ABSTRACT
Objective To investigate the value of intravital fluorescence microscopy in the observation of the changes of hepatic microcirculation in the rat model with hepatic cirrhosis and portal hypertension.Methods Seventy male SD rats were selected.According to the random number table,40 SD rats were randomly divided into the sham operation group,bile duct ligation (BDL) 2 weeks group,4 weeks group and 6 weeks group,there were 10 rats in each group,and the hepatic microcirculation of the rats was observed with intravital fluorescence microscope; the remaing 30 SD rats were randomly divided into the normal saline (NS) group,endothelin-1 (ET-1) group and the S-nitrosoglutathion (GSNO) group at 4 weeks later after the establishment of BDL model.The changes of hepatic microcirculation of the 3 groups were observed.All data were analyzed using the one-way analysis of variance (ANOVA) or paired samples t test.Results Nine rats died in the BDL model groups,and the survival rate was 85.0％ (51/60).All rats in the sham operation group were survived.The hepatic sinusoid diameters were decreased as time passed by.The hepatic sinusoid diameters of the BDL 2 weeks group,4 weeks group and 6 weeks group were (13.6 ± 1.0) μm,(8.8 ± 0.7) μm and (8.0 ± 0.5) μm,respectively,which were significantly shorter than (17.4 ± 1.0) μm of the sham operation group (t =5.86,18.24,15.57,P ＜ 0.05).The hepatic sinusoid densities of the BDL 2 weeks group,4 weeks group and 6 weeks group were (6.8 ±0.8)/ 200 μm,(4.3 ± 1.8)/200 μm and (4.0 ± 1.2)/200 μm,which were significandy lesser than (8.8 ± 0.5)/200 μm (t =3.25,2.77,2.12,P ＜ 0.05).At 15 minutes after injection of NS,the hepatic sinusoid diameter of the NS group was (7.2 ± 1.2) μm,which was significantly different from (6.9 ± 0.5) μm before injection of NS (t =0.89,P ＞ 0.05) ; the hepatic sinusoid density of the NS group before and after injection of NS were (6.6 ± 0.4) / 200 μm and (6.8 ± 1.4)/200 μm,with no significant difference(t =1.12,P ＞0.05).At 15 minutes after injection of ET-1,the hepatic sinusoid diameter of the ET-1 group was (5.4 ±0.5) μm,which was significantly different from (7.9 ± 0.6) μm before injection of ET-1 (t =7.39,P ＜ 0.05) ; the hepatic sinusoid density of the ET-1 group before and after ET-1 injection were (5.8 ± 1.2)/200 μm and (5.4 ± 1.8)/200 μm,with no significant difference(t =0.84,P ＞0.05).At 15 minutes after injection of the GSNO,the hepatic sinusoid diameter of the GSNO group was (11.4 ± 1.3) μm,which was significantly different from (7.5 ± 1.7) μm before injection of GSNO (t =5.95,P ＜ 0.05) ; the hepatic sinusoid density of the GSNO group before and after GSNO injection were(5.6 ± 0.8)/200 μm and (6.4 ± 1.6)/200 μm,with no significant difference (t =0.54,P ＞ 0.05).Conclusions The changes of hepatic microcirculation observed under intravital fluorescence microscope could reflect the progress of hepatic cirrhosis,and the changes of hepatic sinusoid diameters caused by drugs could be dynamically monitored under the intravital fluorescence microscope.
ABSTRACT
A method for counting Infectious pancreatic necrosis virus (IPNV) through epifluorescence microscopy was analyzed in detail. Image processing and statistic considerations are included. The particle size of viruses was compared in different experimental conditions such as the staining of the virus with SYBR-Green I or with antibodies for specific fluorescence labeling of viral proteins. The type of surface used as mounting support was assayed as well. The results indicated that the most suitable method involves the mounting of the viral-containing suspension on a membrane filter followed by the staining with a monoclonal antibody specific for a viral protein combined with a FITC (fluorescein isothiocyanate)-conjugated secondary antibody.
Subject(s)
Aquabirnavirus , Aquabirnavirus/pathogenicity , Birnaviridae Infections/diagnosis , Birnaviridae Infections/genetics , Birnaviridae Infections , Salmonidae , Fluorescent Antibody Technique/methodsABSTRACT
OBJECTIVE To design molecular beacon detecting embB330 codon of ethambutol-resistant Mycobacterium tuberculosis(MTB),meanwhile,and try to detect fluorescence of mutation site of embB330 codon in liquid by fluorescence microscope by compareing the mutation strains and standard strains.METHODS The software,Beacon designer,was used to design molecular beacon detecting embB330codon and detecting fluorescence signal from hybridization between the amplified product and probe by fluorescence microscope,and to confer to the sequencing results.RESULTS The difference between PCR products from standard strain and ethambutol-resistant one was obvious in detecting the fluorescent light by use of fluorescence microscope.We detected fluorescent light signal between the 33 ethambutol-resistant strains and 10 H37RV standard strains.The rate of ethambutol-resistant strains was about 3%,and the rate of sequencing was about 3%.CONCLUSIONS The technology of molecular beacon effectually can detect mutation single base site of embB330codon.Fluorescence microscope owns characteristics such as high sensitiveness to detect the fluorescent light.
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In order to identify novel proapoptotic genes, we screened approximately 1,000 hypothetical genes whose functions are completely unknown. After these genes were transiently expressed in HeLa cells, their nuclei images were captured using automated high-speed fluorescence microscope, through which the ratio of apoptotic nuclei was estimated. We selected genes that induce greater than 3-fold increase in apoptotic nuclei compared to that of the vector control. The candidate proapoptotic genes were sequenced and their effects on cell death were further confirmed by the additional assay, DNA fragmentation ELISA. Finally, we were able to identify 4 full-length hypo-thetical genes with proapoptotic activity.
Subject(s)
Humans , Apoptosis , Cell Death , DNA Fragmentation , Enzyme-Linked Immunosorbent Assay , Fluorescence , HeLa CellsABSTRACT
OBJECTIVE To design molecular beacon probe of 43 Codon of high mutation site in rpsL of STR resistant MTB and its amplification system, meanwhile, to find the way of detecting the fluorescent light and making a qualitative judgment by use of fluorescence microscope and image analysis software. METHODS The software, Beacon designer, was used to design the 43 Codon molecular beacon probe and the amplification system, and the fluorescence microscope and image analysis software were used to detect the fluorescent light and make a qualitative judgment. RESULTS The strap of amplification product was shown clearly. The difference between PCR products from standard strain and STR resistant one was obvious in detecting the fluorescent light by use of fluorescence microscope. The rate of resistant STR detected was about 80% (P
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Objective:To design hair clamp probe and the probes with single extending arm detecting embB306codon of Ethambutol resis-tant Mycobacterium tuberculosis bacterium(MTB),meanwhile,to design fair clamp probe chip and detecting fluorescence signal from hy-bridization between the amplified product and probe by fluorescence microscope.Methods:The software,Beacon designer,was used to de-sign fair clamp probe and the probes with single extending arm detecting embB303codon and detecting fluorescence signal from hybridiza-tion between the amplified product and probe,and confer to the sequencing results.Results:The difference between PCR products from standard strain and ethambutol resistant one was obvious in detecting the fluorescent light with fluorescence microscope.We detected fluo-rescent light signal between the 33 ethambutol resistant strains and 10 H37RV standard strains.The rate of resistant ethambutol detected with hair clamp probe was about 66%,and the rate of sequencing was about 69%.Conclusions:The mutation site of embB306codon of MTB is the main reason of ethambutol resistant MTB.The technology of fair clamp DNA probe chip can effectively detect mutation of single base site.Fluorescence microscope can sensitivily detect the site of hybridization on fluorescence chip.
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By means of the observation of acridine orange-stained lysosomes with fluorescence microscope, we studied the intracellular killing and digestive process after alveolar macrophage (AM) ingested living-yeast. Based on the morphological changes of the cells, we divided the process into three stages: the pre-phagosome-lysosome fusion stage, the phagosome-lysosome fusion and killing stage, and the digestive stage. The results revealed that all the values of phagocytic index, phagocytic rate, fusion index and fusion rate of the isolated AM of rabbits infected with living-BCG were higher than those of normal rabbits (P