鼻咽癌患者糖基化磷脂酰肌醇特异性磷脂酶D活性及其表达水平的初步研究

Primary Study on the GPI-PLD activity and its expression in Nasopharyngeal carcinoma patients

目的①通过研究正常人和鼻咽癌患者的糖基化磷脂酰肌醇特异性磷脂酶D(GPI-PLD)的酶活性和酶促反应的3-D图,探索鼻咽癌患者该酶活性的变化情况及该酶促反应的可能机制。②通过研究正常人和鼻咽癌患者GPI-PLD mRNA的表达水平,探寻鼻咽癌患者该酶活性变化的可能机制,为鼻咽癌的诊断、预后估计提供有效指标。方法①采用我室自制的具完整GPI结构的胎盘型碱性磷酸酶(PLAP)作底物进行酶促反应,测定正常人和鼻咽癌患者GPI-PLD的酶活性并应用韩国新科有限公司生产的Photodiode Array Uv-Vis Spectrophotometer仪器研究了该酶的催化机理,并且利用该机所带软件作出3-D图。②采用逆转录PCR(RT-PCR)检测正常人和鼻咽癌患者的GPI-PLD mRNA的表达水平,以RT-PCR结果的琼脂糖电泳照片的积分光密度值(IA)比值代表GPI-PLD mRNA的表达水平。结果①正常人和鼻咽癌患者血清GPI-PLD的活性水平(均以转化底物百分比表示)分别为17.6%±2.7%和20.6%±2.4%,鼻咽癌患者的血清GPI-PLD活性比正常人血清GPI-PLD活性显著增高(P<0.05)。并且其酶促反应的3-D图显示有直观差异。2.RT-PCR检测GPI-PLD mRNA的表达,结果表明正常人鼻咽上皮细胞中GPI-PLD mRNA琼脂糖电泳照片的平均积分光密度比值(IA比值)是2.6131±0.7653,鼻咽癌患者鼻咽上皮细胞中GPI-PLD mRNA琼脂糖电泳照片的平均积分光密度比值(IA比值)是9.3522±5.2879,鼻咽癌患者的GPI-PLD mRNA的表达水平比正常人鼻咽上皮细胞中GPI-PLD mRNA的表达水平显著性增高(P<0.05)。结论①鼻咽癌患者的血清GPI-PLD活性比正常人显著增高,其酶促反应的3-D图也与正常人有明显差异,提示测定GPI-PLD活性可作为临床诊断鼻咽癌的生化指标。②鼻咽癌患者GPI-PLD mRNA的表达水平高于正常人。GPI-PLD mRNA表达增强是导致鼻咽癌患者GPI-PLD酶活性增高的主要机制。

Objective ①To investigate the glycosylphosphatidylinositol-specific phospholipase D（GPI-PLD） activity and the 3-D fig change of GPI-PLD enzyme reaction in normal controls and Na- sopharyngeal carcinoma （NPC） patients, also to study the possible mechanism of this enzyme reaction. ②To study GPI-PLD expression level of mRNA in normal controls and NPC patients and the possible mecha- nism of this enzyme activity change in NPC. We also try to use GPI-PLD as an item to directly demonstrate and to diagnosis NPC with primary explore. Methods ① We used GPI-anchored placental alkaline phos- phatase （PLAP）, produced by our laboratory, as substrate to determine enzyme reaction, measure the GPI- PLD activity in normal controls and NPC patients. Using the Photodiode Array Uv-Vis Spectrophotometermade in South korea scico limitied company to study mechanism of GPI-PLD enzyme reaction. Using this machine self owned software to describe mechanism in GPI-PLD 3-D figs. ②We used reverse transcrip- tion polymerase chain reaction （RT-PCR） to measure expression level of mRNA of GPI-PLD in normal controls and NPC patients. The results were demonstrated by IA ratio. Results ①The GPI-PLD activity in serum from normal controls and from NPC patients were 17.6% ±2.7% and 20.6% ± 2.4% separately. The results showed higher GPI-PLD activity in serum from NPC patients than normal controls（P〈0.05）. TheGPI-PLD 3-D figs have straight diversity between NPC patients and normal controls. ②The GPI-PLD mRNA levels showed by IA ratio in nasopharyngeal cells from normal controls and from NPC patients were 2.6131±0.7653 and 9.3522±5.2879 separately. GPI-PLD mRNA levels were significantly higher in NPC patients than in normal conSols（P〈0.05）. Conclusions ①GPI-PLD activity were higher in NPC pa- tients than in normal controls （P〈0.05） and GPI-PLD 3-D figs have straight diversity. All these suggested GPI-PLD activity may be used as a biochemical indicator for NPC diagnosis and treatment. ②GPI-PLD mRNA levels were significantly higher in NPC patients than the normal controls. So we can primarily con- clude that the increase of the GPI-PLD mRNA expression is the cause of the increase of GPI-PLD activity in NPC patients.