新型人工肝材料——接枝改性聚丙烯膜体外免疫相容性的实验研究

In vitro immunocompatibility of a novel bioartificial liver reactor material: propylene-acidamide grafted polypropylene membrane

目的 通过体外检测改性前、后聚丙烯 (PP)膜对健康青年人外周血单个核细胞(PBMC)的激活程度 ,来评价聚丙烯酰胺接枝改性聚丙烯 (PP g AAm )膜的免疫相容性。 方法 用生化法检测 2h、6h两种膜材料表面PBMC的乳酸脱氢酶 (LDH)值 ;用流式细胞术检测 2 4h两材料上培养的PBMCCD6 9的表达 ;ELISA法检测培养上清中细胞因子TNFα、IL 1β和IL 6的浓度 ;用扫描电镜 (SEM )观察材料上黏附的PBMC。结果 2h及 6h ,两材料组间LDH差异有显著意义 (P =0 0 0 8,P =0 0 0 0 ) ;PP g AAm膜组CD6 9活化率显著少于PP膜组 (P =0 0 13) ;2 4h两材料组细胞因子的浓度均达到最高峰 ,且PP g AAm膜组TNFα ,IL 1β和IL 6的浓度都小于PP膜组 (P =0 0 0 4 ,P =0 0 0 3,P =0 0 2 2 ) ;SEM观察到PP g AAm膜表面黏附的PBMC较少。结论 PP g AAm膜对PBMC的激活程度较轻 ,具有较好的免疫相容性。

Objective To evaluate the immunocompatibility of a novel bioartificial liver bioreactor material: propylene acidamide grafted polypropylene membrane (PP g AAm) in vitro on peripheral blood mononuclear cells (PBMCs) Methods Fifty milliliters of peripheral blood were collected from 30 healthy young people PBMCs were extracted and inoculated on the 24 well culture plate preset with sterilized PP g AAm membrane and ungrafted polypropylene (PP) membrane Automatic biochemical analyzer was used to detect the lactic dehydrogenase (LDH) value of the PMBCs after 2 and 6 hours The PMBCs on these 2 kinds of membrane were cultured for 6 hours and then added with lipopolysaccharide and cultured continually Six, twelve, and thirty six hours later the supernatant was collected ELISA was used to detect the values of tumor necrosis factor (TNF)α, interleukin (IL) 1β, and IL 6 Flow cytometry was used to detect the expression of CD69 antigen Scanning electron microscopy was used to observe the adhesion of PMBCs on the materials Results After 2 hours′ epimembranous inoculation the LDH value was 43 50 U/L±12 71 U/L in the PP group, significantly higher than that in the PP g AAm group (29 13 U/L±8 74 U/L, P =0 008) and the newly extracted PMBC group (0h group, 19 89 U/L±4 67 U/L, P =0 000) However, the difference between the 0h group and PP g AAm group ( P =0 080) was insignificant After 6 hours' epimembranous inoculation the LDH value was 50 25 U/L±13 38 U/L in the PP group, and 32 50 U/L±9 21 U/L in the PP g AAm group ( P =0 001), both significantly higher than that in the 0h group ( P =0 000,and P =0 019) There was no significant difference between the values 2 hours and 6 hours after inoculation for the two groups No expression of TNFα,IL 1β, and IL 6 was found in the supernatant of the 2 groups without LPS stimulation Expressions of TNFα,IL 1β, and IL 6 could be found at a low level 12 hours after LPS stimulation for both groups and peaked 24 hours after LPS stimulation The expressions of TNFα,IL 1β, and IL 6 were lower in the PP g AAm group than in the PP group ( P =0 004, P =0 003,and P =0 022) The TNFα,IL 1β, and IL 6 levels all decreased after 36 hours The CD69 antigen expression rates were 17 20%±3 45%and 12 02%±2 44% respectively in the PP group and PP g AAm group, both significantly higher than that of the blank control group (3 38%±1 30%, both P =0 000) SEM showed that the PMBCs adhered on the PP Aam membrane were significantly less then those adhered on the PP membrane And the PMBCs adhered on the PP g AAm membrane were smaller and with less microvilli Conclusion PP g AAm membrane has weaker activation capability to PMBCs and has better immunocompatibility in comparison with the PP membrane.