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Objective To analyze the expression characteristics of annexin A2 in dermal papilla cells (DPCs) with aggregative behavior.Methods Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were performed to measure the mRNA and protein expressions of annexin A2 respectively in DPCs with or without aggregative behavior.Results The mRNA expression level of annexin A2 was significantly higher in DPCs with aggregative behavior than in those without aggregative behavior (0.50 ± 0.15 vs.0.35 ± 0.19, t =8.26, P ＜ 0.05).Western blot showed that annexin A2 had two isoforms, including one isoform with a relative molecular mass of 40 000 and the other one with a relative molecular mass of 36 000.The annexin A2 isoform with a relative molecular mass of 40 000 was highly expressed in both DPCs with aggregative behavior and those without aggregative behavior, while the other isoform was only expressed in DPCs with aggregative behavior.Conclusion Annexin A2 may be closely related to the aggregative growth of DPCs.
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Objective To study the expression ot secreted proteins in aggregated dermal papilla cells (DPCs).Methods DPCs were isolated from human scalp tissue and subjected to primary culture and subculture.Aggregated and non-aggregated DPCs served as the subject of this study.Secreted proteins were prepared from these cells and subjected to two-dimensional polyacrylamide gel electrophoresis.Differentially expressed proteins were screened by the PDQuest image analysis software.Protein spots were digested and identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry,and finally analyzed using the National Center for Biotechnology Information (NCBI) non-redundant (Nr) protein database.Results Two-dimensional electrophoresis maps with good repeatability and high resolution were established.Image analysis of 2-D gels revealed that the average number of detected protein spots was 1 134 ± 52 and 1 078 ± 36 in aggregated and nonaggregated DPCs respectively,and the majority of these protein spots were matched between aggregated and nonaggregated DPCs.Twenty-eight protein spots showed more than 5-fold difference between the two groups of cells,and 10 proteins were preliminarily identified as differentially expressed proteins by peptide-mass fingerprinting.Of these differentially expressed proteins,8 proteins including Rhogdi 1,filamin A,cystatin C,fibronectin,cyclophilin A,procollagen C proteinase enhancer 1,tissue inhibitor of metalloproteinase and tissue inhibitor of metalloproteinase-2 were up-regulated,and 2 proteins including neuropolypeptide h3 and matrix metalloproteinase-3/tissue inhibitor of metalloproteinase-1 complex were down-regulated in aggregated DPCs compared with non-aggregated DPCs.Conclusions Differentially expressed proteins between aggregated and non-aggregated DPCs are mainly implicated in cell signaling pathway,cellular proliferation and differentiation,extracellular matrix synthesis and degradation,and so on.
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Objective To detect the expression and function of cysteinyl leukotriene receptors (CysLTRs)in keratinocytes.Methods Human keratinocytes were isolated from the tissue of foreskin by digestion with dispase Ⅱ and trypsin,and subjected to primary culture.By using confocal laser scanning microscopy and reverse transcriptase PCR,the localization and expression of CysLTRs were studied in kemtinocytes.respectively.Some primarily cultured keratinocytes were pretreated with leukotriene D4 (30 nmoi/L),MK571(300 nmol/L),and BAYu9773 for 5 minutes followed by the detection of intmcellular calcium level using the Ca2+ indicator dye Fura-2/AM as well as cell proliferation bv MTT assay.Results The expressions of CysLTR1 and CysLTR2 were observed in cultured keratinocytes,and they were mainly located on cell membrane,partly in cytoplasm and nuclei.Compared with non.stimulated cells,a significant increase Was noted in the expression of CysLTRs,especially in the nuclei of keratinocytes stimulated by LTD4(P＜0.05),together with an elevation in intracellular calcium level(42.27±3.00 mmol/L,P＜0.01)and acceleration in cell proliferation (P＜0.01).However,both MK571 and BAYu9773 could completely block the effect of LTD4 on intmcellular calcium level and cell proliferation.and there was no significant difference in the blocking effect between MK571 and BAYu9773.Conclusions Functional CysLTRs are expressed in human keratinocytes.and they carl increase the intracellular calcium level in,and cell proliferation of,keratinocytes.
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Aim To observe the production of anti-idiotypic antibodies directed against AK auto Ab in the rabbit sera after long-term, high dose allogenic AK auto Ab injection. Methods Allogenic AK auto Ab (5mg/kg)was injected intramuscularly to rabbit once every other day for 90 days. Anti-idiotypic antibodies in rabbit sera were detected by ELISA. Results Rabbits injected with AK auto Ab generated anti-idiotypic antibodies that could react to F(ab′ )2 of AK auto Ab. The titers of anti-idiotypic antibodies reached the highest levels at 4 weeks after the administration of AK auto Ab and then gradually decreased. Conclusion Rabbits could be induced to form immune tolerance when high-does allogenic AK auto Ab is administrated for long-term.