Objective To detect 10 RNA viruses including Alphavirus in Togoviridae, Flavivirus in Flaviridae, Hantavirus and Nairovirus in Bunyavirudae and SARS-CoV in Coronavirudae by using genechip technique. Methods The universal PCR primers of Alphavirus and Flavivirus and the PCR primers specific for HFRSV in Hantavirus and XJHFV in Nairovirus were designed by DNAStar software. PCR primers specific for SARS-CoV were adopted from WHO website. In addition, all the PCR primers specific for each virus were designed inside the regions of universal primers. These specific primers were utilized for amplification of cDNA probes. The concentration of probes, the hybridization temperature and duration, the formulation of hybridization solution and the washing conditions were optimized. Results The specific hybridization signals could be obtained when the concentration of probes was 0.3?g/?l. Good hybridization signals could be obtained for all the 10 RNA viruses when the hybridization solution contained 20% formamide, and the hybridization reaction was conducted at 60℃ for 1.5 hours. Two or four pathogens could be detected simultaneously when the target nuclear acids were amplified by multiplex PCR. Conclusion The results showed that the virus pathogens could be detected by genechip technique, and the key step was to design suitable primers and probes.

BACKGROUND:There are many kinds of ways to obtain osteoblasts at present, but how to get high-purity osteoblasts in a easy and fast way has become a hot research.
OBJECTIVE:To explore a method to get massive and high purified osteoblasts effectively by comparing three common primary osteoblast culture methods, and to observe the biological characteristics of the osteoblasts from the skul of neonatal rabbit.
METHODCalvarias were dissected from newborn New Zealand white rabbits within 24 hours, and osteoblasts were isolated with bone tissue method, col agenase digestion method and modified tryptase and col agenase sequential digestion method respectively, then the cells were subcultured in vitro. Osteoblast proliferation and osteogenic activity were identified by inverted microscope for morphology observation. The rate of living osteobalsts was counted with trypan blue staining. The growth curve of the cells was drawn with MTT method. Alizarin red staining was applied to detect alkaline phosphatase and osteocalcin protein in the cellculture supernatants. Col agen I and col agen III immunohistochemical staining was also performed. RT-PCR was used to determine the expression of osteocalcin and col agen I mRNA expression.
RESULTS AND CONCLUSION:The cultured cells showed highly homogeneous appearance with active proliferation, and they had the typical features of osteoblasts. Alizarin red staining and col agen I immunohistochemical staining were both positive, while col agen III immunohistochemical staining was negative. Alkaline phosphatase and osteocalcin protein expression in the cellculture supernatants can be detected. The expression of osteocalcin and col agen I mRNA was positive in the RT-PCR test. Compared with col agenase digestion method, the modified tryptase and col agenase I sequential digestion method cost less time, presented higher production of osteoblasts and higher cellsurvival rate (P<0.05). Bone tissue method was the easiest method and did the least damage to osteoblasts, but it presented lowest production of osteoblasts and cost the maximum time among the three methods. So it cannot be used in large-scale osteoblast culture. A large quantity of high purity osteoblasts were obtained by modified trypsase and col agenase I sequential digestion method, which can be used as a reliable and efficient way to obtain the original generation osteoblasts in vitro.

Objective To develop a real-time quantitative PCR(RQ-PCR) assay based on TaqMan technology for rapid detection and quantification of tick-born encephalitis virus (TBEV) RNA. Methods According to all the TBEV genome sequences in GenBank, RQ-PCR primers and probes were designed in the conservative regions of TBEV C gene and NS5 gene. In addition, primers for conventional PCR were designed using E gene as target. The detective system was established and validated by using TBEV MDJ01 strain. In order to examine the specificity of the system, other viruses of flavivirus were assayed with the RQ-PCR simultaneously. The TBEV standard curve was drawn respectively by measuring TCID_ 50 titre and copy number. The sensitivity of RQ-PCR and the conventional PCR assays were compared, and TBEV infected mice model was reproduced for evaluation. Results The sensitivity of RQ-PCR assay was 100copies/reaction or 1 TCID_ 50 , which was 10 fold higher than conventional PCR. The results were all negative when used to detect other flavivirus including the yellow fever virus, dengue virus type 1, 2, 3 and 4, Japanese encephalitis virus, West Nile virus. The coefficient of variability was less than 5% from inter- and intra-assay showing that both the repeatability and stability of the system were good. Conclusion A sensitive, specific and convenient RQ-PCR method has been established, which is valuable for early detection of TBEV.

Objective:To establish a multidrug resistant human salivary adenoid cystic carcinoma cell line. Methods:Salivary adenoid cystic cells of the cell line SACC were exposed to 1 ?g/ml of DDP for 48 h at the interval of one month. 6 months later, the cell line was established and named SACC/DDP. MTT method was used to study the drug resistance of SACC and SACC/DDP cells against 4 chemotherapeutic agents(MTX, PYM, VCR, MMC). Flow cytometry was adopted to study the cell cycle distribution of the cells after treatment with 4 agents at different concentrations for 72 h. Results:The drug resistance of SACC/DDP cells against VCR,MTX,PYM and MMC was 2.9,2.4,1.1 and 1.8 times of that of SACC cells. The ratio of G0 cell number to G1 cell number in SACC/DDP cell line was smaller than that in SACC cell line after treatment with the 4 agents at different concentrations respectively.Conclusion:The multidrug resistant human salivary adenoid cystic carcinoma cell line can be estabished by exposing parent cell line SACC periodically to DDP.

Objective To discuss the clinical value of serum tumor specific growth factor (TSGF) and vascular endothelial growth factor (VEGF) in early diagnosis of thyroid cancer.Methods Eighty thyroid cancer patients (thyroid cancer group),120 nodular goiter patients (nodular goiter group),and 80 healthy adult (blank control group) were enrolled in this study.The level of TSGF and VEGF in two groups were detected by ELISA and compared.Results The level of TSGF and VEGF in thyroid cancer group and nodular goiter group were significantly higher than those in blank control group (P ＜ 0.05).Conclusion Serum TSGF and VEGF have some clinical value in early diagnosis of thyroid cancer.

Objective To evaluate the value of HbA2 level determined by capillary electrophoresis (Hb-CE)in screening and diagnosis of thalassemia.Methods HbA2 level of 249 thalassaemia carriers and 142 healthy controls confirmed by molecular biological detection were determined by Hb-CE method.The thalassaemia carrier subjects were divided into different groups and subgroups according to their results of gene detection.The sensitivity,specificity,accuracy,positive predictive value and negative predictive value for the diagnosis ofα-thalassemia,β-thalassaemia,α,β-thalassaemia were calculated under different HbA2 cut-off value.Results Mean value of HbA2 in healthy controls was (3.03±0.27)%.Mean values of HbA2 inα-thalassemia group and its subgroups of silentα-thalassemia,standardα-thalassemia and hemoglobin H disease were (2.38± 0.55)%,(2.61±0.46)%,(2.47 ± 0.32)% and (1.07 ± 0.17)%,respectively.Mean values of HbA2 inβ-thalassaemia group and itsβ0 subgroup,β+ subgroup were (5.65±0.47)%,(5.71±0.48)% and (5.56±0.43)%.Mean value of HbA2 in compoundαandβ-thalassaemia group was (5.7±0.82)%.Compared with healthy controls,HbA2 level inα-thalassemia group,silentα-thalassemia subgroup,standardα-thalassemia subgroup and hemoglobin H disease group decreased signifi-cantly (t values of 11.73,5.02,12.91 and 33.46,respectively,P<0.01).HbA2 level in hemaglobin H disease was signifi-cantly lower than silent and standardα-thalassemia subgroups (t values of 15.62 and 21.31,respectively,P<0.01),but there were no differences in HbA2 level between silent and standardα-thalassemia subgroups (t=1.50,P>0.05).HbA2 level inβ-thalassaemia group,β0 subgroup,β+ subgroup and compoundαandβ-thalassaemia group increased significantly (t values of 55.12,44.33,38.94 and 9.10,respectively,P<0.01),but there were no differences in HbA2 level betweenβ0 andβ+ subgroups (t=1.79,P>0.05).Of 249 thalassemia carriers,all 124β-thalassaemia carriers were distinguished with ele-vated HbA2 level (>3.5%)determined by Hb-CE and only 57 were distinguished from 117α-thalassemia carriers by Hb-CE.Under the cut-off value of 2.5%,the sensitivity,specificity,positive predictive value,negative predictive value and accu-racy for the diagnosis ofα-thalassemia were 48.72%,97.18%,93.44%,69.70%,75.29%,respectively.Under the cut-off value of 3.5%,they were 100.00%,98.59%,98.41%,100%,and 99.25% for the diagnosis ofβ-thalassaemia,respectively. The analysis of ROC curve showed that the optimal HbA2 cut-off values for diagnosis ofα,β-thalassaemia by capillary elec-trophoresis were 2.8% and 3.7%,respectively.Conclusion When no abnormal bands,the elevated HbA2 (>3.7% in this study)determined by Hb-CE could be used as a marker forβ-thalassaemia diagnosis,but theβ-thalassaemia co-existingα-thalassemia could not be differentiated fromβ-thalassaemia diagnosis.Decreased HbA2 level (<1.5% in this study)and HbH band could be used for the diagnosis of hemoglobin H disease.Only HbA2 determination by Hb-CE has no clinical sig-nificance for the screen and diagnosis ofα-thalassemia.

Objective To analyze the antigens of solid cultured germ, liquid cultured germ and secreted composition, looking for the main immunity antigens of Mycobacterium tuberculosis H37Rv.MethodsShowing the difference of the antigens of solid cultured germ, liquid cultured germ and secreted composition by SDS-PAGE, Western blotting, monoclonal and polyclonal antibody techniques.Results Antigens of the solid cultured germ were nearly accordance with the liquid cultured germ and they were difference with secreted proteins. The main immunity antigens of the cultured germ were 79 000, 54 000 ,38 000~50 000, 28 000 and the secreted proteins are 66 000, 55 000~64 000, 30 000~34 000, 24 000, 16 000.The antigens of 66 000, 55 000 and 16 000 were proved that they were secreted proteins by monoclonal antibodies.Conclusions This study demonstrated that the main immunity antigens of the cultured germ in Mycobacterium tuberculosis H37Rv were difference with the secreted one .This is beneficial to the cognition of H37Rv and looking for special antigens for diagnosis of tuberculosis .

Objective To express and purify Japanese encephalitis virus ( JEV) EDⅢprotein and evaluate the possibility of using it as a candidate antigen in JEV diagnostic kit .Methods PCR primers spe-cific for the gene encoding JEV EDⅢprotein were designed and used to amplify the gene fragment by RT-PCR.The cloned gene fragment was then inserted into pET-30a (+) to construct the recombinant expression plasmid.The transformed E.coli BL21 carrying expression plasmid were induced by IPTG to express JEV EDⅢ protein.The expressed JEV EDⅢprotein and a control antigen of tick-borne encephalitis virus protein were deposited in small spots to set up ELISA microarray .The serum samples from patients with Japanese encephalitis and healthy people were detected by Array-ELISA.The results obtained by Array-ELISA were compared with those by using indirect immunofluorescence assay .Results The gene fragment encoding JEV EDⅢprotein was successfully cloned and expressed in E.coli BL21.The recombinant protein could be used in Array-ELISA assay for the detection of serum samples from patients with Japanese encephalitis and healthy subjects .The results were consistent with those by using indirect immunofluorescence assay.Conclusion The recombinant JEV EDⅢprotein can be used as a candidate antigen for the diagnosis of JEV infection .

Objective To express and purify the recombinant nucleoprotein fragments of hemor-rhagic fever with renal syndrome ( HFRS) virus and to evaluate their diagnostic efficacy by using array-ELISA technology .Methods The target genes encoding nucleoprotein fragments of HFRS virus were amplified by PCR, and then inserted into prokaryotic expression vectors to construct the recombinant plasmids of pET -32a (+)/Pn and pET-32a(+)/Pc.The plasmids were transformed into E.coli BL21 ( DE3) to induce the ex-pression of nucleoprotein fragments by IPTG and the expressed products were purified by affinity chromatog -raphy using Ni-NTA agarose.The specificity and sensitivity of the recombinant antigens were evaluated by the assay of array-ELISA using commercial colloidal gold assay kit as a comparison .Results The recombi-nant nucleoprotein fragments of HFRS virus were correctly expressed in E.coli and highly purified by affinity chromatography .Array-ELISA showed that 13 of 16 suspected serum samples were positive by using the His-Pn protein as diagnostic antigen , consistency with the commercial colloidal gold assay kit reaching 94%. Conclusion The recombinant His-Pn protein expressed in E.coli cells could be used for specific serodiag-nosis of HFRS virus as its high antigenicity and sensitivity .The array-ELISA is an effective assay for the de-tection of virus at protein level .

Objective To explore the relationship of DVL2 expression and the development of (CCRCC) by comparing the changes of DVL2 mRNA and protein expression in CCRCC specimens and matched normal renal specimens and its clinical significance. Methods DVL2 mRNA expressions in 22 CCRCC tissues, the matched adjacent normal tissues, and 10 CCRCC tissues alone were examined by semiquantitative RT-PCR and fluorescence quantitative PCR (real-time RT-PCR). Meanwhile, the different expression of the CCRCC between TNM Stage Ⅲ + Ⅳ and Stage Ⅰ +Ⅱ was also examined. Furthermore,immunohistochemistry was employed to examine DVL2 protein expression in 22 CCRCC and the matched adjacent normal tissues, and the other 10 CCRCC tissuses without the matched tissues. Results The DVL2 mRNA expression levels in 17 CCRCC tissues were increased by semi-quantitative RT-PCR and by real time RT-PCR compared with that in corresponding adjacent normal tissues, with the difference being significantly different (t = 2.535, P =0.0197). The DVL2 expression of 8 in 13 Ⅲ + ⅣCCRCC was higher than Ⅰ +ⅡCCRCC. Immunohistochemical examination showed that the DVL2 protein was located in cytomembrane and cytoplasm. Moreover, the positive level of DVL2 protein in CCRCC tissues[81.8 % (18/22)]was significantly higher than those in the adjacent tissues. However the expression was not associated with patients' age, gender, TNM stages (Fisher exact frenquently, P >0.05). Conclusion The DVL2 expression in CCRCC is obviously higher than the corresponding normal tissues in the level of mRNA and protein. And the higher DVL2 expression might be closely associated with the development and progression of CCRCC in the level of mRNA, which may be a potential molecular marker of CCRCC development and metastasis mechanism.