Temporal expression pattern of the insulin-like growth factor II and fibroblast growth factor transcripts in avian embryogenesis

In this study, the abundance of IGF-II and bFGF transcripts was estimated in the chicken embryos using the competitive RT-PCR analysis. Significant enhancements in the abundance of IGF-II mRNA were observed at stages HH1 and 5, and a new accumulation in these levels was observed at stage HH18 in comparison to the basal levels. The abundance of bFGF mRNA increased significantly at stages HH18 and 20, followed by an upregulation in the expression of these transcripts at stage HH26. These findings provided important information about the temporal expression pattern of IGF-II and bFGF transcripts in the whole chicken embryos during in ovo development.

Fatores de crescimento coordenam múltiplas vias de sinalização durante o desenvolvimento embrionário. Neste estudo, a abundância de mRNA dos genes IGF-II e bFGF foi estimada em embriões de galinha por análises de RT-PCR competitiva. Aumentos na abundância de mRNA de IGF-II foram observados nos estádios HH1, 5. Os níveis de mRNA de bFGF exibiram aumentos a partir dos estádios HH18 e 20, seguido por uma acentuada redução a níveis basais no estádio HH24 e por um segundo pico na expressão destes transcritos no estádio HH26. Tais descobertas proporcionam importantes informações sobre o padrão de expressão destes fatores de crescimento durante a embriogênese de aves

Effect of IgA Aggregates on Transforming Growth Factor-beta1 Production in Human Mesangial Cells and the Intraglomerular Expression of Transforming Growth Factor-beta1 in Patients with IgA Nephropathy

BACKGROUND: Transforming growth factor-beta (TGF-beta) stimulates renal fibrosis in various renal diseases including IgA nephropathy. METHODS: We examined whether immunoglobulin A (IgA) stimulated TGF-beta1 synthesis in human mesangial cells (MCs), and whether this effect was mediated through the protein kinase C (PKC) pathway. We measured the intraglomerular TGF-beta1 mRNA expression by using competitive RT-PCR, and this was compared with various parameters in IgA nephropathy patients. RESULTS: The IgA aggregate increased the TGF-beta1 mRNA expression in MCs, while this expression was not affected by the culture media or IgG aggregate. Phorbol 12-myristate 13-acetate and calphostin C did not influence the TGF-beta1 mRNA expression that was increased by the IgA aggregate. Intraglomerular TGF-beta1 mRNA expression was significantly correlated with creatinine clearance (r=-0.764, p=0.027), daily proteinuria (r=0.781, p=0.022), serum creatinine (r=0.884, p=0.004), and tubulointerstitial changes (r=0.809, p=0.015). Glomerular TGF-beta1 mRNA expression was associated with an increased tendency for glomerulosclerosis (r=0.646, p=0.084). After 4 years, patients with a high expression of intraglomerular TGF-beta1 mRNA showed a tendency for an decrease of their renal function. CONCLUSION: The IgA aggregate increased TGF-beta1 mRNA expression in MCs, and this was independent of the PKC pathway. The evaluation of intraglomerular TGF-beta1 mRNA expression could be useful in predicting the progression of IgA nephropathy.

Quantification of IL-10 gene expression in induced sputum of asthmatic patients by competitive PCR / 医学研究生学报

Objective: To investigate the role of anti-inflammatory cytokine IL-10 in the pathogenesis of asthma.Methods:IL-10 internal standards was constructed by restriction digest.The expression levels of IL-10 mRNA were determined in peripheral blood and induced sputum of 23 asthmatic patients by(RT-PCR) with this competitive internal standards. Results:There was no IL-10 mRNA expression in most of the asthmatics.IL-10 mRNA was detected in some catabatic patients.But IL-10 mRNA was readily detected in 29 normal subjects. Conclusion:Airway inflammation of asthma was related to the expression of IL-10 gene.Asthmatic patients may have difficulty in IL-10 synthesis with transcript.

Quantitation of IFN-? mRNA in duck PBMC and its application / 中华传染病杂志

Objective IFN-? is a pleiotropic cytokine with potent immunomodulatory effects and antiviral activity. To study the mechanism of IFN-? clearing duck hepatitis B virus (DHBV) in ducks, it is essential to establish a method to quantify expression of DuIFN-? in immune response. In the present study,a semi-quantitative competitive RT-PCR was developed to quantify expression of duck IFN-?(DuIFN-?) mRNA by PBMCs. Methods Based on ?-actin consensus sequence, fishing the ?-actin gene as house keeping gene from duck PBMC by RT-PCR. A competitive internal control was constructed and the competitive RT-PCR system could be used to quantify the transcription of DuIFN-? mRNA. Results After duck PBMCs were stimulated in vitro with PHA, the peak of DuIFN-? expression was at 24-36h. Then RT-PCR method was applied to detect DuIFN-? mRNA transcription by PBMCs from DHBV infected ducks immunized with DuIFN-? plasmid plus DNA vaccine or DNA vaccine alone. Results showed that expression of DuIFN-? in ducks co-immunized with DuIFN-? plasmid were higher than other groups immunized without DuIFN-? plasmid as adjuvant. Conclusions The results indicated that DuIFN-? gene could be a useful adjuvant to develop vaccines. The semi-quantitation of DuIFN-? mRNA by competitive RT-PCR provides the basis for future study of the mechanism of IFN-? in duck hepatitis B virus persistent infection.

Immunohistochemical Analysis of Transforming Growth Factor (TGF)-beta1 and TGF-beta Receptor II and Quantitative Analysis of TGF-beta1 mRNA during Multistep Hepatocarcinogenesis Induced by Diethylnitrosamine in Sprague-Dawley Rats

Transforming growth factor (TGF)-beta1 plays an important role in hepatocarcinogenesis and has been described as a useful tumor marker and one of the poor prognostic indicators in patients with hepatocellular carcinoma (HCC). To investigate the role and cellular localization of TGF-beta1 during multistep hepatocarcinogenesis we performed a quantitative analysis of TGF-beta1 mRNA and immunohistochemical expression of TGF-beta1 and TGF-beta receptor II (TGF-betarII) in female Sprague-Dawley rats. The experimental groups included neoplastic lesions produced by Solt-Farber's protocol, regenerating liver after partial hepatectomy, and normal control. Quantitative change of TGF-beta1 mRNA was analysed by competitive reverse-transcription polymerase chain reaction (RT-PCR). TGF-beta1 protein and TGF-betarII expression were evaluated by immunohistochemical stain. The discrete tumor nodules were detected on 14th day and then increased in number and size. Three HCCs were induced on 8th or 9th month. RT-PCR demonstrated TGF-beta1 mRNA band in all examples of the normal and regenerating liver, nodules and HCCs. Competitive RT-PCR displayed higher TGF-beta1 mRNA in nodules, HCCs and regenerating liver than in normal controls. Hepatocytes from control and regenerating livers showed weak immunoreactivity for TGF-beta1. In contrast, the cytoplasm of hepatocytes of nodules in 7th, 8th and 9th month and HCCs were intensely stained for TGF-beta1. Some sinusoidal cells showed immunoreactivity for TGF-beta1 in all experimental groups. In early phase of carcinogenesis, the cytoplasm of hepatocytes in liver of 12h, 1d and 3d showed transiently increased immunoreactivity for TGF-beta1 and The immunoreactivity decreased thereafter. TGF-beta1 mRNA was also detected in the neoplastic hepatocytes by in-situ hybridization. Although TGF-betarII expression was correlated with TGF-beta1 immunoreactivity during early phase of carcinogenesis, hepatocytes in most nodules in 7th, 8th, 9th month and carcinomas showed decreased or little immunoreactivity for TGF-betarII. Based on the above results, it is concluded that TGF-beta1 expression increases not only in precancerous nodules but also in HCCs and its increase seems to be correlated with decrease or loss of TGF-betarII expression although its mechanism remains unclear. Hepatocytes may be a major cellular source of TGF-beta1 during hepatocarcinogenesis.

Construction of a multispecific internal control for competitive RT-PCR analysis of genes / 中国免疫学杂志

Objective:To construct a mutispecific internal control for competitive RT-PCR analysis of Fas、FasL、GB、P、TIA-1 and ?-actin.Methods:Invitro synthesized fragments were amplified by PCR,there are two products,of which 145 and 147 bp.The 145 bp one contained 5′ primer sequences of Fas、FasL、GB、P、TIA-1 and ?-actin,another contained 3′ primer sequences of the same genes.The products with restriction sites were inserted into the vector PKF_3.Results:Restriction enzyme analysis and DNA sequencing were used to identify the recombinant plasmid,corresponding internal control was obtained by the amplification of the recombinant plasmid with each primer pair.The mutispecific internal control was then used for quantitative detection of TIA-1 in peripheral blood leukocytes from a patient with acutely rejecting allograft.Our study on Fas、FasL、GB、P、TIA-1 and ?-actin showed that the coamplified templates accumulated in a parallel manner throughout not only the exponential phase.Conclusion:The mutispecific internal control can be used for quantitative detection of the six genes. [

CHANGES IN THE EXPRESSION OF OREXIN AND ITS RECEPTORS IN THE RAT HYPOTHALAMUS DURING PREGNANCY,PARTURITION AND LACTATION / 解剖学报

Objective To investigate the possible role of orexin in reproduction and examine the changes in the expression of orexin and its receptors(OX1R,OX2R) in the rat hypothalamus during pregnancy,parturition and lactation. Methods The expressions of prepro-orexin(prepro-OX),orexin-A,OX1R,and OX2R in the rat hypothalamus during pregnancy,parturition,and lactation were evaluated by competitive reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry assay. Results Orexin-A immunoreactive(ir) neurons and the OX1R subtype in neuronal cell bodies were mainly located in the lateral hypothalamic area(LHA) as well as in the magnocellular neurons of the paraventricular and supraoptic nuclei respectively in pregnant and lactating rats.The prepro-OX and OX1R mRNA levels on the 1st day of lactation were significantly higher than that during late pregnancy and lactation.No significant changes of OX2R expression were observed during the various reproductive phases.Conclusion Orexin might be involved in regulating reproductive function in early lactation through their binding sites in hypothalamic PVN and SON.

CHANGES OF OREXIN AND ITS RECEPTORS IN THE RAT HYPOTHALAMUS DURING ESTROUS CYCLE / 解剖学报

Objective To investigate a possible role of orexin in the regulation of estrous cycle by examining the expression of orexin and orexin receptors(OX1R,OX2R) in the rat hypothalamus during the estrous cycle. Methods The levels of prepro-orexin(prepro-OX),orexin-A,OX1R,and OX2R in the rat hypothalamus during the estrous cycle were evaluated by competitive reverse transcription-polymerase china reaction(RT-PCR) assay. Results Only expression OX1R mRNA during late proestrus was significantly higher than that at metestrus.No significant changes of prepro-OX and OX2R mRNA expression were observed during the various estrous cycle phases.Conclusion Orexin might regulate the secretion of GnRH and/or LH by binding OX1R,contributing to the occurrence of ovulation.