Objective: To investigate the effects of TFPI-2 (tissue factor pathway inhibitor 2) on the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from human liver cancer cells. Methods: The Hep3B cells stably expressing TFPI-2 (Hep3B-TFPI-2 group) and the Hep3B cells transfected with empty vector PCDNA3.1 (Hep3B-V group) or without transfection (Hep3B-P group) were subcutaneously transplanted into nude mice respectively to generate subcutaneous tumor xenografts. The volume of tumor xenograft was measured every three days, and the growth curve of tumor xenograft was drawn when the subcutaneous tumor xenograft was visible. The nude mice were killed three weeks after transplant, the volume of tumor xenograft was measured, and the total RNAs and proteins in tumor xenografts were extracted. The mRNA and protein expressions of TFPI-2 and VEGF (vascular endothelial growth factor) in tumor xenografts were analyzed by RFQ-PCR (real-time fluorescence quantitative PCR) and Western blotting, respectively. The expression of TFPI-2 protein and the MVD (microvessel density) in tumor xenografts were observed by immunohistochemistry. Results: The eventual tumor volume of tumor xenografts in Hep3B-TFPI-2 group was apparently smaller than those in Hep3B-V group and Hep3B-P group (both P < 0.05). The expression of mRNA and abundance of protein of TFPI-2 in Hep3B-TFPI-2 group were significantly higher than those in the other two groups (P < 0.05); while the expression of mRNA and abundance of protein of VEGF in Hep3B-TFPI-2 group were apparently lower than those in the other two groups. Compared with Hep3B-V group and Hep3B-P group, the inhibitory rates of VEGF protein expression in Hep3B-TFPI-2 group were 19.8% and 23.5%, respectively (P < 0.05). The MVD in Hep3B-TFPI-2 group was apparently lower than those in the other two groups (P < 0.05). Conclusion: TFPI-2 can significantly inhibit the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from hepatocarcinoma Hep3B cells. Copyright © 2013 by TUMOR.

Objective To explore the role of endothelial nitric oxide synthase(eNOS) in the regulatory effects of erythropoie‐tin (EPO) on mitochondiral biogenesis in cardiomyocytes exposed to chronic hypoxia .Methods H9c2 cardiomyocytes were cultured in the environment of hypoxia for 1 week(94% N2 ,5% O2 ) ,establishing the chronic hypoxic cardiomyocyte model .All the cells were divided into 3 groups :HC(chronic hypoxic control) ,HE[treated with chronic hypoxia and 20 U/mL recombinant human EPO (rhEPO) ]and HR(cells transfected with eNOS shRNA plasmid and treated with 20 U/mL rhEPO and chronic hypoxia) .Fluores‐cent probe was used to detect the changes of mitochondial number .Mitochondial DNA (mtDNA) relative express level was assayed by RT‐PCR .The expression and phosphorylation of eNOS protein were analyzed with Western blot .Results rhEPO significantly increased the phosphorylation of eNOS and elavated the mitochondialt number and mtDNA (P< 0 .05) .shRNA interference on eNOS significantly blocked all the above changes induced by rhEPO (P<0 .05) .Conclusion Phosphory lation of eNOS is the im‐portant signalling pathway for the enhanced mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia by EPO .

A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase, equipped with a polyhistidine affinity tag, was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108, and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein, the ATP content of bacteria was 9.48×10-16mol/mL, and was identical to the bacteria counts (4500CFU/mL) in order of magnitude. Taken together, our results provided a simple and efficacious method of the preparation of recombinant luciferase, which could be applied in the determination of bacteria via ATP bioluminescence.

Acute Disease ; Cohort Studies ; Humans ; Leukemia ; diagnosis ; Myelodysplastic Syndromes ; diagnosis ; Prognosis

Objective: To establish a molecule binding model of mouse PD-1/PD-L1 protein in vitro, so as to lay a foundation for studying the biological activities of the recombinant protein and to establish a high throughput drug screening model. Methods: Prokaryotic expression plasmid pET28 a (+) /mPDL-1 and pGEX-4T-1/mPD-1 were transformed to E. coli BL21 (DE3), which was then induced with IPTG. The expression products were purified by fast affinity chromatography with FPLC Protein Purification Instrument or gel separation. ELISA and GST pull-down were used to detect the interaction between mPD-1 and mPD-L1. In addition, Alamar blue was used to test the role of protein mixtures in the mixed lymphocyte proliferation. Results: SDS-PAGE and Western blotting analysis showed that the recombinant proteins were successfully expressed. Then purified mPD-1 and mPD-L1 protein was obtained by affinity chromatography, Gel seperation and refolding, etc. ELISA and GST pull-down showed that mPD-1 and mPD-L1 protein had a specific binding activity in vitro. The mPD-1/ mPD-L1 protein had a significantly decreased influence on the proliferation of lymphocytes(P<0. 05). Conclusion: Mouse PD-1/PD-L1 recombinant protein model has been successfully established using purified mPD-1 and mPD-L1 protein expressed prokaryotically, which lays a foundation for high-throughput drug screening.

OBJECTIVETo explore the effects of chronic hypoxia on left and right ventricular function and the expression of cardiac transient receptor potential canonical (TRPC) channels in rats.

METHODSForty eight SD male rats were randomly divided into control group (CON) and chronic hypoxic pulmonary hypertension model group (CH) (n = 24). In CH group, rats were exposed in chronic hypoxia environment (10% +/- 0.2% O2) to induce myocardial hypertrophy. After 3 weeks, mean systemic arterial pressure (mSAP), right ventricular systolic pressure (RVSP), left ventricular systolic pressure (LVSP), left or right ventricular pressure maximum rate of rise (LV/RV + dp/dt(max)), left or right ventricular pressure maximum rate of descent (LV/RV-dp/dt(max)), right ventricular hypertrophy index (RVMI) and left ventricular hypertrophy index (LVMI) were measured. Left and right ventricular myocardium tissue sections were stained by HE and observed under light microscope. Real-time polymerase chain reaction (real-time-PCR) and Western blot were performed to detect the expression of TRPC subfamily.

RESULTSRVSP, RVMI, RV + dp/dt(max) and RV-dp/dt(max) were markedly elevated in CH group (P < 0.01) in comparison to CON group. LVMI was markedly reduced in CH group in comparison to CON group (P < 0.01). LVSP, LV + dp/dt(max) and LV- dp/dt(max) had no significant changes in CH group in comparison to CON group. Right ventricular myocardial cells of CH group became thick, the nuclei stained deeply, the shape of nuclei became not regularity. Left ventricular myocardial fibers did not change significantly. There was significant difference in the levels of mRNA and protein of TRPC1 between CON and CH groups.

CONCLUSIONFor three weeks exposed to chronic hypoxia induced right ventricular hypertrophy specifically, raised the mRNA and protein expression of TRPC1 on right ventricular myocardial cells . TRPC1 might be involved in the development of cardiac hypertrophy.

Animals ; Disease Models, Animal ; Hypertension, Pulmonary ; metabolism ; physiopathology ; Hypoxia ; metabolism ; physiopathology ; Male ; Rats ; Rats, Sprague-Dawley ; Transient Receptor Potential Channels ; metabolism ; Ventricular Function, Left ; physiology ; Ventricular Function, Right ; physiology

Objective To assess the efficacy of combination therapy with all-trans-retinoic acid (ATRA ) and arsenic trioxide (ATO) in treating patients with acute promyelocytic leukemia (APL) .Methods A retrospective study was conducted to evaluate the efficacy of combining ATO with ATRA based induction therapy ,followed by 3 courses of consolidation chemotherapy and 2-year sequential ATO/ATRA maintenance therapy in newly diagnosed APL ,and the efficacy between high risk group and low/inter-mediate risk group ,also between different PML-RARa isoform sub-group were compared .Results In high risk group and low/in-termediate risk group ,the complete remission(CR)rates were 70 .0% and 96 .9% (P=0 .04) ,respectively ;the 3 years overall sur-vival rates(OS) and disease free survival rates (DFS) were(70 .0 ± 14 .5)% ,(96 .9 ± 3 .1)% ,P= 0 .01 and(66 .7% ± 19 .2)% , (93 .8 ± 6 .1)% ,P=0 .08 ,respectively .In BCR1 group and BCR3 group ,the CR were 78 .6% and 95 .6% (P=0 .14) ,respectively ;the rates of 3 years OS and DFS were(95 .7 ± 4 .3)% ,(78 .6 ± 11 .0)% ,P=0 .18 ,and(92 .9 ± 6 .9)% ,(87 .5 ± 11 .7)% ,P=0 .24 , respectively .Conclusion The results indicate that ATO based first-line protocol is highly effective for treatment of newly diagnosed APL ,especially for the PML-RARa BCR3 isoform APL .

Autoimmune Lymphoproliferative Syndrome ; diagnosis ; genetics ; immunology ; therapy ; Biomarkers ; blood ; Child, Preschool ; Humans ; Male ; Mutation ; Prednisolone ; administration & dosage ; therapeutic use ; Ultrasonography, Doppler, Color ; fas Receptor ; genetics

Adolescent ; Bone Marrow Transplantation ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Thalassemia ; surgery ; Transplantation Chimera

Objective Human sperm hyaluronidase in the acrosome is a key enzyme during fertilization.To study and establish a modified substrate film method to improve the diagnotic and treatment of male infertility and investigate the influence of sperm hyaluronidase on male fertility.Methods According to the biochemical feature of sperm hyaluronidase that can dissolve the hyaluronic acid in matrixes,the modified sodium hyaluronate-Gelatin membrane was used as substrate to demonstrate the sperm hyaluronidase activity by incubation and staining.70 human semen samples were selected,categorized and detected for hyaluronidase activity according to the resuh of routine clinical semen examination.The average sperm hyaluronidase activity was statistically anyliezed between the fertile group and infertile group.Results Under general light microscope,the clear and bright digestion circle around the sperm head can be observed against the background of deep purple-blue stained substrate at the positive-reaction area.The amounts of positive reaction and the diameter of the bright circle are positively related to the activity of sperm hyaluronidase.The average hyaluronidase reaction rate and diameter of normal fertile group were 70.84% and 78.17 ?m;What about the infertile group A were 60.02% and 76.92 ?m;The infertile group B were 29.11% and 8.22 ?m; There was statistical difference of HYD activity between the infertile group.B and the fertile group(P