| Objective:Specific IgE play a core role in allergic diseases,and their biological functions are mediated by binding to tetramer high affinity IgE receptors.At present,detection of s IgE is still in content determination rather than functional determination.The correlation with clinical symptoms is poorly,because it can indicate the intensity of IgE binding allergen,but can not reflect the degranulation effect of sensitized cells caused by allergen binding.The expression of high affinity IgE receptor in RBL cells can be used to detect the exocytosis of other inflammatory mediators such as histamine and serotonin caused by allergen-specific IgE and allergen binding.The experimental results showed that the fusion expression of NPY and fluorescent protein could be used to label granules in neuronal cell line PC12,and NPY-m RFP could also be used to label granules in non-neuronal RBL cells for quantitative exocytosis.We hypothesized that RBL cells stably transfected with NPY-EGFP and FcεRIα could be used for sensitization in human serum.Methods:1.Transient cell line: The plasmid and cell practicability were tested by liposome lipo2000 transfection.The gene level,protein quantity,protein membrane expression and function of the FcεRIα were identified by fluorescence quantitative PCR,Western blot,flow cytometry,immunofluorescence and β-hexosaminidase release assay.2.Stable cell line:The transfection conditions of different generations of lentivirus packaging vector plasmid were optimized,the drug screening concentration and the amount of lentivirus infection were determined by cell proliferation experiment,and the stable cell line was established by infecting RBL-2H3 cells.The expression of receptor was identified by flow cytometry and immunofluorescence.3.Preliminary application of stable cell line: The β-hexosaminidase release assay was used to identify the specificity and sensitivity of stable cell lines for the detection of allergen Gal d 6.Results:1.The qPCR results of transient cell lines showed that the overexpression of FcεRIαin the experimental group was 165.91 times higher than that in the control group,which was used to transiently transfect the expression of receptor gene in RBL-2H3 cells.In western blot,an obvious protein band was detected at 50 k D in the experimental group by using specific monoclonal antibody against human FcεRIα.The expression of FcεRIα in the control group and target group was recognized by PE labeled specific monoclonal antibody against human FcεRIα.The positive cell rate of the target group was 83.2 %.Positive and negative serum were used forβ-hexosaminidase release assay,the stimulation positive serum was between 20 %and 60 %,and the negative was less than 10 %.The same serum samples were used to change the new index NPY-EGFP to detect the release rate.The release rate ranges from 20 % to 80 %,which can be distinguished from negative serum.2.The second generation,the third generation and the fourth generation of lentivirus packaging particles were used to package the transfected plasmids for the construction of stable cell lines,and it was found that the second generation had the best packaging effect.Through MTS cell proliferation assay,the screening concentration of puromycin was 1.61 μg/m L.By using MTS cell proliferation test,it was confirmed that the best amount of virus infection was 1.53 times of the original virus solution.The positive clones were detected by flow cytometry,and the positive cell rate was 95.5 %.3.The β-hexosaminidase release assay was used to identify the specificity of stable cell lines to allergen Gal d 6.The results showed that Gal d 6 could effectively stimulate RBL cells sensitized by Gal d 6 allergic serum,but Bos d 5 could not(p <0.0001).The sensitivity of stable cell lines to Gal d 6 was 0.01 μg /m L.Conclusion:The cell line established in this study was based on the detection of allergen stimulation sensitized and stably transformed into RBL-2H3. |
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