OBJECTIVEBy using the RNAi method to inhibit Itk protein expression specificity, to observe lymphocytes proliferation and cytokines production, verify its function as a drug target.

METHODSDesigned siRNA aims at Itk sequence according to its sequence and solid structure, then electrotransfected into mouse spleen lymphocytes, We validated the decrease of Itk protein by Western-Blot, and detected the change of the cell proliferation by MTS and the change of inflammatory cytokines by ELISA.

RESULTSItk protein can be suppressed by Itk-siRNA, there were significantly reduced compared to its control group on cell proliferation as well as cytokine secretion such as IL-2, IL-4, IL-5, IFN-gamma. They all have statistical difference (P < 0.05).

CONCLUSIONItk has an important immunomodulatory effect in mouse spleen lymphocytes proliferation and secretion of inflammatory cytokines.This can supply an experimental basis to regard Itk as drug target for inflammation therapy.

Animals ; Cell Differentiation ; Cell Proliferation ; Cytokines ; genetics ; immunology ; Lymphocytes ; cytology ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Protein-Tyrosine Kinases ; genetics ; immunology ; Spleen ; cytology ; immunology

The purpose of this study is to prepare T7 and TAT dual modified liposomes (T7-TAT-LIP) to penetrate through blood brain barrier and target to brain tumor cells. The liposomes were prepared with CFPE, T7 modified PEG-DSPE, TAT modified PEG-DSPE, soybean phospholipid, PEG-DSPE and cholesterol. The CFPE was used to track the cellular uptake efficiency. The density of T7 and TAT and the length of PEG were optimized, and then the liposomes were characterized by particle size, zeta potential, morphology and stability. Afterwards, the cellular uptake by bEnd.3 and C6 cells were evaluated. The results showed that the optimized parameters were 6% of T7, 0.5% of TAT, the molecular weight of PEG for T7 was 2000 and the molecular weight of PEG for TAT was 1000. After optimization, the particle size of T7-TAT-LIP was 118 nm, the zeta potential was -6.32 mV and the particles were spherical. The turbidity and particle size of liposomes were not obviously changed after 24 h incubation in PBS at 37 °C. The particle size and polydispersity index were also stable during 1 month incubation at 4-8 °C. The cellular uptake by both bEnd.3 and C6 cells of T7-TAT-LIP was higher than that of T7 or TAT modified liposomes, suggesting dual modified liposomes possessed better blood brain barrier targeting ability and brain tumor targeting ability than the single ligand modified liposomes.
Biological Transport ; Blood-Brain Barrier ; Brain Neoplasms ; drug therapy ; Cell-Penetrating Peptides ; pharmacology ; Cholesterol ; Liposomes ; Particle Size ; Phosphatidylethanolamines ; Polyethylene Glycols

Aim To study mRNA and protein expression of eNOS in pulmonary tissue of chronic hypoxic pulmonary hypertension(HPH)rats and chronic hypoxic rats treated with novel KATP opener iptakalim.Methods sixty Sprague-Dawley(SD)male rats were randomly divided into control group, hypoxic group, low dose iptakalim group(0.75 mg·kg~(-1)·d~(-1)), and high dose iptakalim group(1.5 mg·kg~(-1)·d~(-1)).Except the first group, the other three groups were put into hypoxic and normobaric chamber (10%±0.5% O_2,8 h/day and 6 day/week) to establish chronic hypoxic model. After four weeks, the mean pulmonary arterial pressure(mPAP), RV/(LV+S)and the plasma concentration of NO were measured. RT-PCR was performed to analyze the mRNA expression of eNOS in pulmonary tissue. Western blot was performed to analyze the protein expression of eNOS, iNOS in pulmonary tissue. Results ① The level of mPAP and RV/ ( LV + S) were significantly higher in the hypoxic group than those in control group ( P < 0. 05 ) , Low dose iptakalim groupandhighdoseiptakalimgroupdecreased the level of mPAP and RV/( LV + S) significantly (P <0. 05). ② The level of NO was significantly lower in the hypoxic group than those in control group (P<0. 05). Low dose iptakalim group and high dose iptakalim group increased the level of NO significantly (P < 0. 05 ). ③ The mRNA and protein expression of eNOS in the hypoxic group were significantly lower than those in the control group (P < 0. 05 ). Low dose iptakalim group and high dose iptakalim group increased the expression of eNOS significantly ( P < 0. 05). High dose iptakalim group was more significant. Conclusion Pulmonary vascular endothelial dysfunction is induced by chronic hypoxia,and the level of NO, the mRNA and protein expression of eNOS are decreased. Iptakalim can improve the vascular endothelial dysfunction, increase the expression of eNOS and the level of NO and reverse hypoxic pulmonary hypertension.

Novozym 435 was selected from four lipases and two proteinase because of its high catalytic activity and enantiosectivity.For the ammonolysis of (?)-?-methylbenzyl acetate,The effect of ammonia sources,the concentration of enzyme and substrates on the reaction were further explored .under the optimum conditions of this study,after 6h reaction,with the enantiomeric excess of the remaining (-)-?-methylbenzyl acetate was found to be higher than 99%.

The effects of reaction media, water activity, temperature and pH on Novozym 435-catalyzed enantiose-lective ammonolysis of (?) -?-methylbenzyl acetate have been systematically explored. Novozym 435 showed high catalytic activity and enantioselectivity in hexane; the optimum temperature and the initial water activity were 25℃ and 0.33 respectively; The suitable reaction pH was in the range of 6.0 - 7.0.

To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) on CVB3 infection in an animal model by RNA interference technique, we constructed a recombinant lentivirus expressing shRNA-3753 against the viral genome region 3753-3771, then transduced Lenti-sh3753 into mice infected with CVB3. We evaluated the antiviral ability of lenti-sh3753 by cytopathic effect (CPE), viral plaque assay and histological analysis of mice hearts. The results showed that Lenti-sh3753 exhibited a significant protective effect on cell viability and reduction of viral titers in supernatant of cell culture by specific inhibition on viral replication. Lenti-sh3753 also prolonged the mice survival and limited the viral production in mice hearts. These data proposed that Lenti-sh3753 can effectively inhibit CVB3 infection in a coxsackievirus-induced myocarditis model, suggesting its potential role in prevention and therapy of viral diseases.
Animals ; Coxsackievirus Infections ; drug therapy ; virology ; Down-Regulation ; Enterovirus B, Human ; genetics ; physiology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; RNA, Viral ; genetics ; Virus Replication

OBJECTIVETo explore the feasibility and therapeutic effect of thinned posterior tibial artery free perforator flap for the reconstruction of soft tissue defects at dorsum of hands.

METHODSSix fresh adult lower limbs specimens were injected with red latex via arterial cannula and dissected. The number, distribution, branches, and outer diameter of posterior tibia artery perforators were observed. Based on the anatomic study, the perforator flaps were designed to reconstruct soft tissue defects at dorsum of hands and wrists. The redundant fat on the flaps was removed, but preserving the nutrient vascular system. 11 flaps were used with the size ranging from 2 cm x 5 cm to 10 cm x 14 cm.

RESULTS43 skin perforators of posterior tibial artery were observed in six lower limbs, 29 perforators with the outer diameter is greater than 0.5 mm when they threading over the deep fascia plane, on average every 4.8 bundles of sides. The mean outside diameter of perforating artery is (1.8 +/- 0.5) mm, and the length is (44 +/- 15) mm. 6 perforators were founded both in the second and fifth zone which could be used for anastomosis for its better diameters. All flaps survived completely without any complication at donor sites. 7 cases were followed up for 3-12 months. Both satisfactory functional and cosmetic results were achieved with a soft and thinned appearance.

CONCLUSIONSThe thinned posterior tibial artery free perforator flap has a reliable blood supply and good appearance. It is very suitable for the reconstruction of small or medium-sized defects at the dorsum of hands and wrists.

Adolescent ; Adult ; Female ; Hand Injuries ; surgery ; Humans ; Lower Extremity ; anatomy & histology ; blood supply ; Male ; Middle Aged ; Perforator Flap ; blood supply ; Reconstructive Surgical Procedures ; methods ; Tibial Arteries ; anatomy & histology ; transplantation ; Young Adult

Objective To investigate the mitochondrial damage and its effect in early stage of pressure ulcer in rats.Methods Forty rats were randomly divided into 5 groups(n=8), control group(Con group) rats without stress, the experimental group was treated with of 170 mmHg for 2 h and relax 0.5 h as one cycle(1C), experi-mental group was divided into 3C, 6C, 9C and 12C group.The pathological changes of the compressed muscle tissue of the rats in each group were observed by HE staining , Western blot was used to detect the expression of Bcl-2 and Bax in the compressed tissue , and the ultrastructure of muscle fibers and mitochondria were observed by transmission electron microscope .Results There were pathological damage and gradually increased in the ex-perimental groups, with the increase of compression cycle; the expression of Bcl-2 in each experimental group was significantly increased as compared with the control group(P<0.05), in the 3C group reached the peak, and then decreased; the expression of Bax was increased gradually with the increase of compression cycle ( P<0.05) , and in the 12C group reached the peak;with the increase of the compression cycle the muscle fibers of each experimental group appeared gradually increased pathological damage:disorder, dissolution and fracture, the ridge of the mitochondria disappeared, vacuolar degeneration, et al.Conclusions In the early stage of pres-sure ulcer in a rat , it brings occurred mitochondrial damage and induces apoptosis .

OBJECTIVETo study the compatibility of composite herbal medicines of the Ling Gui Zhu Gan Decoction.

METHODEthanol extract test solutions of the different combinations were prepared according to the orthogonal layout L16(4(5)). Pharmacologic experiments, such as the time of surviving of mice in shortage of oxygen in regular air pressure, the antagonizing effect on arrhythmia induced by chloroform and diuresis were carried out with the solutions. Variance analysis, canonical correlation and stepwise regression analysis were applied to interrelate the amount of each drug and the pharmacologic data.

RESULTThe results confirmed that Fuling and Guizhi are the basis, while Baizhuand Gancao are the adjuvans, which is conformed to the theory of TCM.

CONCLUSIONThis study provides a significant try for studying the compatibility of composite herbal medicines.

Animals ; Anti-Arrhythmia Agents ; therapeutic use ; Diuretics ; pharmacology ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; therapeutic use ; Female ; Hypoxia ; drug therapy ; Male ; Medicine, Chinese Traditional ; Mice ; Phytotherapy ; Plant Extracts ; isolation & purification ; therapeutic use ; Plants, Medicinal ; chemistry ; Ventricular Fibrillation ; drug therapy

To improve the targeting of adenovirus vector for gene therapy, a fusion gene sCAR-EGF, in which epidermal growth factor gene was fused to the 3' end of extracellular Coxsackie virus-adenovirus receptor gene, was constructed and cloned into shuttle plasmid pDC315 to obtain a recombinant plasmid pDC315-sCAR-EGF. With the AdMax system, AD-293 cells were co-transfected with pDC315-sCAR-EGF and adenovirus genomic plasmid pBHGloxdeltaE13cre. Through high efficiency site specific recombination, a replication-defective adenovirus Ad5-CMV-sCAR-EGF was constructed. The recombinant adenovirus was analyzed by PCR and Western blotting, the results indicated that Ad5-CMV-sCAR-EGF contained the fusion gene sCAR-EGF, and the adenovirus infected cells was induced to produce and secrete the fusion protein into the supernatant. We have demonstrated that the fusion protein sCAR-EGF is helpful for elevating the infection efficiency of Ad5-CMV-luc with the reporter gene in vitro, which providing a new approach to the gene therapy for tumors overexpressing EGFR.
Adenoviridae ; genetics ; Cell Line ; Coxsackie and Adenovirus Receptor-Like Membrane Protein ; Enzyme-Linked Immunosorbent Assay ; Epidermal Growth Factor ; analysis ; genetics ; Genetic Therapy ; Humans ; Neoplasms ; therapy ; Polymerase Chain Reaction ; Receptors, Virus ; analysis ; genetics ; Recombinant Fusion Proteins ; biosynthesis