The intact Porcine Interleukin- 2 (pIL- 2) cDNA sequence was cloned into pPIC3.5K (expression vector of Pichia pastoris) to construct the expression plasmid pPIC3.5K-IL2.The pPIC3.5K-IL2 was further linearized and transformed into P.Pastoris GS115 strain.After being induced by 1% methanol,the recombinant strain excreted a protein in supernatant of culture,with molecular weight of 20 KD approximately,which was proved to be Porcine Interleukin- 2 through SDS-PAGE and Dot Blotting.Its expression level is up to 80 mg/L and bio-activity unit 8×10~6 U/ml.In a 9 day induction experiment,the daily expression status was measured.The results suggest highest level in the fifth day.And we observed the continual expression status of the recombinant strain in 4 test batches and found a stable,high and continual pIL- 2 expression in opportune concentration.
[Objective] To study effects of traditional Chinese medicine Reduning injection on the secretion of TSLP in human bronchial epithelial cell infected by RSV in vitro.[Method] Hep-2 cell lines were used to pack and generate RSV.The RSV infectious titer was determined by TCID50 technique.An NHBE model of RSV infection in vitro was established and identified.First experiment included RSV infection-NHBE group and normal NHBE group.RSV-infected NHBE group was divided into 5 group based on RSV titers(1000,500,100,50,and 10 TCID50) in which each group also divided to different incubation hours(12 h,24 h,48 h,72 h,96 h,and 120 h).The concentrations of TSLP in the culture supernatants were determined by enzyme-linked immunosorbent assay(ELISA).Second experiment concern the effect of Redunig treatment on TSLP secretion based on cell culture in vitro.According to the way of Reduning delivery,experiments were divided into 3 groups(including precaution,direct deactivation and therapy),accompanying a negative(normal NHBE) and a positive(NHBE-RSV) control group in which each group divided into 5 different incubation period(12 h,24 h,48 h,and 72 h).The concentration of TSLP in the culture supernatants were also determined by ELISA.[Result] The mRNA of RSV was detected in total RNA of NHBE infected with RSV by real-time RT-PCR method,which demonstrated that a model of RSV infection in vitro was established successfully.1.The concentration of TSLP in NHBE cells supernatants infected by RSV significantly increased,compared with non-RSV.In the same post-inoculation time(12 h,24 h,48 h,72 h,96 h,and 120 h),TSLP level increased obviously with raised RSV titers(P 0.05).According to the same RSV titer(10,50,100,500,and 1000 TCID50),TSLP level increased statistically in series infection time,compared with 12 h group(P﹤0.05).2.The TSLP level of NHBE-RSV-Reduning group and NHBE-RSV-Ribavirin group were evidently lower than NHBE-RSV group in all detected points(12 h,24 h,48 h,and 72 h) and in all drug delivery ways(P 0.05),especially in precaution way.(P 0.05).TSLP level of NHBE-RSV-Reduning group are depressed than NHBE-RSV-Ribavirin group by precaution(P 0.05).[Conclusion] TSLP secretion induced by RSV infection obviously increased with raised RSV titer and infected duration.Reduning injection observably inhibits TSLP secretion in RSV-infected NHBE with a positive time-effect relation.The inhibitory effect of precaution group was most significant among three drug delivery ways.The Reduning injection has a notable role in treating the viral diseases.
This study was conducted to determine whether paeoniflorin (PF) could prevent H₂O₂-induced oxidative stress in ARPE-19 cells and to elucidate the molecular pathways involved in this protection.Cultured ARPE-19 cells were subjected to oxidative stress with H₂O₂ in the presence and absence of PF. The preventive effective of PF on reactive oxygen species (ROS) production and retinal pigment epithelium (RPE) cell death induced by H₂O₂ was determined by 2',7'- dichlorodihydrofluorescein diacetate (H₂DCFDA) fluorescence and 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) assay. The ability of PF to protect RPE cells against ROS-mediated apoptosis was assessed by caspase-3 activity and 4', 6-diamidino-2-phenylindole (DAPI) staining. Furthermore, the protective effect of PF via the mitogen-activated protein kinase (MAPK) pathway was determined by western blot analysis.PF protected ARPE-19 cells from H₂O₂-induced cell death with low toxicity. H₂O₂-induced oxidative stress increased ROS production and caspase-3 activity, which was significantly inhibited by PF in a dose-dependent manner. Pretreatment with PF attenuated H₂O₂-induced p38MAPK and extracellular signal regulated kinase (ERK) phosphorylation in human RPE cells, which contributed to cell viability in ARPE-19 cells.This is the first report to show that PF can protect ARPE-19 cells from the cellular apoptosis induced by oxidative stress. The results of this study open new avenues for the use of PF in treatment of ocular diseases, such as age-related macular degeneration (AMD), where oxidative stress plays a major role in disease pathogenesis.
Objective:To study the value of intravascular stent therapy for peripheral vascular disease.Materials and Methods:Sixteen cases of peripheral vascular occlusive disease(15 stenosis or occlusion of femoral artery resulted from atherosclerotic disease and 1 stenosis of subclavian arterial orifice)and 2 cases of traumatic peripheral vascular injuries (1 traumatic aneurysm of brachial artery and 1 traumatic arteriovenous fistula) were studied.Sixteen patients with peripheral vascular stenotic or occlusive underwent intravascular thrombolysis,percutaneous transluminal angioplastic(PTA) and intravascular stents,2 traumatic peripheral vascular injury patients underwent covered metallic stent implantation Results:After intra arterial thrombolysis,PTA and intravascular stents therapy,the vessels in all patients got recanalization,and blood flow were restored without complication.Conclusions:Intravascular stents have significantly therapeutic values for peripheral vascular disease.
ABSTRACT The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long noncoding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. In this study, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 replication and latency. uc002yug.2 potentially enhances HIV-1 replication, long terminal repeat (LTR) activity, and the activation of latent HIV-1 in both cell lines and CD4 + T cells from patients. Further investigation revealed that uc002yug.2 activates latent HIV-1 through downregulating RUNX1b and -1c and upregulating Tat protein expression. The accumulated evidence supports our model that the Tat protein has the key role in the uc002yug.2-mediated regulatory effect on HIV-1 reactivation. Moreover, uc002yug.2 showed an ability to activate HIV-1 similar to that of suberoylanilide hydroxamic acid or phorbol 12-myristate 13-acetate using latently infected cell models. These findings improve our understanding of lncRNA regulation of HIV-1 replication and latency, providing new insights into potential targeted therapeutic interventions. IMPORTANCE The latent viral reservoir is the primary obstacle to curing HIV-1 disease. To date, only a few lncRNAs, which play major roles in various biological processes, including viral infection, have been identified as regulators in HIV-1 latency. In this study, we demonstrated that lncRNA uc002yug.2 is important for both HIV-1 replication and activation of latent viruses. Moreover, uc002yug.2 was shown to activate latent HIV-1 through regulating alternative splicing of RUNX1 and increasing the expression of Tat protein. These findings highlight the potential merit of targeting lncRNA uc002yug.2 as an activating agent for latent HIV-1.
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