ABSTRACT
Guanylyl cyclase C (GC-C) is a member of a family of enzymes that metabolize GTP to cGMP and was first identified as a receptor for heat-stable enterotoxin. Guanylin (GNY) has since been identified as an endogenous ligand for GC-C in the intestine of several mammalian species. The GNY/GC-C system regulates ion transportation and pH in the mucosa. Recently, it was reported that GC-C and GNY are involved in lipid metabolism in rat mesenteric adipose tissue macrophages. To examine the role of GC-C and GNY in lipid metabolism in cattle, we used a bovine mesenteric adipocyte primary culture system and a coculture system for bovine adipocytes and GNY-/GC-C-expressing macrophages. Fat droplets were observed to accumulate in bovine mesenteric adipocytes cultured alone, whereas few fat droplets accumulated in adipocytes indirectly cocultured with macrophages. We also observed that GC-C was present in bovine mesenteric adipose tissue, and that fat droplet accumulation decreased after in vitro GNY administration. Expressions of mRNAs encoding lipogenic factors decreased significantly in adipocytes after either coculture or GNY administration. These results suggest that the GNY/GC-C system is part of the control system for lipid accumulation in bovine mesenteric adipose tissue.
Subject(s)
Animals , Cattle , Humans , Rats , Adipocytes , Adipose Tissue , Coculture Techniques , Enterotoxins , Guanosine Triphosphate , Guanylate Cyclase , Hydrogen-Ion Concentration , In Vitro Techniques , Intestines , Ion Transport , Lipid Metabolism , Macrophages , Mucous Membrane , RNA, MessengerABSTRACT
Objective To investigate the expression of guanylyl eyelasec(GC-C)mRNA in pefipheral blood of colorectal cancer patients and discuss its clinical significance. Methods To detect the GC-C mRNA in peripheral blood of colorectal cancer patients by reverse transcriptase PCR and analysis their clinical and pathological indexes. Results The positive rate of GC-C mRNA in 52 colorectal cancer patients was 50%(26/52),while to patients with extra-intestinal cancer,the positive rate was 0(0/8).There were significant difierences of positive rate of GC-C mRNA associated with tumor infiltration(P＜0.05)and tumor embolus in vessel(P＜0.05).While to CEA level in peripheral blood(P＞0.05),but there were no statistical difference.The positive rate of micrometastasis in coloreetal cancer patients might increase using the GC-C mRNA combined with CEA level in peripheral bood.The positive rate of GC-C mRNA in colorectal cancer patients with Dukes A、B、C and D stages were 0(0/3),47.06%(8/17),52.38%(11/21),63.63%(7/11),The expression of GC-C mRNA increased with the clinical stage chang,there was no statistical difference.Conclusions GC-C expresses selectively in peripheral blood of colorectal cancer patients.Itis a valuable new tumor marker in micrometastasis diagnosis and post-operation follow-up of colorectal cancer,and it can also be used as the location determination of metastasis intestinal tumor.
ABSTRACT
Objectives To establish real-time fluorenscence quantitative polymerase chain reaction ( RFQ-PCR ) for measurement of the expression level of guanylyl cyclase-C(GC-C) in the Peripheral blood mononuclear cell(PBMC)in 30 blood donors, 10 cases colorectal cancer tissue and 1 case T84 human colon cancer cell line. Methods Specific primers and TaqMan probe have been designed,and fluorenscence of the PCR product was detected continuously during amplification. According to the standard curve created by plasmid DNA, the expression level of GC-C in clinical samples has been determined using software, and the results were presented as the ratios of GC-C mRNA to?2-microgluobulin(?2M)mRNA. Results The detection range of the assay was from 101 pg/ml to109pg/ml,the coefficient of variation values of both intra-experimental and inter- experimental reproducibility were 6. 87% to 11. 12% and 8. 86% to 15. 19% . None of 30 blood donors and 11 benign intestinal patients expressed GC-C mRNA,it was expressed in 31/37 colorectal cancer patients. The expression level of GC-C mRNA in colorectal cancer patients was 0. 88?0.06,and the expression level of its in colorectal carcinoma tissue and T84 cells were 0. 86?0.07/ug tissue and 0.0082/per cell. Conclusions This assay had high sensitivity,specificity and reproducibility.
ABSTRACT
Objective:To develop a real-time fluorescent quantitative reverse transcription polymerase chain reaction(RFQ-RT-PCR) system for determination of the expression of guanylyl cyclase-C(GC-C) mRNA in the peripheral blood mononuclear cells(PBMC) in patients with colorectal cancer.Methods: The specific primers and TaqMan probe targeting the high conservative region of GC-C gene were designed for PCR amplification and the fluorescence was monitored in a real time manner.The expression levels of GC-C mRNA in clinical samples(including 30 healthy blood donors,11 patients with benign intestinal lesions and 37 with colorectal cancer) were calculated according to the standard curve.The mRNA levels of GC-C were presented as the ratios of GC-C mRNA to ?_2-microgluobulin mRNA.Results: The linear detection range of this RFQ-RT-PCR system was from 10~1 to 10~9 pg/ml and the coefficient of variation values for intra-experimental and inter-experimental reproducibility ranged from 6.87% to 11.12% and from 8.86% to 15.19%,respectively.GC-C mRNA was expressed in 31/37 colorectal cancer patients,but not in healthy blood donors and patients with benign intestinal lesions.The mean level of GC-C mRNA was(0.88?)(0.06) in the above 31 colorectal cancer patients.GC-C mRNA levels were positively correlated to the Duke stage,lymph node metastasis and liver metastasis but not to patients' age,sex and tumor size.Conclusion: RFQ-RT-PCR has good sensitivity,specificity and reproducibility in determination of GC-C mRNA levels.Determination of GC-C mRNA levels in colorectal cancer patients may play a role in assessing Duke stage,lymph node metastasis,liver metastasis and approaching post-operation therapy.