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【Objective】 To establish a stable human megakaryocytic cell line with low expression of Ley antigen to further study the role of Ley on activation of platelets. 【Methods】 The expression level of the Ley antigen in a human megakaryocytic cell line, DAMI, was determined using Western Blot and flow cytometry. The expression level of genes that encode fucosyltransferase (FUTs), which was involved in the biosynthesis of Ley antigen, was also determined to identify the candidate genes to be knocked out. The candidate FUT gene was knocked out via a CRISPR/Cas 9 gene knockout system and cells with low Ley antigen expression were sorted by flow cytometry. The sorted cell line was cultured and characterized. 【Results】 The Ley was expressed intensively on DAMI cell. FUT1 and FUT4 mRNA was expressed relatively higher, both may be key enzymes for the biosynthesis of the Ley antigen. In the DAMI cell line with the knockout of FUT1 gene, the expression of the Ley adntigen was remarkedly reduced, while cell proliferation was not affected compared to the wildtype control cells. 【Conclusions】 Since various FUTs contributes to the biosynthesis of the Ley antigen, the knockout of the primary one of them cannot totally block its biosynthesis, but only reduce its expression. In this study, a stable FUT gene knockout human megakaryocyticcell line is established using CRISPR/Cas 9 technology, which provides basis for the study of the impact of the Ley antigen on platelet functions.
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Objective To observe the expression change of melanoma-associated antigen(MAGE)-A9 in colorectal cancer (CRC)tissue and to explore its significance.Methods The samples in 23 cases of initially diagnosed CRC in the Affiliated Hospital of Nantong University from January 2006 to December 2008 were collected.The quantitative real-time(qRT)-PCR was adopted to detect MAGE-A9 mRNA expression in cancer tissue and corresponding paracancerous tissue.Its correlation with the clinicopatho-logical features and prognosis was analyzed.Results The positive rate of MAGE-A9 in CRC tissue was significantly higher than that in paracancerous normal tissue(P<0.05).MAGE-A9 protein expression in CRC was related to the clinicopathological features such as tumor differentiation degree(P=0.011),TNM stage(P=0.003),tumor infiltration depth(P=0.001)and lymph node me-tastasis(P=0.003).The survival analysis showed that the expression of MAGE-A9 was closely related to the prognosis of CRC pa-tients.Conclusion MAGE-A9 expression is increased in CRC tissue,suggesting the poor prognosis.
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Objective To explore the expression and clinical significance of sodium borate transporter member 11 of solute carrier family 4 (SLC4A11) and tumor protein P53 (TP53) proteins in gastric cancer (GC).Methods From March 2004 to December 2009,a total of 415 patients with GC,who received operation at Department of General Surgery of Affiliated Hospital of Nantong University,were enrolled.The clinical data and gastric tissues samples of them were collected.At same period,64 patients with non-malignant gastric diseases,who underwent surgery at Department of General Surgery of Affiliated Hospital of Nantong University,were also recruited.The clinical data and gastric precancerous tissues were also collected.The tissues were maken into tissue microarray (TMA).The expression of SLC4A11 and TP53 protein in tissue microarrays was detected by immunohistochemical staining.The relationship between the two proteins and clinicopathological parameters and prognosis of patients were analyzed.Chi square test,and univariate and multivariate analyses were used for statistical analysis.Results The positive rates of protein expression of SLC4A11 and TP53 in GC tissues were 48.19％ (200/415) and 34.94％ (145/415),respectively,which were higher than 17.19％ (11/64) and 6.25％ (4/64) in gastric precancerous lesions,respectively,and the differences were statistically significant (x2 =24.150 and 59.345;both P＜0.01).The results of statistical analysis showed that the expression of SLC4A11 was correlated with human epidermal growth factor receptor 2(Her2) levels,depth of invasion,distant metastasis and TNM staging (x2 =28.056,11.300,8.880,24.943;all P＜0.05).Meanwhile,the expression of TP53 was related with depth of invasion,lymph node metastasis,distant metastasis,TNM staging and preoperative carcinoembryonic antigen (CEA;x2=12.333,7.875,9.347,20.307,10.678;all P＜0.05).The expression of TP53 was significantly positive correlated with SLC4A11 expression (x2 =6.237,P=0.013).The results of univariate analysis showed that SLC4A11,TP53,SLC4A11+/TP53+,Her2,preoperative CEA and cancer antigen 19-9 (CA19-9) levels were correlated with the poor prognosis of GC patients (hazard ratio (HR)=1.947,1.459,1.797,1.419,2.221,1.908;all P＜0.05),while the results of multivariate analysis indicated that positive SLC4A11 and TNM staging were the independent parameters for judging the prognosis of patients with GC (HR =1.954,1.468,both P＜0.05).Conclusions The high expression of SLC4A11 and TP53 is related to the occurrence and development of GC.Combined detection of their changes will contribute to the early diagnosis and prognostic evaluation of patients with GC.
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Objective To screen differentially expressed serum proteins by label-free quantitative proteomics in patients with atopic dermatitis,in order to discover serum protein markers for atopic dermatitis.Methods Serum samples were collected from 6 patients with atopic dermatitis and 6 healthy human controls.Then,the samples from the patients were pooled together,so with those from the controls.Proteins were extracted from the two joint serum samples followed by separation with one-dimensional sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE).Peptides were obtained by enzymolysis in protein gels,which were subsequently identified by high performance liquid chromatography tandem mass spectrometry.The Mascot software was used for database searching,and the Scaffold software for relatively quantitative analysis of proteins.Results Totally,76 proteins with a difference of greater than 1.5 folds were identified between the serum samples of patients with atopic dermatitis and controls.There were 50 up-regulated and 26 down-regulated serum proteins in the patients compared with the healthy controls.Among these differentially expressed proteins,26 were only detected in the sera of the patients,and 14 proteins in those of the controls.Conlusion Differences exist in the expression of serum proteins between atopic dermatitis patients and healthy people.
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Objective To investigate the expressions of guanylyl cyclase-c(GC-C) and caudal-type homeobox transcription factor 2 (CDX2) in human gastric tissues and precursor lesions and its significance. Methods The cancerous and paracancerous (5 cm from cancer lesion )samples from 30 cases of gastric cancer and 32 samples including 23 intestinal metaplasia and 9 dysplasia were collected. The mRNA expressions of GC-C and CDX-2 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and proteins of GC-C and CDX-2 were measured by using Western blot and immunofluorescence methods. Results The mRNA expressions of GC-C and CDX-2 were absent in paracancerous tissues, but were 66.7% and 63.3% in cancerous tissues, respectively(P=0. 000). The Western blot indicated that expressions of GC-C and CDX-2 were 19/30 and 17/30 in cancerous tissues, but absent in paracancerous tissues(P=0. 000). The immunofluorescence examination revealed that GC-C and CDX-2 expressions were 39.1% and 39.1% in intestinal metaplasia, 55.6% and 55.6% in dysplasia, and 56.7% and 60.0% in cancerous tissues, respectively, but absent in paracancerous tissues. Moreover, expressions of GC-C and CDX-2 showed a statistical difference between intestinal-type and diffuse-type of gastric cancer (P＜ 0.05) ,but had no correlation with age, sex, size of the lesion, clinical stage and lymphnode metastasis. The positive correlation was found in expressions of GC-C and CDX2 between intestinal metaplasia and cancerous tissues(r=0. 4524 and 0. 3845, P= 0. 037 and 0. 0408, respectively). Conclusions The over expressions of GC-C and CDX2 in human gastric cancer is associated with precursor lesions and may play an important role in the carcinogenesis of gastric cancer. The examination of GC-C and CDX2 expressions will be helpful in diagnosing gastric cancer and precursor lesions.
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The incretion secretion metrical technique is the important embranchment in clinical diagnosis presently,in which the magnetic separate enzyme immunoassay is the main technique.This paper introduces the principle and design of Chinese enzyme immunoassay instrument,and emphatically discusses the design of nonlinear data processing model.
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Purpose:To Explore the requirements and accuracy for applying Microwave-ELISA Two-Steps-Test to the factory physical examination.Methods:Do the Microwave-ELISA Two-Steps-Test for 1,128 factory physical examination specimens that need the HBsAg and HBeAg inspection;comparing the result to the conventional ELISA test;and retest the specimens with the HBsAg-positive results.Results:There are 119 HBsAg-positive and 32 HBeAg-positive results for the ELISA test;there are 122 HBsAg-positive and 34 HBeAg-positive results for the microwave test;all the HBsAg-positive results are proved to be positive by using the colloid gold HbsAg rapid testing paper.Conclusion:Microwave-ELISA Two-Steps-Test is timesaving,simple and high sensitive for testing Hepatitis-B signs,which can reduce the hook phenomenon and can be used to do the large number of HBsAg and HBeAg checks in factory physical examinations.It can hasten the result report and greatly improve the efficiency.
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Objective To compare the operation modes of AP-960 full automated ELISA system.Methods Two models were tested: beginning with adding specimens(model 1) and beginning with adding enzymes(model 2).The following factors were compared: the consumption of suckers,usage of time,result of test and the usual alarm during the test.Results The consumptions of suckers were 564 and 84 respectively.The needing time of the former mode was 125 minutes and that of the latter mode was 90 minutes.Conclusion The mode beginning with adding enzymes(model 2) is better for the current status of our clinical laboratory.