Objective To construct an eukaryotic expression vector for TAP genes fused with enhanced green fluorescent protein(EGFP)gene,and to analyze the expression and subcellular localization of the fusion protein in A375 human malignant melanoma cells transfected with the eukaryotic expression vector.Methods A375 cells were cotransfected with the combination of plasmid(P)TAP1-EGFP or pTAP2-EGFP and pDsRed2-endoplasmic reticulum(ER),or with pEGFP-TAP1 and-TAP2,or monotransfected with pTAP1-EGFP or pTAP2-EGFP alone.The monoclonal A375 cells stably expressing TAP were obtained by G418 selection.Then.the distribution and expression of fusion proteins were assessed in A375 cells by using fluorescence microscopy and Western blot,respectively.Flow cytometry was used to measure the expression of HLA class Ⅰ on A375 cells.Results Transfection of A375 cells with pTAP1-EGFP or pTAP1-EGFP and pTAP2-EGFP significantly increased the expression of TAP 1 and TAP 2 in as well as HLA class Ⅰ antigen on A375 cells.The green fluorescence of TAP1-EGFP and TAP2-EGFP overlapped with the red fluorescence of ER marker in cotransfected cells.indicating that TAP was localized subcellularly on the ER.Conclusions The expression vector for TAP-EGFP fusion gene has been constructed cuccessfully and expressed in A375 cells,and the expressed fusion protein is subcelluiarly localized to ER.This study will provide a basis for the research into subsequent immune response following induction of TAP expression.

Objective To evaluate the effect of hypoxic-ischemic time on reduction of hypoxic-ischemic brain injury by sevoflurane postconditioning in neonatal rats.Methods Two hundred and ten 7-day-old Sprague-Dawley rats (105 male,105 female),weighing 13-17 g,were randomly divided into 7groups (n=30 each) using a random number table:sham operation group (group Sham),hypoxia-ischemia group (group HI),and sevoflurane postconditioning at different hypoxic-ischemic time point groups (P0,P3,P6,P 12 and P24 groups).Immediately after ligation of the left common carotid artery,and at 3,6,12 and 24 h after ligation,the rats inhaled the mixed gas containing 2％ sevoflurane for 30 min in P0,P3,P6,P13 and P24 groups,respectively.The fatality was recorded within 7 days after establishment of the model.At 7 days after establishment of the model,the rats were sacrificed,the brains were removed,and the right and left cerebral hemispheres were weighed separately,and the left/right cerebral hemisphere weight ratio was calculated.The hippocampal CA1 region and posterior cingulate gyrus were isolated,and the ratio of density of normal neurons in the left to the right was calculated.Results Compared with group Sham,the left cerebral hemisphere weight,left/right cerebral hemisphere weight ratio,and ratio of density of normal neurons were significantly decreased,and the fatality rate was increased in the other six groups (P＜0.05).Compared with group HI,the left cerebral hemisphere weight,left/right cerebral hemisphere weight ratio,and ratio of density of normal ncurons were significantly increased in P0,P3 and P6 groups (P＜0.05),and no significant change was found in the parameters mentioned above in P12 and P24 groups (P＞0.05).Compared with group P6,the left cerebral hemisphere weight,left/right cerebral hemisphere weight ratio,and ratio of density of normal neurons were significantly increased in P0 and P3 groups (P＜ 0.05).There was no significant difference in the parameters mentioned above between group P0 and group P3 (P＞0.05).Conclusion Sevoflurane postconditioning performed within 6 h of hypoxia-ischemia can reduce hypoxic-ischemic brain injury,and it provides no cerebral protection if exceeding 12 h.

Objective To compare imaging quality and radiation dose on full-field digital mammography (FFDM) system and dual-sided reading CR mammography (DSCRM) system.Methods The TRM of ALVIM statistics phantom was exposed by FFDM system and (DSCRM) with the same radiation dose.The exposure parameters and the entrance surface dose (ESD) were recorded.Then,the phantom was exposed by DSCRM system with different mAs and same kV,and the ESD was recorded.The images obtained above were read by three radiologists on the monitor of diagnosis work station with the same window width and window level.The evaluation of the images were given using a five-level confidence scale.ROC curves were drawn and probability Pdet were calculated.Results The ROC analysis of microcalcifications and masses showed A(z) values of 0.730-0.925 and 0.670-0.945 for FFDM,and 0.632-0.815 and 0.575-0.785 for DSCRM when the radiation dose is 1.36 mGy.The radiation dose with DR system could be reduced compared with dual-sided reading CR system at the same probabilities Pdet,the ESD reduced from 1.63 mGy to 1.36 mGy by 19.8％,the AGD reduced from 0.65 mGy to 0.56 mGy by 16.0％.Conclusions The observable details with FFDM system were proved to be superior to DSCRM system at the same radiation dose.With the similar image quality,the radiation dose of FFDM system was less than the DSCRM system.

In order to compare the clinical characteristics of familial and non-familial bullous lichen planus (BLP), the archival data of 36 BLP patients with positive family history and 21 BLP patients with negative family history diagnosed according to the clinical features and histopathology were collected in our department from 1956 to 2003. The clinical features were analyzed and compared. There were significant differences between familial and non-familial BLP in age of onset, duration of disease and extension of eruption (P<0.01). It was concluded that familial BLP appeared to differ from the non-familial form in its earlier age of onset, longer duration of the disease, more extensive eruption and more tendency to involve nails. Hereditary factors may play a role in the pathogenesis of familial BLP.

Objective To investigate the role of B cell lymphoma/leukemia-2 and adenovirus E1B 19 000 interacting protein 3 (BNIP3) in oligodendrocyte cell apoptosis induced by carbon monoxide poisoning (CO poisoning) and the potential signal pathways.Methods Twenty-five male C57BL/6 mice were randomly divided into control group and CO poisoning group.Mice were left to breathe room air (control group) or subjected to 40-minute exposure to 2 500-3 000 ppm CO (CO poisoning group).The mice were sacrificed at 1,3,7 d and 14 d following CO poisoning.We examined the damage of myelin sheath and oligodendrocytes by observing the expression of myelin basic protein (MBP) and oligodendrocyte transcription factor 2 (Olig2) in corpus callosum.Furthermore,we explored the role of BNIP3 and potential signal pathways in the oligodendrocyte cell death following CO poisoning by observing the expression of BNIP3,Bcl-2 associated X protein (Bax) and cysteinyl aspartate specific proteinase 9 (caspase 9).Results Immunohistochemistry showed that the expression of MBP decreased significantly in the corpus callosum from 1 d (0.12±0.02,t=3.357,P＜0.05) to7 d (0.05±0.02,t=9.730,P＜0.01) and increased from 7 d to 14 d (0.13 ± 0.02,t =2.897,P ＜ 0.05) to some degree after CO poisoning compared with the control group (0.16 ± 0.02) and that Olig2 expression increased markedly in 3 d CO poisoning group (72.2 ± 5.45,t =12.211,P ＜ 0.01) compared with the control group (36.6 ± 3.58).The results of MBP and Olig2 in Western blotting revealed that MBP began to decrease from 1 d (0.39 ± 0.02,t =10.391,P＜0.01)to 7 d(0.09 ±0.01,t =34.767,P＜0.01)and increased in 14 d (0.45 ±0.03,t =6.146,P ＜ 0.01) compared with the control group (0.55 ± 0.03),and that O1ig2 increased obviously in 3 d (0.52 ± 0.02,t =16.651,P ＜ 0.01) compared with the control group (0.31 ± 0.02).Western blotting analysis showed that the levels of BNIP3 were increased in 1 d (2.49 ±0.40,t =15.342,P ＜0.01),started to decrease in 3 d (1.90 ± 0.24,t =12.417,P ＜ 0.01) and finally recovered in 14 d (0.24 ± 0.02,t =0.798,P ＞0.05),as compared with the control group(0.25 ±0.03).Moreover,compared with the control group(0.44 ±0.03),Bax was also upregulated in the corpus callosum from 1 d (1.09 ± 0.15,t =9.427,P ＜ 0.01) to 7 d (0.64 ± 0.09,t =4.540,P ＜ 0.05) after CO poisoning.The expression of caspase 9 showed the similar tendency that increased in 1 d (1.10 ± 0.17,t =7.137,P ＜ 0.01),decreased in 3 d (0.79 ± 0.10,t =5.051,P ＜ 0.01)and recovered in 7 d (0.55 ± 0.05,t =0.910,P ＞ 0.05) compared with the control group (0.51 ± 0.08).BNIP3 expression was positively correlated with Bax (r =0.995,P ＜0.01) and caspase 9 (r =0.950,P ＜ 0.01).Conclusion BNIP3 may play an important role in the apoptosis of oligodendrocytes induced by CO poisoning via the pathway of caspase dependent mitochondrial apoptosis.

Objective: To explore deep vein thrombosis (DVT) occurrence in patients after right heart catheterization and the effect of anticoagulant therapy. Methods: A total of 171 consecutive patients with electrophysiological study (EPS) and/or radiofrequency catheter ablation (RFCA) in our hospital from 2015-01 to 2016-05 were enrolled. All patients had supra-ventricular tachycardia and completed a venous surgery, they were randomly divided into 2 groups: Anticoagulation group,n=87 and Non-anticoagulation group,n=84. Lower extremity vascular Doppler ultrasonography was performed at (24-48) h post-operation to compare the incidence of DVT between 2 groups. Results: There were 13/171 patients were excluded for not completing post-operative lower extremity vascular Doppler ultrasonography including 9 patients in Anticoagulation group and 4 in Non-anticoagulation group. 158 patients finished post-operative examination and follow-up study. Anticoagulation group had 7/78 (8.97%) patients suffered from DVT, Non-anticoagulation group had 41/80 (51.3%) patients suffered from DVT,P<0.001. Conclusion: The incidence of DVT was higher after right heart catheterization without anticoagulation; heparin treatment may reduce DVT occurrence in relevant patients.

In order to investigate the expression of endothelin receptor B (ETR-B) in human malignant melanoma (MM) cells A375 and SK-mel-1 and the proliferative effects of endothelin 3 (ET3) on A375 cells, RT-PCR was applied to detect the expression of ETR-B gene in human MM cells A375 and SK-mel-1. MTT method was used to evaluate the growth enhancing effects of ET3 on A375 cell line in vitro. The results showed that ETR-B gene was expressed in both MM A375 and SK-mel-1 cells. ET3 had stronger ability to enhance the proliferation of A375 cells in vitro in a concentration-dependent manner. It was suggested that ET3/ETR-B might play an important proliferative role in MM.
Cell Line, Tumor ; Cell Proliferation/*drug effects ; Endothelin-3/*pharmacology ; Melanoma/*metabolism ; Melanoma/*pathology ; Receptor, Endothelin B/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction

ObjectiveTo investigate the change in the activity of glucosylceramide synthase, the key enzyme in glycosphingolipid metabolism and synthesis, in Huh7 cells infected by hepatitis B virus (HBV) in vitro. MethodsBlood samples were collected from nine previously untreated patients with acute hepatitis B who attended Department of Infectious Diseases, The First Hospital of Lanzhou University, from June to August, 2019, and the blood samples collected from seven healthy individuals who underwent physical examination were established as control. Huh7 cells were inoculated with the high-copy HBV particles (＞9.9×107 IU/ml) in the serum of patients with HBV infection (infection group), and Huh7 cells co-cultured with the serum of healthy individuals were established as control group. The expression levels of HBsAg and HBV DNA in the cytoplasm of HBV-infected Huh7 cells were measured, and the correlation between GCS activity and virus was analyzed. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups, and a Pearson correlation analysis was performed. ResultsCompared with the control group, the infection group had a significant reduction in the number of cells, an increase in cell volume, and cell membrane fragmentation. The infection group had a significant increase in the expression of HBsAg in cytoplasm at 4 hours, 8 hours, 2 days, and 5 days after infection (P＜0.05); the expression level of HBV DNA tended to increase significantly from 4 hours after infection to 8 hours, 2 days, and 5 days after infection (16.67±11.55 IU/ml vs 112.01±25.94 IU/ml/328.01±10350 IU/ml/101.60±49.84 IU/ml, P＜0.001), with the highest level at 2 days after infection. During HBV infection, the activity of GCS gradually increased with the increase in viral replication from 4 hours after infection (126.21±9.59 IU/ml) and reached a peak at 2 days after infection (226.53±36.27 IU/ml), with a significant difference between the infection group and the control group at 2 days after infection (226.53±36.27 IU/ml vs 136.50±1544 IU/ml, t=3.956, P=0.016 7). The activity of GCS was positively correlated with HBV DNA level (r=0.576 8, P=0047 1). ConclusionHuh7 cells are successfully infected with the high-copy HBV particles in the serum of patients with HBV infection, which mimics the characteristics of HBV infection in vitro to a certain degree. The activity of GCS may be associated with HBV infection, suggesting that glycosphingolipid synthesis and metabolism may be closely associated with HBV.

In order to compare the clinical characteristics of familial and non-familial bullous lichen planus (BLP), the archival data of 36 BLP patients with positive family history and 21 BLP patients with negative family history diagnosed according to the clinical features and histopathology were collected in our department from 1956 to 2003. The clinical features were analyzed and compared. There were significant differences between familial and non-familial BLP in age of onset, duration of disease and extension of eruption (P＜0.01). It was concluded that familial BLP appeared to differ from the non-familial form in its earlier age of onset, longer duration of the disease, more extensive eruption and more tendency to involve nails. Hereditary factors may play a role in the pathogenesis of familial BLP.

Objective To study the effect of marein on myocardial fibrosis in diabetic mice.Methods Ten lep-tin receptor gene defective heterozygous(db/m)mice aged 5-6 weeks were selected as the control group and 30 diabetic mice with leptin receptor gene defective db/db were divided into:db/db group(db/db,n=10),metformin(Met)positive group(280 mg/kg daily,n=10)and marein drug intervention group(50 mg/kg,n=10).After continuous administration for 8 weeks,the cardiac morphological changes were observed by HE staining and Masson staining.The distribution and expression of vimentin were detected by immunohistochemis-try method.The expression of fibronectin,vimentin,and transforming growth factor-β1(TGF-β1)protein in cardiac tissue was detected by Western blot.Results Myocardial fiber hypertrophy was observed in db/db group,and myocardial structural damage was improved in metformin group and marein group.Compared with db/m group,the expression of myocardial collagen fiber in db/db group increased(P＜0.01),while the expression of myo-cardial collagen fiber in metformin group and marein group decreased(P＜0.01).Compared with the control group,the expression of vimentin in myocardial tissue of db/db group was significantly increased(P＜0.01),while the expression of vimentin in metformin group and marein group was significantly decreased(P＜0.01).The expression of fibronectin,vimentin and TGF-β1 in db/db group was significantly increased as compared with those in db/m group(P＜0.01),while the expression of fibronectin,vimentin and TGF-β1 in metformin group and marein group were significantly decreased(P＜0.01).Conclusions Marein improves myocardial fibrosis in diabetic db/db mice.