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文献参考#白细胞衍生趋化因子2（LECT2）ELISA试剂盒

TGF-β1-Mediated Leukocyte Cell-Derived Chemotaxin 2 Is Associated With Liver Fibrosis in Biliary Atresia

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

Frozen liver tissues were used to extract total RNA with the kit according to the instructions provided by the manufacturer (Tian Gen, Beijing, China) and tested the purity with Nanoｄrop. The extracted RNA had an absorbance 260/280 ratio of 1.9–2.0. First-strand cDNA was synthesized using the RT reagent kit including gDNA Eraser (Sangon Biotech, Shanghai, China). The expression of LECT2 was measured using the SYBR Green Master Mix kit (SYBR Premix Ex Ta q™ II, Sangon Biotech) on a Light Cycler 480 Real-Time PCR detection system. The internal reference is the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences are listed in Table 1.

TABLE 1

Table 1. Primer sequences used in this study.

Enzyme-Linked Immunosorbent Assay

Leukocyte cell-derived chemotaxin 2 level of the serum collected at the time of admission was detected using enzyme-linked immunosorbent assay (ELISA) kit (RenjieBio, Shanghai, China) according to the protocols provided by the manufacturer. Briefly, the peroxide-conjugated anti-LECT2 (100 μl/well) antibody was added to the microplate with a standard or serum sample (50 μl/well) and incubated at 37.0°C for 1 h. After washing three times, the peroxidase-specific substrate was added to each well and incubated at 37.0°C for 15 min. After that, a termination solution (50 μl/well) was added to stop the reaction. Finally, the color intensity proportional to the concentration of LECT2 was achieved at 450 nm.

Bioinformatics Analysis

The biliary atresia expression profile datasets GSE15235 were downloaded from the Gene Expression Omnibus 1 database. GSE15235 included a total of 47 biliary atresia liver tissues, and the raw data were preprocessed with the “affy” package (10) in R software (version 4.1.2).2 The gene expression matrix data were processed using the CIBERSORT method (11) in the “IOBR” package (12) to obtain the infiltration matrix for 22 immune cell types. Finally, Spearman correlation analysis of LECT2 and infiltrating immune cells was performed.

Immunofluorescence

Immunofluorescent staining was similar to the immunohistochemistry staining (IHC) protocol, the sections of liver tissues were incubated with primary rat monoclonal anti-CD163 and rabbit polyclonal anti-TGF-β1 (1:300, Bioss Beijing, China) at 4°C overnight, and washed completely in phosphate buffer solution (PBS), with the secondary antibodies combined with Alexa488/594 (Bioss Beijing, China) for 2 h. Sections were mounted for observation under the microscope after being washed.

Cell Culture and Treatment

Human normal liver cell line HL-7702 (L-02) (BNCC351907) was purchased from bnbio (BNCC351907, China) and cultured in 1640 DMEM medium (SH30809.01, Jiangsu KeyGEN BioTECH, China), containing 10% fetal bovine serum medium. All cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. L-02 cells were treated with PBS and 100 ng/ml recombinant human TGF-β1 (rh TGF-β1, Bioss Beijing, China), and rhTGF-β1 plus ICG001 (inhibits TCF/β-catenin/CBP mediated tranｓcriptional activity) for 24 h and measured the expression of LECT2.

Data Analysis

The data were analyzed using SPSS 22.0, and non-normal distribution data were expressed as the median. The chi-square test was used to evaluate qualitative data (gender). The unpaired t or Mann–Whitney test was used to evaluate quantitative data in two groups. Comparison between multiple groups was performed by one-way analysis of variance. The correlations were analyzed by Pearson coefficient analysis. The area under the receiver operating characteristic (ROC) curve (AUC) was calculated, and the optimal cutoff values of serum LECT2 were determined. The p-value < 0.05 was considered to be statistically significant.

Results

Baseline Characteristics

A total of 53 patients were enrolled in this study. Among them, 36 patients, including 20 boys and 16 girls, had BA and the median age was 53.0 days, and 17 patients, including 8 boys and 9 girls, had no BA, and the median age was 89.0 days. Gender was tested using the chi-square test and χ2 = 0.335, p = 0.56. There was no statistical difference in gender, but the age was statistically significant (p = 0.02). Beyond CBD, there was no statistical difference in BA, BH, and IBS (p = 0.61; Table 2).