Objective To compare compound staining of Alizarin red S and Alcian blue showing ossified skeletal structures and cartilage in fetal,neonatal and adult rat. Methods All specimens were skinned and fat tissue removed completely and eviscerated,then fixed in 95% ethanol;Acetone was used remove fats; 0.1% Alizarin red S and 0.15%-0.3% Alcian blue were used in ossified bone and cartilage;Differentiation and clearing in 0.5%-2% aqueous KOH. Results Ossified bones showed rose red;cartilage showed blue;the others were transparent.Conclusion The best results of Alizarin red S and Alician blue compound staining could be obtained in all rat skinned,eviscerated,and removed the adipose tissues.

The academic thought of preventing possoble disease is one of the important part of the theoretical system of Chinese medicine. In Pro. WEI Pin-kang's opinion, we must identify the possible diseases prior to preventing it. The article discusses how to identify the possible disease from three aspects, such as indentifying constitution, comprehensive analysis by the four examination methods, drawing assistance from micro-indexex, which to cultivate young physicians' clinical thinking of indentifying possible disease.

Objective To study the effects of calcium dobesilate plus laser on serum vascular endothelial growth factor ( VEGF ) and insulin-like growth factor-1 ( IGF-1 ) in patients with diabetic retinopathy .Methods 82 patients(93 eyes) were randomly divided into two groups ,the control group(n=41 cases,46 eyes) and the obser-vation group(n=41 cases,47 eyes).The control group was treated through laser ,while the observation group was treated through laser plus calcium dobesilate .Serum VEGF and IGF-1 were measured before and after treatment . Results Before treatment,visual acuity had no significant difference between the two groups (t=0.601,P>0.05). After treatment,visual acuity was improved in 30 eyes in the control group with improving rate of 65.2%,visual acuity improved in 38 eyes in the observation group with improving rate of 80.9%.The improving rate of visual acuity in the observation group was significantly higher than that of the control group (χ2 =6.92,P<0.05).The total effective rate of the control group was 73.9%,which was significantly lower than 87.2%in the observation group (χ2 =9.28,P<0.05). Serum VEGF and IGF-1 had no significant differences before and after treatment in the control group ( t=0.613, 0.496,all P>0.05).After treatment,serum VEGF and IGF-1 in the observation group were (91.4 ±25.1)ng/L and (121.6 ±21.7)mg/L,which were significantly lower than those before treatment (t=3.721,5.992,all P<0.05). Conclusion Calcium dobesilate plus laser can significantly decrease serum VEGF and IGF-1 in patients with diabet-ic retinopathy .

Case Method is useful to helping the teacher teach students the knowledge of gynecological diseases and train their capability of clinical thought. The key of the Case Method is choosing the cases and designing teaching steps carefully.

Objective To prepare monoclonal antibodies against VP6 protein of human group A rotavirus(RVA),develop a double-antibody sandwich ELISA detection method,and to verify and preliminarily apply the method,in order to provide more choices for the rapid diagnosis,epidemiological investigation and virus surveillance of RVA in China.Methods VP6protein of human RVA G9P [8] strain was expressed and purified with baculovirus expression system,and then applied to immunize five female BALB/c mice to generate monoclonal antibodies.The median effective concentration(EC_(50)) of the monoclonal antibodies was determined by indirect ELISA,and the binding activity of monoclonal antibodies to RVA was determined by Western blot and indirect immunofluorescence assay(IFA).Using monoclonal antibody pairing,one strain was used as coating antibody and the other strain was labeled with HRP for detection,and a double-antibody sandwich ELISA was developed.The sensitivity,specificity and precision of the method were further verified.RV clinical stool samples were taken and detected by Oxoid ProSpecT commercial kit and the developed double-antibody sandwich ELISA respectively,and the coincidence rate of the detection results was compared.Results The purified soluble VP6 protein was obtained,with a purity of over 90% and an expression level of about 4.5 mg/L.Two strains of RVA VP6 monoclonal antibodies were screened,named 1L3 and 4F22,and the EC_(50) of them binding to VP6 protein was 1.777 and 3.748 ng/mL,respectively,indicating that the two strains of monoclonal antibodies could bind well to RVA.The sandwich ELISA method for detection of human RVA was developed using 1L3 as the capture antibody and HRP labeled 4F22 as the detection antibody.The minimum detectable amount of RVA VP6 protein was 156.25 ng/mL.There was no cross-reaction between the two monoclonal antibodies and human Norovirus(NoV),Sapovirus(SaV) and adenovirus(AdV).The coefficients of variation(CVs) of intra-batch and inter-batch precision verification were both less than 10%.The 192 clinical stool samples of RV were detected by using this method,and compared with the commercial kit results,the positive coincidence rate and negative coincidence rate were 99% and 98%,respectively.Conclusion The double-antibody sandwich ELISA against human RVA developed in this study has good specificity,sensitivity,and precision,which can be used for the detection of human RVA.

Objective To prepare monoclonal antibodies against VP6 protein of human group A rotavirus(RVA),develop a double-antibody sandwich ELISA detection method,and to verify and preliminarily apply the method,in order to provide more choices for the rapid diagnosis,epidemiological investigation and virus surveillance of RVA in China.Methods VP6protein of human RVA G9P [8] strain was expressed and purified with baculovirus expression system,and then applied to immunize five female BALB/c mice to generate monoclonal antibodies.The median effective concentration(EC_(50)) of the monoclonal antibodies was determined by indirect ELISA,and the binding activity of monoclonal antibodies to RVA was determined by Western blot and indirect immunofluorescence assay(IFA).Using monoclonal antibody pairing,one strain was used as coating antibody and the other strain was labeled with HRP for detection,and a double-antibody sandwich ELISA was developed.The sensitivity,specificity and precision of the method were further verified.RV clinical stool samples were taken and detected by Oxoid ProSpecT commercial kit and the developed double-antibody sandwich ELISA respectively,and the coincidence rate of the detection results was compared.Results The purified soluble VP6 protein was obtained,with a purity of over 90% and an expression level of about 4.5 mg/L.Two strains of RVA VP6 monoclonal antibodies were screened,named 1L3 and 4F22,and the EC_(50) of them binding to VP6 protein was 1.777 and 3.748 ng/mL,respectively,indicating that the two strains of monoclonal antibodies could bind well to RVA.The sandwich ELISA method for detection of human RVA was developed using 1L3 as the capture antibody and HRP labeled 4F22 as the detection antibody.The minimum detectable amount of RVA VP6 protein was 156.25 ng/mL.There was no cross-reaction between the two monoclonal antibodies and human Norovirus(NoV),Sapovirus(SaV) and adenovirus(AdV).The coefficients of variation(CVs) of intra-batch and inter-batch precision verification were both less than 10%.The 192 clinical stool samples of RV were detected by using this method,and compared with the commercial kit results,the positive coincidence rate and negative coincidence rate were 99% and 98%,respectively.Conclusion The double-antibody sandwich ELISA against human RVA developed in this study has good specificity,sensitivity,and precision,which can be used for the detection of human RVA.

BackgroundRetinal pigment epithelial (RPE)cell senescence damage the metabolism of photoreceptor,leading to retinal dysfunction and loss of vision.To understand RPE cell senescence mechanism will contribute to the study of age-related macular degeneration ( AMD).ObjectiveThe present study was to prepare the ageing RPE cell model with passage and explore its potential mechanisms.MethodsThis study was approved by the Ethic Committee of Qingdao University Medical College,and the informed consent was obtained from each gravida.Six human eyeballs were obtained from artificial labor fetusl with the gestational age 16-28 weeks.RPE cells were isolated,cultured and passaged in vitro to establish the cell replicative aging model.The third to twelfth cells were collected to be used to this experiment.Human keratin was used to identify the cells by immunochemistry,and MTT method was utilized to assess the proliferation and viability of different generations of cells as the A490 value.The cellular cycles and transmembrane potential (△ψm)of mitochondrion (△ψm) with passage were detected and compared using Flow Cytometry. Results Cultured and passaged cells showed the hexagon in shape with the melanin in 1-2 generations of cells and presented with the brown staining in cytoplasm for human keratin.The melanin was absent in the third generation cells.Vibrant growth statues were seen from the 3-6 generations cells and thereafter the proliferation ability reduced.The cells of G0/G1 phase were gradually elevated with the passage from 3 - 12 generations with a percentage of 68.40％ in the third generation of cells to 87.33％ in the twelfth generation of cells,showing a significant difference among various generations ( F =180.43,P =0.00),and that of the sixth,ninth and twelfth generation of cells was significant higher than the third,sixth and ninth generation respectively (t =4.002,P＜0.05 ; t=12.885,P＜0.01 ;t=16.387,P＜0.01 ).MTT assay showed that of RPE cells were significantly declined with the passage ( F =38.77,P =0.00),and the A490 value of the ninth,twelfth generations of cells was considerably lower in comparison with sixth and ninth generation respectively ( t =5.991,11.983,P＜0.01 ).From 3 through 12 generations of cells,the staining intensity of rhodamine 123 was gradually decreased ( F =121.68,P =0.00 ),and the staining intensity in the sixth,ninth and twelfth generation of cells was significant lower than that the third,sixth and ninth generation respectively(t=6.918,7.620,11.207,P＜0.01 ).Conclusions A replicative aging model can be successfully created by the passage in vitro using human fetal RPE cells.The reduce of transmembrane potential and damage of mitochondria might be one of mechanisms of senescence of RPE cells.

The enzyme,as a biocatalyst,can not be surpassed in speeding up the reactions by other common chemical catalysts under the suitable conditions and can catalyze the reaction mildly,simplify the facilities needed,improve the work conditions,and reduce the production costs,etc.Those are its advantages mentioned above.But while the enzyme is extracted outside the cells,the molecular conformation may be changed with decreasing its bioactivity and stability seriously.As one type of physical energy,(ultrasonic) wave with lower intensity and suitable frequency can make the conformation of enzymatic molecular comformation transform positively and the transforming reactions accelerate so as to facilitate the effective constituents of Chinese materia medica and natural products transforming.

Objective To research the correlation between SOD and TXB2/6-k-PGF1? and relationship with TCM syndrome in stroke patients. Methods A contrast test on blood SOD and plasma TXB2 and 6-k-PGF1? was given separately to two groups, one group was 74 cases with stroke and the control group was 36 cases in health. Results Content of b1ood SOD in stroke group reduced remarkably compared with the health group (P

BACKGROUND: Drug therapy can improve the clinical symptoms and extend the gestational age through the maternal way to treat intrahepatic cholestasis of pregnancy (ICP), however, fetal hypoxia and meconium-stained amniotic fluid can not clearly improved. OBJECTIVE: To explore the effect of replacement of amniotic fluid that contains high concentrations of bile acids by artificial amniotic fluid through amniocentesis in the rat model of ICP. DESIGN, TIME AND SETTING: Random control animal experiments were completed in the Laboratory (first-degree laboratory) of Animal Experimental Center in Chongqing Medical University from March to September 2008. MATERIALS: Forty SD pregnant rats with 15 days of pregnancy were randomly divided into intracavitary injection of S-Adenosyl-L-methionine (SAMe) (IUS), intracavitary injection of sodium chloride (IUN), intravenous injection of SAMe (IVS) and blank control (IC) groups, with 10 rats in each group. METHODS: On day 15 of pregnancy, ICP rat models were induced by daily injection of estradiol benzoate. On day 17 of pregnancy, 1 mg SAMe and 0.3 mL sodium chloride were injected into amniotic cavity of in the IUS and IUN groups through amniocentasis. Meanwhile, 30 mg SAMe were injected though tail veins in IVS group. No treatment was performed in the IC group. MAIN OUTCOME MEASURES: The meconium-stained amniotic fluid rate and the still birth rate were calculated. Total bile acid (TBA) level in amniotic fluid was measured. In addition, the expression of hypoxia-inducible factor 1 ?(HIF-1?) mRNA in the placenta of rats were examined by immunohistochemistry and reverse transcription PCR at the days 20 of pregnancy. RESULTS: The meconium-stained amniotic fluid rate in the experimental group was lower than that of the IC group (P