Objective To observe the effect of Shenfu injection on fluid intake volume of resuscitation therapy for patients with septic shock. Methods The clinic data of 36 patients with septic shock admitted to Department of Critical Care Medicine of the Affiliated Hospital of Hangzhou Normal University from June 2010 to June 2013 were retrospectively analyzed. All the patients were treated with western conventional medicine. Twenty cases treated with western medicine combined with Shenfu injection (intravenous drip 100 mL once daily, half of a month was a therapeutic course) were defined as Shenfu group; the rest 16 cases treated with western medicine only were assigned as control group. The following data after treatment for 6, 24, and 72 hours in the two groups were compared:liquid intake and urine volumes, system vascular resistance index (SVRI), mean arterial pressure (MAP), cardiac index (CI), and case fatality rate in 28 days. Results There were no significant differences in the liquid intake volume in 6 hours after treatment (mL:3 101±219 vs. 3 329±295, P>0.05), the urine volumes in 6, 24 and 72 hours after treatment (mL, 6 hours:701±229 vs. 651±292, 24 hours:1 870±566 vs. 1 697±618, 72 hours:7 396±2 546 vs. 5 987±2 497), and the levels of SVRI in 24 hours after treatment between Shenfu group and control group (kPa·s·L-1·m-2:802±158 vs. 741±106, all P>0.05). The total liquid intake volumes (mL) in 24 hours and 72 hours after treatment in Shenfu group were significantly less than those in the control group (24 hours:4 544±425 vs. 4 996±396, 72 hours:10 985±891 vs. 11 612±807, both P<0.05). The SVRI, MAP, and CI in 72 hours of Shenfu group were significantly higher than those of control group [SVRI (kPa·s·L-1·m-2): 1 361±182 vs. 1 163±183, MAP (mmHg, 1 mmHg = 0.133 kPa): 76.2±6.1 vs. 71.8±6.3, CI (mL·s-1·m-2):76.2±7.5 vs. 70.8±7.2, all P<0.05], and the 28-day mortality rate in Shenfu group was significantly lower than that of control group [25.0%(5/20) vs. 62.5%(10/16), P<0.05]. Conclusion The application of Shenfu injection was favorable to the reduction of liquid intake volume in 72 hours after treatment that may be beneficial to the fluid limitation management in the course of treatment for septic shock.

Objective To investigate the antitumor activity of IL-24 delE5 in human leukemia cell line K562. Methods The expression of mda-7/IL-24 and its splice variant induced by TPA in leukemic cell lines, U937 and HL-60, was evaluated. The effects of IL-24 delE5 in K562 on cell proliferation, colony-forming ability, cell cycle, apoptosis, and tumor growth in vivo by using MTr assay, colony forming assay, flow cytometry, Annexin-V/PI and tumor xenograft models in nude mice were assessed. Meantime, the effects of IL-24 delE-5 and mda-7/IL-2A were compared. Results The expression of IL-24 dciE5 was detected in differentiated U937 and HL-60 cells. Transfection with IL-24 delE5 significantly reduced tumor cell viability, inhibited colony formation. Comparing with the control, G_0/G_1 stage add from (24.46±3.99) % to (42.69±3.04) %, caused cell cycle arrest in G_0/G_1 stage and significantly inhibited the growth of K562 transplantation tumor. No significant differences in the aforementioned antileukemia characteristics between IL-24 delE5 and mda-7/IL-24 was found. Conclusion Similar with mda-7/IL-24, IL-24 delE5 can efficiently inhibit the proliferation of K562 in vitro and in vivo, probably through induction of G_0/G_1 cell cycle arrest.

BACKGROUND: Previous studies suggest that vascular endothelial growth factor 121 may be an optimal target gene for thetreatment of acute myocardial infarction.OBJECTIVE: To investigate effect of direct myocardial injection of adenovirus recombinant human vascular endothelial growthfactor 121 gene (Ad-hVEGF121) on myocardial infracted rat heart structure, function and angiogenesis.METHODS: Totally 78 male SD rats were randomly divided into the sham-surgery (n=18), acute myocardial infarction (n=24),Ad-VEGF121 (n=19) and normal saline (n=17) groups. Among them, left anterior descending coronary arteries of the latter threegroups were ligated to prepare acute myocardial infarction models and rats were randomly selected to receive Ad-hVEGF12 ornormal saline via three points in the cardiac muscle at the 10-15 minutes after ligation. The chest was exposed without ligation inthe sham-surgery group.RESULTS AND CONCLUSION: At 2 weeks after injection, cardiac ultrasound showed that, compared with the sham-surgerygroup, the number of new capillaries, body weight and left ventricular mass / body weight of the acute myocardial infarction,Ad-hVEGF121 and normal saline groups were obviously increased (P < 0.05 or P < 0.01), especially those received transfectedrAd-hVEGF12, had higher density of blood capillaries than those of the normal saline and acute myocardial infarction groups.However, there were no obviously differences between each group in infarct size, cardiac structure or functions. The directmyocardial injection of Ad-VEGF121 can significantly promote the formation of new blood vessels within the myocardium.

Objective To investigate the effects of overexpression of heat shock protein 22(HSP22) in hematopoietic malignant tumor cell lines.Methods A lentiviral system was used to mediate transduction of HSP22 complementary DNA-containing expression vector or empty vector into K562 and Namalwa cells.The transduction effeciency was tested by fluorescence microscope scan and flow cytometry.Semi-quantitative RT-PCR and Western blot were used to identify the expression levels of HSP22 mRNA and protein.Growth curve analysis,cell cycle analysis,colony-forming assay,tumor growth in nude mice and apoptosis analysis were used to evaluate the role of HSP22 in K562 and Namalwa cells.Results Lentivector expression systemmediated delivery of HSP22 into K562 and Namalwa cells can inhibit colony forming of K562 and Namalwa cells,the average numbers of colonies per well were 108,72,125 and 80 for K562-V,K562-H,Namalwa-V and Namalwa-H respectively (P =0.000 16 and 0.000 37 for K562 and Namalwa respectively).HSP22 transduction can also inhibit proliferation of Namalwa cells in vitro (P =0.015,0.042 and 0.048 for day 5,6 and 7 respectively) and K562 cells in vivo (P =0.022 for day 21).No significant difference in cell cycle and apoptosis was found in K562 and Namalwa cells compared with controls (all P ＞ 0.05).Conclusion Overexpression of HSP22 could inhibit the growth of hematopoietic malignant tumor cell lines K562 and Namalwa.

Objective To investigate the anti-inflammatory/immune modulatory effects of high-expressing membrane bound M-CSF-hematopoietic malignant cells on macrophages. Methods After coculturing RAW264.7 and murine macrophage cell line with Namalwa-M, a cell line stably expressing mM-CSF, and companing with Nainalwa-V, a cell line stably transfected with the empty vector as the control, flow cytometry was used to detect the expression of the marker of alternatively activated macrophages, CD206, and intracellular expression of IL-10, IL-12, IL-6 and TNF-α to study the immunophenotype of RAW264.7; phagocytic assays to investigate their functional activity in vitro. Results RAW264.7 cocultured with Namalwa-M consistently showed high-level expression of CD206, which indicated activities of these macrophage cells were increased. Furthermore, these RAW264.7 expressing high levels of IL-10, TNF-a and low levels of IL-12, IL-6, as determined by intracellular staining were suggested that the phagocytic activity was increased. Functionally, RAW264.7 cocultured with Namalwa-M showed a higher level of phagocytic activity. Conclusion Macrophage generated in vitro after cocultured with mM-CSF-expressing hematopoietic malignant cell line could be transformed into abnormal macrophage with an immunophenotype defined as IL-10-high, IL-12-low, IL-6-low and TNF-α-high.

Objective To explore the mechanism of the cell-cycle arrest induced by human melanoma differentiation associated gene-7 (mda-7/IL-24) in chronic myelocytic leukemia cell line K562. Methods Microarray analysis was performed to determine the genes that were differentially regulated by mda-7/IL-24 in K562 cells, and was validated by realtime PCR. The phosphorylated pRb were detected by Western blotting analysis. Results A microarray analysis showed that G_0/G_1 phase-associated genes p21~(WAF-1) and BCCIP were up-egulated, while cdk6 and Smurf2 were down-regulated. The directional change in the expression of the four genes was successfully validated with real-time quantitative RT-PCR. pRb Set~(795) phosphorylation was observed with no modification of the pRb protein level. Conclusion These results suggest that IL-24/mda-7 may inhibit K562 proliferation and induce G_0/G_1 cell cycle angst by up-regulating p21~(WAF-1) and BCCIP, down-regulating cdk6 and Smurf2.

Objective To investigate the expression of FBXW7 during the development of Notch1induced murine leukemia.Methods Notch1 over-expressing murine model of T-cell acute lymphoblastic leukemia was used to study the expression of FBXW7 by real-time PCR methods.Bone marrow mononuclear cells (BMNC) were isolated on different days after transplantation and CD45.2+ GFP+ leukemia cells were sorted by flow cytometry at late stage.The expression changes of FBXW7 were tested by real-time PCR.Results The mouse bone marrow cells both from leukemia and control groups expressed FBXW7.Different expression patterns of FBXW7 were observed during the development of leukemia. The expression of FBXW7 was gradually increased in control group, whereas the expression level of FBXW7 in leukemia group was decreased steadily and reached one-sixth of that in control group on 12th day.Furthermore,lower expression level of FBXW7 was observed in CD45+.2 GFP+ leukemia cells.Conclusion Decreased expression of FBXW7 is observed in Notch1-induced mouse leukemia model,suggesting that the abnormal ubiquitin degradation pathway mediated by FBXW7 might contribute to the leukemogenesis in Notch1-induced murine leukemia model.

Objective To construct the miRNA‐21 eukaryotic overexpression vector pmR‐21 and to explore its regulation effect on the expression of c‐myc gene in HepG2 .2 .15 cells .Methods The miRNA‐21 precursor gene fragment pre‐miRNA‐21 was amplified by PCR ,then connected to the pmR‐mCherry plasmid vector after double enzyme digestion ,the accuracy of the recombi‐nant vector was verified by double enzyme digestion and sequencing ;then the recombinant vector was transfected into HepG2 .2 .15 cells ,the fluorescent protein expression was observed under the fluorescence microscopy at 24 h and the transfection efficiency was detected by flow cytometry ;the expression of miRNA‐21 was evaluated by real‐time quantitative PCR;at 72 h after transfection ,the expression levels of c‐myc gene were detected by RT‐PCR and Western blot ;CCK‐8 was used to detect the cell proliferation in each group .Results The double enzyme digestion and Western blot verified that the target gene fragment was inserted into the pmR‐mCherry vector;at 24 h after transfection ,intracellular strong fluorescence was seen ,the transfection efficiency was higher than 50% ;miRNA‐21 expression level of the pmR‐21 recombinant vector group was significantly increased;c‐myc gene expression was increased in the pmR‐21 recombinant vector group at 72 h after transfection ,the cell proliferation in the pmR‐21 recombinant group was faster than that in the control group(P<0 .05) .Conclusion The pmR‐21 eukaryotic overexpression vector is successfully con‐structed ,this recombinant vector can express miRNA‐21 stably ;miRNA‐21 can up‐regulate c‐myc gene expression ,c‐myc gene is one of miR‐21′s targets for playing a cancer‐promoting action .

Objects To evaluate the immune protective effect of dexmedetomidine on breast cancer dur-ing perioperative radical mastectomy via sevoflurane inhalation general anesthesia. To explore reasonable anesthet-ic strategyfor breast cancer radical mastectomy. Methods Patients were divided into two groups. Patients in ex-perimental group receivedgeneral anesthesia with dexmedetomidine and sevoflurane. Control group means general anesthesia with sevoflurane only. In both groups, the level of cortisol, IL-6, IL-8 and of TNF-αin serum were measured at 5 time points , 30 minutes before anesthesia , after cutting skin , after surgery , 24 h after surgery and 72 h after surgery. Results The amount of anesthetic used to induce general anesthesia in the experimen-talgroup were lower than that of the control group.There is no obvious difference of cortisol , IL-6, IL-8 and of TNF-αin serumat the time of 30 min before anesthesia between two groups.Concentrations ofseveral markersin-creasedafter anesthesia, of which experimentalgroup were lower than that of the control group. Conclusions Dexmedetomidine could be immunoprotective for patient with breast cancer during perioperative radical mastecto-my via sevoflurane inhalationgeneralanesthesia. This study recommends usingmultiple anestheticdrugs to anes-thetize patients of breast cancer when performing radical mastectomy.

Objective To construct the has-miR 146a eukaryotic overexpression vector pmR 146a and to explore its effect on the expression of c-Myc gene in HepG2.2.15 cells.Methods The has-miR-146a precursor gene fragment pre-has-miR-146a was amplified by PCR,then connected to the pmR-mCherry plasmid vector after double enzyme digestion,the accuracy of recombinant vector was verified by colony PCR,double enzyme digestion and sequencing;then the recombinant vector was transfected into HepG2.2.15 cells as the experimental group,meanwhile the empty vector group (transfecting pmR-mCherry empty plasmid group) and blank group(transfecting reagent lip2000+PBS),then the fluorescent protein expression amount was observed under the fluorescence microscopy at 24,48 h;the expression of has miR-146a was evaluated by qPCR;at 24,48 h after transfection,the expression levels of c-Myc gene mRNA were detected by qPCR,and the c-Myc protein expression level after 48 h was detected by Western blot.Results The colony PCR,double enzyme digestion and sequencing verified that the pre-has-miR-146a gene fragment was inserted into the pmR-mCherry vector;at 24,48 h after transfection in the experimental group and empty vector group,intracellular strong fluorescence was seen by fluorescent microscope,the transfection efficiency was at 50％-60％ contrasting without fluorescence;the has-miR-146a expression level in the experimental group was significantly higher than that in the empty vector group and blank group (P＜0.01);the c-Myc mRNA expression at 24,48 h after tranfection was significantly lower than that in the empty vector group and blank group (P＜0.05);the protein expression amount at 48 h after transfection was lower than that in the empty vector group and blank group (P＜0.01).Conclusion The pmR-146a eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miR-146a stably;miR-146a can down-regulate c-Myc cancer gene expression,which can serve as one of potential targets for treating hepatocellular carcinoma.