Analysis of chromosome aberration due to ethidium bromide using AFM

AIM: To study chromosome aberration due to ethidium bromide (EB),a heterocyclic organic compound and an organic fluorescence dye commonly used in biochemical experiment,and to help further understanding the molecular mechanism of tumor or cancer induced by EB and other heterocyclic organic compounds.METHODS: The toxicity action of EB was evaluated from three aspects including DNA,chromosome and embryo stem cells (ESCs) using atomic force microscopy (AFM),and thereinto,the morphology structural difference of ESCs treated with two EB doses was also valuated.RESULTS: The morphological structures of DNA,chromosome and ESCs were dramatically damaged.The average height of DNA decreased 0.5 nm;chromosomal arms were ruptured from centromere location;molecules of cellular membrane congregated and loop-like structure formed,and ES cell masses were collapsed and became dead after large EB doses treatment and mesh-like morphological structure was discernable.CONCLUSION: The toxicity action of EB is strong and destroys the surface structure of DNA and chromosome.EB induces structural aberration of ES cellular membrane and cell death.The results indicate that the action of EB is externalized at gene level and cell level,which is important to study the carcinogenicity of EB.     

Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion

AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34⁺ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F₂JAK2).CD34⁺cells were enriched from cord blood with a MiniMACS system.The purified CD34⁺ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34⁺ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34⁺ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33⁺,CD61⁺ and Gly-A⁺ partial positive;CD38⁺ and HLA-DR⁺ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34⁺ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy.     

The latent gene expression of Epstein-Barr virus in nasopharyngeal carcinoma

AIM: To examine the latent membrane protein 1(LMP1)-DNA sequence in nasopharyngeal carcinoma(NPC) and detect mRNA expression of LMP1,EBNA1,EBNA2,and to explore the relationship between EBV infectious status,expression products and NPC carcinogenesis.METHODS: LMP1 DNA was detected in NPC by PCR.Direct sequence was applied to analyze the difference between NPC-LMP1-DNA and B95-8- LMP1-DNA.mRNA expressions of LMP1,EBNA1,EBNA2 in NPC were detected by nested RT-PCR.RESULTS: LMP1 DNA existed in all 47 NPC tissues.Several single nucleotide variations were found between NPC-LMP1-DNA and B95-8- LMP1-DNA.The notable variation was the lost of XhoⅠrestriction site in NPC.Direct sequence showed 30 bp deletion in NPC.The mRNA expressions of LMP1,EBNA1 and EBNA2 in NPC were 76.6%,80.0% and 74.5% respectively by nested RT-PCR.The expression of EBNA1 in NPC was promoted by Q promoter while the expression of EBNA1 in B95-8 was promoted by C promoter.CONCLUSION: The way of EBV involved in NPC is complex.Latent genes such as LMP1,EBNA1 and EBNA2 as well as early lytic gene BARF1 may all play certain roles in NPC carcinogenesis.     

Histamine receptor antagonist prevent and ameliorate airway remodeling and acid-base imbalance in asthma of guinea pig

AIM:To investigate the effect of histamine receptor antagonist on airway remodeling and acid-base imbalance in asthma of guinea pig. METHODS:Guinea pigs were divided into 5 groups: the normal control group, the asthma model group, the continued asthma model group, histamine group and histamine receptor antagonist group. For each group, the content of histamine, Na+, Cl-, PaO2, PaCO2, pH, AB, SB in serum, and thickness of airway mucosa and smooth muscle cell layer were measured and compared with each other. RESULTS:(1) According to the content of histamine in serum and thickness of airway mucosa and smooth muscle, the order was: the histamine group>continued asthma model group>the asthma model group>the normal control group (P<0.01), and the histamine receptor antagonist groupthe continued asthma model group (P<0.01), but for PaCO2, the order was conversed. Airway remodeling, increase in histamine in serum, respiratory acidosis and metabolic acidosis in asthmatic guinea pig were observed. Exogenous histamine accentuated the change, however, histamine receptor antagonist attenuated it. CONCLUSION:Histamine may take part in the airway remodeling of asthma. Histamine receptor antagonist can prevent and ameliorate airway remodeling and acid-base imbalance in asthma of guinea pig.     

VEGF induces HUVECs to produce extracellular H2O2 and its proliferation role

AIM:To study the effect of VEGF on extracellular H₂O₂ production in HUVECs and the role of H₂O₂ in the VEGF-induced proliferation. METHODS:HUVECs was stimulated with 500 μg/L VEGF. Products of extracellular H₂O₂ was detected by H₂DCFDA staining. MTT method was used to value the influences of 3×10⁶ U/L catalase and 5-20 mmol/L H₂O₂ to VEGF function. RESULTS:After treatment for 15 min with VEGF, HUVECs appeared fluorescence, and continued to become stronger, peaked at 45 min then decreased. HUVECs, which was treated simultaneity with VEGF and 3×10⁶ U/L catalase, only appeared very faint fluorescence. The proliferation of HUVECs by VEGF was restrained when treated with 3×10⁶ U/L catalase. The extrinsic H₂O₂ at concentration of 5-10 mmol/L promoted the proliferation of HUVECs but inhibited the proliferation effect of VEGF on HUVECs (P<0.01). CONCLUSION:These findings indicate that VEGF induces HUVECs to produce extracellular H₂O₂ and plays role in proliferation, but extrinsic H₂O₂ restrains VEGF function.     

Influences of coculture with bone marrow mesenchymal stem cells on dopaminergic neurons in brain slice treated with 1-methyl-4-phenylpytidinium

AIM: To study the protective effects of bone marrow-derived mesenchymal stem cells on damaged dopaminergic neurons induced by 1-methyl-4-phenylpytidinium(MPP+).METHODS: The parkinson disease(PD) models were established in newborn rats. Bone marrow mesenchymal stem cells(MSCs) obtained from adult bone marrow were cultured, isolated and purified. MSCs were co-cultured with brain slice and the immunohistochemical technique, electron microscopy, propidium iodide staining were used to observe the changes of neurons. RESULTS: In the MPP+ treatment group, the neurites grew slowly and sparsely, dead cells were found in all regions. In the co-culture group, the neuritis grew densely, only a few cells were dead, the number of tyrosine hydroxylase(TH)-stained neurons increased and the structure of organellae was normal. CONCLUSION: MSCs may protect dopaminergic neurons against damage induced by MPP+. These results provide some data for cell transplantation therapy to Parkinsons disease.     

Activity of H+-K+-ATPase in gastric parietal cells under stress in rats and analysis of electron microscopic enzyme cytochemistry

AIM: To demonstrate the changes of activity and electron microscopic enzyme cytochemistry staining of H⁺-K⁺-ATPase of gastric parietal cells under stress in rats. METHODS: Twenty-four male SD rats were randomly divided into normal group, stress group and stress+omeprazole (OM) group. Water immersion-restraint stress (WRS) model in SD rats was performed. The ulcer index (UI) of gastric mucosa and H⁺-K⁺-ATPase activity of gastric parietal cells were measured. The changes of ultrastructure and electron microscopic enzyme cytochemistry staining of parietal cells were observed under transmission electron microscope (TEM). RESULTS: Compared with control group, the UI of gastric mucosa and H⁺-K⁺-ATPase activity of gastric parietal cells increased (P<0.01 and P<0.05) in stress group. In stress+OM group, both UI and H⁺-K⁺-ATPase activity decreased (P<0.01) compared with stress group. Parietal cells were in a resting state in control group, and became active in stress group, where plenty of intracellular canaliculi were observed under the TEM. In stress+OM group, the dilated intracellular canaliculi lined with rare microvilli were founded. Enzyme cytochemistry staining showed that there was little black punctate enzyme reactive product scatted in intracellular canaliculi and the apical plasma membrane of parietal cells in control group, and there were large amounts of black enzyme reactive product accumulated at the intracellular canaliculi in stress group. Scarcely deposition of enzyme reactive product in intracellular canaliculi was observed in stress+OM group. CONCLUSION: The results indicate that the H⁺-K⁺-ATPase activity of gastric parietal cells increases under WRS, and is in accordance with ultrastructure changes. These findings suggeste that gastric acid might be one of the most important factors that result in stress ulcer.     

Effect of lung cancer cells transfected with interleukin 18 gene on the immunological activity of dendritic cells

AIM: To study the effect of interleukin 18 gene transfected lung cancer cells on the phenotype and immunological activity of dendritic cells (DC). METHODS: A secretive IL-18 expression vector containing IL-12 P40 signal sequence was constructed and transfected into NCI-H460 lung cancer cells. DC induced from human peripheral blood were divided into 4 groups (NT, PV, GT and PD). DC were stimulated by non-transfected NCI-H460 cells, pure vector transfected NCI-H460 cells and IL-18 transfected NCI-H460 cells respectively for group NT, PV, GT, and non-stimulated DC for group PD. CD54, CD80, CD83 and CD86 on DC in the 4 groups were detected with flow cytometry. T cell proliferation stimulated by DC in the 4 groups was assayed with MTT method. IL-12 release in cultured DC supernatant was measured by ELISA. RESULTS: Sequencing result of the secretive IL-18 was correct. The transfected cells expressed IL-18 fusion gene and 18 kD IL-18 protein. DC in GT group expressed more surface molecules than those in other 3 groups. T cell proliferation and IL-12 secretion in GT group were higher than those in other 3 groups. CONCLUSION: IL-18 gene transfected NCI-H460 cell increases surface molecule expressions on DC. It also enhances immunological activity and IL-12 secretion in DC.     

Distribution and identification of immunocytes in humanized SCID mouse model

AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34⁺ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human β₂M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34⁺ cells were higher than those in mice transplanted with CB MNC. The mRNA of human β₂M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34⁺ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.     

Lethal effect of VEGFR2shRNA on HL60 cells in vitro using lentiviral vector

AIM: To look for harmfulless anti-leukemia drug with selective high performance, lethal effect of small hairpin RNA (shRNA) on VEGFR₂ gene expression of tumor cell line HL60 in vitro.METHODS: The most effective VEGFR₂ siRNA was designed and screened. The shRNA oligo was designed and pU6/VEGFR₂ entry clone was constructed. HL60 was transfected transiently and vascular endothelial growth factor receptor 2(VEGFR₂) expression was tested with MTT assay, RT-PCR and Western blotting. The expression clone was constructed and cotransfected with ViraPowerTM Packaging Mix into 293FTTM cells to produce Lentiviral vectors harboring Lenti6/shVEGFR₂. The virion supernatant was added into HL60 cells and VEGFR₂ gene inhibitory effect was determined. RESULTS: The inhibitory rates of VEGFR₂ siRNA c were high. VEGFR₂ expression in HL60 was inhibited by using pU6/VEGFR₂ entry clone constructed with shRNA and pENTRTM/U6. For HL60 cells, the inhibitory rate was 84.9%. The expression of VEGFR₂ mRNA and protein decreased significantly. 48 hours after transfection of pU6/shVEGFR₂ entry clone and transduction of Lenti6/shVEGFR₂ expression clone, the cell inhibitory rates were similar. Cell growth inhibitory rate of entry clone descended rapidly after this time point, the expression clone changed slowly, reaching the peak at 96 hours, dropped slightly, having no significance deviation. CONCLUSION: in vitro, VEGFR₂ shRNA using lentiviral vector blocks VEGF/VEGFR₂ self-secretion in HL60 cells, which inhibits leukemia development.