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Application of nadh dehydrogenase flavoprotein 2 in the preparation and detection of drugs

An enzyme flavoprotein, dehydrogenation technology, applied in the direction of recombinant DNA technology, DNA/RNA fragment, microorganism determination/examination, etc., can solve clinical complications, no direct or indirect correlation of NADH dehydrogenase flavoprotein reports, large sampling bias, etc.

Inactive Publication Date: 2011-12-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The clinical diagnosis method of liver fibrosis has always been an irreplaceable liver puncture technique, but this method itself has the following serious disadvantages: there is a certain mortality rate and the possibility of clinical complications; at the same time, there is a large sampling deviation. Come, a coefficient of variation of 55% must be accepted (Gressner OA et al., Clinical Chemistry Literature, 2007, 381: 107-113)
[0008] So far, there is no report on the direct or indirect relationship between NADH dehydrogenase flavoprotein 2 and liver fibrosis

Method used

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  • Application of nadh dehydrogenase flavoprotein 2 in the preparation and detection of drugs
  • Application of nadh dehydrogenase flavoprotein 2 in the preparation and detection of drugs
  • Application of nadh dehydrogenase flavoprotein 2 in the preparation and detection of drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of normal liver and immune liver fibrosis liver non-parenchymal cell protein samples

[0028] Urea, thiourea, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), 3-[(3-cholamidopropyl)-diethylammonium]-1- Propanesulfonic acid (CHAPS) was purchased from Sigma, DNase was purchased from Takara, and Percoll was purchased from Pharmacia.

[0029] In this example, a modified Percoll centrifugation method was used to first separate hepatic non-parenchymal cells, and then the protein of hepatic non-parenchymal cells was obtained by enzymatic hydrolysis. details as follows:

[0030] Cut the normal liver or immune liver fibrosis liver tissue into pieces in ice-cold saline, digest the tissue fragments with 0.05 mg / ml type IV collagenase for 3 hours, filter the digested suspension through a 200-mesh nylon membrane, and take the filtered For a good cell suspension, centrifuge at 100g for 5min at 20°C, take the cell pellet, resuspend in PBS, centrifuge...

Embodiment 2

[0033]Example 2 Screening of Differentially Expressed Proteins

[0034] 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.

[0035] The cleaved proteins were separated by two-dimensional gel electrophoresis, analyzed by ImageMaster 2D Platinum 6.0 software to find out the differentially expressed proteins, and the obtained differentially expressed proteins were analyzed by Diana upgraded liquid chromatography Ultimate3000 series high-capacity ion Trap mass spectrometry (LC-MS / MS) for analysis and identification. Specific steps are as follows:

[0036] 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, 5h...

Embodiment 3

[0043] Example 3 Verification of differential expression of NADH dehydrogenase flavoprotein 2

[0044] The oligonucleotide primers used in this example were synthesized by Invitrogen, Trizol was purchased from Invitrogen, chloroform, isopropanol, and ethanol were purchased from Sigma, Glycogen, kit ReveryTra-Plus-TM, and SYBR Green dye were purchased from ToYoBo company.

[0045] Extract RNA from hepatic fibrotic nonparenchymal cells, perform reverse transcription reaction, and then amplify the reverse transcription product of NADH dehydrogenase flavoprotein 2 by polymerase chain reaction (PCR), compare it with the internal reference GAPDH, and obtain NADH dehydrogenation The content of flavoprotein 2 mRNA is as follows:

[0046] Add 1mL Trizol to the cells, pipette thoroughly for 10min, add 200uL chloroform and mix vigorously, place the sample at room temperature for 5min, centrifuge at 13000r / min for 15min at 4°C, take the uppermost supernatant, add an equal volume of isopr...

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Abstract

The invention belongs to the technical field of biology, and relates to application of an NADH dehydrogenase flavoprotein 2 to preparation of a detection medicament. According to the invention, proteins with differential expressions in immunological hepatic fibrosis non-parenchymal liver cells and normal liver non-parenchymal liver cells are screened to find out a high expression protein in immunological hepatic fibrosis non-parenchymal liver cells. A reverse-transcription polymerase chain reaction has further proved that the NADH dehydrogenase flavoprotein 2 indeed has a high transcription level in immunological hepatic fibrosis non-parenchymal liver cells. As a correlation between the NADH dehydrogenase flavoprotein 2 and immunological hepatic fibrosis, the protein can be applied to preparation of medicament for detecting immunological hepatic fibrosis. The application of the NADH dehydrogenase flavoprotein 2 provides a brand new approach for detection of immunological hepatic fibrosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the application of NADH dehydrogenase flavoprotein 2 in the preparation and detection of drugs, in particular to the application of NADH dehydrogenase flavoprotein 2 in the preparation and detection of immune liver fibrosis drugs. Background technique [0002] Liver fibrosis refers to the excessive deposition of extracellular matrix in tissues caused by chronic liver cell injury. Its further development will lead to serious adverse consequences such as liver cirrhosis and primary hepatocellular carcinoma. Often, the onset of liver fibrosis is clinically difficult to detect, and the associated morbidity and mortality occur mainly after cirrhosis. Therefore, it is of great significance to prevent or reverse the occurrence and development of liver fibrosis through early diagnosis. [0003] The clinical diagnosis method of liver fibrosis has always been an irreplaceable liver puncture te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/32C12N15/11
Inventor 张丽军袁正宏马芳贾小芳姚亚敏
Owner FUDAN UNIV
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