OBJECTIVETo develop an improved substrate for indirect immunofluorescence test (IIF) for detecting anti-Ro60/Sjogren's syndrome A (Ro/SSA) autoantibodies.

METHODS60-kDa Ro/SSA autoantigens (Ro60) cDNAs were obtained from human placental cDNA library using polymerase chain reaction (PCR) and were cloned into the mammalian expression vector-pEGFP-C1. Then, the recombinant plasmids were transfected into HEp-2 cells. We confirmed the overexpression, localization and antigenicity of fusion proteins in transfected cells by means of immunoblotting, confocal fluorescence microscopy and IIF. HEp-2 and HEp-Ro60 were analyzed by IIF using a panel of 10 precipitin-positive anti-Ro human sera simultaneously.

RESULTSStable expression of Ro60-green fluorescent protein (Ro60-GFP) fusion proteins were maintained ten more generations. Ro60-GFP kept the antigenicity of Ro while demonstrating its own characteristic immunofluorescent pattern in HEp-Ro60 cells. The transfectants dramatically increased the sensitivity of IIF testing (a mean increase of 6.7-fold in endpoint titer). Eight over ten (8/10) positive anti-Ro sera showed characteristic immunofluorescent patterns for HEp-Ro60, including two sera that were anti-nuclear antibodies (ANA) negative for untransfected HEp-2. IIF-ANA in all healthy sera was negative for HEp-Ro60.

CONCLUSIONSAs a new substrate for IIF, the Ro60 transfectants can be used to detect anti-Ro antibodies. In addition, transfected HEp-2 cells keep the immunofluorescent properties of HEp-2 cells in IIF-ANA tests and can be employed as a substrate for routine IIF-ANA detection.

Antibodies, Antinuclear ; blood ; Autoantigens ; Cell Line ; Fluorescent Antibody Technique, Indirect ; Humans ; Molecular Weight ; RNA, Small Cytoplasmic ; Recombinant Fusion Proteins ; immunology ; Ribonucleoproteins ; immunology ; Transfection

OBJECTIVE: To explore the effects of nuclear protein-like transcription factor nuclear receptor subfamily 2 group E member 1 (NR2E1) on the growth, division, and proliferation of neuroblastoma cell line IMR32. METHODS: A NR2E1 shiRNA plasmid vector was constructed and transfected into neuroblastoma cell line IMR32 using lipofedamine™2000. Subsequent cell growth was measured by cell counting and the protein expression of somatic nuclear division was examined by immunofluorescent staining. RESULTS: At 48 h after the neuroblastoma cells IMR32 were transfected with NR2E1-shiRNA vector, the related nuclear division protein and the proliferation of the transfected cells IMR32 were remarkably depressed. CONCLUSION Cells division and proliferation of neuroblastoma cell line IMR32 is inhibited through transfection with the NR2E1-shiRNA plasmid vector.
Cell Division ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Neuroblastoma ; pathology ; RNA, Small Interfering ; genetics ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Transfection

This study is purposed to investigate the effects of praeruptorin A (PA) and praeruptorin C (PC) on UGT1A1 in HepG2 cells through hCAR pathway. PA and PC were incubated with HepG2 cells for 24 h and 48 h, mRNA and protein expressions of UGT1A1 were determined by real-time PCR and Western blotting assays. Additionally, effects of PA and PC on UGT1A1 mRNA and protein expressions were also measured after transient transfection of a specific CAR siRNA for 72 h in HepG2 cells. UGT1A1 mRNA and protein expression levels were significantly increased by PA and PC after incubation for 48 h. Moreover, the mRNA and protein up-regulations of UGT1A1 were attenuated by transient transfection of a specific CAR siRNA, suggesting the induction was mediated by CAR. The results suggest that PA and PC can significantly up-regulate UGT1A1 expression partially via the CAR-mediated pathway.
Apiaceae ; chemistry ; Coumarins ; isolation & purification ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Glucuronosyltransferase ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Signal Transduction ; Transfection

OBJECTIVETo investigate the effect of gene Af116609 on gastric cancer multi-drug resistance (MDR) by introducing it into gastric cancer multi-drug resistant (MDR) cell line SGC7901/VCR.

METHODSGene Af116609 was cloned from SGC7901/VCR by RT-PCR and its differential expression between gastric cancer MDR cells and its parental cells was displayed by Northern blot. The gene was introduced to gastric cancer cells by transfection of recombinant eukaryotic expression vector by electroporation. MTT assay in vitro was applied to investigate its effect on multi-drug resistance phenotype of gastric cancer cells.

RESULTSThe full length CDS of gene Af116609, as long as 327 bp, was cloned from gastric cancer MDR cell line SGC7901/VCR and its sequence was coincident with the hypothetical gene Af116609 in GenBank. It was overexpressed in MDR cells than its parental cells at mRNA level. In the MTT assay in vitro, the drug sensitive cells transfected with sense eukaryotic expression vector showed upregulated targeted gene, with increased resistance to vincristine, 5-fliorouracil and arabinoside, and decreased resistance to adriamycin, but no influence on resistance to methotrexate. However, the drug resistant cells transfected with anti-sense eukaryotic expression vector, showed down regulated targeted gene, with less resistance to all the five anticancer drugs to different degrees.

CONCLUSIONGene Af116609 is related to MDR phenotype of gastric cancer cells and may become a candidate molecular target to reverse the MDR of gastric cancer.

Antineoplastic Agents, Phytogenic ; pharmacology ; Autoantigens ; genetics ; Cell Line, Tumor ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Humans ; RNA, Small Cytoplasmic ; genetics ; Ribonucleoproteins ; genetics ; Stomach Neoplasms ; genetics ; pathology ; Vascular Endothelial Growth Factor A ; biosynthesis ; Vincristine ; pharmacology