肾病综合征患儿外周血单个核细胞内地塞米松浓度与细胞膜表面P-糖蛋白170表达的相关性研究

目的分析对糖皮质激素（GC）敏感（SS）和耐药（SR）的原发性。肾病综合征（NS）患儿的外周血单个核细胞（PBMC）内地塞米松浓度和P-糖蛋白170（P—gp170）表达与GC耐药的关系，探讨P—gp170在GC耐药Ns患儿中的作用及介导耐药的可能机制。方法收集2006年1至10月在中南大学湘雅二医院儿科诊断为原发性NS的初治患儿为研究对象，依据对GC的反应分为SSNS亚组和SRNS亚组，并设同期行查体的健康儿童为对照组。应用地塞米松刺激SSNS和SRSS亚组患儿的PBMC并进行培养，以高效液相色谱法测定培养前（0 h）及培养后12、24和36h时PBMC内地塞米松浓度；蛋白质免疫印迹检测培养各时间点PBMC膜表面P—gp170的表达量；分别对不同培养时间点PBMC内地塞米松浓度与P—gp170的表达量行相关性分析。结果研究期间SSNS亚组纳入16例，SRNS亚组纳入8例，对照组纳入10例。①PRMC内地塞米松浓度随培养时间延长SSNS亚组呈增高趋势（P〈0．05），SRNS亚组变化不显著（P〉0．05）；各培养时间点PBMC内地塞米松浓度SSNS亚组和SRNS亚组均高于对照组（P〈0．05）。②SSNS亚组P—gp170表达随培养时间的延长而逐渐降低，各培养时间点差异有统计学意义（P〈0．05），且均高于对照组（P〈0．05）。SRNS亚组各培养时间点P—gp170表达差异无统计学意义（P〉0．05），但高于SSRN亚组（P〈0．05）。③对照组不同培养时间点PBMC内地塞米松浓度与P—gp170表达呈显著负相关（12h：r=-0．852，24h：r=-0．794，36h：r=-0．847；尸均〈0．05）；SSNS亚组也呈显著负相关（12h：r=-0．728，24h：r=-0．785，36h：r=-0．842；P均〈0．05）；SRNS亚组无显著相关性。结论①PBMC膜表面P—gp170表达增高与Ns患儿GC耐药有关，P—gp170表达增高介导了部分Ns患儿的GC耐药；②应用GC可诱导NS患儿PBMC膜表面P—gp170的表达增高，部分Gc耐药患儿与GC应用所致P—gp170增高有关；⑧NS患儿PBMC膜表面P—gp170的表达增高为GC耐药标志之一.

Correlations between intracellar dexamethasone concentration and expression of P-gp170 in peripheral blood mononuclear cells of children with primary nephrotic syndrome

Objective To probe into the correlation between P-gp170 expression level of peripheral blood mononuclear cells （PBMC） and intracellular dexamethasone （Dex） concentration by testing P-gp170 expression and intracellular Dex concentration at different time point, in order to analyze the role P-gp170 may play in SRNS and the mechanism involved. Methods 24 children with NS without any glucocorticoid treatment before admission were chosen as subjects, namely SSNS and SRNS, and 10 healthy children serving as control. High performance liquid chromatography was used to test intracellular Dex concentration at different time, namely 0, 12,24 and 36 h, and Western blotting assay was adopted to test P-gp170 expression level of peripheral blood mononuclear cell membranes, and correlation analysis of Dex concentration and expression of P-gp170, then regression analysis on those having correlations were performed. Results Intracellular Dex concentration of children with SSNS varied with different time, the longer the period of time cells cultured, the higher the Dex concentration, and a significant difference was found between each time point according to statistics（P 〈 0.05 ）, and there was also notable difference between SSNS group and control group （P 〈0.05）, and intracellular Dex concentration of children with SRNS varied with different time as well, but there was no significant difference between each time point statistically（ P 〉0.05 ） ; No change was found in P-gp170 expression level of PBMC of healthy blank control at different time, thus no significant difference was found between groups according to statistics（ P 〉 0.05 ） ; P-gp170 expression level of PBMC of Dex groups in SSNS decreased by and by, with a significant difference between different time point statistically（P 〈 0. 01 ） ;P-gp170 expression level of PBMC of Dex groups decreased with time passing by, and with a significant difference between different time point statistically （P 〈 0.05 ） ;there was significant P-gp170 expression level of each time point（ P 〈 0.05 ） ; No change was found in P-gp170 expression level of PBMC of children with SSNS at different time （ P 〉 0.05）; P-gp170 expression level of PBMC of SSNS Dex groups decreased with time passing by, with a significant difference between different times statistically （P 〈 0.05 ） ;P-gp170 expression levels of different time points were all higher compared with healthy Dex control（P 〉 0.05 ） ; P-gp170 expression levels of PBMC of SSNS Dex groups were all higher than those of SSNS blank control （P 〈 0. 05 ） ;No change was found in P-gp170 expression level of PBMC of children with SRNS at different time（ P 〉 0.05 ） ; and there was a significant difference of P-gp170 expression level compared with healthy blank control statistically（ P 〈 0.05 ） ;No change was found in P-gp170 expression level of PBMC of children with SRNS Dex group at different time （ P 〉 0.05 ） ; and statistically there was a significant difference of P-gp170 expression level compared with SRNS （ P 〈 0.05 ） ; P-gp170 expression levels of different time points were all lower compared with SRNS blank control（P 〉0.05） and P-gp170 expression levels in Dex group of different time points were all lower, a significant difference were found between groups according to statistics（ P 〉0.05 ）, No change of P-gp170 expression level of peripheral blood mononuclear cells of children with SRNS in Dex group were found at different time compared with SRNS bank control （ P 〉 0. 05 ） ; A negative correlation was found between intracellular Dex concentration and expression of P-gp170 of Dex groups in healthy control, r was - 0. 852, - 0. 794, - 0. 847 , respectively（ P 〈 0.05） ;and the same was between those of SSNS, r was - 0. 728, - 0. 785, - 0. 842,respectively（ P 〈 0.05 ） ; No correlation was found between those of SRNS according to statisticas （P 〉 0.05）. Conclusions ①Expression of P-gp170 was found both in children with NS and control with the later higher than the former, thus P-gp170 may play different roles physiologically and pathologically in NS children. ②Mechanisms in some children with SRNS may attribute to up-regulating the expression of P-gp170 primarily, while others developing SRNS may resulting from a large amount of GC administration during a long period, which was the mechanism of second GC resistant, GC administration may lead to higher expression of P-gp170 in cell membrane, a large amount of GC administration during a long period clinically may result in GC resistance by way of up-regulating the expression of P- gp170, consequently an inhibition of GC from outside into inside of the cells. ③Up-regulating the expression of P-gp170 was one of the markers of GC resistance in NS children.