Objective:To investigate the changes of reactive oxygen species(ROS)and Glutathione(GSH) and their chronological order in the apoptosis of K562 cells induced by simvastatin to explore the apoptosis mechanism.Methods:K562 cells cultivated in routine method for 24h were exposed in the simvastatin 20?mol/L.Cell morphological analysis were conducted at 48h and the vitalities of K562 cells were cultivated detected with MTT at different times(12,24,48,72 h) respectively.Fluorochrome flow cytometry was performed to detect cell apoptotic ratio and changes of intracellular ROS.The content of intracellular GSH in K562 cells was measured by colorimetric method.Results:K562 cells presented typical apoptosis morphological changes the treatment of simvastatin for 48h.cell inhibition ratio at different times(12,24,48,and 72 h) were 0,(19.02?0.92)%,(56.4?3.20)%,(74.4?5.54)% respectively and cell growth of K562 was inhibited.The apoptotic ratio were(2.55?0.25)%,(6.1?0.35)%,(14.15?0.42)%,and(30.70?0.65)% respectively.Furthermore,the changes of fluorescence intensity of ROS were also significantly increased(P

OBJECTIVE:To observe the changes of mitochondrial membrane potential(MMP)at different time in the process of simvastatin-induced apoptosis of K562 cells.METHODS:K562 cells cultivated by routine method for 24h were treated in the 20? mol? L-1 simvastatin for 12,24,48,and 72h,respectively.And theh cell morphological changes were observed.Vitality of K562 cells was detected with MTT;flow cytometry were performed to detect cell apoptotic ratio and changes of mitochondrial membrane potential,and the detection results were compared with those in solvent control group.RESULTS:As compared with control group,the morphology changes of K562 cells such as karyopyconosis,nuclear fragmentation and apoptotic body emerged after treatment with 20? mol? L-1 simvastatin for 48h;the apoptotic ratio at 12,24,48,and 72h increased by(2.55? 0.35)%,(6.1? 0.35)%,(14.15? 0.42)%,and(30.70? 0.65)%,respectively;and the apoptotic ratio of K562 cells increased time-dependently.The cell membrane potential was decreased by(0.7? 0.24)%,(39.6? 4.80)%,(24.4? 2.45)%,and(6.0? 1.62)%,respectively,with that at 24h showing the highest decreasing rate.CONCLUSION:The change of MMP is the early events of apoptosis.The potential mechanism of K562 cells apoptosis induced by simvastatin might be related to the collapse of MMP.

Objective:To develop an inducible system for expression of GraB in E.coli for the purpose of investion of its function on genome DNA fragment.Methods:Amplified granzyme B cDNA by RT-PCR via extracting lymphocyte total RNA.Identified with endonuclease cut and DNA sequence analysis,the gene was inserted into E.coli expression vector pTrcHis2A,the pokaryotic expression plasmid pTrcHis2A/GraB was constructed and transformed into TOP10F.Results:After 8 h of IPTG in duction, the GraB was expressed 15% of total proteins by SDS-PAGE.Western blot assay proved the expressed hGraB to be of good antigenicity and high specificity.The recombinant protein purified by affinity chromatography has effect on GraB substrate.GraB was found to work directly on the genome DNA fragments,promoting its split.Conclusion:It is found that there may exist certain novel pathway for GraB-induced apoptosis.

Objective To evaluate the ethical management status quo of Capital Medical Research Fund in 2007.Methods Cross-sectional study was applied to analyze the ethics management and ethics self-evaluation in applications.Results There were 652 applications,covering with 14 districts and 2 counties in Beijing,from 128 Hospitals.Applicants have some knowledge of the ethical issues in clinical research accounted for 88.7％ ; informed consent of subjects considered in 72.5％ ;the potential risk and protection involved in application accounted for 62.0％; personal privacy protection of subject accounted for 49.4％.Unfortunately,the benefit vs risk assessment was only involved in 28.7％ applications.Applicants from general hospitals had more ethical issue knowledge (90.2％) than those from community hospitals (84.6％).Applicants from university hospitals had more ethical issue knowledge (93.9％) than those from military hospitals (80.2％).Applicants aged 55 0r over had less ethical issue knowledge (70.0％) than other applicants (89.2％).The applicants to study on descriptive research and etiological research had less ethical issue knowledge than others.578 applications filled out the approval from ethics committee or research management department.62.6％ of the written were acceptable.Conclusion The applicants for clinical research had preliminary understanding for ethical issues in Beijing in 2007.Hospital ethics committees or research management departments had conducted clinical research ethical review applications for funds management.It has taken place the external conditions to carry out the ethical management in clinical research fund management.

Objective To carry out further analysis and assessment of the radiation risk from DSA examinations and related factors.Methods All the online radiation dose values and associated parameters of the 107 patients examined were collected for purpose of statistical analyses according to their classification.Results The analyses of DSA examinations indicated:for coronary angiography,DAP was (22 285.5±18 682.7)μGy·m2,ESD was (2 942.1 ±2 557.3) mGy;for head angiography,DAP (25 929.6±8 302.7) μGy·m2,ESD (1 288.8 ±682.3) mGy;for abdominal angiography,DAP (12 129.7 ± 10 646.1) μGy· m2,ESD (730.1 ± 584.7) mGy.Conclusions Among total accumulated radiation dose,the dose arising form coronary angiography is the highest,followed by the head angiography,and the dose from abdominal angiography is the lowest.

Objective To investigate the distribution of five indicators of hepatitis B(HBV-M)and its relationship with liver function parameters and HBV-DNA load in patients with chronic HBV infection in different stages.Methods The serum samples were collected from 456 patients infected with HBV.The HBV-M,liver functional parameters and HBV-DNA level were quantita-tively detected.According to the stages of disease,the patients were divided into 3 groups including chronic hepatitis B group(inclu-ding mild subgroup,moderate subgroup and severe subgroup),liver cirrhosis group(including compensatory subgroup,decompen-sated subgroup)and hepatocellular carcinoma goup.Results The ratio of HBsAg,HBeAg,HBcAb positive pattern(135 pattern) and HBsAg,HBeAb,HBcAb positive pattern(145 pattern)in the three groups were statistically different(P <0.05).In each chron-ic hepatitis B subgroup,both ALT and AST levels of 135 pattern were significantly higher than the other two patterns(P <0.05). In each liver cirrhosis subgroup and hepatocellular carcinoma group,there were statistically significant differences in ALB and TBIL levels between the three patterns(P <0.05 ).In each group,the HBV-DNA level of 135 positive pattern were significantly higher than the other two patterns(P <0.05).Conclusion With the advancement of chronic hepatitis B,there is a downtrend in the ratio of 135 pattern and increasing trend in the ratio of 145 pattern.If the stage of hepatitis B is discriminated,ALT,AST,ALB TBIL and HBV-DNA level were closely related to HBV-M pattern.

Objective To study the effects of 5-aza-dc on the expression of IGFBP7 in and proliferation of melanoma cell lines A375 and M14.Methods Reverse transcription-PCR and immunocytochemistry were performed to detect the mRNA and protein expression of IGFBP7 in A375 cells and M14 cells after treatment with 5-aza-dc of 10μmol/L for 48 hours,and MTT assay to measure the proliferation of both cell lines treated with 4 different concentrations (2.5,5,10,20μmol/L) of 5-aza-dc for various durations.Results The treatment with 5-aza-dc restored IGFBP7 expression at both mRNA and protein levels.The four concentrations of 5-aza-dc inhibited the proliferation of A375 and M14 cells in a dose-dependent (F=561.12,271.43,respectively,both P<0.01) and time-dependent (F=141.35,549.33,respectively,both P<0.01) manner.Conclusions DNA methylation may be involved in the modulation of aberrant IGFBP7 gene expression in melanoma,and 5-aza-dc could inhibit the proliferation of A375 and M14 cells.

Objective To construct the eukaryotic expression plasmids of short hairpin RNA (shRNA) specific for mouse Bcl-2-assoeiated athanogene 1 (BAG-1) and to observe their inhibitory effects on the expression of BAG-1 gene in mouse melanoma B16FI0 ceils. Methods Plasmids named pRNAT-U6.1/Neo-BAG-1, were designed and constructed to target the mouse BAG-1 mRNA coding region. LipofectaminTM 2000 was used to transfect plasmids into BI6F10 cells. Negative plasmid-transfected and tmtransfected B16F10 cells served as negative and blank controls respectively. Forty-eight hours following transfection, G418 was used to select the resistant cells. The mRNA and protein expression of BAG-1 gene was measured by reverse transcription-PCR and Western blot respectively about 1 month after the transfection. Results The eukaryotic expression plasmids, pRNAT-U6.1/Neo-BAG-1, were constructed, and verified by restriction enzyme digestion and DNA sequencing. The transfection rate in B16F10 cells was 20% -30%. Compared with the blank control, the mRNA and protein expression of BAG-1 in BI6FI0 cells was significantly inhibited by BAG-1 shRNA (both P<0.05), and the inhibition rates were (77±4)% and (62 ±2)%, respectively. Conclusions These results indicate that the eukaryotic expression vectors containing shRNA against BAG-1 gene, pRNAT-U6.1/Neo-BAG-1, are successfully constructed, and can significantly inhibit the expression of BAG-1 gene in mouse melanoma B16F10 cells.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is one of important cytokines to promote the maturation of dendritic cells. Blockage of TNF-α action by binding with soluble tumor necrosis factor receptor 1 (sTNFR1) may arrest dendritic cells in an immature state and induce stable, long-term tolerance. OBJECTIVE: To construct the lentiviral vectors carrying sTNFR1 gene and investigate sTNFR1 expression in immature dendritic cells. METHODS: Total RNA of human peripheral blood mononuclear cells was taken as a template. The sTNFR1 gene fragment was amplified by RT-PCR, subcloned to the lentiviral vectors pXZ208, and ligated to the enhanced green fluorescent protein (eGFP) reporter gene to establish lentiviral vector, called pXZ9-sTNFR1. DNA sequencing was performed for lentiviral vector identification. Lentivirus was prepared by transfection of 293 FT cells with pXZ9-sTNFR1. Viral titer was determined by eGFP expression. C57BL/6 mouse bone marrow-derived dendritic cells were in vitro cultured with low-dose granulocyte-macrophage colony stimulating factors and interleukin 4. On day 5 of culture, immature dendritic cells were transfected with pXZ9-sTNFR1 recombinant lentiviral supernatant, sTNFR1 transcription was detected by RT-PCR, sTNFR1 protein expression by Western blot analysis. Following sTNFR1 gene modification and lipopolysaccharide stimulation, the phenotype characteristics of dendritic cells were observed. RESULTS AND CONCLUSION: Recombinant plasmid pXZ9-sTNFR1 was successfully constructed. Twenty-four hours after 293 FT cell transfection, eGFP expression was observed and viral titer was over 10<'6> U/L. RT-PCR demonstrated that pXZ9-sTNFRl-transfected immature dendritic cells showed sTNFR1 positive expression. Western blot analysis revealed that sTNFR1 protein appeared in the immature dendritic cells and supernatant following 293 FT cell transfection. On day 5 of culture, dendritic cells expressed low level of class Ⅱ major histocompatibility complex (MHC Ⅱ), as well as CD40, CD86, CD80, molecules. However, following lipopolysaccharide stimulation, dendritic cells expressed high level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules, exhibiting the phenotype characteristics of mature dendritic cells. But after sTNFR modification, the expression level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules was not altered obviously. Lentiviral vectors carrying sTNFR1 gene and eGFP reporter gene were successfully constructed, and recombinant lentiviral plasmids with high titer were acquired. Following high efficacy of lentiviral gene transfection, immature dendritic cells stably express sTNFR1 mRNA and protein, which prevents immature dendritic cells from activation by exogenous lipopolysaccharide and maintains the immature state.

Objective:To explore the key factors affecting the formulation of treatment and prognosis of medullary thyroid carcinoma.Methods:Patient data, clinical characteristics and the results of follow-up of typical cases of 23 patients with medullary thyroid carcinoma admitted to Hunan Provincial People’s Hospital Breast and Thyroid Surgery from Apr. 2007 to Mar. 2020 were retrospectively analyzed. The therapeutic schedule and prognosis of medullary thyroid carcinoma were discussed in combination with ATA guidelines and others.Results:Of the 23 patients with MTC, 22 (95.65%) had elevated serum calcitonin, 15 (65.22%) had elevated carcinoembryonic antigen, 3 (13.04%) had suspected abnormal lymph nodes, and 2 (8.70%) had capsule invasion. Thyroid lobectomy, thyroid lobectomy with lateral lymph node dissection in level VI, total thyroidectomy, total thyroidectomy with lateral lymph node dissection in level VI, total thyroidectomy with bilateral lymph node dissection in level VI, total thyroidectomy with bilateral lymph node dissection in level VI with lymph node dissection in level I, II, III, IV, V or VII were performed in 1, 2, 3, 1, 13, 3 cases respectively. 8 cases had postoperative recurrence (34.78%) , of which 7 cases were caused by the first operation. The level of Ctn increased significantly in 2 cases before operation, who underwent total thyroidectomy with bilateral lymph node dissection in level VI, and no recurrence was found after operation.Conclusions:The key to the biological cure of medullary thyroid carcinoma is standardized surgical treatment. The surgery method cannot be determined simply by calcitonin. The modern treatment of medullary thyroid carcinoma needs to follow the principle of standardization and individualization at the same time.