新型荧光衍生法测定细胞凋亡关键酶活性

发布时间：2018-01-15 14:42

本文关键词：新型荧光衍生法测定细胞凋亡关键酶活性　出处：《复旦大学》2010年硕士论文　论文类型：学位论文

【摘要】：细胞凋亡是细胞的一种基本生物学现象,在多细胞生物去除不需要的或异常的细胞中起着必要的作用。它在生物体的进化、内环境的稳定以及多个系统的发育中起着重要的作用。尽管凋亡过程的详细机制尚不完全清楚,但是已经确定Caspase即半胱天冬蛋白酶在凋亡过程中是起着必不可少的作用,细胞凋亡的过程实际上是Caspase不可逆有限水解底物的级联放大反应过程,到目前为止,至少已有14种Caspase被发现,Caspase分子间的同源性很高,结构相似,都是半胱氨酸家族蛋白酶。 Caspase酶是细胞凋亡关键酶,因此其活性检测就成为判断细胞凋亡程度的重要标准。最近,我们课题组发现多肽能够与儿茶酚和高碘酸钠在pH7.5进行缩合反应,形成单一的荧光产物,激发最大波长在390nm附近,发射波长在490nm左右。更为重要的是,自由氨基酸、葡萄糖和半乳糖等糖类化合物、精胺等多胺化合物以及DNA碱基如腺嘌呤、鸟嘌呤、胸腺嘧啶和胞嘧啶等相同反应条件下均不产生荧光。因此,新型荧光衍生方法检测caspase酶的活性专属性强,操作简便,利于推广,具有一定的应用前景。课题第一部分首先对寡肽荧光衍生条件进行了优化,在此基础上再对高效液相分析条件进行了优化。优化后的荧光衍生体系为：供试液100μL,儿茶酚(10mmol/L) 100μL,高碘酸钠(12 mmol/L)50μL,硼酸(300 mmol/L,pH 7.0)50μL,加热温度120℃,加热时间10 min。优化后的色谱条件为：色谱柱：CAPCELL PAK C18 MGS5 (5 u m,150 mm X 4.6 mm I.D.);流动相：A(250 mmol/L硼酸,氢氧化钠调pH值到7.5,20%)：B(水,80-0%)：C(甲醇,0-80%)(v/v/v),梯度时间40 min；流速为1.0 mL/min;柱温：30℃；荧光检测波长：激发波长390 nm,发射波长490 nm；进样量：20μL 课题第二部分用新型荧光衍生方法测定亮氨酸脑啡肽并与紫外检测方法、OPA衍生法进行了比较。亮氨酸脑啡肽是一种非常重要的内源肽,其氨基酸顺序和结构为：酪氨酸-甘氨酸-甘氨酸-苯丙氨酸-亮氨酸Tyr-Gly-Gly-Phe-Leu)。体外试验中,保留时间为25min,线性范围为0.5～50.0 mg/L(r=0.9999),最低定量限为0.25 mg/L；模拟血浆实验中,内源物质不干扰测定,线性范围为0.5～50.0mg/L(r=0.9998),最低定量限为0.5 mg/L。血浆中内源性干扰物质多,紫外检测方法和OPA衍生法都不能有效检测亮氨酸脑啡肽的含量。课题第三部分是本课题重点,利用新型荧光衍生方法来检测两种细胞系人宫颈癌细胞(Hela细胞)和人肺腺癌细胞(A549细胞)受诱导凋亡后的caspase-3和caspase-8的活性。每种细胞系均采用三种诱导凋亡方法：低温辅助紫外照射诱导凋亡,丝裂霉素C诱导凋亡和喜树碱诱导凋亡。Caspase酶底物设计为在特异识别序列N端加上具有亲水性氨基酸丝氨酸,这可以增加底物水溶性,N端延长序列同时能增加底物专属性,C端加上实验证明衍生效率较高的两种寡肽片段LG和ALG。本部分采用市场上常用的caspase-3,8活性检测试剂盒作为新型荧光衍生方法的对照,结果表明,两种方法的活性检测相似(r=0.9556,P0.01),表明新方法具有可靠性,同时,重组caspase酶实验表明新方法具有更好的专属性,因此新型荧光衍生方法可以作为检测caspase酶活性的可靠方法。
[Abstract]:Apoptosis is a basic biological phenomenon of cells, plays a necessary role in multicellular organisms to remove unwanted or abnormal cells. It is in the evolution of organisms, plays an important role in the stability of the environment and the development of multiple systems. Although apoptosis detailed mechanism still is not entirely clear. But Caspase has been confirmed that caspase in the apoptotic process is play an essential role, the process of apoptosis is irreversible proteolytic cascade Caspase co amplification reaction process, so far, has at least 14 Caspase was found between Caspase molecules have high homology and similar structure, all cysteine proteases.
Caspase was a key enzyme of apoptosis, therefore its activity detection has become an important standard to judge the extent of apoptosis. Recently, our research group found that peptides can condensation reaction with catechol and sodium periodate in pH7.5, forming a fluorescent single product, stimulate the maximum wavelength in the vicinity of 390nm, the emission wavelength at about 490nm more. It is important that the free amino acid, glucose and galactose in carbohydrate, spermine and polyamine compounds such as DNA bases adenine, guanine, thymine and cytosine etc. under the same reaction conditions had no fluorescence. Therefore, a new fluorescence derivatization method for detection of caspase enzyme activity of strong specificity, simple operation, conducive to the promotion. It has a certain application prospect.
The first part of the paper oligopeptide fluorescence derivatization conditions were optimized based on the HPLC analysis conditions were optimized. The optimized system for fluorescent derivative: test solution 100 L, catechol (10mmol/L) 100 L, sodium periodate (12 mmol/L) 50 L, boric acid (300 mmol/L, 7 pH) 50 L, heating temperature of 120 DEG C, heating time 10 min. chromatographic conditions were optimized: the chromatographic column: CAPCELL PAK C18 MGS5 (5 u m, 150 mm X 4.6 mm I.D.); mobile phase: A (250 mmol/L boric acid, sodium hydroxide to adjust the pH value to 7.5,20%:B) (water, 80-0%):C (methanol, 0-80%) (v/v/v), gradient time 40 min; the flow rate was 1 mL/min; column temperature: 30 C; fluorescence detection wavelength: excitation wavelength 390 nm, emission wavelength of 490 nm; sample size: 20 L
The second part of the study with new fluorescence derivatization method for the determination of leucine enkephalin and detected with UV method, compares the OPA derivative method. Leucine enkephalin is an important endogenous peptide, amino acid sequence and structure: Tyr Gly Gly phe - leucine Tyr-Gly-Gly-Phe-Leu). In vitro, retention time is 25min, the linear range was 0.5 ~ 50 mg/L (r=0.9999), the lowest detection limit of 0.25 mg/L; plasma simulation experiment, endogenous substances did not interfere with the determination, the linear range of 0.5 ~ 50.0mg/L (r=0.9998), the lower limit of quantification was 0.5 mg/L. in plasma endogenous interference substances, the content of UV detection method and OPA derivative method is not effective in detecting leucine enkephalin.
The third part is the emphases of this paper, the derivative method to detect two cell lines of human cervical cancer cells by using fluorescent (Hela cells) and human lung adenocarcinoma cells (A549 cells) induced by apoptosis after caspase-3 and caspase-8. The activity of each cell line were used three methods: apoptosis induced by hypothermia apoptosis UV irradiation induced apoptosis induced by mitomycin C, and camptothecin induced apoptosis of.Caspase enzyme substrate is designed with hydrophilic amino acid serine in the specific recognition sequence of N terminal, which can increase the water solubility of the substrate, N can also increase the end of an extended sequence of substrate specificity, were added to C - the results show that the two widow LG ALG. derived peptide fragments and high efficiency by adopting the market commonly used caspase-3,8 activity detection kit as a new fluorescence derivatization method. The results show that the two methods have similar activity (r= 0.9556, P0.01) It indicates that the new method is reliable. Meanwhile, the recombinant caspase enzyme experiment shows that the new method has better specificity. Therefore, the new fluorescence derivatization method can be used as a reliable method to detect caspase enzyme activity.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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