Objective To construct eukaryotic expression plasmid of human B7-H1 extracellular region-hIgG1Fc-pCI-neo eukaryotic expression vector,and express the functional fusion protein in mammalian CHO cell.Methods Full-length human B7-H1 encoding sequence was amplified from human activated T cell cDNA library by PCR,fused with hIgG1Fc,then transformed into pCI-neo expression vector and verified by sequencing.The validated recombinant was transfected into mammalian CHO cell by lipofectamine reagent.The supernatant of the cultured cell was collected and analyzed by the sandwhich ELISA to detect if there was the fusion protein,and the fusion protein was purified by HiTrap recombination protein Protein A affinity chromatography.The concentrated supernatant or purified fusion protein were used for Western blotting after SDS-PAGE to identify the molecular weight and immune activity of the fusion protein of B7-H1.Results The extracellular region of hB7-H1 about 727 bp was cloned from human T cell cDNA library and was inserted with hIgG1Fc into the eukaryotic expression vector pCI-neo.After the transfection of recombinant into mammalian CHO cell by lipofectamine reagent,the expression of B7-H1 fusion protein was detected in the cultured CHO cell supernatant by the sandwhich ELISA.The immune activity of the fusion protein was verified by Western blotting,and its molecular weight was about 51.76?10~(3),very close to the expected value.Conclusion The hB7-H1-Fc chimeric molecule was successfully constructed and the expression of its functional fusion protein in mammalian CHO cells lays a foundation for further research on the role of B7-H1 in immune tolerance,autoimmune diseases.

Purpose To study the expression of Ech1 in hepatocarcinoma and its clinical significance, and to explore the relationship between subcellular location of Ech1 and malignant biologic behaviour of hepatocarcinoma. Methods Immunohistochemical analysis was used to detect Ech1 expression in the 20 cases of normal liver tissue and the 30 cases of hepatocarcinoma. Subcellular location of Ech1 in Hca-F and FEch1-down cell was observed with subcellular protein extraction. Results The expression of Ech1 in primary hepa-tocarcinoma was increased compared to that of normal liver tissues ( P<0. 05 ) , while there was no significant difference between the expression of Ech1 with the age, AFP, and with or without liver cirrhosis or hepatitis virus in hepatocarcinoma (P>0. 05). Ech1 was found expressed almost at the same location although the expression level one is normal and the other is downregulation. Ech1 expres-sion was found in cytosol, membrane fraction, and its expression was higher in cytosol than other fractions both in Hca-F cell and Ech1 downregulated Hca-F cell. Conclusions The expression of Ech1 in primary hepatocarcinoma was increased, which may indicate that Ech1 is a critical factor in the development of primary hepatocarcinoma, but the subcellular location of Ech1 has not much contribution to that.

PURPOSE: The aim of this study was to investigate whether propofol could attenuate hypoxia/reoxygenation-induced apoptosis and autophagy in human renal proximal tubular cells (HK-2) by inhibiting JNK activation. MATERIALS AND METHODS: HK-2 cells were treated with or without propofol or JNK inhibitor SP600125 for 1 hour and then subjected to 15 hours of hypoxia and 2 hours of reoxygenation (H/R). Cell viability and LDH release were measured with commercial kits. Cell apoptosis was evaluated by flow cytometry. The expressions of p-JNK, cleaved-caspase-3, Bcl-2, and autophagy markers LC3 and p62 were measured by Western blot or immunofluorescence. RESULTS: HK-2 cells exposed to H/R insult showed higher cell injury (detected by increased LDH release and decreased cell viability), increased cell apoptosis index and expression of cleaved-caspase-3, a decrease in the expression of Bcl-2 accompanied by increased expression of p-JNK and LC3II, and a decrease in expression of p62. All of these alterations were attenuated by propofol treatment. Similar effects were provoked upon treatment with the JNK inhibitor SP600125. Moreover, the protective effects were more obvious with the combination of propofol and SP600125. CONCLUSION: These results suggest that propofol could attenuate hypoxia/reoxygenation induced apoptosis and autophagy in HK-2 cells, probably through inhibiting JNK activation.
Anoxia ; Apoptosis ; Autophagy ; Blotting, Western ; Cell Survival ; Flow Cytometry ; Fluorescent Antibody Technique ; Humans ; Propofol

Objective To evaluate the role of thioredoxin?interacting protein(TXNIP)∕oligomer?ization domain?like receptor family pyrin domain?containing 3(NLRP3)signaling pathway in renal ische?mia?reperfusion(I∕R)injury in diabetic rats. Methods Pathogen?free healthy male Sprague?Dawley rats, aged 8-12 weeks, weighing 200-220 g, were used in the study. Diabetes mellitus was induced by intrap?eritoneal injection of 1% streptozotocin 65 mg∕kg and confirmed by blood glucose≥16.7 mmol∕L 3 days lat?er. Twenty?four diabetic rats were divided into 3 groups(n=8 each)using a random number table: sham operation group(group S), renal I∕R group(group I∕R)and resveratrol(TXNIP inhibitor)group (group R). Resveratrol 10 mg∕kg was intraperitoneally injected every day for 7 consecutive days starting from 3rd week after successful establishment of the model in group R. At 4th week after successful establish?ment of the model, renal I∕R was produced by occlusion of bilateral renal pedicles for 25 min followed by reperfusion in anesthetized rats in group R. The animals were sacrificed at 48 h of reperfusion, and renal specimens were obtained for microscopic examination of pathologic changes and for measurement of malondi?aldehyde(MDA)content, superoxide dismutase(SOD)activity and superoxide anion scavenging capa?bility(using colorimetric method), interleukin?1beta(IL?1β)and IL?18 contents(by enzyme?linked immunosorbent assay), cell apoptosis(using TUNEL)and expression of TXNIP, NLRP3 and caspase?1 in renal tissues(using Western blot). Blood samples were obtained from the left ventricle for determination of serum urea nitrogen(BUN)and creatinine(Cr)concentrations. Results Compared with group S, the serum Cr concentration and apoptosis index were significantly increased, superoxide anion scavenging capability in renal tissues was decreased, and the expression of TXNIP, NLRP3 and caspase?1 was up?reg?ulated in I∕R and R groups, and the serum BUN concentration and contents of MDA, IL?1β and IL?18 in renal tissues were increased, the SOD activity was decreased(P<0.05), and the pathological changes of renal tissues were aggravated in group I∕R. Compared with group I∕R, the serum BUN and Cr concentra?tions were significantly decreased, the contents of MDA, IL?1β and IL?18 and apoptosis index were de?creased, the SOD activity and superoxide anion scavenging capability were increased, the expression of TXNIP, NLRP3 and caspase?1 was down?regulated(P<0.05), and the pathological changes of renal tis?sues were significantly attenuated in group R. Conclusion The pathophysiological mechanism of renal I∕R injury is associated with the activation of TXNIP∕NLRP3 signaling pathway in diabetic rats.

Objective:To evaluate the performance of key performance indicator (KPI) lean teaching management system in clinical medical laboratory.Methods:Combining lean teaching management in universities with KPI system of enterprises, an innovative KPI lean teaching management system was developed and applied in Clinical Medical Laboratory of Guangdong Provincial People′s Hospital (GDPH). A total of 106 teachers, who had worked from January 2021 to December 2022 in GDPH, joined this study. Teachers were divided into 8 professional groups. Firstly, we quantified the teaching workload by class hours, evaluated the teaching outcomes base on national, provincial, school, and college levels to assign different teaching points, and linked the teaching KPI scores to the evaluation, salary, and professional title of teachers. Then, we analyzed the overall teaching points and teaching points for each professional group (2021-2022). Finally, we asked teaching managers, teachers, and colleagues to evaluate the KPI lean teaching management system and compared the effects before and after the implementation of this system.Results:Compared with 2021, the teaching scores of 106 teachers increased significantly from 1.0 (0.2, 2.7) to 3.8 (2.3, 6.0) in 2022 ( Z=8.1, P<0.01). The teaching scores of clinical molecules, clinical coagulation, clinical immunology, clinical microbiology, Huifu laboratory, and clinical biochemistry group were significantly higher in 2022 than the scores they got in 2021 (all P<0.05). Compared with 2021, there were 3 new set up of educational reform projects, 2 submitted teaching articles, 3 new competition awards, 7 outstanding teachers, and 5 outstanding students in 2022. After application of KPI lean teaching management, the evaluation scores of teaching work by teaching managers, teachers, and colleagues are all significantly improved ( P<0.05). Conclusion:KPI lean teaching management system could effectively enhance teachers′ work initiative, improve teaching efficiency and outcome, and promote the teaching quality. Therefore, based on the performance of KPI lean teaching management system in our study, it is possible to realize its potential in terms of lean management in clinical medical laboratory.

Objective: To determine whether intrauterine infection with hepatitis B virus (HBV) occurs in early pregnancy and to characterize associated virulence factors. Methods: Villi tissues and blood samples of 45 HBV surface antigen (HBsAg)-positive pregnant women were collected during the first trimester and HBV DNA loads were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression of GCM1, HBsAg and hepatitis B core antigen (HBcAg) in villi tissues were detected by immunohistochemical method. Results: Data from qRT-PCR showed that HBV DNA was detected in 14 of 45 villi tissues (positive rate of 31.11%), and 24 of 45 blood samples (positive rate of 53.33%), further statistical analysis showed that the positive rates of HBV DNA between blood samples and villi tissues were not significantly different (χ²=4.555, P=0.054). Among them, 12 samples were consistently positive between the villi and blood specimens, and HBsAg, HBeAg, HBeAb, HBV DNA from peripheral blood in these pregnant women were significantly higher than those of the other women (P value was 0.007, 0.004, 0.000, and 0.000 respectively). The multivariate logistic regression analysis showed that blood HBV DNA greater than 106 IU/ml was independently associated with HBV DNA positive in villi, and the HBsAg, HBeAg, villi tissues HBV DNA positive rates of these pregnant women were significantly higher than those of the other pregnant women (all P value were 0.000). Immunohistochemistry results showed that all 45 cases were positive for GCM1 expression in the cell nucleus. Nine cases also had HBsAg expression in the cytoplasm. Only one case was found to express HBV core antigen (HBcAg) in the nucleus. Conclusions HBV DNA and HBsAg can be detected from villi tissues harvested during the first trimester in HBsAg-positive pregnant women, and the results suggest an early occurrence of intrauterine infection of fetuses with high HBV levels.