Aim To investigate the effects of lentivirus mediated nerve growth factor ( NGF) gene silencing on pheochromocytoma cells ( PC12 ) and the possible mechanisms .Methods The NGF shRNA expression vector was constructed .PC12 cells were randomly divi-ded into five groups (n=3 each) as follows: negative control group ( NC ) , control lentivirus group ( LV CON) , lentivirus NGF shRNA1 group ( LV shNGF1 ) , lentivirus NGF shRNA2 group(LV shNGF2), lentivir-us NGF shRNA3 group(LV shNGF3).The cells in NC group were cultured in DMEM/HG and polybrene me-dium, while others were cultured in DMEM/HG, poly-brene and corresponding lentivirus medium .After the treatment, the infection efficiency was determined by fluorescent microscope .Relative expression of NGF , extracellular signal-regulated kinase ( ERK1/2 ) and p-ERK1/2 were assessed by Western blot .The expres-sion of NGF mRNA was analyzed by quantitative re-verse transcription polymerase chain reaction ( qRT-PCR) .The differentiation degree was valued according to the length of neuritis and max diameter of cells .The cell viability was detected by CCK-8.Results The in-fection efficiency in PC12 cells reached over 90%. Compared with NC group , the relative expression of NGF mRNA and NGF protein was significantly down-regulated ( P<0.05 ) .There was no difference in the expression of ERK1/2 protein and cell viability .The expression of p-ERK1/2 protein was markedly down-regulated in LV shNGF3 group ( P<0.01 ) .The cells morphology was changed , and the length of neuritis and max diameter of cells were strained in LV shNGF 3 group than those in NC group ( P<0.01 ) .Conclusion Lentivirus-mediated NGF gene silencing inhibits the differentiation of PC12 cells through suppressing the activation of ERK1/2.

To prepare the polyclonal antibody against the 3D polymerase of foot and mouth disease virus (FMDV), the 3D polymerase gene of this virus was amplified by PCR and doubly digested with BamH I and Nde I. Then, it were cloned into expression vector pET-30a(+) to obtain the recombinant plasmid pET-3D and this plasmid was transformed to E.coli BL21(DE3) with induction by IPTG.The target protein was identified and purified with SDS+PAGE, and the inclusion bodies were extracted. The purified target protein was used as antigen to immunize New Zealand rabbits to prepare the polyclonal antibody against 3D polymerase of FMDV, which was then characterized by indirect ELISA and Western blotting. As demonstrated by SDS-PAGE, the target protein with a molecular weight at 46 ku was expressed. The polyclonal antibody showed high affinity and obvious specificity and its titer was above 1:8 000. This polyclonal antibody may lay a foundation for the further studies on the biological functions and epitopes of the 3D polymerase of FMDV.

Objective To evaluate the role of μ opioid receptor in morphine preconditioning-induced reduction of myocardial ischemia-reperfusion (I/R) injury in rats with chronic heart failure.Methods Adult male Sprague-Dawley rats,weighing 170-230 g,in which chronic heart failure was induced by injecting doxorubicin via the tail vein,were studied.The rats were sacrificed and their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95％ O2-5％ CO2 at 37 ℃.Forty isolated rat hearts with I/R injury were randomly divided into 4 groups (n=10 each):group I/R,morphine preconditioning group (group MP),μ opioid receptor antagonist CTOP plus morphine preconditioning group (group CTOP+MP) and CTOP group.Myocardial I/R was induced by occlusion of the left coronary artery for 30 min followed by 120 min of reperfusion.In group MP,the hearts were perfused with K-H solution for 15 min,with K-H solution containing 1 μmol/L morphine for 5 min and with K-H solution for 5 min,3 cycles in total,and then the model of myocardial I/R was established.The hearts were perfused with K-H solution containing 1 μmol/L CTOP starting from 10 min before morphine preconditioning until 5 min of ischemia in group CTOP + MP.The hearts were perfused with K-H solution containing 1 μmol/L CTOP starting from 40 min before ischemia until 5 min of ischemia in group CTOP.The coronary effluent was collected at 15 min of equilibration (baseline) and 5 and 10 min of reperfusion to detect the activity of lactate dehydrogenase (LDH).Myocardial infarct size (IS) and the area at risk (AAR) were measured by 2,3,5-triphenyl-tetrazolium staining,and IS/AAR percentage was calculated.The expression of Bcl-2 and Bax mRNA was determined using uantitative real-time polymerase chain reaction,and the ratio of Bcl-2/Bax was calculated.Results Compared with group I/R,the IS and IS/AAR percentage were significantly decreased,the activity of LDH in coronary effluent was decreased,the expression of Bax mRNA was downregulated,the expression of Bcl-2 mRNA was up-regulated,and the Bcl-2/Bax ratio was increased in group MP (P＜0.05),and no significant change was found in the IS or IS/AAR percentage in CTOP and CTOP+ MP groups (P＞0.05).Compared with group MP,the IS and IS/AAR percentage were significantly increased,the activity of LDH in coronary effluent was increased,the expression of Bax mRNA was up-regulated,the expression of Bcl-2 mRNA was down-regulated,and the Bcl-2/Bax ratio was decreased in group CTOP+MP (P＜0.05).Conclusion The mechanism by which morphine preconditioning reduces myocardial I/R injury may be related to activating μ opioid receptors and thus maintaining the balance between Bcl2 and Bax gene expression in the rats with chronic heart failure.

Objective: To evaluate the role of morphine preconditioning on necroptosis during myocardial ischemia-reperfusion (I/R) injury in the rats with heart failure. Methods: Clean-grade adult male Sprague-Dawley rats, weighing 200-230 g, were injected with 2 mg/kg doxorubicin via the tail vein once a week for 6 consecutive weeks to establish the chronic heart failure model.Thirty rats with chronic heart failure at the end of 8th week were divided into 3 groups (n=10 each) using a random number table method: sham operation group (group S), I/R group and morphine preconditioning group (group MPC). Myocardial I/R was induced by occlusion of anterior descending branch of left coronary artery for 30 min followed by 120 min reperfusion in each group except group S. In group MPC, the rats were subjected to 3 cycles of 5-min infusion of 0.1 mg/kg morphine via the femoral vein at 5 min intervals before ischemia.The animals were sacrificed at the end of reperfusion, and the myocardial specimens were obtained for determination of the area at risk (AAR), infarct size (IS), expression of Fas mRNA (by quantitative real-time polymerase chain reaction) and expression of Fas, receptor-interacting protein 1 (RIP1) and RIP3 (by Western blot). The IS/AAR ratio was calculated. Results: Compared with group S, the IS and IS/AAR ratio were significantly increased at the end of reperfusion, and the expression of Fas protein and mRNA, RIP1 and RIP3 was up-regulated in group I/R (P<0.05). Compared with group I/R, the IS and IS/AAR ratio were significantly decreased at the end of reperfusion, and the expression of Fas protein and mRNA, RIP1 and RIP3 was down-regulated in group MPC (P<0.05). Conclusion The mechanism by which morphine preconditioning reduces myocardial I/R injury is related to inhibiting necroptosis in the rats with heart failure.

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×10^9.1 TCID₅₀/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.
Adenoviridae/genetics* ; Adenoviruses, Human/genetics* ; Animals ; Antibodies, Viral ; Capsid/metabolism* ; Capsid Proteins ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus/genetics* ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins/genetics* ; Serogroup ; Swine ; Viral Proteins ; Viral Vaccines/genetics*