表面增强拉曼光谱技术检测隐球菌脑膜炎患者血清中CD93^+外泌体及其意义

Detection of Serum CD93^+ Exosomes in Patients with Cryptococcal Meningitis by Surface-Enhanced Raman Scattering Technology and Its Significance

目的利用表面增强拉曼光谱技术(SERS)检测隐球菌脑膜炎患者血清中CD93^+外泌体,以优化该病的监测手段。方法收集2013年12月~2017年3月在上海长海医院和长征医院确诊的38例隐球菌脑膜炎患者的血清作为实验组,同期体检的38例健康者血清作为对照组。用ELISA法检测血清中的IFN-γ,TNF-α的蛋白表达水平;用超高速离心法分离血清外泌体;用磁珠连接抗体法捕获CD93^+外泌体并进行标记,于拉曼光谱仪下检测样本的SERS信号。实验组与对照组计量资料间的比较采用两独立样本的t检验,用Origin75软件对两连续变量进行线性拟合,两变量之间的相关性以Pearson系数表示。结果 SERS检测隐球菌脑膜炎患者血清中外泌体最低浓度为5 500个/ml,最低量为110个/ml,log(SERS信号)与log(外泌体浓度)进行线性拟合的R值为0.995 96,P<0.0001;SERS检测实验组与对照组血清外泌体CD93的信号值取对数分别为2.75±0.59vs 2.47±0.15,差异具有统计学意义(t=2.835,P=0.005 9);SERS检测实验组血清CD93^+外泌体得到的数据与ELISA法检测实验组血清中IFN-γ,TNF-α的结果呈正相关,差异有统计学意义(r=0.389,0.478,P<0.05)。结论该方法可初步提高隐球菌脑膜炎患者血清标本检测的灵敏度,为该病监测提供新的技术思路。

Objective To detect the serum CD93＋ exosomes of patients with cryptococcal meningitis by Surface-enhanced Raman Scattering technology（SERS）in order to optimize the measures to monitor this disease.Methods The serum samples of 38 patients with cryptococcal meningitis diagnosed in Changhai Hospital and Changzheng Hospital were collected from December 2013 to March 2017.In the same period,the serum samples of 38 health subjects were collected as control group.The protein expression level of IFN-γ,TNF-αin serums were examined by ELISA method.The exosomes in serum were separated by the ultracentrifuge method.The CD93~＋ exosomes were captured by magnetic bead-connected antibodies.Under the Raman spectroscopy,the SERS signals of samples was acquired.The comparison of measure data between experimental and controlled groups was measured by two independent samples＇ t test,Origin 75 software was used to do the linear fitting for two continuous variables,and the relationship between the two variables was measured by Pearson coefficient.ResultsThe lowest concentration of serous exosomes in patients with cryptococcal meningitis that could be tested by SERS was 5500 particles/ml,the lowest counts were 110 particles/ml.The Rvalue for linear fitting of log（SERS signal）and log（exosomes＇ concentration）was 0.995 96（P〈0.000 1）.The log values of SERS signals tested for CD93 on serous exosomes in experimental and controlled groups were 2.75±0.59 vs 2.47±0.15（t=2.835,P=0.005 9）,with statistical difference.The results from the CD93＋ exosomes in serum samples of experiment group were positively related with the ELISA results of IFN-γand TNF-αin serum samples of experiment group and there was statistical difference（r=0.389,0.478,P〈0.05）.Conclusion This method can primarily improve the sensitivity of the detection of serum samples in patients with cryptococcal meningitis and may provide a new technological way to detect this disease.