This showed us a clue to study and prevention and cure the ultrafiltration failure of peritoneal dialysis in peritonitis.