应用生物发光法监测CD4^+细胞三磷酸腺苷水平评估肝移植术后患者的免疫功能

Application of adenosine triphosphate-bioluminescence assay in monitoring immune function of CD4^+ cell following liver transplantation

目的:评估三磷酸腺苷生物发光法检测肝移植受者早期CD4+细胞免疫功能的变化。方法:选择2004-06/10在解放军第二军医大学长征医院器官移植研究所进行的30例肝移植受者手术前后(0,1,2,4周)和50例健康志愿者。采用三磷酸腺苷生物发光法通过植物凝血素刺激后CD4+细胞三磷酸腺苷量的变化来监测细胞的免疫功能。①三磷酸腺苷标准品的检测:用培养基将三磷酸腺苷标准品配制成系列浓度:1,10,1×102,1×103和1×104μg/L,加入等量的Celltiter-GloTM荧光试剂混匀,取200μL进行测定,取对数制作标准曲线。②样本三磷酸腺苷检测:采集两组人员全血样本(肝素抗凝),置于细胞培养板过夜,加入含有洗涤过的抗人CD4单抗免疫磁粒悬浮液,用磁珠强效分离磁架将CD4+细胞吸于培养孔的一侧,用磷酸盐缓冲液洗3～5次,加入培养基(+体积分数为0.1的胎牛血清),加入Celltiter-GloTM试剂,1h内用荧光仪(发射波长562nm)读取荧光发光信号,计算其平均数值。③外周血T淋巴细胞亚群检测:肝素钠抗凝血加相应的抗体,放于Q-prep,workstation上处理后上机,计算出阳性细胞的百分率;或标记后的标本同样处理后,加混匀的Flow-Count标准荧光小球到标本管2h内上机,计算出CD4+细胞的绝对计数。④检测他克莫司血药浓度。结果:①在三磷酸腺苷浓度为1×(100～104)μg/L之间时,三磷酸腺苷标准品与荧光发光信号呈线性关系。直线方程为Y=0.75X+2.37(r=0.99484)。②移植组患者三磷酸腺苷明显低于健康人群犤(350±191),(735±370)μg/L,P<0.05犦,术后1周显著下降为(161±107)μg/L,而后逐渐提高。表明肝移植受者的细胞免疫功能术后第1周明显下降,以后逐渐恢复,第4周仍低于术前水平。③反应细胞免疫功能的三磷酸腺苷量与免疫抑制剂他克莫司血药浓度缺乏直接的相关性(r2=0.02)。结论:三磷酸腺苷生物发光法有利于正确评估肝移植后患者的细胞免疫功能。

AIM： To assess the application of adenosine triphosphate （ATP）-bioluminescence assay in measuring CD4＋ cell immune function early post-liver transplantation. METHODS： Thirty patients who undergone liver transplantation at Institute of Organ Transplantation of Changzheng Hospital, Second Military Medical University of Chinese PLA between June and October 2004 were chosen to assay the immune response before （0 week） and after transplantation （1, 2 and 4 weeks after transplantation）, and 50 healthy volunteers were used as controls. ATP-bioluminescence assay was used to determine the immune response of CD4＋ cells by ATP syntheses which stimulated by phytohemagglutinin（PHA-L）. （1）Detection of ATP standard specimen： The ATP standard substance were diluted with nutrilit at different concentrations： 1,10,1×10^2,1×10^3 and 1×10^4 μg/L. 200μL of each diluted standard specimen was measured after mixed with the same volume CelltiterGlo^TM reagent, then ATP calibration concentrations vs. RLU values were plotted on a log-log scale, and linear regression analysis generated a calibration curve. （2） Detection of ATP specimen： The whole blood of two groups with or without PHA-L was incubated overnight. Suspension with anti-human CD4 monoclonal body coated magnetic particles was added. Phosphate buffer solution （PBS） was used to .wash the CD4＋ cells selected on a strong magnet. Culture medium （＋ 0.1 volume fraction fetal bovine serum） and Celltiter-GloTM were added. Within one hour, fluorometer （emission wavelength 562 nm） was used to read the intensity of luminescence signaling and calculated the mean value. （3） Measurement of peripheral blood T lymphocyte subsets： PHA-L and corresponding antibody were treated on Q-prep, workstation and the percent of positive cells was calculated; Specimen after labelling was treated as the same, and Flow-Count standard fluorescent micro-ball was added in specimen tube. Absolute counting of CD4＋ cells was calculated within 2 hours.（4）The tacrolimus level in blood was routinely assayed. RESULTS： （1）There was a linear relationship between log ATP and the log luminescence in a range of 1×（10^0-10^4） μg/L . The liner equation was Y=0.75X＋2.37（r=0.99484）. （2） The liver transplant recipients had the significant lower cell mediated immune function than normal healthy volunteers[（350±191 ） vs （735±370） μg/L,P 〈 0.05]. ATP level decreased sig- nificantly in the first week postransplantation[（161 ±107）μg/L], then increased gradually. It indicated that the immune function of transplant recipients declined sharply in the first week post-transplant then increased gradually. However, immune function in the fourth week was lower than that before transplant. （3） ATP level which reflected immune function lacked of direct correlation with the taerolimus level in blood（r^2=0.02）. CONCLUSION： ATP-biolumineseenee assay is a better measurement in assessing immune function post-transplantation exactly.