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Objective To investigate the effect of lentivirus-stromal cell-derived factor-1α-green fluorescent protein(LV-SDF-1α-GFP) on the cardiac fibroblasts, the optimum conditions of infection, the expression and secretion of the target protein. Methods The cardiac fibroblasts of neonatal rats were primarily isolated and cultured by differential adherence methods, and were observed and identifi with immunofluorescence. LV-SDF-1α-GFP with different titers and conditions was transfected into cardiac fibroblasts. The expression of fluorescence and the optimal transfection conditions were observed. LV-SDF-1α-GFP target gene virus and negative control C0N145 virus were transfected into cardiac fibroblasts. The growth curve was drawn, and the effect of transfection on the proliferation of cardiac fibroblasts was explored. The cardiac fibroblasts were transfected with the optimum transfection dose, and the expression of SDF-1α was detected by Dot-blotting. The measurement data underwent statistical analysis. Results There was no statistical difference between the cardiac fibroblasts with SDF-1α transfected lentivirus and without no-transfected SDF-1α lentivirus. The peak of the expression of SDF-1α appeared in culture day 4 and statistical analysis showed significantly difference (P<0.05). Conclusion The LV-SDF-1α-GFP vector is of higher transfection efficiency to cardiac fibroblasts with the both low cytotoxicity and ability of secreting SDF-la protein.
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Objective To investigate the clinical value of the probe melting curve analysis‐based PCR assay for the detection of three types of αT .Methods A total of 149 blood and prenatal archival DNA samples (6 of which were amniotic fluid samples)with three knownαT genes ,which included 63 carriers with Hb CS ,22 cases with Hb QS ,43 individuals with Hb WS and 1 double heter‐ozygote with Hb CS and Hb WS) as well as 20 samples with normalα‐globin gene sequence that had been confirmed by RBD com‐bined with DNA sequencing were selected to test the specificity of probe melting curve analysis by blind analysis .Results The probe melting curve analysis accurately detected 100 of the DNA samples previously characterized by S RBD combined with DNA sequencing .Conclusion Probe melting curve analysis‐based PCR assay for the detection ofαT is featured with rapidity and accuracy and can be applied to clinical and prenatal diagnosis .
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Objective To improve the purifying method of Jo-1 antigen from rabbit thymus used for detection of anti-Jo-1 antibody by dot-blotting immunoassay(DB).Methods The rabbit thymus glands were cut into pieces,homogenized and extracted by PBS.Total protein was precipitated by acetone to get acetone powder(RTAP).The RTAP was solved in PBS and separated by an by anti-Jo-1 IgG affinity column.Results 5～7 g RTAP was obtained from 100g rabbit thymus glands.There was 19%～24% of protein in RTAP.Jo-1 antigen was enriched around 1900 folds through affinity chromatography,with 2.5% recovery of antigenic activity.In this preparation,there were several bands on SDS-PAGE,but only one band about 50 ku,reacted with anti-Jo-1 antisera on immunoblotting.Dot-blotting also showed that the antigen only reacted with Jo-1 antisera.The purified Jo-1 antigen was not stable for long time,but the antigenic activity could maintain for a long time when there was MgCl2 in the solution.Conclusion Affinity chromatography was a simple and easy method for purifying Jo-1 antigen from rabbit thymus.The antigen purified by affinity chromatography could meet the requirement for detecting Jo-1 antibody bydot-blotting.
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Objective:To study the infection rate of Chongqing duck hepatitis B virus(DHBV)in Chongqing and its seasonal difference in adult ducks,in order to optimize DHBV research model.Methods:560 serum specimens of Chongqing ducks were collected randomly from a duck farm in Spring and Summer of 2009.Duck hepatitis B virus DNA was detected by dot blotting,with DNA probe labelled with digoxigenin.Results:The infection rate of duck hepatitis B virus was 1.82% in Spring and 5.26% in Summer,with the lattre being significantly higher than the former(P
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Objective To investigate the clinical significance of quantitation of TGF-?1 mRNA of peripheral blood mononuclear cell (PBMC) in type 2 diabetic patients at different stages of nephropathy (DN). Methods TGF-?1 mRNA transcription in PBMC was analyzed quantitatively by RT-PCR and dot blot in 93 cases of type 2 diabetic patients and 35 normal controls. Correlation of TGF-?1 mRNA in PBMC with 24 hours urinary albumin excretion rate (UAER) was also analysed. Results TGF-?1 mRNA level of PBMC in type 2 diabetic patients was significantly higher than that in normal controls (1.56?1.00 vs 1.03?0.25, P
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Objective To determine the lumbrukinase contents and its hydrolysates in processed earthworm,set up a new evaluating method of dot blotting to evaluate the qualities of those processed Chinese medicines as earthworm. Methods Contents of lumbrukinase and its hydrolysates in processed earthworm were determined by dot blotting with rabbit antibody of anti-lumbrukinase as the first antibody. Blotting signal was analyzed by Quantity One software. Results Contents of lumbrukinase and its hydrolysates in processed earthworm of Pheretima aspergilum,P. pingi,Drawida japonica,and D. gisti were 125.23,118.34,81.05,and 72.13 ?g/g,respectively. Conclusion There was no significant difference between the processed earthworms of P. aspergilum and P. pingi in the content of lumbrukinase and its hydrolysates,both of which were higher than those of the other two species in Shandong. Theoretically,the processed products of P. pingi could be used as the succedaneum of P. aspergilum in the clinical of traditional Chinese medicine. Dot blotting,as a convenient and accurate method,could be broadly used for the evaluation of the processed products of Chinese crude drugs similar to earthworm.
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Pterygium, a disease of unknown origin and pathogennesis, is a chronic condition characterized by the encroachment of triangular portion of the bulbar conjunctiva onto the cornea. We have studied the expression of extracellular matrix genes and oncogenes in cultured pterygium using Northernm dot, and slot blot hybridizations. Northern hybridization with total RNA isolated from passaged (4-8 passages) cultures demonstrated expression of genes for alpha1(I) and alpha1(III) procollagen, fibronectin, and c-Ha-ras, but no expression of gene for c-myc was observed. The pterygium exhibited significantly increased expression of alpha1(I) and alphal(III) procollagen genes when compared with normal control cells(p<0.01). And We observed there were no differences between pterygium and normal control cells in the expression of genes for fibronectin and c-Ha-ras. According to these results we thought that the causes of pterygium may be related to the increased expression of alpha1(I) and alphal(III) procollagen genes but may not be related to c-Ha-ras, c-myc, and fibronectin genes.