Molecular marker for hepatopathy aggravation and application thereof
A biomarker and severe disease technology, applied in the field of liver disease diagnosis, to achieve the effect of optimization and simplicity
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Embodiment 1
[0033] Example 1: Screening of Liver Fibrosis Biomarkers
[0034] In the GEO and ArrayExpress databases, "NAFLD liver fibrosis" was used as the keyword to search for fatty liver-associated liver fibrosis gene chip data. Data requirements:
[0035] 1) The selected data set must be the expression profile data of the whole genome;
[0036] 2) The selected data set contains fibrosis and control expression profiles; all samples are from human liver tissue.
[0037] 3) The selected data set must include more than 4 samples.
[0038] The GSE49541 dataset was first chosen as an exploratory cohort because it contained the largest number of NAFLD samples with clear fibrosis stages. The data of the validation cohort came from E-MEXP-3291, GSE48452 and GSE59045; the other 5 datasets were fatty liver samples, but without clinical information annotations such as fibrosis (GSE17470, GSE24807, GSE37031, GSE46300 and GSE63067). The positive control uses the HBV-related liver fibrosis GSE840...
Embodiment 2
[0043] The dye method PCR quantitative detection of embodiment 2, EFEMP1, THBS2, LUM
[0044] Research objects: 20 patients with fatty liver disease, and the diagnosis of fatty liver was in accordance with the "Guidelines for the Diagnosis and Treatment of Nonalcoholic Fatty Liver Disease in China (2010 Revised Edition)". Tissue samples after liver puncture were collected in the Affiliated Hospital of Hangzhou Normal University. Liver biopsies were routinely stained with HE, and professional pathologists scored for fibrosis staging. Among them, there were 12 cases of fibrosis stage 0-1, and 8 cases of stage 2-4.
[0045] experimental method:
[0046] 1. RNA extraction
[0047] 1) About 50 mg of liver tissue was placed in a 1.5 ml EP tube with small steel balls, 1 ml of Trizol solution was added, put into a grinder, and grind rapidly. In the centrifuge, centrifuge at 12000r / min, 4°C for 5min, after centrifugation, let stand at room temperature for 5min.
[0048] 2) Separat...
Embodiment 3
[0072] Embodiment 3, universal probe expression detection
[0073] 1. Standard product construction
[0074] According to the sequence of the coding region of the human THBS2 gene (Genbank gene accession number is NM_003247), the full-length cDNA vector containing THBS2 was constructed in the pGEM-T vector and synthesized by Qingke Bio. The plasmid standard was named pGEM-THBS2.
[0075] According to the coding region sequence of human LUM gene (Genbank gene accession number is NM_002345), the full-length cDNA vector containing LUM was constructed in pGEM-T vector and synthesized by Qingke Bio. The plasmid standard was named pGEM-LUM.
[0076] According to the sequence of the coding region of human EFEMP1 gene (Genbank gene accession number is NM_001039349), the full-length cDNA vector containing EFEMP1 was constructed in pGEM-T vector and synthesized by Qingke Bio. The plasmid standard was named pGEM-EFEMP1.
[0077] The plasmid DNA was digested with ApaI to linearize the...
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