Objective To study the efficacy of complement depletion with CVF in preventing a-cute rejection after heart allotransplantation in inbred strain rats. Methods Inbred male Lewis rats were used as recipients and Brown-Norway rats as donors, and the heterotopic heart transplantation model was established. The allografts were divided into 2 groups (n=8 in each group). After infusing low-dose CVF 20?g/kg to the CVF-treated group, cardiac allograft survival time was observed on 4 rats of each group. The remaining 4 rats in each group were killed respectively at day 1,3,5 and 6, the pathological grade for acute rejection, the complement activity in serum, the deposition of C3 on tissue, and the extent of infiltration by CD3+ T cells were compared. Results The mean survival time of heart allograft was (11. 69?0. 72) days and (6. 65?0. 35) days in CVF-treated group and control group respectively (P

Objective To investigate the inhibitory effect of recombinant LIGHT-Fe gene on the proliferation of human esophageal carcinoma cell line Eea109. Methods LIGHT-Fc expression vector was transfected into human esophageal squamous carcinoma cell line Eca109 by using DOTAP liposomal transfection reagents. The effects of LIGHT-Fc gene on the proliferation of esophageal carcinoma cell line Eca109 in vitro were detected by cell growth curve and MTr assay. Forty-five nude mice were equally divided into Eea109/Wt group, Eca109/neo group and Eca109/LIGHT group. Carcinogenesis and pathological expression of the esophageal carcinoma tissues were observed. Results The expressions of LIGHT receptors were detected in Eca109 cells. The proliferation of Eca109 cells was inhibited after trasfecting LIGHT-Fc gene into Eca109 cells. The numbers of tumors generated in Eea109/Wt group, Eca109/neo group and Eca109/LIGHT group were 12, 11 and 5, with statistical significance between Eca109/LIGHT group and the other two groups (X2 =6.652, 4.821, P <0.05). The result of histopatholagical examination indicated that the tissue necrosis appeared significantly in tumors derived from Eea109/LIGHT cells. Conclusions The growth of esophageal squamous carcinoma cell line Eca109 can be suppressed by LIGHT-Fc gene whether in vitro or in vivo.

Objective To construct eukaryotic expression plasmid of human B7-H1 extracellular region-hIgG1Fc-pCI-neo eukaryotic expression vector,and express the functional fusion protein in mammalian CHO cell.Methods Full-length human B7-H1 encoding sequence was amplified from human activated T cell cDNA library by PCR,fused with hIgG1Fc,then transformed into pCI-neo expression vector and verified by sequencing.The validated recombinant was transfected into mammalian CHO cell by lipofectamine reagent.The supernatant of the cultured cell was collected and analyzed by the sandwhich ELISA to detect if there was the fusion protein,and the fusion protein was purified by HiTrap recombination protein Protein A affinity chromatography.The concentrated supernatant or purified fusion protein were used for Western blotting after SDS-PAGE to identify the molecular weight and immune activity of the fusion protein of B7-H1.Results The extracellular region of hB7-H1 about 727 bp was cloned from human T cell cDNA library and was inserted with hIgG1Fc into the eukaryotic expression vector pCI-neo.After the transfection of recombinant into mammalian CHO cell by lipofectamine reagent,the expression of B7-H1 fusion protein was detected in the cultured CHO cell supernatant by the sandwhich ELISA.The immune activity of the fusion protein was verified by Western blotting,and its molecular weight was about 51.76?10~(3),very close to the expected value.Conclusion The hB7-H1-Fc chimeric molecule was successfully constructed and the expression of its functional fusion protein in mammalian CHO cells lays a foundation for further research on the role of B7-H1 in immune tolerance,autoimmune diseases.

Objective To clone the full length cDNA of human LIGHT and to construct the recombinant eukaryotic expression plasmid pCI neo LIGHT for the stable expression on 293T cells. Methods Human LIGHT cDNA was cloned from a normal human activated T cell cDNA library phAD.CAD by PCR. After sequencing, LIGHT cDNA was inserted into plasmid pCI neo for the construction of the eukaryotic expression. The recombinant was transfected into 293T cells by electroporation. The expression of LIGHT on the surface of 293T cells was detected by flow cytometry after screening with G418. Results Sequencing confirmed that ORF of LIGHT gene was intact and right. Restrictive enzyme digestion proved that LIGHT gene was inserted into the recombinant plasmid of LIGHT pCI neo correctly. FACS analysis revealed that about 78.69% 293T cells expressed LIGHT protein on the cell surfaces at 3 months after screening with G418. Conclusion LIGHT gene has been cloned successfully, and a 293T cell line expressing LIGHT protein on its membrane surface has been obtained.

Objective To construct eukaryotic expression plasmid of hCD137 Fc gene and express hCD137 Fc fusion protein with high biological activity. Methods PCR technique was employed to clone the human CD137 cDNA from a normal human activated T cells cDNA library, and then clone its extramembrane encoding region. The extramembrane sequence together with human IgG1 Fc cDNA were inserted into the eukaryotic expression plasmid pcDNA3. CD137 Fc gene was expressed transiently in 293T cells, and then CD137 Fc protein was purified by recombinated protein A affinity chromatography column. At last, the MW, purity and antigenicity of CD137 Fc were identified by sandwich ELISA, SDS PAGE and Western blotting, respectively. Results The ORF of CD137 Fc gene was coincident with what we expected. ELISA and SDS PAGE confirmed protein expression in 293T cells. Western blotting proved the antigenicity of the purified CD137 Fc protein. Conclusion We obtained a purified recombinated CD137 Fc protein with biological activity and the expected MW. This lays the foundation for further studies of CD137 such as its role in immune homeostasis and other biological functions.

The aim of the present study was to evaluate the active and passive mechanical properties and wall collagen and elastin contents of mesenteric small arteries (MSAs) isolated from rats of 28-day simulated microgravity (SUS), countermeasure [S + D: SUS plus 1 h/d -G(x) to simulate intermittent artificial gravity (IAG)] and control (CON) groups. Three mechanical parameters were calculated: the overall stiffness (β), circumferential stress (σ(θ))-strain (ε(θ)) relationship and pressure-dependent incremental elastic modulus (E(inc,p)). Vessel wall collagen and elastin percentage were quantified by electron microscopy. The results demonstrate that the active mechanical behavior of MSAs differs noticeably among the three groups: the active stress-strain curve of SUS vessels is very close to the passive curve, whereas the active σ(θ)-ε(θ) curves of CON and S + D vessels are shifted leftward and display a parabolic shape, indicating that for MSAs isolated from S + D, but not those from SUS rats, the pressure-induced myogenic constriction can effectively stiffen the vessel wall as the CON vessels. The passive mechanical behavior of MSAs does not show significant differences among the three groups. However, the percentage of collagen is decreased in the wall of SUS and S + D compared with CON vessels in the following order: SUS < S + D < CON. Thus, the relationship between passive mechanical behavior and compositional changes may be complex and yet depends on factors other than the quantity of collagen and elastin. These findings have provided biomechanical data for the understanding of the mechanism of postflight orthostatic intolerance and its gravity-based countermeasure.
Animals ; Biomechanical Phenomena ; Collagen ; metabolism ; Elasticity ; Hindlimb Suspension ; physiology ; Male ; Mesenteric Arteries ; physiology ; ultrastructure ; Muscle, Smooth, Vascular ; physiology ; ultrastructure ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical ; Vasoconstriction ; physiology ; Weightlessness Simulation

This article introduces a new method using the servo motor which is controlled by ARM microcontroller to provide power for a pulsatile blood pump to beat. This method is featured with straightforward structure, accurate control, excellent timeliness, stable performance and small noise. And it can adjust the rate of beat, the rate of flow and the compression ratio according to actual demand.
Algorithms ; Heart-Assist Devices ; Models, Cardiovascular ; Pulsatile Flow

The aim of the present study was to investigate the effect of a short-term (3-day) simulated microgravity with and without daily dorsoventral gravitation (-G(x)) for 1 h on myogenic tone and vasoconstrictor responsiveness of the middle cerebral artery and mesenteric third-order small artery in rats. The tail-suspension (SUS) model was used to simulate cardiovascular deconditioning due to microgravity. Daily restoring to normal standing (STD) posture for 1 h was adopted to provide -G(x) as the countermeasure. Segments of middle cerebral artery and mesenteric third-order small artery were isolated and cannulated. Vascular diameters in response to increased intraluminal pressure (from 20 mmHg to 120 mmHg, by 20 mmHg steps) of isolated arteries under no-flow conditions were recorded by a Pressure Myograph System in both physiologic salt solution (PSS) (active diameter, Da) and calcium-free PSS (passive diameter, Dp). The myogenic tone was calculated by (Dp-Da)/Dpx100%. Vasoconstrictor responsiveness of the isolated middle cerebral artery to serotonin and that of small mesenteric artery to phenylephrine were assessed in the PSS under an intraluminal pressure of 40 mmHg. The results showed that SUS induced an enhancement of the myogenic tone and vasoconstrictor responsiveness in the isolated middle cerebral artery but a depression of those in the small mesenteric artery. Daily STD for 1 h prevented the depression of myogenic tone and vasoconstrictor responsiveness in the small mesenteric artery, but did not prevent the functional enhancement in the middle cerebral artery. These data suggest that a short-term simulated microgravity may result in different alterations in the function of the cerebral artery and the resistance vessel in the hind-body. Moreover, only the decrease of function in these resistance vessels, not in the cerebral arteries, can be prevented by such a countermeasure of daily STD for 1 h.
Animals ; Cerebral Arteries ; pathology ; Hindlimb Suspension ; Mesenteric Arteries ; pathology ; Pressure ; Rats ; Serotonin ; pharmacology ; Vascular Resistance ; Vasoconstriction ; Weightlessness Simulation

Recently, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is suggested as a new agent in the fighting against fibrogenesis. In tumor, DJ-1 is identified as a negative regulator of PTEN. But the expression of DJ-1 and the regulation of PTEN in fibrosis are unclear. Renal fibrosis was induced in 5/6 subtotal nephrectomy rat model. Human proximal tubular epithelial cells (HKC) were treated with transforming growth factor-beta 1 (TGF-β1), or transfected with DJ-1 or PTEN. Confocal microscope was used to investigate the localization of DJ-1 and PTEN. The selective phosphoinositide-3 kinase (PI3K) inhibitor, LY294002, was administered to inhibit PI3K pathway. The DJ-1 and PTEN expression, markers of epithelial-mesenchymal transition (EMT) and Akt phosphorylation were measured by RT-PCR, Western blotting or immunocytochemistry. In vitro, after HKC cells were stimulated with 10 ng/mL TGF-β1 for 72 h, the expression of DJ-1 was increased, and that of PTEN was decreased. In vivo, the same results were identified in 5/6-nephrectomized rats. In normal HKC cells, most of DJ-1 protein localized in cytoplasm, and little in nucleus. TGF-β1 upregulated DJ-1 expression in both cytoplasma and nuclei. In contrary, TGF-β1 emptied cytoplasmic PTEN protein into nucleus. Overexpression of DJ-1 decreased the expression of PTEN, promoted the activation of Akt and the expression of vimentin, and also led to the loss of cytoplasmic PTEN. Contrarily, overexpression of PTEN protected HKC cells from TGF-β1-induced EMT. In conclusion, DJ-1 is upregulated in renal fibrosis and DJ-1 mediates EMT by suppressing cytoplasmic PTEN expression and Akt activation.

OBJECTIVE: To determine the relationship between the nitroglycerin tolerance and the stimulation of radical oxygen species (ROS) production, and the therapeutical effect of 3,4,5,6-tetrahydroxyxanthone. METHODS: Vasodilator responses to nitroglycerin were examined in the isolated thoracic aorta. The contents of ROS,and cGMP were determined in the cultured human umbilical vein endothelial cells. RESULTS: 3,4,5,6-tetrahydroxyxanthone could significantly reduce the inhibition of relaxation by nitroglycerin. 3,4,5,6-tetrahydroxyxanthone could significantly inhibit the ROS increase and increase the cGMP level. CONCLUSION Nitroglycerin tolerance is associated with the stimulation of ROS production,and the reversal of nitroglycerin tolerance with 3,4,5,6-tetrahydroxyxanthone is related to the reduction of ROS.
Animals ; Antioxidants ; pharmacology ; Aorta, Thoracic ; cytology ; Cells, Cultured ; Drug Tolerance ; Endothelium, Vascular ; cytology ; Humans ; Male ; Nitroglycerin ; pharmacology ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Umbilical Veins ; cytology ; Xanthones ; pharmacology