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Efavirenz hepatotoxicity molecular marker and application thereof

A molecular marker, efavirenz technology, applied in the field of preparation and detection of liver cytotoxicity molecular marker, D-3-phosphate glycerol dehydrogenase, can solve the problem of low drug resistance barrier

Inactive Publication Date: 2013-07-24
CENT SOUTH UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the barrier to drug resistance of EFV is low [1], and HIV-1 reverse transcriptase (RT) single-site mutant strains are resistant to efavirenz. For this reason, clinical and scientific researchers have carried out a large number of viral drug resistance gene detection work; In addition, EFV has more serious liver damage, skin allergic reaction and neurotoxicity 1，2

Method used

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  • Efavirenz hepatotoxicity molecular marker and application thereof
  • Efavirenz hepatotoxicity molecular marker and application thereof
  • Efavirenz hepatotoxicity molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of Untreated and EFV-treated Hepatocyte Mitochondrial Protein Samples

[0023] The cell culture medium DMEM used in this example was purchased from Invitrogen Company, dextran T500, polyethylene glycol 3350, urea, thiourea, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT) , 3-[(3-cholamidopropyl)-diethylammonium]-1-propanesulfonic acid (CHAPS) were purchased from Sigma, and EFV was purchased from U.S. Pharmacopeia.

[0024] In this example, different concentrations of EFV were used to detect ROS at different times. The results showed that the production of ROS was time- and concentration-dependent with EFV (such as figure 1 shown), the present embodiment selects 2.5 μg / L and 10 μg / L of EFV to treat the cells of liver cancer cells (Huh7) for 1 hour to carry out the experiment: collect the cell homogenate and break it up, separate the mitochondria with the method of two-phase centrifugation, and obtain Add lysis buffer (8mol / L urea, 4% CHA...

Embodiment 2

[0025] Example 2 Screening for differentially expressed proteins

[0026] Acrylamide, N,N'-methylenebis(acrylamide), glycine, sodium dodecyl sulfate (SDS), tris (Tris), urea, and glycerol used in this example were purchased from Amresco, USA, ammonium persulfate (AP), TEMED were purchased from Bio-Rad.

[0027] The cleaved proteins were separated by two-dimensional gel electrophoresis, analyzed by ImageMaster 2D Platinum 6.0 software to obtain differentially expressed proteins, and the obtained differentially expressed proteins were analyzed using a Diana upgraded liquid chromatography Ultimate3000 series high-capacity ion trap Mass spectrometry (LC-MS / MS) for separation and identification. Specific steps are as follows:

[0028] The first isoelectric focusing electrophoresis of two-dimensional gel electrophoresis uses a pH 3-10 non-linear gel strip. The electrophoresis program setting: hydration 30V, 12h; electrophoresis: 500V, 1h; 1000V, 1h; 8000V, 30min, gradient; 8000V, ...

Embodiment 3D-3

[0035] Embodiment 3D-Verification of differential expression of 3-glycerol phosphate dehydrogenase

[0036] The primers used in this example were synthesized by Invitrogen.

[0037] The RNA of hepatocytes was extracted, reverse-transcribed into cDNA, and PCR experiments were carried out using Invitrogen fluorescence quantification. The results were compared with the internal reference GAPDH. The relative quantitative formula was: 2-ΔΔCt(ΔCt=Ct(gene-GAPDH), ΔΔCt=ΔCt( Treatment group-untreated group).Finally determine the difference multiple of D-3-glycerol phosphate dehydrogenase in EFV treatment group and untreated group according to 2-ΔΔCt (results such as image 3 shown).

[0038] image 3 Shown in , compared with the content of D-3-glycerol phosphate dehydrogenase in the untreated group, the content in the EFV treatment group was significantly increased, indicating that D-3-glycerol phosphate dehydrogenase in EFV-treated hepatocytes There is a higher level of expression,...

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Abstract

The invention belongs to the biology technical field, and relates to an efavirenz hepatotoxicity molecular marker and an application, and especially relates to an application of D-3-phosphoglycerol dehydrogenase in preparation of the molecular marker for detecting hepatotoxicity. A cell model of hepatotoxicity, a proteomics method used for screening, fluorescent quantitation PCR and immunoblotting experiments are used to obtain the D-3-phosphoglycerol dehydrogenase which is positively related to the hepatotoxicity. The protein in EFV processed groups with different concentrations enables high expression by comparing with unprocessed protein, and the D-3-phosphoglycerol dehydrogenase is positively related to the EFV hepatotoxicity. The result shows that a transcript of protein can be taken as one molecular marker to perform quantitative determination on transcriptional level, so that the EFV hepatotoxicity can be detected. The specific primers and antibody of the D-3-phosphoglycerol dehydrogenase can be used for preparing a preparation to detect EFV hepatotoxicity, and can be used to prepare a kit for detecting the EFV hepatotoxicity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to efavirenz (EFV) liver cytotoxicity molecular markers and applications thereof, in particular to the use of D-3-phosphate glycerol dehydrogenase in the preparation and detection of liver cytotoxicity molecular markers . Background technique [0002] The full name of AIDS is "Acquired Immunodeficiency Syndrome" (Acquired Immunodeficiency Syndrome, AIDS). By the end of 2010, there were about 370,000 HIV-infected people and patients diagnosed in my country, of which 15,982 were newly diagnosed with AIDS in 2010, which had a great impact on my country's politics and economy. Highly Active Antiretroviral Therapy (HAART) is currently the most important program for the treatment of AIDS, and it is the main way to improve the prognosis and quality of life of patients. Currently, there are 5 types of drugs available for clinical selection: nucleoside reverse transcriptase inhibitors (NRTI), no...

Claims

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Application Information

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IPC IPC(8): C12Q1/32C12Q1/68G01N33/573
Inventor 张丽军马芳贾小芳周宏灏
Owner CENT SOUTH UNIV
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