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Application of protein c in the treatment of hepatic portal hypertension

A kind of use, liver cell technology, applied in the field of biomedicine, can solve the problems of lack of research on the pathogenesis and treatment mechanism of portal hypertension

Active Publication Date: 2021-07-06
首都儿科研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Overall, there is still a lack of in-depth research on the pathogenesis and treatment mechanisms of portal hypertension

Method used

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  • Application of protein c in the treatment of hepatic portal hypertension
  • Application of protein c in the treatment of hepatic portal hypertension
  • Application of protein c in the treatment of hepatic portal hypertension

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Detection of relative content of activated protein C (PROTEIN C, PROC).

[0032] 1. Experimental group

[0033] Control group: whole blood samples from 6 normal children

[0034] Experimental group: Whole blood samples from 6 children with low protein C levels after portal vein cavernous degeneration Rex operation

[0035] 2. ELISA experiment

[0036] Set blank wells, standard wells and sample wells to be tested respectively. Blank wells (do not add samples, enzyme-labeled reagents and biotin-labeled anti-PROTEIN C antibodies, and the rest of the steps are the same), add 50 μL of standard products to the standard wells, add 400 μL of the sample to be tested, and then add biotin-labeled Anti-MCP3 antibody 10μL, shake gently to mix. The experimental steps were carried out according to the ELISA experimental operating procedures. The optical density (OD value) of each well was measured with a microplate reader at a wavelength of 450 nm.

[0037] 3. Experim...

Embodiment 2

[0039] Embodiment 2, detect the relative content of miRNA and PROC mRNA

[0040] 1. Primer design

[0041] Table 1 Primer Sequence

[0042]

[0043]

[0044] 2. Extraction of RNA

[0045]1. Use TRI REAGENT BD kit to extract RNA in plasma, add 0.25mL plasma and 1-4μL polyacrylamide carrier to TRI reagent, mix thoroughly and place at room temperature for 5min.

[0046] 2. Add 0.2mL chloroform or 0.1mL BCP to each TRI reagent, shake well for 15s, let stand at room temperature for 2-5min, centrifuge at 12000g for 15min at 4°C, and absorb the upper aqueous phase.

[0047] 3. Add 0.5mL isopropanol to each TRI reagent and mix well, place at room temperature for 5-10min, centrifuge at 12000g for 10min at 4°C, discard the supernatant, and the RNA will sink to the bottom of the tube.

[0048] 4. Add 1mL of 75% ethanol to each TRI reagent, shake the centrifuge tube, suspend the precipitate, centrifuge at 7500g at 4°C for 5min, discard the supernatant, and dry at room temperature...

Embodiment 3

[0076] Embodiment 3, verify the double luciferase experiment of PROC target gene

[0077] 1. Experimental group

[0078] (1) NC mimics+Luc-PROCwt. (2) miR-506mimic+Luc-PROCwt. (3) miR-124mimic+Luc-PROCwt. (4) NC mimics+Luc-PROCmut. (5) miR-506mimic+Luc-PROCmut. (6) miR-124mimic+Luc-PROCmut.

[0079] After miRNA mimics, firefly luciferase and Renilla luciferase plasmids were co-transfected into 293T cells for 48 hours, Luciferase activity was detected.

[0080] 2. Experimental steps

[0081] 1. Discard the medium, wash with 1x PBS, and discard the PBS.

[0082] 2. Add 100 μL 1x PLB (1 volume of 5X Passive Lysis Buffer plus 4 volumes of double-distilled water) to each well, and place on a shaker at room temperature for 15 minutes.

[0083] 3. After the cells are fully lysed, add the lysate to the 96-well plate, and add 20 μL to each sample.

[0084] 4. Add 100 μL LAR II to each well of the sample to be tested (use Luciferase Assay Buffer II to resuspend Luciferase Assay...

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Abstract

This application relates to the application of protein C in the treatment of hepatic portal hypertension. This application relates to the correlation research between protein C and portal hypertension, and also relates to protein C inhibitors, including miRNA. Protein C inhibitor (miRNA) can inhibit the proliferation of hepatocytes and promote the apoptosis of hepatocytes, which can reduce the proliferation of hepatic sinusoids, so that the portal blood flow is smooth and the portal pressure is reduced. This has opened up new research prospects for the treatment of portal hypertension (especially portal cavernous degeneration), and provided new ideas for the in-depth development and clinical treatment of miRNA drugs.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of protein C in the treatment of diseases related to hepatic portal hypertension. Background technique [0002] Portal hypertension (PH), also known as portal hypertension, portal hypertension, and portal hypertension, is defined as portal hypertension (PH) when the portal pressure increases more than 5 mmHg relative to systemic blood pressure. The basic pathological and physiological characteristics of PH are obstruction and / or increased blood flow in the portal vein system, continuous increase in hydrostatic pressure in the portal vein and its branches, and the formation of collateral circulation. Cirrhosis of the liver due to various causes is its most common cause. The main clinical manifestations are gastroesophageal varices (GOV), esophagogastric variceal bleeding (EVB), ascites, and hepatic encephalopathy, among which EVB is the most lethal complication of PH, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63A61K31/7105A61P9/12
CPCA61K31/7105A61P9/12C12N15/113
Inventor 张金山
Owner 首都儿科研究所
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