Objective To introduce a multifunctional thermostated instrument applied in pre-floats,floats,roast,special staining,immunohistochemical staining,situ hybridization and frozen sections in order to enhance speed and quality of slice-making.Methods Three lengths of vats were nested and overlaid each other to make up a thermostat box which could stretch out and contract back.Its control part consisted a timer,precise temperature control and adjustable power controller.Source of heat was provided by durable electric heat plate to keep the constant temperature for different operations.Results The equipment provided constant temperature and possesses all the necessary functions.It can be used for the pathological operation and compensate for the shortcomings of medical equipment.Conclusion With small volume and light weight,the equipment is easy to carry.Besides,it has less power consumption,so it is secure and easy to operate.The equipment can meet conventional pathology techniques both in peacetime and wartime.

Objective: To clone cDNA of human interleukin-29(IL-29) and express it in cos-7 cells, and to study its anti-HBV activity in vitro. Methods: Total RNA was extracted from PBMCs which had been infected with vesicular stomatitis virus(VSV) in vitro.IL-29 cDNA was amplified using one-step RT-PCR technique. The recombinant expressing plasimd of psectagB/His-myc-IL29 was constructed by inserting IL-29 cDNA into the vector and was then transfected into cos-7 cells. Stable expression stains were screened by Hygromycin B and limited dilution method. The target protein was purified through Ni2+-chelating Sepharose Fast Flow. Anti-viral bioactivity of the recombinant IL-29 fusion protein was analyzed through an in vitro model of production of HBV by the HepG2.2.2.15 cell line.ELISA was used for detection of the viral titers in the cell cultural supernants. Results: IL-29 was cloned and stably expressed in cos-7 cells successfully. SDS-PAGE and Western blot analysis showed multiple bands of the target protein with the molecular weights between 20 000 and 33 000, and the major band was located at about 33 000, indicating the fused IL-29 modified by additional glycosylation. The rhIL-29 was shown to dose-dependently inhibit secretion of HBsAg and HBeAg accompanied by the reduction of HBV genomic DNA in the cells tested. The inhibition ratio of HBsAg and HBeAg production was attained 85% at a concentration of 160 ?g/L of rhIL-29. Conclusion: The rhIL-29 with anti-HBV activity has been obtained.

Objective To study the characteristics of immune function,Epstein-Barr virus(EBV) antibodies and EBV-DNA in children with different clinical types of EBV infection,which provide basis for prevention and treatment of EBV infection.Methods Clinical data of 103 patients suffering from EBV infection were retrospectively analyzed in Xijing Hospital of the Fourth Military Medical University.A total of 103 children were divided into infectious mononualeosis(IM) group(n=68),chronic active Epstein-Barr virus infection(CAEBV) group(n=13) and Epstein-Barr virus-related hemophagocytic lymphohistiocytosis(EBV-HLH) group(n=22).The changes of EBV antibodies,EBV-DNA,immunoglobulin levels,lymphocyte subpopulation and complement series were detected and compared among the three groups.A total of 26 healthy children at the same stage were enrolled as a control group,immunoglobulin levels,lymphocyte subpopulation and complement series were detected in control group,then compared with the rest of the three groups.Results The levels of C3 and C4 in CAEBV group and EBV-HLH group were significantly decreased than those in the control group(P<0.05).The levels of IgA,IgG and IgE in EBV-HLH group,CAEBV group,IM group and control group gradually increased(P<0.05,respectively).The levels of IgA,IgG and IgE in EBV-HLH group and CAEBV group significantly decreased than those in control group(P<0.05,respectively).The levels of IgA,IgG and IgE in IM group decreased than those in control group,while there were no statistically significant differences(P>0.05).CD8+T cells in IM group significantly increased than those in the rest of the three groups(P<0.05,respectively).T cells,CD8+T cells,CD4+ T cells,CD4+/CD8+ ratios,NK cells,B cells of EBV-HLH group significantly decreased than those in the rest of the three groups(P<0.05).The positive rates of EBV antibodies in CAEBV group and IM group were significantly higher than those in EBV-HLH group(P<0.05).EBV-DNA in EBV-HLH group were significantly higher than those of CAEBV group and IM group(P<0.05).Conclusion EBV-DNA levels in the serum are positively correlated with disease types and severity,the pathogenesis of IM,CAEBV and EBV-HLH induced by EBV infection are associated with immune dysfunction.Dynamic monitoring of EBV load and cell immune function can reflect disease status and progress risk.

Objective_To explore the influence and mechanism of IL-31 on the expression of VEGF, EGF and EG-FR in 16HBE cells.Methods_16HBE cells were cultured and treated with IL-31 with or without SB203580 or SP600125, real-time PCR and Western blot were applied to determine the mRNA and protein expression of VEGF, EGF and EGFR respectively.Meanwhile, Western blot was used to examine the changes of P38 MAPK and JNK signaling pathways.Results_Compared with control group, the mRNA expression of VEGF, EGF and EGFR was increased markedly under the stimulation of IL-31 ( P<0.01 ) , the expression of p-P38 MAPK and p-JNK signifi-cantly increased ( P<0.01) .Compared with IL-31 group, the expression of p-P38 MAPK significantly decreased in IL-31 combined with SB203580 or SB203580 group ( P <0.01 ) , while the expression of p-JNK markedly decreased in IL-31 combined with SP600125 or SP600125 group( P<0.01) .Compared with IL-31 group, the expression of VEGF was significantly decreased in IL-31 combined with SB203580 or SP600125 group ( P <0.01 ) , while the expression of EGF and EGFR was markedly declined in IL-31 combined with SB203580 group ( P<0.01 ) .Conclusions_IL-31 may up-regulate the expression of VEGF through activating P38 MAPK and JNK signaling pathways and up-regulate the expression of EGF and EGFR through activating P38 MAPK signaling path-way in16 HBE cells.

Objective:To construct Interleukin 21 (IL 21) expression plasimd pCDNA3.1 IL21 and express it in Hela cell and analysis its acitivity of costimilating T cell proliferation with anti CD3 monoclonal antibody in vitro.Methods:The CDs of IL 21 was amplified by PCR using the pMD IL21 as templet.The expression plasimd pCDNA3.1 IL21 was constructed by inserting the sequence of coding region of the IL 21 into pCDNA3.1/Hismyc B containing CMV promoter and transfected into Hela cells.The stability expression stain was screened by the condition media containing 400 mg/L G418 and cloned through the limited dilution method.The target protein was purified through Ni 2+ chelating Sepharose Fast Flow.The bioactivity was confirmed by MTT method on costimulating the T cell proliferation with anti CD3.SDS PAGE and Western blot analysis the rhIL 21 expressed.Results:hIL 21 was expressed in Hela cell successfully.SDS PAGE analysis showed the IL 21 fusion protein with Mr 18 000 or so was expressed in supernatant of the cells.The rhIL 21 has significant proliferation effect on mature human T cells in the presence of anti CD3 monoantibody.Conclusion:The rhIL 21 with bioactivity has been obtained,which may help researcher study its new function and effects. [

Purpose To explore the antiviral effect of interleukin 29(IL-29) on hepatitis B virus in vitro.Methods To study the antiviral effect of IL-29 against hepatitis B virus by the amount of HBV mRNA detected .Through the quantity of mRNA translated from genes of MxA,2′,5′-OAS,PKR and RNase L as well as the signal pathway induced by IL-29,we used RT-PCR and Western blot to discuss the anti-hepatitis B virus mechanism which was stimulated by IL-29.Results The amount of HBV mRNA in HepG2.2.15 cells was reduced by stimulation of IL-29.The expression of MxA and 2′,5′-OAS was up-regulated,as well as P-ERK and P-AKT were activated by IL-29.Conclusion These findings showed that IL-29 had obvious antiviral activity towards HBV in HepG2.2.15 cells.

Objective To study the effect of transfection of antisense MBD1 gene eukaryotic expression vector on the expression of MBD1 gene in human cholangiocarcinoma cell line QBC-939.Methods The(constructed) antisense MBD1 gene eukaryotic expression vector was transfected into the human(cholangiocarcinoma) cell line QBC-939 using lipofectamine transfection reagents,and positive cell clones were obtained using G418 selection after transfection.The constructed recombinant vector was transfected into(QBC-939) cells successfully and was confirmed by amplifying the exogenous neo~R gene with PCR method.The expression level of MBD1 gene mRNA and protein was detected by RT-PCR and FCM methods respectively.Results Following the transfection,the MBD1 gene mRNA level in human cholangiocarcinoma cell line QBC-939 decreased from 0.912?0.022 to 0.215?0.017,and the MBD1 gene protein level also(decreased) from(80.19?5.05)% to(35.11?4.05)%.There were very significant differences on the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression vector transfection group(P

Objective To explore the expression and relationship of PTEN and mTOR in the development of cholangiocarcinoma,determine the effect of the PI3K/PTEN/AKT/mTOR signal transduction on the(development) of cholangiocarcinoma.Methods The expression of mTOR and PTEN in the cholangiocarcinoma was detected by the immunohistochemistry and RT-PCR method.Results Compared to normal tissues,the(expression) of mTOR gene in cholangiocarcinoma significantly increased,but the expression of PTEN gene(decreased).There was a negative correlation between mTOR gene and PTEN gene expression in(cholangiocarcinoma). Conclusions The expression of mTOR gene in cholangiocarcinoma was increased and the expression of PTEN was decreased.It suggested that the mTOR gene and PTEN gene could play an important role in the process of development of cholangiocarcinoma.

Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1 gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912 +/- 0.022 to 0.215 +/- 0. 017, and the protein level of MBD1 gene also decreased from (80.19 +/- 5.05) % to (35.11 +/- 4.05) %. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression plasmid transfection group (P < 0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.
Biliary Tract Neoplasms/*metabolism ; Biliary Tract Neoplasms/pathology ; Cell Line, Tumor ; DNA Methylation ; DNA-Binding Proteins/*biosynthesis ; DNA-Binding Proteins/genetics ; Eukaryotic Cells/metabolism ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Oligonucleotides, Antisense/*genetics ; Plasmids/genetics ; Transcription Factors/*biosynthesis ; Transcription Factors/genetics ; Transfection

Hypermethylation in the promoter region is an important epigenetic mechanism for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferases are considered to be the main cause of promoter hypermethylation. In order to explore the roles of two methyltransferase members (DNMT1 and DNMT3b) in the cholangiocarcinoma tumorigenesis, antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene was constructed respectively, and were co-transfected into the human cholangiocarcinoma cell line QBC-939 to observe their biological effects on the cell growth and proliferation ability, apoptosis, cell cycle alteration, and the tumorigenesis ability in the subcutaneous tissue of nude mouse. The results demonstrated that co-transfection with antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene and single transfection with antisense eukaryotic expression plasmid of DNMT1 gene can suppress the growth and proliferation of QBC-939, block the cell cycle at G1 phase, increase the apoptosis rate, minimize the tumor size in the subcutaneous tissue of nude mouse. The suppressing biological effect of co-transfection is stronger than single transfection with antisense DNMT1. Meanwhile, single transfection with antisense eukaryotic expression plasmid of DNMT3b gene has no effects on the biological characteristics of QBC-939. This study suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support its function in inactivation of tumor suppressor genes. Combination DNMT1 and DNMT3b will increase their biological effects and have the synergistic effect on suppressing the growth of human cholangiocarcinoma cell line QBC-939.
Apoptosis ; Biliary Tract Neoplasms/*metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cholangiocarcinoma/*metabolism ; DNA (Cytosine-5-)-Methyltransferase/*biosynthesis ; DNA (Cytosine-5-)-Methyltransferase/genetics ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Mice, Nude ; Neoplasm Transplantation