Using antisense nucleic acid technology to study the influence of POLκ on genetic stability

AIM:To establish cell line FL-POLκ^- and to study the role ofPOLκ(polymerase kappa) on genetic stability.METHODS:A mammalian expression vector expressing antisense POLκ gene fragment pMAMneo -amp^--POLκ was constructed by cloning the 1 690-1 918 fragment of POLκ gene into the mammalian expression vector pMAMneo-amp^- in antisense orientation. FL cells were fransfected with this antisense RNA expressing vector and selected by G418. Based on the shuttle-plasmid pZ189, the mutation assay was made.RESULTS:The spontaneous mutation frequency of supF tRNA gene in the plasmid replicated in the FL- POLκ^- was 11.2×10^-4, while it was 4.9×10^-4 and 3.7×10^-4 in the control cells FL and FL-M, respectively.CONCLUSION: POLκ playes an important role in maintenance of genetic stability.     

High-efficient genetic transfection of CD41+,UT7, U937 and MDA-MB-435 cells with a recombined murine stem cell retroviral vector

AIM: Gene transduction with a recombined murine stem cell retroviral vector has been investigated to find an effective way of gene transduction and to offer theory and experimental basis for the recombined murine stem cell retroviral vector used for gene transduction. METHODS: 1. Construction of retrovirus vector: EC1-4 gene (repeats 1-4 of cadher in-5 extracellular domain) and mutant (Ser 222A) MEK1 gene were cloned into retrovirus vector pMSCV after cut by Bgl Ⅱ and EcoR 1 restriction endonuclease. 2. Obtaining CD41⁺ cells and cell culture: Cells expressing CD34⁺ from cord blood were isolated. The inducement of cells expressing CD41 from CD34⁺ cells was performed by using TPO and cells CD41⁺ were selected by FACS. NIH 3T3 cells were cultured in high sugar DMEM medium and U937 in RPMI 1640 medium. UT7 cells which is cytokine-dependent cell line were grown in Iscove's modified Dulbeco's medium supplemented by GM-CSF. 3. Determination of viral titers: Retroviral vectors were transferred to packaging cell line 293. Retroviral containing supenatant was collected after transfection. The viral titers was tested on infection of NIH 3T3 cells by FACS analysis. 4. Western blot: Transduced CD41⁺, UT7, U937 and MDA-MB-435 cells were analyzed by western blot to examine expression of transduced genes. RESULTS: A packaging cell line 293 produces high-titer MEK1 pMSCV retroviruses (3.1×10⁷) and EC1-4 pMSCV retroviruses (1.0×10⁸). With 8-folds dilution retroviruses, 60.73% GFP positive cells have been obtained in MEK1 pMSCV transduced UT7 cells, 72.56% in U937 cells and 30.57% in CD41⁺ cells, respectively. GFP positive cells have reached up 97.54 % in EC1-4 pMSCV transducted MDA-MB-435 cells. Phosphorylated MEK1 has been decreased in experiment group when TPO has stimulated CD41⁺ and UT7 cells or serum has stimulated U973 cells. This indicates that is a dominant negative effect of mutation MEK gene. EC1-4 gene transduced MDA-MB-435 cells have expressed EC1-4. CONCLUSION:The recombined murine stem cell retrovirus can effectively mediate gene transduction of CD41⁺,UT7,U937 and MDA-MB-435 cells,and transduced genes can be stably expressed.     

Apoptosis induced by berbamine in K562 cells correlates with the expression levels of bcr/abl gene and P210

AIM: To explore the effect of berbamine(BER) on apoptosis in K562 cells and its possible molecular mechanisms. METHODS: The apoptosis rate was measured by flow cytometry while electron microscopy and DNA electrophoresis were used to evaluate the characteristic changes of apoptosis, RT-PCR and Western blot were used to examine the expression levels of apoptosis related gene bcr/abl and BCR/ABL protein. RESULTS: By FCM, the apoptosis rate of K562 cells treated with 8.0 mg/L BER for 24 h and 72 h increased from (29.20±3.82)% to (61.77±4.35)% (P＜0.01); The typical apoptosis morphologic changes and the DNA ladder were more clearly observed. After treated with BER 0 to 16.0 mg/L for 24 h, the expression levels of bcr/abl mRNA and P210 (semiquantity value) decreased quickly from 1.19±0.02 to 0.73±0.02 (P＜0.01) and from 1.04±0.02 to 0.63±0.01 (P＜0.01), respectively. CONCLUSION: BER induces apoptosis of K562 cells in a time-and-concentration-dependent manner, the decline of bcr/abl mRNA and P210 may play an important role in the apoptotic effect of BER in K562 cells. BER could be used as a new clinical trials for bcr-abl⁺ diseases such as CML.     

Cellular biological effects of matrine on K562 and K562/Vin cells

AIM: To investigate the cellular biological effects of matrine on K562 and K562/Vin cells and discuss the anticancer mechanism of matrine. METHODS: MTT assay was used to detect the IC₅₀ of matrine on these two cell lines and the reversal effect of matrine on K562/Vin cell's resistance to vincristine. In addition, the growth curve of cells was drawed. The p-glycoprotein (P-gp) expression was determined by immunohistochemistry analysis. The morphological changes of cells under light microscopy and the structural changes under transmission electron microscope were observed. RT-PCR assay was used to detect the hTERT-mRNA expression. RESULTS: The IC₅₀ of matrine was 3.4, 4.6 mmol·L ^-1 for K562 and K562/Vin cells, respectively. Matrine (4.0 mmol·L ^-1) inhibited the growth of K562, K562/Vin cells, 2.0 mmol·L ^-1 matrine inhibited expression of P-gp and with 492.4 reversal index. Matrine killed K562 cells by inducing the apoptosis and the same effect on K562/Vin cells was also observed. The hTERT-mRNA expression of K562 cells were also inhibited by matrine. CONCLUSIONS: Matrine enhanced the cytotoxicity of vincristine in K562/Vin cells, induced the apoptosis of K562 and K562/Vin cells, also inhibited the hTERT-mRNA expression in K562 cells. It shows that matrine would be an effective anticancer medicine.     

Sca-1+ cells from mouse fetal liver can differentiate into neurons in vitro

AIM: To study whether Sca-1⁺ cells from fetal liver can be induced to differentiate into neuronal cells in vitro. METHODS:Sca-1⁺cells from 14 5-days-old murine fetal liver were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco s modif ied Eagle s medium(DMEM)/F12 supplemented with 10%fetal bovine serum(FBS), and passaged at a rat io of 1 3 when cells reached more than 80%confluence.The 5 passage cells were induced by 10^-3mol/Lβ-mercaptoethanol(β-ME)and 5×10^-7 mol/L all-trans-retinoic acid(RA)for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days.The characteristics of treated cel s were assayed by immunocytochemistry staining analysis at 5 hours, or 5 days.RESULTS: Cells treated with β-ME and RA exhibited neuronal phenotype and expressed neuron-specific protein such as neuron-specific nuclear protein (NeuN), neuronfilament-M, and neuron-specific tubulin-1 (TuJ-1) but not tau, MAP-2, or the astrocyte-specific marker glial fibrillary acidic protein (GFAP).CONCLUSION: Sca-1⁺ cells from fetal liver, of which most are regarded as hematopoietic stem cells, could differentiate into early immature neuronal cells in vitro. These findings suggest that Sca-1⁺ cells from fetal liver may be an alternative source in cell therapy and gene therapy of neural dysfunction.     

Construction of atherosclerosis full-length fully human antibody libraries by mammalian cell surface display technique

AIM: To construct atherosclerosis (As) full-length fully human antibody libraries by mammalian cell surface display technique for looking for the atherosclerotic therapeutic antibody. METHODS: The vector pcDNA-DHL was constructed by remounding the vector pcDNA5/FRT. The human total RNA from human peripheral blood mononuclear cells was isolated, and then reverse transcripation of RNA to cDNA was performed. The genes encoding the heavy chain variable regions and kappa light chains of the antibodies were amplified by PCR, and the product of PCR was connected to the vector pcDNA-DHL and the vector pDHL-As. The library DNAs were transfected into Flp-In^TM-CHO (FCHO) cells, the expression of the full-length human antibodies on the surface of cells was analyzed by flow cytometry. The cells that were successfully transfected with pDHL-As were screened with hygromycin, and then the full-length antibody library of atherosclerosis was obtained. RESULTS: The vector pcDNA-DHL were constructed successfully. The heavy chain gene library constructed showed a diversity of 1.79×10⁵, and the kappa light chain gene library had a diversity of 1.80×10⁵. Theoretically, the diversity of antibody library is 2.32×10¹⁰. The antibody library of atherosclerosis pDHL-As expressed on the surface of Flp-In^TM-CHO cells. CONCLUSION: The full-length human antibody cell library of atherosclerosis was constructed successfully, and 45 FCHO cells that expressing anti-oxidized low density lipoprotein antibodies were selected.     

Mesenchymal stem cell-induced CD8α+ Jagged2high CD11bhigh regulatory dendritic cells prevent and treat mouse aGVHD

AIM: To investigate the role of mesenchymal stem cell-induced regulatory dendritic cells (MSC-DCregs) in mouse acute graft-versus-host disease(aGVHD) model. METHODS: Bone marrow cells from BALB/c (H-2^d) mice were isolated and were induced to differentiate into DCs. The DCs were selected by flow cytometry, and after 10 d co-culture with MSCs, they were induced to be MSC-DCregs. Male 8-week-old C57BL/6 (H-2^b) mice were used as donor mice. The female 8-week-old BALB/c (H-2^d) mice, who had received 100 cm source-skin distance, 30 cm×30 cm radiation field, 700 cGy total body irradiation (TBI) pretreatment were used as recipient mice. The recipients were divided into 5 groups: control group, TBI group (injected with medium only), bone marrow transplantation group (injected with 1×10⁷ bone marrow cells), aGVHD group (injected with 1×10⁷ bone marrow cells and 1×10⁷ spleen cells), and MSC-DCregs group (injected with 1×10⁷ bone marrow cells, 1×10⁷ spleen cells and 1×10⁶ MSC-DCregs). The white blood cell count, recipients' chimerism, clinical evaluation of aGVHD, survival analysis and pathological changes were determined. RESULTS: Hematopoieic recovery was seen at 10 d after transplantation. The recipients' chimerism was parallel to the donors' at 30 d. The median survival time of the mice in aGVHD group and MSC-DCregs group was 27 d and 33 d, and the survival rates at 30 d were 20% and 100% (P<0.01), respectively. The clinical scores of the mice in MSC-DCregs group were lower than those in aGVHD group (P<0.01). Moreover, the pathological changes in the skin and liver of the mice in MSC-DCregs group were less serious than those in aGVHD group. CONCLUSION: The MSC-DCregs induce an aGVHD tolerance in vivo, and further research of its mechanism is still in great necessary.     

PMA specifically inhibits mRNA expression of BCR/ABL and Fyn in K562 cells

AIM: To study the effects of phorbol 12-myristate 13-acetate (PMA) on the mRNA expression of BCR/ABL and Fyn in K562 cells, and to explore their relationship. METHODS: The K562 cells were stimulated by PMA at a series of concentrations (1~250 μg/L) for 24 h, and the mRNA expression levels of BCR/ABL and Fyn in K562 cells were detected by real-time fluorescence quantitative PCR. The relative changes of both mRNA expression were measured using 2^-ΔΔCt formula. RESULTS: PMA significantly inhibits the mRNA levels of BCR/ABL and Fyn in a dose-dependent manner, and the correlation of these inhibitory effects were significant. Compared with gene Molt-4 cells, the inhibition by PMA was specific for K562 cells. The K562 cells were induced to differentiate to be pseudopodium-like cells. CONCLUSION: The PMA downregulates the mRNA level of Fyn by inhibiting BCR/ABL fusion gene.     

Knockdown of RALA by siRNA inhibits cell proliferation and induces apoptosis in human leukemic K562 cells

AIM: To investigate the effect of siRNA-induced knockdown of v-ral simian leukemia viral oncogene homolog A(RALA) on proliferation and apoptosis of chronic myelogenous leukemia(CML) K562 cells. METHODS: The chemically synthesized siRNA targeting to RALA gene was transfected into K562 cells using Lipofectamine^TM 2000. The proliferation and viability of K562 cells were detected by MTT assay and trypan blue dye exclusion. The expression levels of RALA mRNA and protein were determined by quantitative real-time PCR and Western blotting,respectively. The cell apoptosis was analyzed using flow cytometry by double staining with annexin V and propidium iodide, and the apoptotic morphological changes were detected by Hoechst 33258 staining. RESULTS: RALA siRNA significantly down-regulated RALA mRNA and protein expression in K562 cells(P<0.05). The proliferation of K562 cells in RALA siRNA group was inhibited compared with control group(P<0.05). The apoptotic rate was much higher in RALA siRNA group than that in negative control group(P<0.05). The apoptotic morphological changes were observed in the nuclei of K562 cells transfected with RALA siRNA. CONCLUSION: The siRNA-mediated knockdown of RALA results in inhibition of proliferation and induction of apoptosis in K562 cells, indicating that RALA might be used as a potential therapeutic target in chronic myelogenous leukemia.     

Imperatorin increased sensitivity of lung cancer CD133+ cell subsets to gefitinib by down-regulating c-met expression

AIM: To investigate the role of imperatorin in reversing the resistance of the PC9 CD133⁺ cell subsets to gefitinib. METHODS: MTT assay was performed to evaluate the viability of PC9 cells treated with imperatorin and gefitinib. The expression of c-met, activation of caspases and phosphorylation of epidermal growth factor receptor (EGFR), PI3K and AKT in the PC9 cells treated with imperatorin and gefitinib were determined by Western blot. The percentage of CD133⁺ cell subsets population and the apoptotic rate of the PC9 cells treated with imperatorin and gefitinib were analyzed by flow cytometry. RESULTS: The sensitivity of the PC9 CD133⁺ cell subsets to gefitinib was significantly lower than that of the PC9 CD133^- cell subsets. Treatment with gefitinib alone significantly inhibited the protein levels of EGFR/PI3K/AKT in the PC9 CD133^- cell subsets but not the PC9 CD133⁺ cell subsets. Treatment with gefitinib alone increased the percentage of CD133⁺ cell subsets population in the PC9 cells. However, combination of gefitinib with imperatorin significantly inhibited the enrichment of CD133⁺ cell subsets population. Imperatorin down-regulated c-met expression, suggesting the c-met was the target of imperatorin in the PC9 CD133⁺ cell subsets. The results of MTT assay, Western blot analysis and flow cytometry indicated that imperatorin increased the gefitinib induced inhibition of PI3K/AKT protein levels by down-regulating the expression of c-met, which subsequently induced the cleavage of caspases and apoptosis in the PC9 CD133⁺ cell subsets.CONCLUSION: Imperatorin increases the sensitivity of lung cancer CD133⁺ cell subsets to gefitinib by down-regulating the expression of c-met, and the synergistic anti-tumor effect exists between imperatorin and gefitinib.     

The transdifferentiation of Sca-1+ cells from murine fetal liver

AIM:To explore transdifferentiation potential of Sca-1⁺ cells from murine fetal liver. METHODS:2×10³ of Sca-1⁺ cells from male murine fetal liver were transfused into female mouse irradiated lethally with γ ray from ⁶⁰ Co source (10 Gy) via tail vein. Two months later, FISH and immunohistochemistry were used to detect the situation for transdifferentiating of the donor cells (male cells) in tissues of female recipient mouse. RESULTS:The renal tubular epitheliocyte-like and neurocyte-like cells with Y chromosome were found on the sections of renal and brain tissues from female recipient mice. These cells have phenotype characteristics of RCA⁺/CD₄₅^-F_4/80^- and NueN⁺/CD₄₅^-F_4/80^-, respectively. CONCLUSION:The evidence is provided for Sca-1⁺ cells from murine fetal liver to transdifferentiate into both renal and brain tissue cells.     

Antisense drug targeting with VEGF mRNA designed by computer aid and inhibitory effects on K562 cell line in vitro

AIM:The effective antisense sequences targeted VEGF mRNA with computer software would be screened and designed, and effect of them on growth K562 cells and protein expression of VEGF were studied with experiments.METHODS:Seven antisense sequences were selected and synthesized, which consisted of 18-20 deoxynucleotide acid and were modified with phosphorothioate, according to principle of low free energy of overall △G₃₇ Overall. Cell growth was assayed by trypan blue dye exclusion assay and level of VEGF protien in the media was determined by ELISA.RESULTS:Six of seven sequences were capable of inhibing growth of K562 cells and downregulating the VEGF protein expression significantly, compared with Scrambed control group. It was found that there was a close correlation between low level of overall △G₃₇ and antisense effectiveness (r=0.887,P<0.01).CONCLUSION:VEGF mRNA antisense oliogdeoxynucleotides, which were designed by computer software of RNAstructure, were able to inhibit growth of K562 and its protein expression. The VEGF mRNA may be new target attached by drugs. At same time, the computer aided design is useful methods to obtain the effective antisense.     

Inhibition of K562 cell growth by antisense drug targeting with VEGF mRNA in vitro

AIM: To investigate inhibition of K562 cell growth by antisense drug targeted VEGF mRNA. METHODS: X7, 20-mer antisense sequences were selected, synthesized and modified with phosphorothioate. The drug was transfected into K562 cells in the present of lipofection. Cell growth was assayed by trypan blue dye exclusion assay and MTT. The level of VEGF protein in the media was determined by ELISA. The morphology of apoptotic cells were observed by Giemsa staining, and the propotion of apoptotic cells was detected by flow cytometry. RESULTS: The antisense drug inhibited growth of K562 and downregulated expression of VEGF protein significantly, compared with Scrambed control group and showed dose-dependent relation. Signs of apoptosis of K562 cells were not observed. CONCLUSION: Inhibition of K562 cell proliferation, but not cells apoptosis induction is the mechanism of inhibing growth of K562 cells by antisense drug targeted VEGF mRNA. At same time, VEGF has function of promoting K562 cell proliferation, and VEGF mRNA may be a new target attached by drugs.     

Fetal liver-derived Sca-1+ cells can differentiate into both neurons and astrocytes in vivo

AIM: To determine whether Sca-1⁺ cells from fetal liver can differentiate into neural cells. METHODS: The sex of 14.5-day-old murine fetuses was determined by PCR analysis of sry gene, and Sca-1⁺ cells from male fetal liver were isolated with a magnetic cell sorting kit, 2×10³ of which were then transplanted into lethally irradiated female mice. The donor cells and their characteristics in recipient brains were identified and detected by FISH and immunohistochemistry double-staining analysis at 60, 120, 180 days after transplantation. RESULTS: There existed many male cells in brains of female recipients, some of them express neuron-specific nuclear protein (NeuN), and some of them express the astrocyte-specific marker, i.e. glial fibrillary acidic protein (GFAP). CONCLUSION: Sca-1⁺ cells from fetal liver, which contain hematopoietic stem cells, can differentiate into neuronal cells and astrocytes in the brains of adult mice.     

Salinomycin inhibits proliferation and induces apoptosis of Gleevec-resistant chronic myeloid leukemic cell line K562/Glv

AIM: To study the effect of salinomycin on inhibiting proliferation and inducing apoptosis of Gleevec-resistant chronic myeloid leukemia cell line K562/Glv. METHODS: The inhibitory effect of salinomycin on the growth of K562/Glv cells was detected by CCK-8 assay in vitro. Flow cytometry was used to observe apoptosis, mitochondria membrane potential (ΔΨ_m), reactive oxygen species (ROS) and the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in K562/Glv cells. The activity of caspase-3, -8 and -9 was measured by the method of colorimetry. The levels of cytochrome C, Bcl-2, Bax, β-catenin and phosphorylated low-density lipoprotein receptor-related protein 6 (p-LRP6) were determined by Western blotting. RESULTS: Salinomycin inhibited the growth of K562/Glv cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L inhibited the growth of the cells with the inhibitory rate of (36.70±2.31)%. The cell apoptotic rate was (19.66±2.23)%. Salinomycin at concentration of 0.2 μmol/L decreased the level of ΔΨ_m, and increased the levels of ROS, cytochrome C and[Ca²⁺]_i in the cells. Salinomycin also increased the activity of caspase-3, -8 and -9 in the cells, reduced the ratio of Bcl-2/Bax, and attenuated the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin induces the apoptosis of Gleevec-resistant myeloid leukemia cell line K562/Glv via Bcl-2/Bax and mitochondria-dependent pathways, and inhibits the cell growth through Wnt signal pathway.     

Yangxue qingnao-containing serum inhibits rat vascular smooth muscle cell proliferation induced by lysophosphatidic acid

AIM: To observe the effects of Yangxue qingnao-containing serum on rat vascular smooth muscle cell (VSMC) proliferation induced by lysophosphatidic acid (LPA). METHODS: The [³H]-TdR incorporation and mitogen-activated protein kinasc (MAPK) activity were measured in cultured VSMC. End product of lipid peroxidation-MDA levels were also detected. RESULTS: 1×10^－9,1×10^－8 and 1×10^－7 mol/L LPA enhanced the cultured VSMC [³H]-TdR incorporation, increased MAPK activity and MDA content in a concentration-dependent manner. 5％, 10％ and 15％ Yangxue qingnao-containing serum concentration-dependently inhibited the increase in VSMC [³H]-TdR incorporation, MAPK activity and MDA content induced by LPA. CONCLUSIONS: LPA has a stimulating effect on VSMC proliferation. The LPA-induced intracellular signal transduction may be related to MAPK activity. Yangxue-qingnao can efficiently inhibit LPA -induced VSMC proliferation,MAPK activity and lipid peroxidation.     

The cytotoxicity of nitric oxide induced by inflammatory cytokine in combination with LPS in endothelial cells

AIM: To explore the mechanism underlying inducible nitric oxide (NO) caused injury of endothelial cells during inflammation. METHODS:The activity of iso-enzymes of NO synthase (NOS), NO level and iNOS expression were examined using NADPH method, Griess reaction and RT-PCR, respectively. Furthermore, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content were also measured. RESULTS:Co-administration of cytokines (TNF-α 5×10⁵ U/L, IL-1β 2×10⁵ U/L, INF-γ 2×10⁵ U/L) and LPS (10 mg/L) caused an obvious increase in NOS activity, NO levels (about two-fold) and a significant injury of the cells. At the same time, a significant increase in iNOS mRNA was also detected. Wheareas, treatment of the cells separately with cytokines or LPS for 24 h had no significant effect on NOS activity and NO level in cell lysates, however, it caused a significant increase in LDH release and MDA content. Also, the effect of cytokines and LPS on cell viability was concentration-and time-dependent. L-NMMA, a inhibitor of NOS, can suppress inducible NO production and protect cells against NO induced injury. CONCLUSION:Co-administration of cytokines (TNF-α, IL-1β and INF-γ) and LPS significant activated iNOS and NO production which, in turn, induced oxidative reaction in endothelial cells.     

Influence of SDD and caecostomy/colonic irrigation on gut endotoxin/bacteria translocation following acute severe pancreatitis

AIM: To observe the influence of the selective decontamination of the digestive tract (SDD) and caecosomy/colonic irrigation on gut endotoxin/bacteria translocation following acute severe pancreatitis (ASP). METHODS: Twenty three pigs weighing 16-22 kg were divided into four groups. Group I (n=5): sham-control; Group Ⅱ (n=6): ASP-control; Group Ⅲ (n=6): gntamicin [(8.55×10⁵±5.70×10⁴)units/time] and nystatin [(1.37×10⁵±9.00×10³)units/time]were fed orally every 8 h for 1 week before the induction of ASP; Group Ⅳ (n=6): caecostomy was performed before the induction of ASP. ASP was induced by infecting 1 mL/kg BW of combined solution of 5% sodium taurocholate and (8-10)×10⁶ BAEE units/L of trypsin into pancreas via pancreatic duct. Systemic plasma endotoxin levels were quantified by the chromogenic limulus amebocyte lysate (LAL) technique. Specimens of tissue from mesenteriolum and mesocolon lymph nodes, lung, lymph nodes in hilus pulmonis, pancreas and the samples of both portal and systemic blood were collected before and at 72 h following ASP and cultured for aerobic as well as anaerobic bacteria growth. Positive specimens were subcultured and the bacteria identified by standard procedure. RESULTS: Preventive SDD not only effectively reduced the amount of bacteria in stool (P<0.01), but also significantly reduced the levels of plasma endotoxin and the magnitude of bacteria translocation to the portal and systemic blood and the remote organs and tissues, for instance, mesenteriolum and mesocolon lymph nodes, lung, lymph nodes in hilus pulmonis, pancreas. Early caecostomy/colonic irrigation also significantly reduced the levels of translocated origin-endotoxin and bacteria after ASP. CONCLUSIONS: SDD and caecostomy/colonic irrigation effectively reduce the levels of plasma endotoxin and the magnitude of bacteria translocation to the portal and systemic blood and the remote organ, especially the latter will be of a great importance in the future clinical practice.     

Morphological study of macrophages infected with constructed recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain

AIM: To construct a recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain,which can infect mouse bone marrow-derived macrophages (BMM). METHODS: Co-transfection of shuttle and backbone plasmids of AdMax system into 293Ad5⁺ cells was performed, followed by viral packaging, propagation and purification. These viruses were subject to Karber TCID₅₀ titration. The expression of gp120 protein in 293Ad5⁺ cells was determined by ELISA. The viral titration was validated by a multiplicity of infection (MOI) test with BMM. RESULTS: The titers of the outcome viruses, including AdMax-HIV-1 gp120 (Ad-gp120) and its vector control Ad-GFP, were 10^8.3 and 10^8.1 TCID₅₀/mL, respectively. Both recombinant adenoviruses infected BMM with similar capacity of 293Ad5⁺ cell infection, which validated the TCID₅₀ titration.The gp120 protein was positive in 293Ad5⁺ cell lysates. BMM activation was observed morphologically after Ad-gp120 infection as compared with Ad-GFP-infected cells. CONCLUSION: Functional adenovirus containing HIV-1 gp120 of prevalent strains in China was successfully constructed. Infection of Ad-gp120 causes BMM activation.     

Effects of angiotensin II and losartan on collagen synthesis in rat hepatic stellate cells

AIM: To investigate the effects of angiotensin II (Ang II) and AT_1a receptor antagonist (losartan) collagen synthesis in rat hepatic stellate cells (HSCs). METHODS: ① Rat HSCs were isolated, cultured and identified. ② Rat HSCs were incubated in the medium with different concentrations of AngII or losartan, then the quantity of collagen was examined by -proline release assay. RESULTS: ① The yield of HSCs was 2×10⁷-3×10⁷/per rat, their viability and purity was more than 95% and 90%, respectively. ② The yield of collagen in HSCs significantly got a rise in a concentration-dependent manner when HSCs were incubated with AngII (10^－6mol/L-10^－10 mol/L) (P<0.05). While HSCs were influenced by the antagonist of AT_1a (10^－6 mol/L-10^－9 mol/L), the quantity of collagen dropped greatly (P<0.05). CONCLUSIONS: Ang II stimulates HSCs to produce more collagen. Losartan inhibits the cell to synthesize collagen via AT_1a receptor (P<0.05). The results indicate that Ang II may play an important role in the development of hepatic fibrosis, and using AT_1a antagonist may offer a new strategy to prevent hepatic fibrosis.