Administration, Oral ; Anti-Bacterial Agents ; administration & dosage ; Cytomegalovirus Infections ; drug therapy ; immunology ; virology ; Erythromycin ; administration & dosage ; Female ; Humans ; Infant ; Male ; T-Lymphocyte Subsets ; immunology ; Virus Replication

Objective To construct the prokaryotic expression vector of mouse CD25 extracellular domain and to express it in E coli.Methods Total RNA was isolated from splenocytes of Balb/c mice.The CD25 extracellular domain gene was amplified by RT-PCR and cloned into the PET-32a vector.A positive recombinant,PET-32a-CD25e,was identified by enzyme cleaving and sequencing before its expression in E.coli,and transferred into E.coli BL21(DE3) plysS for its expression.After purification with Ni+ resin and renaturation in vitro,a relative molecular mass(Mr) of the interesting protein was detected by SDS-PAGE and Wes-tern blotting.Effect of the purified interesting protein on the proliferation of splenocytes from T cell vaccine-immunized syngeneic mice was detected by MTT assay.Results The cloned CD25 extracellular domain gene was identified to be functional by sequencing and expression.The purified interesting protein could significantly induce the proliferation and IL-4 secretion of splenocytes from T cell vaccine-immunized mice in vitro.Conclusion Mouse CD25 extracellular domain gene can be successfully expressed in prokaryotic cells with biological activity,which lays a foundation for further relative studies.

The clinical practice on real patients is more and more difficult in the present condition of the hospitals.Then,the modern medical simulating teaching is the main direction of the development in this field due to its characteristics,based on high- technology,simulating the real clinical circumstance,and being applicable in practice and avoiding the risk of clinical miscarriage. The significance and main development direction of modern medical simulated teaching will be discussed in this article.

OBJECTIVETo investigate the effect of total flavonoids of Hedysarum polybotry on the proliferation, cell cycle, and expressions of p21(Ras) and proliferating cell nuclear antigen (PCNA) gene in erythroleukemia cell line K562.

METHODSThe effect of total flavonoids of Hedysarum polybotry on K562 cell line survival was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay. The time- and dose-dependent manner was also observed. The cell cycle and apoptosis were analyzed with flow cytometry (FCM). The immunocytochemistry method was applied to quantitatively analyze the effects of flavonoids of Hedysarum polybotry on changes p21(Ras) and PCNA gene expressions.

RESULTSFlavonoids of Hedysarum polybotry (20-100 μg/mL) significantly inhibited the proliferation of K562 cells in a time- and dose-dependent manner. After K562 cells were cultured for 48 h, total flavonoids of Hedysarum polybotry had no significant effect on the apoptosis of K562 cells but showed significantly inhibition (P<0.01), indicating that total flavonoids of Hedysarum polybotry could induce K562 cells arrested at G(0)/G(1) and G(2)/M phases. Compared with the control group, p21(Ras) and PCNA gene expressions were decreased significantly in K562 cells treated with total flavonoids of Hedysarum polybotry (40 and 80 μg/mL, respectively) for 48 h.

CONCLUSIONThe inhibitory effect on proliferation of K562 cells was observed in the groups treated with flavonoids of Hedysarum polybotry, which might be related to cells arresting.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Flavonoids ; pharmacology ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; K562 Cells ; Leukemia, Erythroblastic, Acute ; drug therapy ; genetics ; pathology ; Oncogene Protein p21(ras) ; genetics ; Proliferating Cell Nuclear Antigen ; genetics ; Ranunculaceae ; chemistry

Aim To screen a more suitable transfection recep-tor,and improve the efficiency of constructing cell lines highly expressing human peptide transporters 1 (hPepT1 ).Methods The recombinant plasmid pcDNA3.1 (+)-hPepT1 was transfect-ed into MDCK cells and HeLa cells by LipofectamineTM 2000 transfection reagent,respectively.The monoclonal cells were se-lected and cultured.Expression of hPepT1 mRNA and protein were determined by qRT-PCR and Western blot,respectively. The uptake capacity of Glysar in transfected cells was examined. Results Compared with wild type cells,the expression of hPepT1 and the uptake of Glysar in transfected MDCK cells and HeLa cells significantly increased (P <0.05).Although the up-take of Glysar in HeLa cells was higher than that of MDCK cells,on the contrary,the expression of hPepT1 and the uptake of Glysar in MDCK-hPepT1 cells was higher than that of HeLa-hPepT1 cells.Conclusion MDCK cells may serve as a more suitable transfected receptor for the construction of a cellular model with high expression of hPepT1 ,which would make the construction of a cell model highly expressing hPepT1 more effi-cient.

BACKGROUND: Paraquat (PQ) is an effective herbicide and is widely used in agricultural production, but PQ poisoning is frequently seen in humans with the lung as the target organ. Clinically pulmonary pathological changes are often used to predict the severity and prognosis of the patients. In this study, we observed the expression of heat shock protein 70 (HSP70) in rat lung after PQ poisoning and to investigate the therapeutic effects of ulinastatin. METHODS: Seventy-two adult healthy SD rats were randomly divided into a control group (group A, n=24), a poisoning group (group B, n=24), and an ulinastatin group (group C, n=24). The rat models of acute PQ poisoning were established by intra-gastric administration of 80 mg/kg PQ to rats of groups B and C, and the rats of group C were intra-peritoneally injected with 100000 IU/kg ulinastatin 30 minutes after poisoning. The expression of HSP70 in lung tissue was observed, and W/D and histopathological changes in the lung tissue were compared 12, 24, 48 and 72 hours after poisoning. The expression of HSP70 in the lung tissue was assayed by using RT-PCR. All quantitative data were processed with one-way analysis of variance to compare multiple sample means. RESULTS: Compared to group A, the expression of HSP70 in the lung of rats in groups B and C increased significantly at all intervals (P<0.05). The pathological changes in lung tissue of rats with PQ poisoning included congestion, leukocytes infiltration and local hemorrhage, whereas those of group C were significantly lessened. CONCLUSION: Ulinastatin may ameliorate acute lung injury to some extent after PQ poisoning in rats by enhancing the expression of HSP70.

BACKGROUND: Paraquat (PQ) is an effective herbicide and is widely used in agricultural production, but PQ poisoning is frequently seen in humans with the lung as the target organ. Currently, there are many studies on lung injury after PQ poisoning. But the kidney as the main excretory organ after PQ poisoning is rarely studied and the mechanisms of this poisoning is not very clear. In this study, we observed the expression of caspase-3 and livin protein in rat renal tissue after PQ poisoning as well as the therapeutic effects of ulinastatin. METHODS: Fifty-four Sprague-Dawley (SD) rats were randomly divided into three experimental groups: control group (group A), paraquat poisoning group (group B) and ulinastatin group (group C), with 18 rats in each group. Rats in group B and group C were administered intragastrically with 80 mg/kg PQ, rats in group C were injected peritoneally with 100000 U/kg ulinastatin once a day, while rats in group A were administered intragastrically with the same volume of saline as PQ. At 24, 48, 72 hours after poisoning, the expression of livin in renal tissue was detected by Westen blotting, the expression of caspase-3 was detected by immunohistochemistry, and the rate of renal cell apoptosis was tested by TUNEL detection. The histopathological changes were observed at the same time. RESULTS: Compared to group A, the expression of caspase-3 in the renal tissue of rats in groups B and C increased significantly at any time point. Compared with group B, the expression of caspase-3 in renal tissue of rats in group C decreased. Compared with group A, the expression of livin in renal tissue in rats of groups B and C increased significantly at any time point (P<0.01), especially in group C (P<0.01). TUNEL method showed that the rate of renal cell apoptosis index was higher in group B at corresponding time points than in group A (P<0.01), and was lower in group C at corresponding time points than in group B (P<0.01). CONCLUSION: UTI has a protective effect on the renal tissue of rats after paraquat poisoning through up-regulating the expression of livin and down-regulating the expression of caspase-3, but the regulation path still needs a further research.

Objective To evaluate the efficacy of haploidentical hematopoietic stem cell transplantation (HSCT) with supplemental umbilical cord blood (UCB) infusion in treatment of malignant hematological diseases.Method Clinical data of 66 patients with hematological malignancies treated with HSCT in our hospital between January 2010 and May 2013,were retrospectively analyzed.Among them 25 cases received infusion of human UCB before HSCT (experimental group) and other 41 cases had no UCB injection before HSCT (control group).Results There were no differences in age,gender,donor type,disease categories,disease status before transplant between two groups (P ＞ 0.05).There was a significant difference in conditioning regimes between two groups (P ＜ 0.05),but no clinical implication.The infused mononuclear cell (MNC) count in experimental group was higher than that in control group (9.94 ± 2.88 × 108/kg vs.7.80 ±0.82 × 108/kg,P =0.00),while there were no difference in infused CD34 + cell count (5.46 ±3.54 × 106/kg vs.3.54 ± 1.60 × 106/kg,P =0.16).Neutrophil recovery time in experimental group was shorter than that in control group (13.7 ±2.9 d vs.16.6 ±2.9 d,P =0.023).The incidences of grade Ⅲ-Ⅳ acute graft versus host disease (aGVHD,P =0.036),bacterial infection (P =0.001) and fungal infection (P =0.001)and hemorrhagic cystitis (P =0.00)in experimental group were lower than those in control group.There were no significant differences in platelet recovery time(P =0.43),the incidence of grade Ⅰ-Ⅱ aGVHD (P =0.27),implanted syndrome (P =0.24),sinusoidal obstruction syndrome (P =0.57)and viraemia (P =0.31)between two groups.Conclusion HSCT with supplemental infusion of human UCB may alleviate the degree of aGVHD,but the long-term outcome remains to be studied.

Objective To explore the efficiency and prognosis of hematological malignancies treated by haploidentical hematopoietic stem cell transplantation.Methods 70 patients who received haploidentical hematopoietic stem cell transplantation were analyzed retrospectively.According to tumor burden before transplantation,the patients were divided into three groups,the low tumor burden group,the mediate tumor burden group and the high tumor burden group.And then the effection of the tumor burden to survival was analyzed,and the engraftment,GVHD,infection,conditioning related toxicity,relapse and survival rate were also observed.Results The follow-up was terminated on January 1,2014.Follow-ups were performed for a median of 34.05 (7.4-83.6) months after transplantation.All patients achieved engraftments.The cumulative incidence of GVHD of grades 2-4 was 47.14 ％ (33/70) and that of grades 3-4 was 21.4 ％ (15/70).The chronic extensive GVHD was 20.0 ％ (14/70).The overall survival was 68.6 ％.Transplant-related mortality was 12.8 ％ and the relapse was 18.6 ％.The overall survivals in low tumor burden group,mediate tumor burden group,high tumor burden group were 91.67 ％,72.7 ％,33.3 ％ respectively.By SPSS 20.0,tumor burden was the high risk factor affecting the survival (low tumor group vs high tumor group,mediate tumor group vs high tumor group,low tumor group vs high tumor group,P =0.000,P =0.038,P =0.016).Conclusions Haploidentical hematopoietic stem cell transplantation in hematological malignancies is safe and effective.And for hematological malignancies with poor prognosis disease,it should be accepted the HSCT as soon as possible after remission in order to reduce the recurrence rate of malignancy.

Abstrate:Aim To construct HEK293 cell line with stable and high expression of sodium taurocholate cotransporting polypeptide (NTCP ) efficiently and rapidly.Method Vector expressing EGFP-NTCP fusion protein was constructed and verified by DNA sequencing.The pEGFP-NTCP expression vector was transfected into HEK293 cells by FuGENE 6 transfection reagent. The transfected cells with high expression of green fluorescent protein were selected using fluorescence microscope for screening of G418 for 14 days to obtain cell lines stably and highly expressing NTCP.NTCP expression was detected by RT-PCR,qRT-PCR, Western-blot and the uptake experiment of taurocholic acid.Re-sult RT-PCR,qRT-PCR,Western-blot and the uptake experi-ment revealed that compared to the control cells,the expression of NTCP was significantly positive (P<0.01)in stable trans-fected cells showing green fluorescence (P<0.05 ).Conclusion The HEK293 cell line with stable and high expression of NTCP has been established efficiently and rapidly,which provides a cellular model for the study of the mechanism of the uptake of bile acid derivatives.