疫苗诱导的特异性细胞杀伤活性检测方法的建立

目的建立一种基于流式细胞术的评价疫苗诱导的特异性细胞杀伤活性的方法,以完善疫苗及基因治疗药物的评价方法。方法用羧基荧光素二乙酸盐琥珀酰亚胺酯(Carboxyfluorescein diacetate,succinimidyl ester,CFSE)标记淋巴细胞,利用肿瘤坏死因子(Tumor necrosis factor,TNFα)模拟杀伤淋巴细胞,用碘化丙啶(Propidium iodide,PI)染色,流式细胞仪检测,确定CFSE和PI的浓度及作用时间。利用已确知有较强细胞免疫作用的治疗性乙型肝炎疫苗免疫小鼠,分离特异性淋巴细胞并用特异性肽刺激,分离未免疫小鼠的淋巴细胞作为靶细胞,并用特异性抗原肽致敏,并从靶细胞的标记、效应细胞培养时间,效靶作用时间、效靶比几方面进行优化,确定试验方法的操作流程。结果采用CFSE/PI双标记能有效分离实验所需各组群,细胞分为CFSE+PI-、CFSE+PI+、CFSE-PI+、CFSE-PI-4个组群,可区分活细胞和凋亡细胞。CFSE标记靶细胞的时间为6 h;效应细胞培养时间为72 h;效靶作用时间为6 h;效靶比可使用100∶1和50∶1。结论建立了基于流式细胞术的评价疫苗诱导的特异性细胞杀伤活性的方法,该方法可有效和精确地评价CTL杀伤效应,完善了疫苗及基因治疗药物的评价方法。     

N-琥珀酰亚胺-4-~(18)F(氟甲基)苯甲酸酯的合成

合成了^18F标记蛋白常用中间体N-琥珀酰亚胺-4-^18F（氟甲基）苯甲酸酯（^18F-SFMB），用操作简单的Sep-Pak硅胶柱代替了繁琐耗时的HPLC分离。探讨了反应溶剂、温度、K/222与K2CO3的摩尔比及反应时间等诸因素对^18F-SFMB放化合成的影响。得出较佳标记条件：以无水乙腈为溶剂，K/222与K2CO3摩尔比为1，80℃下反应5min，标记率为57%，比文献值提高了3倍以上。对IgG进行了标记，在室温下反应15min，标记率可达88%。     

胰高血糖素样肽-1在乙醇中的聚乙二醇修饰

以乙醇为溶剂,单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC)为修饰剂,对胰高血糖素样肽-1(GLP-1)进行了修饰反应条件的优化,同时对单修饰产物Mono-PEG-GLP-1进行分离纯化,并考察了其体外稳定性和体内活性. 得到的优化反应条件为：GLP-1浓度1 mg/mL, mPEG-SC与GLP-1摩尔比1:1, 37℃下反应24 h,该产物最高转化率达77%. 采用冷冻离心方式对Mono-PEG-GLP-1进行初步分离和浓缩,然后经反相液相色谱进一步高效纯化,纯度可达97%以上. 体外实验表明,Mono-PEG-GLP-1在血清中具有更好的稳定性及更强的抗胰蛋白酶消化能力. 体内动物实验表明,Mono-PEG-GLP-1具有更好的降血糖作用.     

A Realtime and Continuous Assessment of Cortisol in ISF Using Electrochemical Impedance Spectroscopy

This study describes the functioning of a novel sensor to measure cortisol concentration in the interstitial fluid (ISF) of a human subject. ISF is extracted by means of vacuum pressure from micropores created on the stratum corneum layer of the skin. The pores are produced by focusing a near infrared laser on a layer of black dye material attached to the skin. The pores are viable for approximately three days after skin poration. Cortisol measurements are based on electrochemical impedance (EIS) technique. Gold microelectrode arrays functionalized with Dithiobis (succinimidyl propionate) self-assembled monolayer (SAM) have been used to fabricate an ultrasensitive, disposable, electrochemical cortisol immunosensor. The biosensor was successfully used for in-vitro measurement of cortisol in ISF. Tests in a laboratory setup show that the sensor exhibits a linear response to cortisol concentrations in the range 1 pm to 100 nM. A small pilot clinical study showed that in-vitro immunosensor readings, when compared with commercial evaluation using enzyme-linked immunoassay (ELISA) method, correlated well with cortisol levels in saliva and ISF. Further, circadian rhythm could be established between the subject's ISF and the saliva samples collected over 24 hours time-period. Cortisol levels in ISF were found reliably higher than in saliva. This Research establishes the feasibility of using impedance based biosensor architecture for a disposable, wearable cortisol detector. The projected commercial in-vivo real-time cortisol sensor device, besides being minimally invasive, will allow continuous ISF harvesting and cortisol monitoring over 24 hours even when the subject is asleep. Forthcoming, this sensor could be interfaced to a wireless health monitoring system that could transfer sensor data over existing wide-area networks such as the internet and a cellular phone network to enable real-time remote monitoring of subjects.     

胰高血糖素样肽-1的聚乙二醇定点修饰

采用分子量为5 kDa的单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC)修饰胰高血糖素样肽-1(GLP-1),建立了GLP-1的定点修饰反应及分离纯化工艺,考察了修饰反应各因素对单修饰产物得率及体外活性的影响,得到优化修饰条件为：pH 7.5, 50 mmol/L Na2HPO4-NaH2PO4缓冲液,GLP-1浓度1 mg/mL, PEG/GLP-1摩尔比2:1, 4℃下反应1 h. 在此条件下,单修饰PEG-GLP-1得率达50%. 反相高效液相色谱分离纯化所得单修饰产品纯度达98%,体外活性保留为未修饰GLP-1的86%,其在Sprague-Dawley远交群大鼠体内的循环半衰期为60 min,比原肽提高了20倍.     

三种苯甲酸琥珀酰亚胺酯的合成

以对甲氧基苯甲酸、3,4-二甲氧基苯甲酸、3．4,5－三甲氧基苯甲酸为原料,以三氟乙酸N-琥珀酰亚胺酯为活化剂合成了3种苯甲酸琥珀酰亚胺酯,经^1HNMR、IR对其结构进行了表征,并对反应投料比及反应时间进行了优化。     

新型罗丹明类荧光标记探针的研究(Ⅰ)--氨基标记产物的质子化互变异构

罗丹明B-N-琥珀酰亚胺酯可用于生命体系物质氨基标记的新型荧光探针。研究了标记产物在不同的pH介质中的紫外一可见及荧光光谱特性，结合质子化互变异构机理，对互变异构现象与光谱变化的相关性进行了考察，确认了仲酰胺在中性和碱性条件下形成无荧光性的螺环内酰胺结构。     

Rapid detection of E. coli O157:H7 on turnip greens using a modified gold biosensor combined with light microscopic imaging system

This research aims to demonstrate the feasibility of a modified gold biosensor to detect E. coli O157:H7 in leafy turnip greens. The gold biosensor was modified with dithiobis-succinimidyl propionate (DSP) and/or protein A or G. The gold biosensor modified with DSP (Gold-DSP) was combined with a light microscopic imaging system (LMIS). The optimal concentration and specificity of anti-E. coli O15 polyclonal antibodies (pAbs) on the biosensor were determined. The reliability of Gold-DSP biosensor was investigated by determining the sensitivity, specificity, and limit of detection (LOD) of the Gold-DSP combined with LMIS. The Gold-DSP combined with LMIS was applied to turnip greens for E. coli O157:H7 detection. The modification of Gold biosensor with DSP significantly increased the detected number of E. coli O157:H7. The specificity of pAbs was sufficient to react with target E. coli O157:H7 among the tested bacterial culture. The optimum concentration of pAbs was determined as 200 μg/mL. The sensitivity, specificity, and LOD of Gold-DSP combined with LMIS were determined as 100%, 90%, and 10(3) CFU/25 mm(2) , respectively. When applied to turnip greens, the Gold-DSP combined with LMIS could detect 2641 ± 394 and 15383 ± 3853 cell/mm(2) with the initial concentrations of 10(1) and 10(2) CFU/25 g turnip greens, respectively, after 10 h-enrichment. Overall, this research suggested that the Gold-DSP combined with LMIS could be used to detect E. coli O157:H7 on turnip greens qualitatively and quantitatively.