摘要
目的:观察病程早期原发性高血压患者外周血单核细胞黏附活性及分泌活性,分析此时期单核细胞是否处于预激活状态。方法:①选择2003-01/2004-10华中科技大学同济医学院附属同济医院心血管内科门诊就诊原发性高血压患者30例。男16例,女14例;病程6～56个月。均为高血压Ⅰ～Ⅱ级,且对检测项目知情同意。选择同期本院血压正常的健康体检者30人为对照组,男女各15人;平均年龄(45±8)岁。均对检测项目知情同意。②分离出纳入对象静脉外周血单核细胞,培养后,调整细胞数至4×107L-1,接种到24孔培养板上。每份标本设3孔,分别为基础分泌孔、血管紧张素Ⅱ刺激孔(加入血管紧张素Ⅱ1×10-8mol/L)、氯沙坦预处理孔(在1×10-8mol/L血管紧张素Ⅱ刺激前先将外周血单核细胞与氯沙坦共同孵育30min)。③培养人脐静脉内皮细胞,待细胞生长至对数增长期后,每孔加入100μL外周血单核细胞悬液,37℃下分别孵育2,4h,经显微镜-计算机图像分析系统计数黏附细胞。高倍镜下每孔计数40个视野,取其平均值。④采用双抗体夹心酶链免疫吸附法,严格按照试剂盒说明测定外周血单核细胞培养上清肿瘤坏死因子α、白细胞介素1β及白细胞介素6水平。⑤采用反转录聚合酶链反应检测外周血单核细胞细胞因子基因表达。⑥组间计量资料差异性比较采用t检验。结果:原发性高血压患者30例和健康体检者30人均进入结果分析。①孵育2和4h,外周血单核细胞与内皮细胞黏附数:高血压组在基础状态下均与对照组相当(P>0.05);血管紧张素Ⅱ刺激后两组均升高,且高血压组明显多于对照组(t=2.445,5.656,P<0.05,0.01);氯沙坦预处理后两组又降至同一水平(P>0.05)。②外周血单核细胞培养上清肿瘤坏死因子α、白细胞介素1β及白细胞介素6水平:基础状态时,两组均较低,且差异不明显;经血管紧张素Ⅱ刺激后,高血压组明显高于对照组(t=2.537～6.984,P<0.05～0.01);氯沙坦预处理后两组均有不同程度降低,组间差异不明显。③外周血单核肿瘤坏死因子α、白细胞介素1β及白细胞介素6mRNA表达:在基础条件下两组处于同一水平;血管紧张素Ⅱ刺激后高血压组明显高于对照组(t=2.381～3.974,P<0.05～0.01);氯沙坦预处理后两组均减低,但差异不明显。结论:病程早期原发性高血压患者外周血单核细胞处于预激活状态。
AIM：To observe the activities of adhesion and secretion in PBMC of patients with essential hypertension （EH） during early stage, and analyze whether the PBMC is in pre-activated state.
METHODS ： ① Thirty outpatients with EH were selected from the Cardiovascular Department of Tongji Hospital Affiliated to Tongji Medical college, Huazhong University of Science and Technology between January 2003 to October 2004, including 16 males and 14 females with the course of disease of 6-56 months. All of them suffered from EH of grade Ⅰ - Ⅱ, and agreed with the detection. Meanwhile, 30 people with normal blood pressure, who received health examination in the hospital were taken as controls, including 15 males and 15 females with an mean age of （45＋8） years, and all agreed with the detection. ②The PBMC in vein of enrolled subjects were isolated. Number of cells after culture were regulated to 4×10^7 L^-1, and then incubated into 24-pore cultural board. There were 3 pores in each sample： pore of basal secretion, pore of angiotensin Ⅱ stimulation （angiotensin Ⅱ 1× 10^-8 mol/L was added） and pore of pretreatment with losartan （PBMC and losartan were incubated for 30 minutes before being stimulated by 1×10^-8 mol/L AGT Ⅱ ）.③Human umbilical vein endothelial ceils （HUVEC） were cultured. 100 μL of PBMC suspension was added in each pore after the increased logarithmic phase of cell growth, and incubated at 37 ℃ for respectively 2 and 4 hours. The cells of adhesion were counted by microscope-computer image analytical system. There were 40 fields in each pore under high-power lens, and the average value was calculated. ④Double antibody sandwich enzyme linked immunosorbent assay （ELISA） was used to measure the levels of tumor necrosis factor-α,interleukin-1β and interleukin-6 in supernatant of PBMC strictly according to the instruction of kits. ⑤ Reverse transcription-polymerase chain reaction （RT-PCR） was adopted to detect the gene expression of cell factor in PBMC. ⑥ Differences of measurement data among groups were compared with t test.
RESULTS：A total of 30 EH patients and 30 normal control were involved in the analysis of results.①At 2 and 4 hours after incubation, the adhesion number of PBMC and endothelial cells： those in the EH group under basic state were similar to those in the control group （P 〈 0.05）, and those in both groups were increased after stimulation by angiotensin Ⅱ, and those in the EH group were significantly higher than control group （t=2.445, 5.656, P 〈 0.05, 0.01）. After the pretreatment with losartan, those of both group were decreased to the same level （P 〈 0.05）. ②Levels of tumor necrosis factor-α,interleukin-1β and interleukin-6 in PBMC; under basic state, those in the two groups were much lower and differences were not significant. After the stimulation of angiotensin Ⅱ, those in the EH group were significantly higher than those in the control group （t=2.537-6.984,P 〈 0.05-0.01）, and levels of both groups after pretreatment with losartan were decreased to different extend, while differences among groups were not remarkable. ③ Expressions of mRNA in tumor necrosis factor-α, interleukin-1β and interleukin-6 of PBMC： those in both groups under basic condition were in the same level, whereas those in the EH group were remarkably higher than the control group after stimulation of angiotensin Ⅱ （t=2.381-3.974, P 〈 0.05-0.01）. Those in both groups after pretreatment of losartan were decreased, while the differences were not marked.
CONCLUSION ：The PBMC of EH patients during early stage are in prectivated state.
出处
《中国临床康复》
CAS
CSCD
北大核心
2006年第24期75-78,共4页
Chinese Journal of Clinical Rehabilitation
基金
湖北省科技攻关资助项目(2003AA301C47)~~
