Objective To develop an efficient recombinant expression system of alanine aminotransferase ( ALT ) in order to build the foundation for the preparation of feedstock related ALT reference materials.Methods A new human ALT gene was synthesized by optimizing the codons of the nucleic acid sequence encoding human ALT using bioinformatic tools, and then it was cloned into pRSF-Duet expression vector.The recombinant plasmid pRSF-Duet-ALT was transformed into E.coli BL21 and the target protein expression was induced by 2 mmol/L isopropyl β-D-1-thiogalactopyranoside ( IPTG ) .The expression condition for soluble protein was optimized by changing the inducer concentration, shaking speed and induction temperature. The soluble protein was purified by nickel ion affinity chromatography and dextran molecular sieve chromatography, and identified by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis( SDS-PAGE) and Western blot analysis.The activity and stability of recombinant proteins in serum matrix under different storage conditions were detected.Results The usage frequency in E.coli of ALT codons was more than 10%after codon optimization.The expressions of soluble proteins were increased by optimizing the induced expression conditions, including a final concentration of 2 mmol/L of IPTG, and continued incubation with shaking at 150 rpm for 8 h at 25℃.The purified protein was identified as ALT by SDS-PAGE and Western blot with ALT activities of up to 80 000 U/L.Recombinant ALT could be stable for 2-8 d at 2-8 ℃or 25 ℃with a relative standard deviation of less than 5%.Conclusions An efficient recombinant expression system of ALT was developed successfully by codons optimization.The obtained recombinant protein could achieve the requirements of reference material feedstock.

This paper briefly introduces the working procedure of in vitro diagnostic products (IVD) industrial standards, and elaborates the importance of professional standards for production and supervision. Based on the analysis of working progress during the past 10 years, some problems and countermeasures on project setting, participation, standard material, personnel training, work cycle are put forward, which are helpful for the future development of the IVD.
Diagnostic Techniques and Procedures ; standards ; Humans ; Reference Standards

OBJECTIVETo observe the inhabitive effect and mechanism of polypeptide extract from scorpion venom (PESV) on repopulation in H22 tumor cell during chemotherapy.

METHODH22 tumor cells were injected into 96 mice subcutaneously, then mice were divided into 4 groups radomly: Model, low-dose-PESV, high-dose-PESV, and control. Reppulation model was established by 5-Fu treating mice with H22. Four groups was treated differently, 6 mice of each group was sacrificed every 7 days, measured tumor volume twice one week. The expression of vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), CD105 microvessel density (CD105-MVD) and platelet derived growth factor (PDGF) in H22 tumor issue was observed by using immunohistochemistry and grey analysis, the relation of VEGF and MVD was affirmed by correlation analysis.

RESULTIn control group tumor volume of H22 increased quickly in 13-24 day, and all mice died before 27 day. In model tumor volume increased quickly before 17, in 17-22 day slowly, after 22 day quickly again, and all the mice died before 31 day. In low and high dose PESV, tumor volume added slowly, and only in 17 day there was significant difference between these two groups. Immunohistochemistry showed, PCNA expression of model group in 31 day was higher than in 21, 28 day, the expression level of high and low PESV group was lower than model group all the time, only in 17 day there was significant difference between high and low PESV group. Immunohistochemitry showed, compared with high and low dose PESV group, CD105-MVD of model group was higher in 21, 28 day (P < 0.05) and in 35 day (P < 0.01), and no difference was found between high and low dose PESV. VEGF expression of model group in 35 day was higher than in 21, 28 day (P < 0.01), and model group higher than high and low dose PESV in 21, 28, 35 day. The expression of PDGF in model decreased gradually, in high and low dose PESV, the expression was lowest in 21 day. In day 35 high dose PESV higher than low dose PESV. There was positive correlation (r = 0.669) between VEGF expression and CD105-MVD.

CONCLUSIONPESV can inhabit repopulation of H22 tumor cell during chemotherapy, and the mechanism maybe is through anti-angiogenesis and nomalizing tumor vessels.

Angiogenesis Inhibitors ; therapeutic use ; Animals ; Antigens, CD ; metabolism ; Carcinoma, Hepatocellular ; drug therapy ; metabolism ; Cell Line, Tumor ; Immunohistochemistry ; Liver Neoplasms ; drug therapy ; metabolism ; Mice ; Peptides ; therapeutic use ; Platelet-Derived Growth Factor ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism ; Scorpion Venoms ; chemistry ; Vascular Endothelial Growth Factor A ; metabolism

Objective To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells.Methods Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group,Nc-shRNA group and Notch1-shRNA group.The Nc-shRNA group was a negative control RNAi lentivirus group,and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group.The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1.The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting.Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay.Apoptosis was detected by Annexin V/7-AAD double staining.Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA),B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1.Results The results of qRT-PCR showed that the relative expression levels of Notch1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were 1.000 ± 0.000,0.937 ± 0.025,0.490 ± 0.036 and 1.000 ± 0.000,1.077 ± 0.070,0.373± 0.038,with statistically significant differences (F =359.707,P ＜0.001;F =210.455,P ＜0.001),further paired comparison,the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P ＜ 0.05).Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results.MTT assay showed that the 24 h A values of A549 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.209 ± 0.005,0.219 ± 0.009,0.159 ±0.006,48 h A values were 0.293 ± 0.004,0.302 ± 0.004,0.205 ± 0.005,72 h A values were 0.450 ± 0.003,0.430 ± 0.012,0.348 ± 0.017,with statistically significant differences (F =79.487,P＜0.001;F =508.664,P ＜0.001;F =57.156,P ＜0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 24,48,72 h (all P ＜ 0.05).The 48 h A values of SPC-A-1 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.438 ±0.022,0.412 ± 0.015,0.364 ± 0.010,72h A values were 0.540 ± 0.016,0.519 ± 0.009,0.438 ± 0.019,with statistically significant differences (F =15.667,P =0.004;F =37.299,P ＜ 0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P ＜ 0.05).The sphere sizes of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667 ± 6.506) μm,(136.667 ± 7.095) μm,(86.676 ± 7.638) μm,with statistically significant difference (F =65.940,P ＜ 0.001).The sphere sizes of the three groups in SPC-A-1 cells were (118.667 ± 6.658) μm,(128.000 ± 7.000) μm,(60.675 ± 4.509) μm,with statistically significant difference (F =105.372,P ＜0.001).Further paired comparison,the sphere size of Notch1shRNA group was significandy smaller than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).The apoptosis rates of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1cells were (0.489 ± 0.014)％,(0.633 ± 0.021)％,(1.683 ± 0.221)％ and (1.323 ± 0.194)％,(1.690 ± 0.188) ％,(3.017 ± 0.356) ％,with statistically significant differences (F =77.660,P ＜ 0.001;F=32.200,P =0.001),further paired comparison,the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).Western blotting showed that the expressions of PCNA,Bcl-2 and Hes-1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F =155.343,P ＜ 0.001;F =22.576,P =0.002;F =70.108,P＜0.001),and the expressions of PCNA,Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F =49.419,P ＜0.001;F =28.090,P =0.001;F =12.040,P =0.007).Further paired comparison,the expressions of PCNA,Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells,and the differences were statistically significant (all P ＜0.05).Conclusion Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis,which may be related to the down-regulation of its downstream gene Hes-1.

Objective To discuss the expression of caspase-7 (CASP7) and its prognostic role in colon cancer. Methods CASP7 expression data was retrieved from Ualcan database, and the survival data of CASP7 down-regulated colon cancer patients was retrieved from Ualcan database and Oncolnc database. Results In colon cancer, the expression of CASP7 was down-regulated and indicated a relatively worse prognosis. The expression of CASP7 was correlated with KRAS and TP53I3. Conclusions CASP7 is down-regulated in colon cancer and indicates a worse prognosis.