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Objective To prepare the mAbs against hK6 for establishing a sandwich enzyme-linked immunosorbent assay(ELISA) of hK6,and exploring its clinical value.Methods The hybridoma technique was used to prepare mAbs against hK6.The mAbs were purified and labelled with horseradish peroxidase for the sandwich ELISA method.The sandwich ELISA method was used to detect the serum hK6 concentrations in patients with malignant gastric neoplasm.Then the best antibody pair was selected from coating antibody and enzyme-linked antibody to establish a sandwich ELISA method through the chessboard titrations.Compared with CEA,we explored the feasibility of hK6 as gastric cancer biomarkers.Results A sandwich ELISA method was established for quantifying hK6 in serum.The results showed that the optimal concentration of coating antibody was 5 μg/mL.The optimal concentration of enzyme-linked antibody was 1:2 000.Serum hK6 in the patients with gastric cancer groups[(5.78±1.66)ng/mL] than healthy individuals groups[(3.35±0.67)ng/mL] and those in gastric ulcer groups[(3.59±1.02)ng/mL],the difference was statistically significant(P0.05).The hK6 positive rate of gastric cancer was 69.70％,and CEA was 45.46％.In the combined detection,the positive rate was 78.79％.Conclusion A sandwich ELISA is established successfully.As a favorable serum biomarker for gastric cancer,the detection of hK6 together with CEA is helpful in the diagnosis of gastric cancer.
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AIM:ToinvestigatetheprotectiveeffectofquercetinonangiotensinⅡ(AngⅡ)-inducedcardio-myocyte hypertrophy and its possible mechanism .METHODS: Cardiomyocyte hypertrophy was induced by AngⅡ ( 100 nmol/L) in primary neonatal cardiomyocytes and H 9c2 cells.The cells were treated with different concentration of querce-tin (10 μmol/L, 20 μmol/L and 40 μmol/L) for 48 h and then the cardiomyocyte surface areas were measured by immu-nofluorescence .Proteasome activity was detected by fluorescent peptide substrate .The phosphorylated levels of GSK-3α/βand Akt in H9c2 cells were determined by Western blot .RESULTS:Compared with control group , the cardiomyocyte sur-face areas were both increased in primary cultured neonatal cardiomyocytes and H 9c2 cells, while the surface areas were significantly decreased by quercetin , especially at concentration of 20 μmol/L compared with Ang Ⅱgroup (P<0.05). Compared with control group , the chymotrypsin-like, trypsin-like and caspase-like activities of proteasome were all in-creased in H9c2 cells (P<0.05).The trypsin-like and caspase-like activities of proteasome were inhibited by 20 μmol/L and 40 μmol/L quercetin , while chymotrypsin-like activity was inhibited only at 20 μmol/L of quercetin compared with AngⅡgroup (P<0.05).In addition, phosphorylated levels of GSK-3α-Ser21, GSK-3β-Ser9 and Akt-Ser473 in AngⅡgroup were all increased compared with control group , which were obviously inhibited by in 20 μmol/L and 40 μmol/L quercetin ( P<0.05 ) .CONCLUSION: Quercetin decreases cardiomyocyte hypertrophy through proteasome inhibition , which may be related to the inhibition of Akt and therefore increasing activation of GSK -3α/βin H9c2 cells.