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Objective To establish a kind of mesenchymal stem cells(MSCs) model that could express hCD4 and hCCR5 to study the field of human immunodeficiency virus type 1 (HIV-1).Methods Lentiviral vectors containing the genes of hCD4 and hCCR5 under the transcriptional control of cytomegalovirus promoter were designed.MSCs were transfected by the lentiviral vectors at optimum multiplicity of infection.Transduction efficiencies of hCD4 and hCCR5 in MSCs were analyzed by quantitative real-time polymerase chain reaction(RT-PCR),Western blot,and immunofluorescence staining.Subsequently,the transfected human MSCs were infected with HIV-1,and the expression of HIVRNA in the MSCs was detected by RT-PCR.Results The MSCs model con-taining hCD4 and hCCR5 and supporting normal HIV-1 infection was constructed.qRT-PCR showed that MSCs upon infection with lentiviral vectors were highly expressed in hCD4 and CCR5 mRNA sequences(P＜0.01).Western blot detection showed the positive bands of 55.0 × 103 (hCD4) and 40.6 × 103 (hCCR5).The results of immunofluorescence staining revealed that hCD4 and CCR5 were expressed on the surface of MSCs.Such results were not found in cells infected with empty lentiviral vectors.And the susceptibility of the hCD4/CCR5 transgenic MSCs to the HIV-1 was further indicated by the detection of HIV-1 RNA in the culture supernatants and cell lysates(P＜0.05).Conclusion The MSCs model that could highly express hCD4 and hCCR5 was established to support normal HIV-1 infection,which can be used to investigate the development of new therapies and vaccines against HIV.
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Objective To investigate the anti-fibrosis mechanism from effects of difference in iron load levels on activation and apoptosis of hepatic stellate cells(HSCs). Methods According to the difference in iron load levels in HSCs,HSC-T6 cells were divided into four groups:blank control,iron deposition model,50μmol/L desferrioxamine and 25 μmol/L desferrioxamine groups. Quantitative polymerase chain reaction(PCR)was applied for the detection of collagen type Ⅰ and transforming growth factor-β1(TGF-β1)mRNA expressions of HSC-T6 cells. Immunohistochemical assay was used for the detection of α-smooth muscle actin(α-SMA)expression. The method of terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)was used for the examination of apoptosis of HSC-T6 cells. Under electron microscope,the ultrastructures of HSC-T6 cells were observed. Results Compared with the blank control group,despite the TGF-β1 mRNA expression in iron deposition model group was increased,no statistical significant difference was seen(1.594±0.168 vs. 1.477±0.126, P>0.05),whereas collagen type I mRNA expression was significantly enhanced(1.354±0.076 vs. 1.197±0.104, P<0.01). Both 50μmol/L and 25μmol/L desferrioxamine could down-regulate collagen type I and TGF-β1 mRNA expressions,and the action of 50μmol/L desferrioxamine was superior to that of 25μmol/L desferrioxamine(collagen typeⅠmRNA:0.391±0.076 vs. 0.688±0.060,TGF-β1 mRNA:0.421±0.068 vs. 0.714±0.090,both P<0.01). Iron deposition could induce HSCs to expressα-SMA in great amount,while apoptosis could be seen scarcely in iron deposited HSCs. By desferrioxamine therapy,α-SMA expression of HSCs was decreased significantly,but some of the cells underwent apoptosis. Conclusion Different iron load levels inside HSCs can induce activation or apoptosis of the cells,showing that iron plays an important role in regulating the process of HSCs activation and apoptosis and revealing that desferrioxamine possesses the potential action for treatment of liver fibrosis.
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ObjectiveTo observe the virological and immunological items in peripheral blood of Chinese-origin Rhesus macaques infected with SIVmac251.The normal levels of WBC and CD4+T cell ratio for healthy Chinese-origin Rhesus macaques that suitable for simian AIDS modeling also investigated.MethodsThirty-six Chinese-origin Rhesus macaques were intravenously infected with SIVmac251.Blood samples were collected at 10 time points respectively include 1 day before SIV infection,every week within 1-8th week and then in tenth week post infection.Blood routine,peripheral blood T lymphocyte subsets,plasma viral load were tested.ResultsThe most significant changes of the tested items were appeared in 1 or 2 weeks post SIV infection,while the WBC counts didn't show marked changes in all the time points tested.WBC counts ranged from 4×106/ml to 10×106/ml and the CD4+T cells ratio high than 25％ are the suitable levels for simian AIDS modeling.ConclusionThis research provides necessary and beneficial informations to the usage of Chinese-origin Rhesus macaques in AIDS research.
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Objective To investigate the effects of Compound Ganduqing Decoction(CGD) on activation and apoptosis of iron overloading hepatic stellate cells(HSC).Methods The cultured HSC-T6 cells were used for experimental cells,and were divided into normal control group,model group and CGD treatment group(in the dose of 0.08g/L).HSC model of iron overloading was induced by incubation with ammonium iron citrate.Immunohistochemical assay was used for the detection of alpha smooth muscle actin(?-SMA) expression.Quantitative polymerase chain reaction(PCR) was applied for the detection of transforming growth factor beta 1(TGF-?1) mRNA expression.TUNEL technique was used for the examination of apoptosis of HSC-T6.Electron microscope was used for the observation of ultrastructure of HSC-T6.Results In the normal control group and the model group,there showed large amount of ?-SMA expression in HSC-T6,but little apoptosis.However,in CGD group ?-SMA expression was decreased obviously,TGF-?1 mRNA expression was reduced,and apoptosis of HSC-T6 was obvious(P
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Objective To evaluate the in-vivo effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on duck hepatitis B virus(DHBV).Methods Thirty ducks with congenital infection of DHBV,which were DHBV-DNA positive confirmed by polymerase chain reaction(PCR),were randomly divided into 5 groups: DHBV control group,high-,middle-,and low-dosage CPU Ⅱgroups(in the dose of 33.6,18.6 and 8.4 g-1?d-1,respectively),and Lamivudine group(20 mg?kg-1?d-1).The serum DHBV-DNA level of all ducks was detected by quantity real-time fluorescence PCR before and after medication.Results On the 7th,14th,21st,and 28th day of medication and on the 5th day of suspension of medication,the serum DHBV-DNA level in both of the high-and middle-dosage CPU Ⅱ groups was obviously lower than that before medication and than that in the DHBV control group(P