高迁移率族蛋白B1对小鼠巨噬细胞免疫功能影响的受体机制

目的探讨高迁移率族蛋白B1(HMGB1)对巨噬细胞免疫功能影响的可能受体机制。方法分离培养小鼠腹腔巨噬细胞,HMGB1刺激后,采用激光扫描共聚焦显微镜和流式细胞术检测细胞表面晚期糖基化终末产物受体(RAGE)的表达。实验分为正常对照组、HMGB1组、HMGB1 抗RAGE抗体组、HMGB1 rmRAGE/Fc组,分别观察巨噬细胞摄取中性红能力、对L1210细胞的杀伤作用(MTT法)、趋化活性(采用Transwell小室趋化装置)、细胞表面I-Ak抗原表达(流式细胞术)的变化。结果100μg/L的HMGB1作用于巨噬细胞48 h后,巨噬细胞表面受体RAGE表达明显上调(P<0.01);HMGB1组巨噬细胞吞噬功能、杀伤活性、趋化活性及I-Ak抗原表达均显著高于对照组、HMGB1 抗RAGE组与HMGB1 rmRAGE/Fc组(P<0.01)。结论HMGB1可诱导RAGE表达增强,RAGE是HMGB1作用于巨噬细胞免疫功能的主要受体之一。     

高迁移率族蛋白B1对严重烧伤大鼠枯否细胞的影响及晚期糖基化终末产物受体的作用

目的 探讨高迁移率族蛋白B1(HMGB1)对严重烧伤大鼠枯否细胞(KC)分泌促炎性细胞因子的影响及晚期糖基化终末产物受体(RAGE)在该过程中的作用. 方法 将32只SD大鼠背部浸于98℃水中12 s,制成30％ TBSAⅢ度烫伤(以下称烧伤).伤后24 h,处死大鼠分离肝脏KC(32个样本),以每孔1×106个接种至24孔板中.(1)取部分细胞按随机数字表法分为2组,每组样本数为8.对照组,加入1 mL PBS液培养;HMGB1组,加入1 mL浓度为100 ng/mL HMGB1刺激.培养48 h后,采用蛋白质印迹法检测细胞表面RAGE的表达(结果以灰度值比值表示).(2)取部分细胞采用随机数字表法分为4组,每组样本数为8.对照组,加入1 mL PBS液培养;HMGB1组,加入1 mL浓度为100 ng/mL HMGB1刺激;HMGB1+抗RAGE抗体组,经1 mL浓度为20 μg/mL抗大鼠RAGE单克隆抗体孵育2h,加入1 mL浓度为100 ng/mL HMGB1刺激;HMGB1+重组大鼠RAGE/Fc嵌合体(rrRAGE/Fc)组,将0.5 mL浓度为100 ng/mL HMGB1与0.5 mL浓度为5μg/mL rrRAGE/Fc孵育2h后,作用于细胞.培养48 h后,采用ELISA法检测细胞培养上清液中TNF-α和IL-1β的含量,RNA印迹法检测细胞内TNF-α和IL-1β mRNA的表达水平(结果以灰度值比值表示).对数据进行单因素方差分析、t检验及LSD检验. 结果 (1)培养48 h后,HMGB1组细胞RAGE表达水平(1.036 ±0.101)明显高于对照组(0.191 ±0.024,t=-23.158,P=0.000).(2) HMGB1组、HMGB1+抗RAGE抗体组以及HMGB1+ rrRAGE/Fc组细胞培养上清液中TNF-α含量分别为(10.59±1.39)、(9.91±1.68)、(11.51±2.27) ng/mL,IL-1β含量分别为(2.49±0.33)、(2.08±0.32)、(2.42±0.42) ng/mL;细胞内TNF-α mRNA水平分别为0.311 ±0.009、0.301±0.047、0.326±0.016,IL-1βmRNA水平分别为0.237±0.021、0.244±0.041、0.245±0.013,3组间比较差异均无统计学意义(P值均大于0.05),均显著高于对照组[细胞培养上清液中TNF-α和IL-1β含量分别为(2.69±0.14)、(0.43±0.05) ng/mL,细胞内TNF-α和IL-1β mRNA水平分别为0.140±0.022、0.077±0.005,P值均小于0.01]. 结论 HMGB1可引起严重烧伤大鼠KC分泌促炎性细胞因子TNF-α和IL-1β,但RAGE在这一过程中未起主导作用.     

半胱天冬蛋白酶-3在高迁移率族蛋白B1诱导小鼠巨噬细胞凋亡中的作用

目的 观察半胱天冬蛋白酶(Caspase)-3与核转录因子(NF)-KB在高迁移率族蛋白B1(HMGB1)诱导小鼠腹腔巨噬细胞凋亡中的作用.方法 分离培养小鼠腹腔巨噬细胞,随机分为对照组、HMCB1组与抑制剂组(HMGB1作用前1 h加入Caspase-3抑制剂Z-DEVD-FMK).采用Hoeehst33342染色观察凋亡的形态学变化、流式细胞术检测巨噬细胞凋亡百分率的变化;应用原位荧光染色及流式细胞术检测Caspase-3活性的变化;同时应用酶联免疫吸附试验检测NF-KB p65蛋白的变化.结果 HMGB1组巨噬细胞凋亡率为(38.21±4.85)%、抑制剂组为(16.47±3.91)%,均显著高于对照组(4.21±1.64)%(P＜0.01).经Hoechst33342染色,荧光显微镜观察HMGB1组凋亡细胞明显增多;原位荧光染色观察到HMGB1组Caspase-3荧光强度明显增强,Caspase-3阳性细胞百分率(29.74±4.55)%显著高于抑制剂组(3.02±1.91)%与对照组(7.19±2.46)%(P＜0.01).同时,HMGB1组、抑制剂组细胞核NF-KB活性较对照组显著降低(P＜0.05或P＜0.01).结论 Caspase-3和NF-KB是HMGB1诱导巨噬细胞凋亡的重要途径,Caspase-3抑制剂可部分阻断HMGB1诱导的巨噬细胞凋亡.     

利多卡因对LPS诱导大鼠腹腔巨噬细胞NF-κB活性的影响

目的 探讨利多卡因对LPS诱导大鼠腹腔巨噬细胞NF-κB活性的影响.方法 取wistar大鼠腹腔巨噬细胞,以2 × 106/ml的密度接种于12孔培养板,每孔1 ml.纯化处理后随机分为5组,每组10孔.正常对照组(C组)加入RPMI-1640培养液1 ml,L组加入含100 ng/ml LPS的RPMI1640培养液1 ml,LL1组、LL2组和LL3组分别加入含有2、20、200μg/ml利多卡因+100 ng/ml LPS的RPMI-1640培养液1 ml.孵育24 h后,收集上清液,测定高迁移率族蛋白B1(HMGB1)浓度;取细胞沉淀,测定HMGBl mRNA表达水平和NF-κB活性.结果 与C组比较,其他各组HMGB1浓度、HMGB1mRNA表达和NF-κB活性均升高(P＜0.05);与L组及LL1组比较,LL2组和LL3组上述指标降低(P＜0.05).LL3组HMGB1 mRNA表达水平低于LL2组(P＜0.05).结论 利多卡因可抑制LPS诱导大鼠腹腔巨噬细胞NF-κB活化,从而抑制HMGB1的合成与释放.     

丹参酮ⅡA对兔纤维环细胞能量代谢的保护作用

[目的]考察丹参酮ⅡA(TSⅡA)对白细胞介素-1β(IL-1β)诱导的兔纤维环细胞能量代谢障碍的保护作用.[方法]藻酸盐串珠立体培养兔纤维环细胞,将细胞分为7组,在培养过程中加入不同浓度的药物:A组为空白对照不加入药物,B组加入4 μg/ml TSⅡA,C组加入10ng/ml IL-1β,D～G组在给予10 ng/mlIL-1β同时分别加入0.5、1、2和4 μg/ml TSⅡA.于培养3 d后行Na+-K+-ATP酶活性检测、,琥珀酸脱氢酶活性检测、MTT法细胞增殖情况检测以及细胞凋亡的流式细胞仪检测.[结果]G组Na+-K+-ATP酶活性(3.23±0.28U/mgprot)较C组(1.118±0.15U/mgprot)明显增高(P<0.01),与A组接近(3.57±0.15 U/mgprot)(P>0.05).G组琥珀酸脱氢酶活性(12.48±0.97U/mgprot)较c组(3.03±0.60 U/mgprot)明显增高(P<0.01),与A组接近(14.24±1.56 U/mgprot)(P>0.05).G组MTT试验吸光度(0.77±0.06)较C组(0.31±0.07)明显增高(P＜0.01),随着TSⅡA浓度的升高,D～G组吸光度随着TsIIA上升而上升.G组细胞死亡细胞比例和凋亡细胞比例分别为21.08±1.46%和8.99±0.33%,均显著低c组(43.11±2.7,P<0.01和11.71±0.32,P<0.01).[结论]TSIIA能够减轻IL-1β对纤维环细胞能量代谢的抑制作用,从而改善纤维环细胞的增殖、死亡及凋亡.     

血必净促进内毒素/脂多糖刺激调节性T淋巴细胞凋亡并介导辅助性T淋巴细胞漂移的作用

目的 了解血必净注射液促进LPS刺激CD4+CD25+调节性T淋巴细胞(Treg细胞)凋亡过程及介导辅助性T淋巴细胞(Th)漂移的调节作用.方法 免疫磁珠法分选获得大鼠脾脏CD4+CD25+Treg细胞,分为常规培养对照组、抗CD3/CD28组、抗CD3/CD28+LPS组、抗CD3/CD28+血必净组和抗CD3/CD28+LPS+血必净组,培养3 d后应用流式细胞术检测Treg细胞凋亡率及叉头翼状螺旋转录因子3(Foxp3)表达.将CD4+CD25+Treg细胞与CD4+CD25+T淋巴细胞1:1培养,伴刀豆球蛋白A刺激68 h,检测上清液中Th1分泌的γ干扰素(IFN-γ)、Th2分泌的IL-4、Th17分泌的IL-17水平.结果 抗CD3/CD28+LPS+血必净组Treg细胞凋亡率为(45.1±2.7)%,明显高于抗CD3/CD28+LPS组[(29.4±1.6)%,P＜0.01];2组Foxp3平均荧光强度分别为95±9、140±18,差异有统计学意义(P＜0.01).同时,抗CD3/CD28+LPS+血必净组IFN-γ分泌水平显著高于抗CD3/CD28+LPS组(P＜0.01),IL-4则呈相反变化(P＜0.05),抗CD3/CD28+LPS+血必净组IFN-γ/IL-4较对照组升高(P＜0.01);抗CD3/CD28+血必净组IL-17分泌水平较抗CD3/CD28组明显下降(P＜0.05).结论 CD4+CD25+Treg细胞活化介导了Th1向Th2功能性极化;血必净对LPS诱导的T淋巴细胞免疫功能有重要调节作用,可促进CD4+CD25+Treg细胞凋亡并介导Th2向Th1漂移,从而缓解细胞免疫抑制状态.     

大鼠精索静脉曲张模型及其高位结扎术后睾丸生精细胞凋亡及其IL-1和NO含量的变化

目的:研究大鼠左侧精索静脉曲张(VC)模型及其高位结扎术后睾丸生精细胞凋亡及白细胞介素-1(IL-1)和一氧化氮(NO)含量的变化。方法:选用雄性SD大鼠60只,均选择左侧精索静脉作为研究对象,建立VC模型。将大鼠随机分为3组:假手术组(SO)15只,VC后高位结扎组(VCT)组15只和VC模型对照组30只。模型对照组中随机选取15只大鼠作为VC1组,余下15只大鼠作为VC2组,分别测定VC1组和SO组、VC2组和VCT组大鼠精液质量及睾丸组织中IL-1和NO的含量并加以比较,采用TUNEL检测睾丸生精细胞凋亡情况。结果:所有大鼠均建模成功,VC1组精子浓度[(1.54±1.16)×10~6/ml]和精子活力[(44.23±15.46)%]均显著低于SO组[2.80±1.62)×10~6/ml、(72.34±12.62)%](P0.05),VCT组精子浓度[1.82±1.34)×10~6/ml]和精子活力[(51.21±12.62)%]较VC2组有显著提高[(1.04±1.21)×10~6/ml、(39.23±13.21)%](P0.05)。大鼠左侧睾丸NO[(0.172±0.030)ng/ml]、IL-1[(1.468±0.080)mg/ml]含量VC1组明显高于SO组[(0.134±0.021)ng/ml、(0.782±0.079)mg/ml](P0.05),VC2组左侧睾丸NO[(0.198±0.020)ng/ml]、IL-1[(1.994±0.090)mg/ml]含量明显高于VCT组[(0.141±0.010)ng/ml、(0.781±0.036)mg/ml](P0.05),而右侧睾丸2组比较差异无显著性(P0.05),而且NO与IL-1含量之间呈正相关关系(r=0.492,P0.01)。VC1组大鼠双侧睾丸生精细胞大量凋亡,左、右侧睾丸生精细胞凋亡指数差异有统计学意义(P0.05),SO组左、右侧睾丸生精细胞凋亡指数无明显差异(P0.05),2组间同侧睾丸生精细胞凋亡指数差异显著,均有统计学意义(P0.01);VCT组大鼠左、右侧睾丸生精细胞凋亡指数差异有统计学意义(P0.05);VC2组左、右侧睾丸生精细胞凋亡指数无明显差异(P0.05);2组间同侧睾丸生精细胞凋亡指数差异显著,均有统计学意义(P0.01)。VC2组、VCT组组内同侧睾丸生精细胞凋亡指数无明显差异(P0.05),但VC2组、VCT组2组间同侧睾丸生精细胞凋亡指数差异显著,均有统计学意义(P0.01)。结论:VC致睾丸组织中NO和IL-1含量升高,并加重睾丸生精细胞凋亡,可能是其致睾丸损伤、影响睾丸生精功能障碍的原因之一。     

Toll样受体4在高迁移率族蛋白B1影响小鼠调节性T细胞免疫功能中的作用

目的 探讨腹腔注射高迁移率族蛋白B1(HMGB1)后不同基因型小鼠天然调节性T细胞(CD4~+CD25~+Treg)免疫功能的改变及其受体作用机制.方法 分别给C3H/HeN和C3H/HeJ[分别为Toll样受体4(TLR4)野生型(TLR4~(+/+))和天然突变型(TLR4~(-/-)]小鼠腹腔注射不同剂量HMGB1(0、10、20μg/只),饲养48 h后采用免疫磁珠法分离小鼠脾脏CD4~+CD25~+Treg.体外培养12 h后采用流式细胞仪检测CD4~+CD25~+Treg表达细胞毒性T淋巴细胞相关抗原-4(CTLA-4)表达强度,并应用酶联免疫吸附试验(ELISA)检测CD4~+CD25~+Treg生成白细胞介素(IL)-10量.结果 与对照组比较,20 μg HMGB1攻击后C3H/HeN小鼠CD4~+CD25~+Treg表达CTLA-4水平显著下降(78.70±11.42与60.76±7.64,P<0.01),同时细胞牛成IL-10量也明显降低[(96.89±6.25)ng/L与(47.11±4.25)ng/L,P<0.01];但不同剂量HMGB1攻击可引起C3H/HeJ小鼠CD4~+CD25~+Treg表达CTLA-4明显上调和生成IL-10量不同程度地增加(P<0.01).结论 HMGB1攻击可显著影响CD4~+CD25~+Treg介导的免疫状态,TLR4在HMGB1诱导CD4~+CD25~+Treg免疫活性过程中发挥了重要负向调控作用.     

胸腺肽α1对脂多糖刺激后人外周血单核细胞和淋巴细胞免疫功能的影响

目的:观察胸腺肽α_1对脂多糖刺激后人外周血单核细胞人类白细胞组织相容性抗原-DR(HLA- DR)表达率及淋巴细胞凋亡率的影响。方法:分离、培养正常人外周血单核细胞和淋巴细胞,空白对照组仅加1640培养液,内毒素(LPS)组加LPS刺激,LPS 胸腺肽α_1组先用不同浓度(100、10、1、0.1、0.01μg/ml)胸腺肽α_1预处理细胞,5min后再加入LPS(100 ng/ml)刺激。培养12 h后用流式细胞术检测CD14~ 单核细胞HLA-DR表达率和外周血淋巴细胞凋亡率。结果:LPS单独刺激单核细胞时,CD14~ 单核细胞HLA-DR表达率[(11.14±5.09)%]较空白对照组[(74.00±3.50)%]明显降低,用胸腺肽α_1预处理后,HLA-DR表达率随着胸腺肽α_1浓度的增高而增高(P均<0.01)。LPS单独刺激淋巴细胞时,淋巴细胞凋亡率[(8.16±0.51)%]较空白对照组[(4.40±0.53)%]明显增加,而用胸腺肽α_1预处理后,淋巴细胞凋亡率随着胸腺肽α_1浓度的增高而降低,胸腺肽α_1浓度为10μg/ml和100μg/ml时,淋巴细胞凋亡率[(4.65±0.75)%和(4.50±0.36)%]与空白对照组比较差异无显著性(P均<0.01)。结论:胸腺肽α_1对LPS引起的人外周血单核细胞和淋巴细胞免疫功能下调有显著抑制作用。     

琥珀酸对人外周血中性粒细胞凋亡的影响

目的 了解琥珀酸对人外周血中性粒细胞(PMN)凋亡的影响,探讨其致病机制.方法 体外孵育PMN,将PMN浓度调至5×106个/ml.在细胞中加入琥珀酸刺激,根据所加琥珀酸浓度分为5、10、20、30 mmol/L琥珀酸组;对照组加入常规培养液.取各组细胞培养上清液,检测其活性氧含量.5、20 mmol/L琥珀酸组细胞于处理前及处理后2、4、6、8、10 h分别取细胞悬液1 ml,行半胱氨酸天冬氨酸蛋白酶3(caspase-3)活性及细胞凋亡率检测.结果 5、10、20、30 mmol/L琥珀酸组活性氧含量(0.437±0.056、0.432±0.024、0.395±0.049、0.386±0.010)均低于对照组(0.505±0.028,P＜0.05).随着孵育时间延长,各组细胞caspase-3活性均逐渐增加,但与对照组比较,5 mmol/L琥珀酸组caspase-3活性降低,20 mmol/L琥珀酸组则升高(P＜0.05).对照组细胞处理前凋亡率为(6.1±1.1)%,处理后2 h为(13.2±2.0)%,10 h达(27.7±3.7)%;5 mmol/L琥珀酸组细胞凋亡率除处理后4 h外其余时相点均低于对照组(P＜0.05);20 mmol/L琥珀酸组细胞处理后4～10 h凋亡率均高于对照组(P＜0.05).结论 低浓度琥珀酸抑制PMN凋亡,而高浓度琥珀酸可促进PMN凋亡.细菌感染时,通过代谢产物琥珀酸抑制PMN免疫功能.     

信号转导及转录激活子1和3抑制剂对高迁移率族蛋白B1诱导鼠巨噬细胞合成肿瘤坏死因子α的影响

目的探讨信号转导及转录激活子1(STAT1)和3抑制剂对高迁移率族蛋白B1(HMGB1)诱导巨噬细胞合成肿瘤坏死因子α(TNFα)的影响。方法取正常Wistar大鼠腹腔巨噬细胞置24孔培养板中(1×106细胞/孔),培养3d后以HMGB1刺激,采用氟达拉滨(Fludarabine,STAT1特异性抑制剂)及雷帕霉素(Rapamycin,STAT3特异性抑制剂)进行干预。观察HMGB1刺激与肿瘤坏死因子αmRNA表达和蛋白释放的时效、量效关系,Fludarabine和Rapamycin处理对TNFαmRNA表达和蛋白释放的影响。结果(1)HMGB1可导致大鼠腹腔巨噬细胞TNFα基因表达明显升高,于攻击后24h达峰值,至36h减弱。HMGB1的用量为10μg/ml时,TNFα基因表达明显增强;(2)HMGB1可诱导大鼠腹腔巨噬细胞TNFα蛋白早期释放,4h即可达到高峰,8h后减弱。随着HMGB1刺激剂量从5μg/ml增大到25μg/ml,TNFα蛋白释放持续增强;(3)Fludarabine和Rapamycin可抑制大鼠腹腔巨噬细胞TNFα基因表达,但不能影响TNFα蛋白的释放。结论STAT1和STAT3抑制剂可显著下调巨噬细胞由HMGB1诱导的TNFα基因表达,但不能影响其早期(<24h)蛋白释放。     

Irradiation with 780 nm diode laser attenuates inflammatory cytokines but upregulates nitric oxide in lipopolysaccharide-stimulated macrophages: implications for the prevention of aneurysm progression

BACKGROUND AND OBJECTIVES: Low level laser irradiation (LLLI) has been shown to reduce inflammation in a variety of clinical situations. We have shown that LLLI (780 nm) increases aortic smooth muscle cell proliferation and matrix protein secretion and modulates activity and expression of matrix metalloproteinases. Inflammation is a major component of arteriosclerotic diseases including aneurysm. Macrophage recruitment and secretion of pro-inflammatory cytokines and the vasodilator, nitric oxide (NO), are central to most immune responses in the arterial wall. The present study was designed to determine the effect of LLLI on cytokine gene expression and secretion as well as gene expression of inducible nitric oxide synthase (iNOS) and NO production in lipopolysaccharide (LPS)-stimulated macrophages. STUDY DESIGN/MATERIALS AND METHODS: Murine monocyte/macrophages (RAW 264.7) were irradiated with a 780 nm diode laser (2 mW/cm(2), 2.2 J/cm(2)) during stimulation with LPS (0, 0.1, and 1 microg/ml). Gene expression of chemokines, cytokines, and iNOS were assessed by RT-PCR. Secretion of interleukin (IL)-1beta and monocyte chemotactic protein (MCP)-1 and NO were assessed by ELISA and the Griess reaction, respectively. RESULTS: LLLI reduced gene expression of MCP-1, IL-1alpha, IL-10 (P<0.01), IL-1beta, and IL-6 (P<0.05) when cells were stimulated by 1 microg/ml LPS. LLLI reduced LPS-induced secretion of MCP-1 over non-irradiated cells by 17+/-5% and 13+/-5% at 12 hours (0.1 and 1 microg/ml LPS; P<0.01 and P=0.05, respectively), and reduced IL-1beta by 22+/-5% and 25+/-9% at 24 hours (0.1 and 1 microg/ml LPS, P=0.01 and P=0.06, respectively). However, LLLI increased NO secretion after 12 hours (LLLI vs. Control: without LPS, 1.72+/-0.37 vs. 0.95+/-0.4 microM, P<0.05; 0.1 microg/ml LPS, 7.46+/-1.62 vs. 4.44+/-1.73 microM, P=0.06; 1 microg/ml LPS, 10.91+/-3.53 vs. 6.88+/-1.52 microM, P<0.05). CONCLUSIONS: These properties of LLLI, with its effects on smooth muscle cells reported previously, may be of profound therapeutic relevance for arterial diseases such as aneurysm where inflammatory processes and weakening of the matrix structure of the arterial wall are major pathologic components.     

Diversity in peritoneal macrophage response of CAPD patients to 1,25-dihydroxyvitamin D3

A major complication of continuous ambulatory peritoneal dialysis (CAPD) is peritonitis. Increasing the activity of the peritoneal macrophages, the predominant cell type found in the peritoneal cavity, may be a promising treatment for this infection. The effect of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] on the activity of peritoneal macrophages from CAPD patients and nonuremic controls was studied. 1,25(OH)2D3 had a biphasic effect on superoxide generation in the concentration range of 2.5 10(-9) M to 5 x 10(-6) M with a peak at 2 x 10(-8) M. The addition of 2 x 10(-8) M 1,25(OH)2D3 to nonuremic control macrophages for 24 hours caused a significant twofold increase in superoxide generation in response to phorbol myristate acetate (PMA), from 2.21 + 0.2 to 4.1 + 0.2 nmol/10(6) mac (P less than 0.001), and enhanced the bactericidal activity from 60 + 7% to 85 + 9% (P less than 0.005). CAPD patients were divided into two groups: Group A, patients with high peritonitis incidence (HPI); group B, patients with low peritonitis incidence (LPI). Macrophages from HPI patients show a lower bactericidal activity (37 +/- 5%) and were not affected by 1,25(OH)2D3 after 24 hours of treatment. The increase in macrophage activity was seen only after three days of incubation with the hormone. Macrophages from this group generated a high amount of prostaglandin E2 (PGE2) during the first 24 hours in culture (7.8 +/- 0.52 ng/ml as compared with 0.35 +/- 0.03 ng/ml in the controls).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of weight loss on renal function in patients with severe obesity

Severe obesity is associated with increased renal plasma flow (RPF) and glomerular filtration rate (GFR). The aim of the present study was to examine whether weight loss may reverse glomerular dysfunction in obese subjects without overt renal disease. Renal glomerular function was studied in eight subjects with severe obesity (body mass index [BMI] 48.0 +/- 2.4) before and after weight loss. Nine healthy subjects served as controls. GFR and RPF were determined by measuring inulin and PAH clearance. In the obese group, GFR (145 +/- 14 ml/min) and RPF (803 +/- 39 ml/min) exceeded the control value by 61% (90 +/- 5 ml/min, P = 0.001) and 32% (610 +/- 41 ml/min, P < 0.005), respectively. Consequently, filtration fraction was increased. Mean arterial pressure, although normal, was higher than in the control group (101 +/- 4 versus 86 +/- 2 mmHg, P < 0.01). After weight loss, BMI decreased by 32 +/- 4%, to 32.1 +/- 1.5 (P = 0.001). GFR and RPF decreased to 110 +/- 7 ml/min (P = 0.01) and 698 +/- 42 ml/min (P < 0.02), respectively. Albumin excretion rate decreased from 16 microg/min (range, 4 to 152 microg/min) to 5 microg/min (range, 3 to 37 microg/min) (P < 0.01). Fractional clearance of albumin decreased from 3.2 x 10(-6) (range, 1.1 to 23 x 10(-6)) to 1.2 x 10(-6) (range, 0.5 to 6.8 x 10(-6)) (P < 0.02). This study shows that obesity-related glomerular hyperfiltration ameliorates after weight loss. The improvement in hyperfiltration may prevent the development of overt obesity-related glomerulopathy.     

Does size matter? Larger Blalock-Taussig shunt in the modified Norwood operation correlates with better hemodynamics.

OBJECTIVE: Excess pulmonary to systemic blood flow ratio (Qp/Qs) correlates with hemodynamic instability and mortality after modified Norwood operation. Studies suggest that maximal oxygen delivery occurs at a Qp/Qs of around 1. The use of a rather small modified Blalock-Taussig shunt (MBTS) is believed to achieve this goal. However, optimal MBTS size with respect to postoperative hemodynamics remains unclear. METHODS: Between 2/2002 and 2/2004, 20 consecutive patients underwent Norwood operation; there were 19 operative survivors: nine with a normalized MBTS area (NSA) > or = 3.3 mm2/kg (group 1) and 10 with NSA < 3.3 mm2/kg (group 2). Mean arterial pressure (MAP) and common atrial pressures (CAP), arterial and superior vena cava oxygen saturations, urinary output and inotropes recorded for the postoperative hours 0, 6, 12, 18, 24 and 48 were analyzed. RESULTS: Hospital mortality was 11.1% (1/9) in group 1 and 30% (3/10) in group 2 (P = 0.6). For group 1 significantly higher MAP of 52+/-1.3 versus 46+/-0.8 mmHg (P < 0.001), higher urinary output of 6.2+/-0.5 versus 4.2+/-0.5 ml/kg per h (P < 0.01), lower CAP of 8+/-0.3 versus 10+/-0.4 mmHg (P < 0.001), and lower heart rate of 145+/-2.6 versus 160+/-1.6 bpm were recorded than for group 2. In group 1, lower doses of adrenaline (0.03+/-0.01 versus 0.15+/-0.01 microg/kg per min, P < 0.05) and noradrenaline (0.01+/-0.01 versus 0.13+/-0.04 microg/kg per min, P < 0.01) were needed. Although Qp/Qs was more often calculated to be > 1.5 in group 1 (51 versus 31%), arteriovenous oxygen difference and oxygen excess factor were not significantly different, indicating similar oxygen delivery. CONCLUSIONS: Monitoring of the central venous oxygen saturations and application of afterload reduction in cases of high Qp/Qs allows the insertion of a larger MBTS without association with lower oxygen delivery. In fact, better hemodynamic status with less inotropic support was noted with a larger MBTS early after Norwood operation.     

高迁移率族蛋白B1对小鼠调节性T细胞与CD4+CD25-T细胞相互作用的影响

目的 观察高迁移率族蛋白B1(HMGB1)对调节性T细胞(Treg)与CD4+CD25-T细胞相互作用的影响,并初步探讨其影响Treg抑制功能的机制.方法 免疫磁珠法分离正常BALB/c小鼠脾脏CD4+CD25+Treg及CD4+CD25-T细胞.采用固相包被抗CD3/可溶性抗CD28进行辅助活化,以不同时间及浓度HMGB1刺激Treg,ELISA法分析HMGB1刺激对Treg分泌IL-10的影响.将HMGB1(1000μg/L)刺激后的Treg与CD4+CD25-T细胞共培养,MTT法观察其对Treg抑制CD4+CD25-T细胞反应的作用,并分析HMGB1刺激后的Treg对CD4+CD25-T细胞IL-2生成及细胞功能极化的影响.结果 经抗CD3/CD28辅助活化的CD4+CD25+Treg在HMGB1作用下IL-2生成无显著差异(P＞0.05),但随时间延长及剂量增加,IL-10生成明显减少(P＜0.05).经HMGB1刺激的Treg对CD4+CD25-T细胞增殖的抑制反应减弱,同时诱导CD4+CD25-T细胞IL-2产生及细胞功能极化的能力均下降(P＜0.05).结论 HMGB1可通过诱导Treg抑制功能的下调,从而影响CD4+CD25-T细胞功能,进而调节炎症反应过程.     

镧对内毒素/脂多糖诱导的巨噬细胞核因子κB活化的抑制作用

目的 了解稀土元素镧对内毒素／脂多糖（LPS）诱导的小鼠腹腔巨噬细胞（Mφ）核因子KB（NF-κB）活化的影响，并探讨其机制。方法分离培养小鼠腹腔Mφ，分为：空白对照组，无刺激因素：LPS组，用1μg／mlLPS刺激30min；镧离子（La^3＋）组，用2，5μmol／L La^3＋刺激30min；La^3＋＋LPS组，先后用2，5-μmol／L La^3＋、1μg／ml LPS各刺激30min；La^3＋／L PS组，2．5μmol／L La^3＋刺激30min后，洗涤细胞，再加入1μg／ml LPS刺激30min。采用细胞免疫荧光染色法观察NF-κB在各组细胞中的分布与表达；酶联免疫吸附测定（ELISA）法检测胞核中NF-κB／p65蛋白活性；蛋白质印迹法检测细胞核内NF-κB／p65蛋白及胞质中核因子抑制蛋白α（IκBα）的表达量。ELISA法测定各组细胞培养上清液中肿瘤坏死因子α（TNF-α）的含量。结果 （1）免疫荧光染色显示，空白对照组、La^3＋组、La^3＋＋LPS组和La^3＋／LPS组M小绿色荧光多分布于胞质，胞核荧光强度分别为42±7、73±30、48±11和67±19；LPS组绿色荧光集中于胞核，荧光强度为116±14，明显高于其余4组（P〈0.01）。（2）胞核NF-κB／p65蛋白活性：LPS组吸光度值为0．435±0．066，与空白对照组（0．048±0．027）、La^3＋组（0．062±0．049）、La^3＋＋LPS组（0.066±0.031）、La^3＋／LPS组（0．108±0.017）比较．明显偏高（P〈0．01）。（3）LPS组M由胞核NF-κB／p65蛋白表达水平明显高于其余4组，胞质IκBα蛋白表达水平明显低于其余4组。（4）TNF-α含量：La^3＋组培养上清液中TNF-α含量低于试剂盒检测下限（25pg／m1）及空白对照组（P〈0.05），La^3＋＋LPS组和La^3＋／LPS组低于LPS组（P〈0．01），但高于空白对照组。结论 LPS可激活小鼠腹腔M由NF-κB／p65核移位，并使胞核中NF-κB／p65的表达量和活性升高、胞质IκBα蛋白表达下调，导致TNF-α分泌增多。镧可抑制上述效应。     

Malnutrition-induced macrophage apoptosis

BACKGROUND: Human and murine studies suggest protein-calorie malnutrition (PCM) results in significant host immunosuppression resulting in increased morbidity and mortality. Apoptosis has been implicated as an important mediator in the immunosuppression observed in several disease states. This study was designed to characterize macrophage apoptosis in a murine model of PCM and investigate components that regulate the apoptotic process, such as protein kinase C (PKC) and Bcl-2 activity. METHODS: Swiss-Webster mice (n = 50) were randomly assigned to receive either a control (24% protein) or a PCM diet (0% protein) for 7 days. Peritoneal macrophages were harvested and detection of apoptosis was performed by terminal deoxy-transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) and propidium iodide DNA staining under baseline and pro-apoptotic conditions. Pro-apoptotic conditions included cells treated with tumor necrosis factor-alpha (TNF-alpha) (10 ng/mL), interferon-gamma (IFN-gamma) (10 ng/mL), and a combination of both agents. In addition, levels of PKC activity and expression of Bcl-2 and p53 protein were measured. RESULTS: Peritoneal macrophages from PCM mice had a significantly greater amount of apoptosis at baseline and under stimulated conditions compared with controls. Levels of PCM apoptosis were elevated at baseline by TUNEL staining compared with macrophages from the control group (16.5% +/- 1.4%, versus 4.5% +/- 1.1%, P <.01). In addition, peritoneal macrophages from the malnourished animals were significantly more susceptible to the apoptotic effect of TNF-alpha and the effects of INF-gamma (27.3% +/- 2.1% and 31% +/- 1.4%) compared with control mice (5.5% +/- 0.7% and 7.2% +/- 0.5%, P <.01), respectively. Again, an increase in the baseline apoptosis rate was demonstrated in peritoneal macrophages from PCM mice compared with control fed mice (13.2% +/- 4.4% versus 4.3% +/- 3.1%, P <.01) as measured by propidium iodide staining. The combination of agents, TNF-alpha and INF-gamma, resulted in an additive apoptotic effect in the malnourished host compared with the control animals (43.4% +/- 4.7% versus 10.5% +/- 2.2%, P <.01), respectively. Furthermore, there was a significant decrease in the mean total PKC activity in the malnourished macrophages compared with results in controls (110,000 +/- 8000 versus 60,000 +/- 4000 cpm, P <.01). Similar changes were also observed in PKC cytosolic and membrane activity between both groups. In addition, Bcl-2 protein expression was significantly decreased in PCM animals compared with control animals. CONCLUSIONS: Thus, peritoneal macrophages from PCM mice exhibit significantly greater levels of apoptosis at baseline and when stimulated with pro-apoptotic agents compared with controls. The propensity of macrophages from PCM mice to undergo apoptosis may be attributable in part to decreased PKC activity and Bcl-2 protein expression. These findings may help to explain the associated immune dysfunction observed in malnutrition.