背景:外周血单核细胞预激活是动脉粥样硬化形成的重要早期事件和促进因素之一,丹参酮是从传统中药丹参中提取的一种脂溶性成分,具有确切的抗动脉粥样硬化作用.目的:通过检测外周血单核细胞的黏附活性与分泌活性,分析原发性高血压患者病程早期是否存在外周血单核细胞预激活,并探讨丹参酮对体外培养条件下外周血单核细胞激活的抑制作用.设计:病例对照分析.单位:华中科技大学同济医学院附属同济医院急诊科与中医药研究室.对象:选取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收治的未经治疗或已停用降压药物2周以上的原发性高血压患者30例,男16例,女14例;平均年龄(44.6±7.4)岁;体质量指数(26.2±4.5) kg/m2;平均病程(38.5±16.9)个月;对本实验均知情同意.根据1999年WHO/ISH的高血压诊断标准,均为高血压Ⅰ～Ⅱ级.排除继发性高血压、器质性心脏病、高脂血症、糖尿病、肝肾功能障碍、感染等临床情况以及高血压所致的心、脑、肾、血管等靶器官损害.另选择30例血压正常的健康体检者作为正常对照组.人脐静脉内皮细胞(武汉大学物种保存中心);丹参酮注射液(雅安三九药业有限公司,批号020724).方法:①入选者均抽取静脉血4.0～5.0 mL,采用Ficoll-Hypaque密度梯度离心法和塑料吸附法分离单个核细胞,37 ℃孵育40～60 min后收集贴壁细胞即为外周血单核细胞.瑞氏染色后镜下观察细胞形态符合单核细胞特征,锥虫蓝拒染试验测定活细胞数＞95%.将新鲜分离的外周血单核细胞重悬,按4×107 L-1接种到24孔培养板上,每份标本设3孔:第1孔为基础分泌孔,第2孔为血管紧张素Ⅱ刺激孔,第3孔为丹参酮预处理孔.在血管紧张素Ⅱ刺激前先将外周血单核细胞与丹参酮共同孵育30 min.血管紧张素Ⅱ、丹参酮终浓度分别为1×10-8 mol/L和1×10-8 g/L,外周血单核细胞终浓度为2×107 L-1.37 ℃下置于体积分数为0.05的CO2培养箱内24 h,分别收集培养上清和细胞成分.②制备外周血单核细胞悬液,密度调整至2.5×109 L-1.在24孔培养板上用含体积分数为0.1小牛血清的M199培养基培养人脐静脉内皮细胞,待细胞生长至对数增长期后,铺板汇成单层,每孔加入100 μL外周血单核细胞悬液,37 ℃下分别孵育2,4 h,去除未黏附细胞,戊二醛固定后计数黏附细胞,高倍镜下每孔计数40个视野,取其平均值.③采用双抗体夹心ELISA法和反转录-聚合酶链技术检测外周血单核细胞培养上清中肿瘤坏死因子α、白细胞介素1β、白细胞介素6的浓度及其mRNA的表达水平.主要观察指标:①外周血单核细胞黏附活性的变化.②外周血单核细胞培养上清中细胞因子浓度及其mRNA的表达.结果:①孵育2,4 h时外周血单核细胞与内皮细胞的黏附数,在基础状态下高血压组和正常对照组基本相似(t=1.153～1.577,P均＞0.05);血管紧张素Ⅱ刺激后高血压组明显多于正常对照组(t=3.842～4.536,P均＜0.01);丹参酮预处理后两组又降至同一水平(f=0.855～1.702,P均＞0.05).②基础状态时,两组培养上清中肿瘤坏死因子α、白细胞介素1β、白细胞介素6的浓度均较低(t=0.981～1.829,P均＞0.05);血管紧张素Ⅱ刺激后,高血压组各细胞因子浓度均明显高于正常对照组(t=2.442～5.075,P＜0.01,0.01,0.05);丹参酮预处理后两组各细胞因子浓度均有不同程度降低,但差异无显著性意义(f=1.227～1.940,P均＞0.05).两组外周血单核细胞中各细胞因子mRNA的表达与浓度变化基本相似.结论:①基础状态下原发性高血压患者外周血单核细胞与内皮细胞的黏附数及各炎性细胞因子浓度和mRNA表达处于正常人群水平,经血管紧张素Ⅱ刺激后显著升高,提示原发性高血压患者病程早期外周血单核细胞处于预激活状态.②丹参酮干预可使原发性高血压患者外周血单核细胞的黏附活性与分泌活性降至正常水平,证明丹参酮能够抑制预激活的外周血单核细胞进一步激活,一定程度上可防止动脉粥样硬化的发生.
揹景:外週血單覈細胞預激活是動脈粥樣硬化形成的重要早期事件和促進因素之一,丹參酮是從傳統中藥丹參中提取的一種脂溶性成分,具有確切的抗動脈粥樣硬化作用.目的:通過檢測外週血單覈細胞的黏附活性與分泌活性,分析原髮性高血壓患者病程早期是否存在外週血單覈細胞預激活,併探討丹參酮對體外培養條件下外週血單覈細胞激活的抑製作用.設計:病例對照分析.單位:華中科技大學同濟醫學院附屬同濟醫院急診科與中醫藥研究室.對象:選取2003-01/2004-10華中科技大學同濟醫學院附屬同濟醫院心血管內科收治的未經治療或已停用降壓藥物2週以上的原髮性高血壓患者30例,男16例,女14例;平均年齡(44.6±7.4)歲;體質量指數(26.2±4.5) kg/m2;平均病程(38.5±16.9)箇月;對本實驗均知情同意.根據1999年WHO/ISH的高血壓診斷標準,均為高血壓Ⅰ～Ⅱ級.排除繼髮性高血壓、器質性心髒病、高脂血癥、糖尿病、肝腎功能障礙、感染等臨床情況以及高血壓所緻的心、腦、腎、血管等靶器官損害.另選擇30例血壓正常的健康體檢者作為正常對照組.人臍靜脈內皮細胞(武漢大學物種保存中心);丹參酮註射液(雅安三九藥業有限公司,批號020724).方法:①入選者均抽取靜脈血4.0～5.0 mL,採用Ficoll-Hypaque密度梯度離心法和塑料吸附法分離單箇覈細胞,37 ℃孵育40～60 min後收集貼壁細胞即為外週血單覈細胞.瑞氏染色後鏡下觀察細胞形態符閤單覈細胞特徵,錐蟲藍拒染試驗測定活細胞數＞95%.將新鮮分離的外週血單覈細胞重懸,按4×107 L-1接種到24孔培養闆上,每份標本設3孔:第1孔為基礎分泌孔,第2孔為血管緊張素Ⅱ刺激孔,第3孔為丹參酮預處理孔.在血管緊張素Ⅱ刺激前先將外週血單覈細胞與丹參酮共同孵育30 min.血管緊張素Ⅱ、丹參酮終濃度分彆為1×10-8 mol/L和1×10-8 g/L,外週血單覈細胞終濃度為2×107 L-1.37 ℃下置于體積分數為0.05的CO2培養箱內24 h,分彆收集培養上清和細胞成分.②製備外週血單覈細胞懸液,密度調整至2.5×109 L-1.在24孔培養闆上用含體積分數為0.1小牛血清的M199培養基培養人臍靜脈內皮細胞,待細胞生長至對數增長期後,鋪闆彙成單層,每孔加入100 μL外週血單覈細胞懸液,37 ℃下分彆孵育2,4 h,去除未黏附細胞,戊二醛固定後計數黏附細胞,高倍鏡下每孔計數40箇視野,取其平均值.③採用雙抗體夾心ELISA法和反轉錄-聚閤酶鏈技術檢測外週血單覈細胞培養上清中腫瘤壞死因子α、白細胞介素1β、白細胞介素6的濃度及其mRNA的錶達水平.主要觀察指標:①外週血單覈細胞黏附活性的變化.②外週血單覈細胞培養上清中細胞因子濃度及其mRNA的錶達.結果:①孵育2,4 h時外週血單覈細胞與內皮細胞的黏附數,在基礎狀態下高血壓組和正常對照組基本相似(t=1.153～1.577,P均＞0.05);血管緊張素Ⅱ刺激後高血壓組明顯多于正常對照組(t=3.842～4.536,P均＜0.01);丹參酮預處理後兩組又降至同一水平(f=0.855～1.702,P均＞0.05).②基礎狀態時,兩組培養上清中腫瘤壞死因子α、白細胞介素1β、白細胞介素6的濃度均較低(t=0.981～1.829,P均＞0.05);血管緊張素Ⅱ刺激後,高血壓組各細胞因子濃度均明顯高于正常對照組(t=2.442～5.075,P＜0.01,0.01,0.05);丹參酮預處理後兩組各細胞因子濃度均有不同程度降低,但差異無顯著性意義(f=1.227～1.940,P均＞0.05).兩組外週血單覈細胞中各細胞因子mRNA的錶達與濃度變化基本相似.結論:①基礎狀態下原髮性高血壓患者外週血單覈細胞與內皮細胞的黏附數及各炎性細胞因子濃度和mRNA錶達處于正常人群水平,經血管緊張素Ⅱ刺激後顯著升高,提示原髮性高血壓患者病程早期外週血單覈細胞處于預激活狀態.②丹參酮榦預可使原髮性高血壓患者外週血單覈細胞的黏附活性與分泌活性降至正常水平,證明丹參酮能夠抑製預激活的外週血單覈細胞進一步激活,一定程度上可防止動脈粥樣硬化的髮生.
배경:외주혈단핵세포예격활시동맥죽양경화형성적중요조기사건화촉진인소지일,단삼동시종전통중약단삼중제취적일충지용성성분,구유학절적항동맥죽양경화작용.목적:통과검측외주혈단핵세포적점부활성여분비활성,분석원발성고혈압환자병정조기시부존재외주혈단핵세포예격활,병탐토단삼동대체외배양조건하외주혈단핵세포격활적억제작용.설계:병례대조분석.단위:화중과기대학동제의학원부속동제의원급진과여중의약연구실.대상:선취2003-01/2004-10화중과기대학동제의학원부속동제의원심혈관내과수치적미경치료혹이정용강압약물2주이상적원발성고혈압환자30례,남16례,녀14례;평균년령(44.6±7.4)세;체질량지수(26.2±4.5) kg/m2;평균병정(38.5±16.9)개월;대본실험균지정동의.근거1999년WHO/ISH적고혈압진단표준,균위고혈압Ⅰ～Ⅱ급.배제계발성고혈압、기질성심장병、고지혈증、당뇨병、간신공능장애、감염등림상정황이급고혈압소치적심、뇌、신、혈관등파기관손해.령선택30례혈압정상적건강체검자작위정상대조조.인제정맥내피세포(무한대학물충보존중심);단삼동주사액(아안삼구약업유한공사,비호020724).방법:①입선자균추취정맥혈4.0～5.0 mL,채용Ficoll-Hypaque밀도제도리심법화소료흡부법분리단개핵세포,37 ℃부육40～60 min후수집첩벽세포즉위외주혈단핵세포.서씨염색후경하관찰세포형태부합단핵세포특정,추충람거염시험측정활세포수＞95%.장신선분리적외주혈단핵세포중현,안4×107 L-1접충도24공배양판상,매빈표본설3공:제1공위기출분비공,제2공위혈관긴장소Ⅱ자격공,제3공위단삼동예처리공.재혈관긴장소Ⅱ자격전선장외주혈단핵세포여단삼동공동부육30 min.혈관긴장소Ⅱ、단삼동종농도분별위1×10-8 mol/L화1×10-8 g/L,외주혈단핵세포종농도위2×107 L-1.37 ℃하치우체적분수위0.05적CO2배양상내24 h,분별수집배양상청화세포성분.②제비외주혈단핵세포현액,밀도조정지2.5×109 L-1.재24공배양판상용함체적분수위0.1소우혈청적M199배양기배양인제정맥내피세포,대세포생장지대수증장기후,포판회성단층,매공가입100 μL외주혈단핵세포현액,37 ℃하분별부육2,4 h,거제미점부세포,무이철고정후계수점부세포,고배경하매공계수40개시야,취기평균치.③채용쌍항체협심ELISA법화반전록-취합매련기술검측외주혈단핵세포배양상청중종류배사인자α、백세포개소1β、백세포개소6적농도급기mRNA적표체수평.주요관찰지표:①외주혈단핵세포점부활성적변화.②외주혈단핵세포배양상청중세포인자농도급기mRNA적표체.결과:①부육2,4 h시외주혈단핵세포여내피세포적점부수,재기출상태하고혈압조화정상대조조기본상사(t=1.153～1.577,P균＞0.05);혈관긴장소Ⅱ자격후고혈압조명현다우정상대조조(t=3.842～4.536,P균＜0.01);단삼동예처리후량조우강지동일수평(f=0.855～1.702,P균＞0.05).②기출상태시,량조배양상청중종류배사인자α、백세포개소1β、백세포개소6적농도균교저(t=0.981～1.829,P균＞0.05);혈관긴장소Ⅱ자격후,고혈압조각세포인자농도균명현고우정상대조조(t=2.442～5.075,P＜0.01,0.01,0.05);단삼동예처리후량조각세포인자농도균유불동정도강저,단차이무현저성의의(f=1.227～1.940,P균＞0.05).량조외주혈단핵세포중각세포인자mRNA적표체여농도변화기본상사.결론:①기출상태하원발성고혈압환자외주혈단핵세포여내피세포적점부수급각염성세포인자농도화mRNA표체처우정상인군수평,경혈관긴장소Ⅱ자격후현저승고,제시원발성고혈압환자병정조기외주혈단핵세포처우예격활상태.②단삼동간예가사원발성고혈압환자외주혈단핵세포적점부활성여분비활성강지정상수평,증명단삼동능구억제예격활적외주혈단핵세포진일보격활,일정정도상가방지동맥죽양경화적발생.
BACKGROUND: Preactivation of peripheral blood mononuclear cells (PBMCS) is one of the most important eerly events and facilitating factor for the formation of atherosclerosis. Tanshinone is a lipolytic component extracted from traditional Chinese medicine of denshen, it has definite anti-atherosclerotic effect.OBJECTTVE: To analyze whether PBMCS preactivation existed at early essential hypertension, and investigate the effects of tanshinone on inhibiting the PBMCS activation cultured in vitro by detecting the adhesion and excretory activities of PBMCS.DESTGN: A case-controlled analysis.SETTING: Department of Emergency and Research Room of Traditional Chinese Medicine, Tongji Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology.PARTTCTPANTS: Thirty patients with untreated essential hypertension or with withdrawal from antihypertensives for at least 2 weeks were selected from the Department of Cardiology, Tongji Hospital affiliated to Tongji Medical College,Huazhong University of Science and Technology from January 2003 to October 2004, including 16 males and 14 females, aged (44.6±7.4) years, body mass index of (26.2±4.5) kg/m2, average disease course of (38.5±16.9) months.Informed contents were obtained from all the subjects. Their hypertension was grade Ⅰ-Ⅱ according to the diagnostic standards for hypertension by WHO/ISH in 1999. Secondary hypertension, organic heart disease, hyperglyceridemia,diabetes mellitus, liver and kidney dysfunction, heart, brain, kidney, vessel and other target damaged induced by infection and other clinical conditions and hypertension were excluded by history, physical examination and assistant examination.Another 30 healthy physical examinees with normal blood pressure were enrolled as the normal control group. Human umbilical vein endothelial cells (Species Reserving Center of Wuhan University); Tanshinone injection (Yaan Sanjiu Pharmaceutical, Co., Ltd., batch number: 020724);METHODS: ① Venous blood samples (4.0-5.0 mL) were drawn from all the subjects, and mononuclear cells were separated by means of Ficoll-Hypaque density gradient centrifugation and plastic adhesion, then incubated at 37 ℃ for 40-60 minutes, and the adherent cells were the PBMCS. These cells (viability ＞ 95%, Trypan blue staining) had the characteristics of mononuclear cells (Wright staining). The newly separated adherent PBMCS were resuspended, and then inoculated to the 24-well plate (4×107 L-1). There were 3 wells for each sample: the first was for basic excretion, the second for angiotensin Ⅱ stimulation, and the third for tanshinone pretreatment. The PBMCS were co-incubated with tanshinone for 30 minutes before angiotensin Ⅱ stimulation. The terminal concentration was 1×10-8 mol/L and 1×10-8 g/L for angiotensin Ⅱ andtanshinone respectively, and that of PBMCS was 2×107 L-1. The cells were cultured in the incubator (CO2 of 0.05 in volume fraction) at 37 ℃ for 24 hours, then the supernatant and cell ingredients were collected respectively. ② The PBMCS suspension was preparedl, and the cellular density was adjusted to 2.5×109 L-1. The human umbilical vein endothelial cells were cultured on the 24-well plate with M199 medium containing fetal bovine serum (0.1 in volume fraction), and spread to monolayer after the cells entered the logarithm phase. Each well was added with PBMCS suspension (100 μL), incubated at 37 ℃ for 2 and 4 hours respectively. The unadherent cells were removed, and the adherent ones were counted after fixed with 20 g/L glutaral, 40 visual sights were counted for each well under high power microscope, and the average value was used. ③ The double-antibody sandwich enzyme-linked immunoabsorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the concentrations of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) in supernatant of PBMCS, and the expressions of their mRNA.MATN OUTCOME MEASURES: ① Changes of PBMCS adhesion activity; ② Concentrations of cytokines and their mRNA expressions in supernatant of PBMCS.RESULTS: ① At 2 and 4 hours of inoculation, the numbers of PBMCS adhered to endothelial cells under basic conditions were similar between the hypertension group and normal control group (t =1.153-1.577, P ＞ 0.05); After angiotensin Ⅱ stimulation, the adherent cells were obviously more in the hypertension group than in the normal control group (t =3.842-4.536, P ＜ 0.01); The numbers of the adherent cells were decreased to the same levels after tanshinone pretreatment (t =0.855-1.702, P ＞ 0.05). ②Under basic state, the concentrations of TNF-α, IL-1β and IL-6 in supernatant were all lower in both groups (t =0.981-1.829, P ＞ 0.05); The concentrations of the cytokines after angiotensin Ⅱ stimulation were obviously higher in the hypertension group than in the normal control group (t = 2.442, 5.075, P ＜ 0.01,0.01, 0.05); The concentrations of the cytokines after tanshinone pretreatment were all decreased to different extent, and there were no significant differences (t =1.227-1.940, P ＞ 0.05). Similar changes were observed in the mRNA expressions of the cytokines in PBMCS in the two groups.CONCLUSTON: ① The number of PBMCS adhered to endothelial cells, the concentrations and mRNA expressions of the cytokines under basic state in patients with essential hypertension were at the levels of normal subjects, and they were significantly increased after angiotensin Ⅱ stimulation, suggesting that the PBMCS were at preactivation at early essential hypertension. ② Tanshinone could decrease the adhesion and excretory activities of PBMCS in patients with essential hypertension to the normal levels, it is proved that tanshinone can inhibit the further activation of the preactivated PBMCS, and can prevent the occurrence of atherosclerosis to some extent.
