| Objectives:Gliomas are the most common central nervous system tumors of the body,whose incidence accounting for more than half of the central nervous system tumors.According to different pathological manifestations,they are generally divided into four levels(Ⅰ ~ Ⅳ).Although the overall incidence of gliomas among human tumors is low,they still have a high mortality rate on account of difficulty to completely removing with surgery and easily postoperative recurrence.At present,it is difficult to diagnose gliomas.The traditional diagnostic methods currently still depend on the surgical biopsy.The pathological tissues from the operation are dehydrated and soaked in paraffin,sliced into slices of wax,sliced,and stained oil microscope observation,and the diagnosis of staging.Pathological tissues in the operation are from a invasive operation,which causes greater damages to patients,and at the same time different pathologist level leads to different diagnosis of gliomas.Because taking pathologicatissue in the operation is an invasive operation,not only it does harm to patients,Severe cases can cause postoperative bleeding leading to intracranial hypertension and endanger the lives of patients,and some patients can not take the pathological tissue biopsy due to the complexity of the disease.For example: patients have bleeding tendency,severe hypertension,intracranialinfection.Metabolomics reveal the intricate mechanism of biological regulation in biological systems,with the methods of the qualitative and quantitative analysis of metabolites of small biological molecules.Metabolites are the final products of a variety of physiological and pathological processes that most directly reflect the state of the human body.The occurrence of gliomas is closely related to the development of intracellular metabolism.The purpose of our study is to carry through preliminary diagnosis of the malignancy of the neoplasm and reveal the link between the metabolites and the disease development and assist in the clinical diagnosis of glioma malignancy without the need for biopsy,and thereby reduce the patient’s economic and psychological burdens with the methods of metabonomics and the combination of liquid chromatography mass spectrometry(LC-MS).Our study is aiming atrelationship of metabolites and benign and malignant gliomas from the perspective of metabonomics.Methods:Metabolites associated with the malignancy of gliomas require two different derivatization methods to derivative of the sample,respectively,using different derivatizing agents.In our experiment,Acc QTagderivatization kits,N-ethylmaleimide(NEM)were used to derive the hypotaurine metabolic pathway and reduced glutathione.hypotaurine metabolic pathway Glioma cells(U251)were analyzed by liquid chromatography-mass spectrometry(LC-MS):Sample derivation depending on Acc QTagderivatization kits were used to remove the samples from lyophilization to room temperature and to the lyophilized samples 70μL of borate buffer,vortexed for 30 s on the vortexer,20μL of derivatization reagent added to the sample,vortexed for 10 s,centrifuged at 14000 prm centrifuge for 30 s,taken out for 1 minute at room temperature,room temperature and to the lyophilized samples 70μL of borate buffer,vortexed for 30 s on the vortexer,20μL of derivatization reagent added to the sample,vortexed for 10 s,centrifuged at 14000 prm centrifuge for 30 s,taken out for 1 minute at room temperature,and then placed Into an electronic water bath at 55 ℃ water bath derived 10 min.Detection of reduced glutathione Glioma cells(U251)were analyzed by liquid chromatography-mass spectrometry(LC-MS): First,0.0061 g of reduced glutathione was dissolved in 1 ml of 50% aqueous methanol and weighed 0.030 g of N-ethylmaleimide(NEM)was dissolved in 1ml of 50% aqueous solution of methanol,and the two were mixed and vortexed for 30 s on a vortexer,Than 45 minutes at room temperature.With the method established,we need to make a standard curve and evaluate the intraday and daytime precision as well as the intracellular standard recovery,Intraday precision generally controlled within 5%,but not more than 10%.The daytime precision controlled at 15%.Spike recovery is the pretreatment and equipment testing the entire process of testing substances pretreatment conditions(Including whether all of them are extracted from the sample or whether they have been lost or contaminated during the pretreatment),in order to determine the feasibility of the experiment method.Spike recovery equals standard cell mass after spiking minus standard cell mass before spiking divided by spike mass,which is necessarybeween90% and 110% according to our laboratory requirements.The validation data showed that the method had good linear correlation coefficient(0.9927~0.9997)for 5 AQC derivatized amino acids;reproducible RSD was 2.5%~8.6%;intraday and inter-day precision RSD were 3.41%~9.53% and 6.23% ~ 9.23%;Cell spike recovery rate of 93% ~ 106%.The correlation coefficients of GSH and GSSH were 0.9997 and 0.9869,respectively.The intraday precision RSD was 5.41% and 9.53%,and the intraday precision RSD was 7.23% and 8.46%,respectively.The spiked recoveries were 103% and 96%,respectively.Results: Through the methodological evaluation of this experimental method,The linear regression analysis of experimental methods,intraday precision and daytime precision,and a comprehensive assessment of intracellular standard recovery rate.Our experimental method can carry through absolute quantitative detection for reduced glutathione and oxidized glutathione,hypotaurine,taurine,cystine,cysteine,cysteaminesulfinic acid of glioma cells(U251)metabolites.Conclusion: Metabonomics can provide a new method for assessing the malignant degree of gliomas.The preliminary diagnosis of glioma malignancy can be made by measuring the changes of the corresponding metabolites in the cells.At the same time,it can be used to evaluate the curative effect of patients with glioma and have an initial judgment on prognosis.It can also provide a new idea for clinical evaluation of benign and malignant gliomas. |
PDF Full Text Request