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Objective@#To establish a Tohoku hospital pediatrics-1 (THP-1) cell line with G protein-coupled recep- tor108 ( GPR108) deletion and explore its functions．@*Methods@#According to the requirements of the clustered regularly interspaced short palindromic repeats ( CRISPR) / CRISPR-associated protein 9 ( Cas9 ) system ，two single guide RNAs (sgRNA1 and sgRNA2) paring to the flanking fragments of human GPR108 gene were designed and synthesized．The two oligonucleotides were inserted in the pL-CRISPR. EFS．GFP vector to generate the new recombinant vectors ( pL-CRISPR. EFS．GFP-sgRNA1 and pL-CRISPR. EFS．GFP-sgRNA2 ) ．The recombinant vectors and packaging plasmids (pMD2. G and psPAX2) ，were co-transfected into 293T cells to generate virus for infecting THP-1 cells．The GFP + cells were screened and isolated in 96-well culture plates by flow cytometry to obtain single-cell clones．PCR and Western blot were used to detect whether GPR108 was successfully knocked out in THP- 1 cells．Both GPR108 + / + and GPR108 －/ － THP-1 cells were treated with lipopolysaccharide (LPS) ．Interleukin 8 (IL-8) derived from the THP-1 cells，which were treated by LPS，was detected with Western blot and cytometric bead array ( CBA) analysis. @*Results@#The recombinant lentiviral vector pL-CRISPR. EFS．GFP-sgRNA was successfully constructed and single-cell clone F9 was obtained by flow cytometric sorting after transfection of THP-1 cells．PCR and Western blot both confirmed that F9 was a GPR108 －/ － THP-1 single-cell clone． LPS stimulated GPR108 －/ － and GPR108 + / + THP-1 cells，both Western blot and CBA results showed a significant decrease in IL- 8 synthesis and secretion in GPR108 －/ － THP-1 cells．@*Conclusion @#The GPR108 －/ － THP-1 cell clone is success- fully obtained based on the CRISPR / Cas9 system．GPR108 deletion in THP-1 cells treated by LPS leads to a decrease of IL-8 expression and secretion．It lays the foundation for further research on the molecular mechanisms of GPR108 in the immune inflammatory response.
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Objective@#To explore the effect of complement C3 a-C3a receptor in the kidney immune inju-ry in trichloroethylene-sensitized mice by using C3a receptor specific antagonist C3aRA and discuss the patho-genesis of kidney injury in occupational dermatitis medicamentosa-like of trichloroethylene (ODMLT) .@*Methods@#42 female 6~8 weeks old BALB/c mice of specific pathogen free were randomly divided into blank control group (5) , solvent control group (5) , TCE treatment group (16) and TCE+C3aRA treatment group (16) . The TCE treat-ment group and TCE+C3aRA treatment group were further divided into the sensitized group and the non-sensi-tized group according to the skin sensitization test score. Renal function was detected by biochemical detection kit; expression of C3aR in kidney tissue was detected by qPCR; expression of IL-1β and TNF-α protein were de-tected by immunohistochemical.@*Results@#Compared with solvent control group and corresponding non-sensitized group, CRE and BUN in TCE sensitized group and TCE + C3aRA sensitized group were significantly increased (P<0.05) . Compared with TCE sensitized group, CRE and BUN in TCE+C3aRA sensitized group were signifi-cantly decreased (P<0.05) . Compared with solvent control group and TCE non-sensitized group, the expression level of C3aR gene in kidney tissue in TCE sensitized group was significantly increased (P<0.05) . There was a large number of IL-1β and TNF-α protein expression in kidney tissue in TCE sensitized group and TCE+C3aRA sensitized group. Compared with the TCE sensitized group, the expression level of IL-1β and TNF-α protein in kidney tissue in TCE+C3aRA sensitized group was significantly decreased (P<0.05) .@*Conclusion@#C3a-C3aR may be involved in the kidney immune injury in TCE sensitized mice, C3aRA has a protective effect on the kid-ney immune injury in TCE sensitized mice.
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Objective@#To explore the expression of CD55 in liver tissue of trichloroethylene-sensitized mice and discuss the role of CD55 in the liver immune injury of trichloroethylene-sensitized mice.@*Methods@#6-8 weeks specific pathogen free female BALB/c were randomly divided into blank control group, solvent control group and TCE treatment group to establish BALB/c mice sensitized model. According to mouse skin sensitization reaction score, TCE treatment mice were divided into sensitized and non-sensitized group at 24 h after the last challenge. At 48 h after the last challenge, the blood and aseptic livers were collected. The level of serum ALT was tested by automatic biochemical analyzer and pathology of the liver was observed. C5b-9 deposition was studied by immunohistochemistry (IHC) . CD55 protein expression level in liver tissue was studied by immunohistochemistry and Western blotting. The expression of CD55 mRNA in liver tissue was detected by qRT-PCR.@*Results@#Liver function test result showed level of serum ALT in TCE sensitized group was significantly higher than solvent control group and TCE non-sensitized group (P<0.05) . There was ballooning degeneration and necrosis of liver cells in TCE sensitized group. IHC demonstrated that TCE sensitized group had obviously increased content of C5b-9 but had reduced content of CD55 compared with solvent control group and TCE non-sensitized group (P<0.05) . Western blotting also showed that TCE sensitized group had lower expression of CD55 than solvent control group and TCE non-sensitized group (P<0.05) . qRT-PCR showed that CD55 mRNA expression level in liver tissue of TCE sensitized group was apparently lower than solvent control group and TCE non-sensitized group (P<0.05) .@*Conclusion@#Complement activation may be involved in TCE-induced liver injury, and the expression change of complement regulatory protein CD55 may play essential role in the process.
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Objective To investigate changes in serum levels of Th22 cell ? related cytokines and complements in patients with drug eruption before and after treatment, and to explore their possible roles in the occurrence and development of drug eruption. Methods This study included 35 patients with drug eruption, and 35 sex?and age?matched healthy controls. Five milliliters of peripheral blood samples were collected from the controls and patients before and after treatment. Enzyme?linked immunosorbent assay(ELISA)was performed to measure serum levels of interleukin 22(IL?22)and IL?13, and the cytometric bead array(CBA)system was used to determine serum levels of tumor necrosis factor?α(TNF?α) and complement components C3a, C4a and C5a. Results Before treatment, the patients with drug eruption showed significantly higher serum levels of IL?22(40.85 ± 14.56 vs. 29.09 ± 8.66 ng/L, t=5.549, P 0.05). Correlation analysis showed positive correlations between complement C3a and C4a serum levels(r = 0.660, P < 0.05), between C3a and C5a serum levels(r = 0.404, P < 0.05), between C4a and C5a serum levels(r = 0.501, P < 0.05), and between IL ? 22 and TNF ? α serum levels(r = 0.573, P = 0.005), but negative correlations between IL ? 22 and complement C3a serum levels(r = -0.490, P = 0.005), in patients before treatment. Conclusion The activation of Th22 cell?related cytokines and complements may play important roles in the occurrence and development of drug eruption, and IL?22 may participate in the regulation of complements.