OBJECTIVETo investigate the alteration of inflammasome and receptor during IgG promoter transfected to HepG2 cells.

METHODBy assay of Elisa to evaluate the secretion of IL-1 beta, IL-8, TNF-alpha and MCP-1 after puerarine and LPS administration, and by assay of real time PCR to evaluate the expression of mRNA of IL-1 beta, IL-8,TNF-alpha and MCP-1, as well as the receptors of TLR2, 4 and NOD2, MyD88.

RESULTIgG promoter did not active innate immunity and enhance the expression and secretion of inflammasome in HepG2. Puerarine did not active the inflammasome either. LPS activated the innate immunity and increased the secretion of IL-8, TNF-alpha and MCP-1.

CONCLUSIONIgG-HepG2 cells could be used specifically as the model of allergy type II for ingredients screening. It is suggested that puerarine was suite for the activator for this type of allergy as positive control.

Allergens ; analysis ; immunology ; Drugs, Chinese Herbal ; chemistry ; Gene Expression Regulation ; immunology ; Hep G2 Cells ; Humans ; Immunity, Innate ; immunology ; Immunoglobulin G ; genetics ; Inflammasomes ; immunology ; Promoter Regions, Genetic ; genetics ; Transfection

In this study, the trypsin gene (bgtryp-1) from the German cockroach, Blattella germanica, was cloned via the immunoscreening of patients with allergies to cockroaches. Nucleotide sequence analysis predicted an 863 bp open reading frame which encodes for 257 amino acids. The deduced amino acid sequence exhibited 42-57% homology with the serine protease from dust mites, and consisted of a conserved catalytic domain (GDSGGPLV). bgtryp-1 was determined by both Northern and Southern analysis to be a 0.9 kb, single-copy gene. SDS-PAGE and Western blotting analyses of the recombinant protein (Bgtryp-1) over-expressed in Escherichia coli revealed that the molecular mass of the expressed protein was 35 kDa, and the expressed protein was capable of reacting with the sera of cockroach allergy patients. We also discussed the possibility that trypsin excreted by the digestive system of the German cockroach not only functions as an allergen, but also may perform a vital role in the activation of PAR-2.
Allergens/analysis/chemistry/*genetics ; Amino Acid Sequence ; Base Sequence ; Blattellidae/*genetics ; Blotting, Western ; Catalytic Domain/genetics ; DNA, Complementary/genetics ; Escherichia coli/metabolism ; Female ; *Genes, Insect ; Humans ; Male ; Molecular Sequence Data ; Molecular Weight ; Receptor, PAR-2/metabolism ; Recombinant Proteins/analysis/biosynthesis/chemistry ; Research Support, Non-U.S. Gov't ; Sequence Alignment ; Sequence Homology, Amino Acid ; Trypsin/analysis/chemistry/*genetics

OBJECTIVETo study the allergen in key processes during the production of Fufang Kushen injection by IgG promoter-HepG2 cells in vitro.

METHODBy transfecting a IgG promoter-regulating the expression of green fluorescent protein(GFP) plasmid into HepG2 cells, this transferred cells were incubated with common allergens (like puerarin, ovalbumin, LPS or Sal typhoid vi polysaccharide vaccine), excipients using in Fufang Kushen injection (NaOH, acetic acid, Tween-80 and ethanol) and samples from the key production processes of the injection for 30 minutes . Fluorescent photographs were analyzed the fluorescence intensity of the cells by using an image analysis software.

RESULTAll of common allergens significantly increased the IgG expression. Two of the excipicents, acetic acids and Tween-80 were shown to increased the IgG expression, while others had no effect on IgG expression. In the 8 samples from the key processes in the production of Fufang Kushen injection, two of them stimulated IgG expression.

CONCLUSIONIgG promoter-HepG2 cells are highly sensitive and specific to allergens, and thus can be applied to rapid screening of allergens in components and injections in transcriptional level. It is possible to use the IgG-promoter HepG2 cells in a real-time monitoring of allergens in the production processes of Chinese medicine injections.

Allergens ; analysis ; immunology ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Gene Expression Regulation ; immunology ; Hep G2 Cells ; Humans ; Immunoglobulin G ; genetics ; Injections ; Medicine, Chinese Traditional ; standards ; Promoter Regions, Genetic ; genetics ; Quality Control