Objective To study the possible molecular mechanism of the damage of HepG2 cell line induced by low concentration of ethanol and ultraviolet irradiation. Methods Hepatoma cell line (HepG2) was exposed to low concentration of ethanol and ultraviolet irradiation, then the changes in cell cycle, the expression of p53 and p21, the correlation between expression of p53 and p21 and cell cycle were analyzed. Results The expression of p53 and p21 was greatly increased, and the cell cycle was found to be blocked at G_0-G_1/G_2-M after ultraviolet irradiation. The cell cycle block of G_2-M and the increased expression of p21 were observed when exposed to low concentration of ethanol, while the expression of p53 showed no significant changes. Cell apoptosis was increased when the cells were exposed to the both injury factors. Conclusion Low concentration of ethanol and ultraviolet irradiation can affect cell cycle and the process of cell apoptosis through activation of different molecular mechanism.

Objective To screen the down-regulated genes in HepG2 cells transfected by pcDNA3.1(-)-IFN-? to further investigate the biological mechanism of IFN-?. Methods pcDNA3.1(-)-IFN-? was transfected into HepG2 cells as treatment group and the pcDNA3.1(-) transfected cells as the control to perform suppression subtractive hybridization (SSH). The subtractive library for down-regulated genes was obtained when pcDNA3.1(-) transfected HepG2 as tester and pcDNA3.1(-)-IFN-? transfected cells as driver. A housekeeping gene, G3PDH, was used to estimate the subtractive efficiency. Genes with lower expressions in IFN-? transfected HepG2 cells were obtained from the library after being randomly sequenced and analyzed. Among the obtained genes, regulation of IFN-? on heat shock 90kD (Hsp90) mRNA was further investigated by RT-PCR. Results G3PDH was subtracted efficiently indicating that the subtractive library was constructed successfully. 50 positive clones were randomly isolated for PCR identification. Results showed that most of the plasmids in the clones contained 200-1 000bp inserts. The down-regulated genes obtained were as follows: eukaryotic translation elongation factor 1 beta, ferritin, RAD23 homolog B, signal sequence receptor 2 (SSR2), tissue factor pathway inhibitor, heat shock 90kD protein 1, and adenosylmethionine decarboxylase 1. RT-PCR showed that hsp90 mRNA had a reduced expression in pcDNA3.1 (-)-IFN-? transfected HepG2 cells. Conclusion The down-regulated cDNA subtractive library in HepG2 cells after pcDNA3.1 (-)-IFN-? transfection was constructed successfully. IFN-? could down-regulate the hsp90 mRNA expression.

To study the effect of heparin on the ConA induced acute liver injury inKunming mice,twenty four mice were randomly divided into groups A,B and C.To the mice in group A, 0 2ml of normal saline (NS) was intravenously administered, while those in group B were given ConA 18mg/kg instead of NS in order to induce severe acute liver injury. Heparin was injected subcutaneously in a dose of 100U per animal at the same time as ConA challenge in group C. Compared with group B, prior injection of heparin in group C significantly decreased the mice death rate and the peak levels of serum ALT within 8h. At the same time, intrasinusoidal congestion and hepatic inflammation were alleviated significantly,and MDA level in liver homogenate was lowered markedly. It suggested that heparin is efficient in protecting the mice from liver injury induced by ConA.

To observe whether there is an interaction between hepatitis virus B (HBV) and tumor suppressor p53, plasmid PCMVp53 was transfected or cotransfected with pCMVHBV a (wild type HBV) or PCMVHBV b (mutation type HBV) into the hepatoma cell line 7721 by phosphate calcium precipitation. Apoptosis cells were labeled by annexin Ⅴ FITC and detected by flow cytometry. Another experiment was performed by cotransfecting the cells with reporter plasmid p21 luc in each group mentioned above, then the luciferase activity was measured. The results showed that the cells transfected by pCMVp53 alone exhibited high luciferase activity and high apoptosis rate. Meanwhile, the luciferase activity and apoptosis rate were further higher in cells cotransfected by pCMVp53 and PCMVHBV a , but remained unchanged in cells cotransfected by PCMVp53 and PCMVHBV b. The results indicated that P53 could induce of 7721 cell apoptosis by activating p21 transcription, and such effect could be enhanced by HBV.

Objective To explore the prognostic influence of combination therapy with mild hypothermia and edaravone for acute cerebral infarction.Methods Two hundred and forty-five cases of diagnosed as acute cerebral infarction within 72 hours of onset were randomly divided into four groups according to the doctor visiting time.All the groups were treated with routine drugs.Combined therapy group (65 cases ) was treated with mild hypothermia combined with edaravone.Mild hypothermia group (59 cases ) was treated with local mild hypothermia.Edaravone group (58 cases) was treated with edaravone.Control group (63 cases) was only treated with routine drugs.The European Stroke Scale (ESS) score was performed before treatment,30 days after treatment.The activity of daily living (ADL) scores were evaluated before treatment,30 and 90 days after treatment.Results ESS scores were (45.22 ± 16.94),(46.88 ± 22.54),(47.13 ± 10.92),(46.94 ± 16.41 ) scores before treatment in combined therapy group,mild hypothermia group,edaravone group,control group respectively.ALD scores were (20.54 ± 14.65 ),(20.94 ± 10.93),(21.83 ± 14.71),(23.61 ± 18.91 ) scores before treatment in combined therapy group,mild hypothermia group,edaravone group,control group respectively.There were no differences in ESS and ADL scores before treatment among the groups.ADL scores were higher 30,90 days after treatment in combined therapy group [(59.57 ± 30.99),(74.46 ± 25.61) scores] than those in mild hypothermia group [(43.91 ±27.61),(58.13 ±26.62) scores) and control group [(34.58 ±27.75), (45.56 ±26.10) scores] (P ＜ 0.05) and higher after 90 days treatment than that in edaravone group [(62.83 ± 28.74) scores] (P ＜ 0.05 ).ESS scores 30 days after treatment in combined therapy group [(72.24 ± 14.54) scores] were higher than those in mild hypothermia group [(65.88 ± 17.76) scores],edaravone group [(65.27 ± 18.02) scores],control group [(60.62 ± 14.97) scores] (P ＜ 0.05 ).The effectiveness in combined therapy group [63.08％ (41/65)] was higher than that in mild hypothermia group [42.37％(25/59)],edaravone group [41.38％ (24/58)] and control group [23.81％ ( 15/63 )] ( P ＜ 0.05 ).The mortality rate in combined therapy group [9.23 ％ (6/65)] was lower than that in mild hypothermia group[10.17％(6/59)],edaravone group[12.07％(7/58 )] and control group [12.70％ (8/63)],but there was no significant difference (P ＞ 0.05 ).Conclusion The combination therapy with mild hypothermia and edaravone can improve the prognosis of acute cerebral infarction.

Objective To study methylation status of the Runx3 gene in occurrence and development of children malignant lymphoma.Methods The bone marrow specimens of 48 children diagnosed as malignant lymphoma were included into experimental group.20 children with non-malignancy were included into control group.Methylation-specific polymerase chain reaction (MS-PCR) was used to detect methylation status of Runx3 gene in bone marrow cells.The reverse transcription-polymerase chain reaction (RT-PCR) was used to detect mRNA expression of Runx3 gene.Results MS-PCR assay results showed that 31 cases expressed Runx3 gene methylation in experimental group,the positive rate was 64.6 ％ (31/48),but no one was detected in control group (20 cases),the difference between two groups was significant (x2 =15.7,P ＜0.05).Dynamic observation of 42 cases in experimental group showed the declining of Runx3 methylation positive rate.RT-PCR assay results showed that all 20 cases in the control group expressed Runx3 gene mRNA,all cases with methylation status of the Runx3 gene in the experimental group didn’ t expressed Runx3 gene mRNA.In experimental group,9 cases of clinical remission expressed Runx3 gene mRNA,and 1 case of relapsed didn’ t expressed Runx3 gene mRNA.Conclusion Runx3 gene shows a high methylation status in bone marrow cells of malignant lymphoma children,so that blocked the expression of Runx3 gene,which is closely related to the occurrence and development of children malignant lymphoma.

Objective To investigate the clinical efficacy and side reaction of brucea javanica oil ( BJO) combined with 125I and chemotherapy on stageⅢ?Ⅳpatients with non?small cell lung cancer ( NSCLC) . Methods One hundred and twenty cases on stageⅢ?Ⅳpatients with NSCLC were randomly divided into two groups,60 cases received BJO combined with 125I and chemotherapy treatment(observation group),the other 60 cases received 125I combined with chemotherapy treatment(control group). Results The objective response rate(ORR) and disease control rate (DCR) were 71. 7%,86. 7% of observation group and 66. 7%,85. 0% of control group,there were no significant difference(χ2=0. 352,0. 069;P>0. 05) . The improvement rate of KPS score in observation group was significantly superior to that in control group, the difference was significant (76. 7% vs. 55. 0%;χ2=6. 261,P<0. 05) . The incidence of myelosuppression and gastrointestinal adverse e?vents in observation group was significantly lower that in control group ( 68. 3% vs. 83. 3%,41. 7% vs. 61. 7%;χ2=3. 883,4. 805;P<0. 05) . Conclusion BJO combined with 125I and chemotherapy for treating on stageⅢ?Ⅳ patients with NSCLC can reduce the toxicity and side effects caused by chemotherapy,and significantly im?prove the clinical symptoms and quality of life of patients.

Objective To examine the benefits of ECMO for potential organ donors with hemodynamic instability after brain death.Methods Three brain-dead potential donors who presented with hemodynamic instability despite maximal medical management,finished a declaration of brain death,that were supported by extracorporeal circulation membrane oxygenation (ECMO).Results Donor organs,including six kidneys,and two livers,were harvested from the three donors under ECMO support,leading to 8 successful transplantations.The organs functioned well and the recipients made full recoveries.Conclusion Our experience indicates that ECMO allows for the maintenance of abdominal organ tissue perfusion without warm ischemia before organ procurement,providing sufficient time for safe organ donation procedures and reducing the risk of unpredictable cardiac arrest that could result in the donor death and graft loss.

Objective:To investigate therapeutic effect of reinfusion of tumor-infiltrating lymphocyte(TIL)with clustered regularly interspaced short palindromic repeats/CRISPR-associated 9(CRISPR/CAS9)knockout programmed death-1(PD-1)on colon cancer in mice.Methods:Subcutaneous injection of CT26 was used to establish mouse colon cancer model.TIL was extracted from tumor tissue of three model mice,and peripheral blood lymphocytes were extracted.PD-1 gene was knocked out of TIL.Reinfusion experiments were divided into control group(Control),lymphocyte group(Lym),tumor-bearing mouse TIL group(TIL),lentivirus empty empty group(pVSV-G-PX458-NC)and PX458-PD-1-sgRNA1 group(PD-1-sgRNA1),with 10 mice in each group.Tumor tissue quality and tumor inhibition rate were detected in each group.TUNEL was used to detect cell apoptosis in tumor tissues of mice.ELISA was used to detect contents of TNF-α and IFN-γ in tumor tissues of mice.Immunohistochemistry was used to detect expressions of CD4+T and CD8+T cells in tumor tissue.Immunofluorescence was used to detect expressions of proliferating cell nuclear antigen-67(Ki-67)and vascular endothelial growth factor(VEGF).Western blot was used to detect expressions of PD-1 and its ligand PD-L1 in tumor tissues.Results:PD-1-sgRNA1 could significantly inhibit growth of mouse tumor cells in vivo,inhibit expressions of Ki-67 and VEGF in tumor tissues,as well as expressions of PD-1 and PD-L1,elevate apoptosis rate,contents of TNF-α and IFN-γ in tumor tissues,and expressions of CD4+T and CD8+T cells(all P<0.05).Conclusion:Reinfusion of TIL with CRISPR/CAS9 knockout PD-1 can significantly inhibit expressions of Ki-67 and VEGF in colon cancer mice,enhance infiltration of CD4+T and CD8+T cells,induce tumor cell apoptosis and inhibit tumor growth.

OBJECTIVE: To analyze transforming growth factor-β1( TGF-β1)-induced differentially expressed genes( DEGs) in human embryonic lung fibroblast( IMR-90) using microarray,and to screen the key genes and signaling pathways related to trans-differentiation of fibroblast.METHODS: The gene chip GSE17518,attained from TGF-β1 stimulated IMR-90 cells,was downloaded from the Gene Expression Omnibus database.The DEGs were screened by GENE-E software.Then,the DEGs were imported into the DAVID online database for Gene Ontology( GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes( KEGG) pathway enrichment analysis.The proteinprotein interaction( PPI) network was constructed and the hub genes were screened using STRING database and Cytoscape software.RESULTS: A total of 394 DEGs related to TGF-β1 stimulation were identified,including 171 down-regulated genes and 223 up-regulated genes.The results of GO analysis showed that the DEGs were widely distributed in cytoplasm,cell membrane,extracellular matrix( ECM) and exosomes,regulating biological functions such as ECM organization,cell migration and adhesion,cell proliferation and apoptosis.The results of the KEGG pathway analysis indicated that most of DEGs were enriched in cell focal adhesion,ECM-receptor interaction and phosphoinositide 3 kinase-Protein kinase B( PI3K-Akt) signaling pathways.The PPI network screened 10 core genes,included nucleolar protein 2( NOP2),succinate dehydrogenase B,glutamyl-prolyl-tRNA synthetase( EPRS),FtsJ homolog 3( FTSJ3),prefoldin subunit 4,Ras-related C3 botulinum toxin substrate 2,signal recognition particle receptor subunit beta,succinate-Co A ligase GDPforming beta subunit,pumilio RNA binding family member 3( KIAA0020),and general vesicular transport factor p115.NOP2,EPRS,FTSJ3,KIAA0020 were mainly distributed in M1 module.The NOP2 is the core gene with the highest number of nodes in M1 module.CONCLUSION: A total of 10 core differential genes and 7 signaling pathways related to TGF-β1 stimulation were screened.Among them,focal adhesion,ECM-receptor interaction,PI3K-Akt and NOP2,EPRS,FTSJ3,KIAA0020 may provide new direction for research of mechanisms of abnormal activation of fibrotic diseases including silicosis in incidence and development of multiple lung fibrotic diseases.