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1.
Electron. j. biotechnol ; 51: 50-57, May. 2021. ilus, graf
Article in English | LILACS | ID: biblio-1343384

ABSTRACT

BACKGROUND: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. RESULTS: In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. CONCLUSIONS: In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.


Subject(s)
Gene Expression Regulation , Optogenetics/methods , Light , Neurons/metabolism , Immunoblotting , Gene Expression , Fluorescent Antibody Technique , Lentivirus
2.
Article in Chinese | WPRIM | ID: wpr-880076

ABSTRACT

OBJECTIVE@#To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1.@*METHODS@#The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry.@*RESULTS@#The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×10@*CONCLUSION@#Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.


Subject(s)
Cell Line, Tumor , Genetic Vectors , Humans , Interleukin-3 Receptor alpha Subunit , K562 Cells , Lentivirus/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Plasmids , Transfection
3.
Chinese Journal of Biotechnology ; (12): 979-991, 2020.
Article in Chinese | WPRIM | ID: wpr-826878

ABSTRACT

Adoptive immunotherapy based on chimeric antigen receptor-modified T cells (CAR-T) is one of the most promising strategies to treat malignant tumors, but its application in solid tumors is still limited. Glypican-3 (GPC3) is a meaningful diagnostic, therapeutic, and prognostic biomarker for hepatocellular carcinoma (HCC). The second/third generation GPC3-targeted CAR-T cells are generated to treat HCC. In order to improve the therapeutic effect, we constructed a fourth-generation lentiviral vector to express GPC3 CAR, human interleukin-7 (IL-7) and CCL19. Then the lentiviral vector and packaging plasmids were co-transfected into HEK293T cells to generate CAR lentiviral particles. Human T lymphocyte cells were transduced with CAR lentiviral to develop the fourth-generation GPC3-targeted CAR-T cells (GPC3-BBZ-7×19). In vitro, we used cell counting, transwell assay, luciferase bioluminescence assay and flow cytometry to compare the proliferation, chemotaxis, cytotoxicity and subtype distribution between GPC3-BBZ-7×19 CAR-T cells and the second generation GPC3-targeted CAR-T cells (GPC3-BBZ). In vivo, we established GPC3-positive HCC xenograft model in immunodeficient mice, then untransduced T cells (non-CAR-T) or GPC3-BBZ-7×19 CAR-T cells were injected. Tumor growth in mice was observed by bioluminescence imaging. Results showed that compared with GPC3-BBZ CAR-T, GPC3-BBZ-7×19 CAR-T cells had stronger proliferation, chemotactic ability, and higher composition of memory stem T cells (Tscm) (P values<0.05). However, there were no significant difference in cytotoxicity and cytokine secretion between them. In addition, GPC3-BBZ-7×19 CAR-T cells could significantly eliminate GPC3-positive HCC xenografts established in immunodeficient mice. Therefore, the fourth-generation GPC3-targeted CAR-T cells (secreting IL-7 and CCL19) are expected to be more durable and effective against HCC and produce tumor-specific memory, to provide a preclinical research basis for future clinical trials.


Subject(s)
Animals , Carcinoma, Hepatocellular , Cell Line, Tumor , Chemokine CCL19 , Metabolism , Glypicans , Metabolism , HEK293 Cells , Humans , Interleukin-7 , Metabolism , Lentivirus , Genetics , Liver Neoplasms , Mice , Receptors, Chimeric Antigen , Metabolism , T-Lymphocytes , Metabolism , Xenograft Model Antitumor Assays
4.
Article in Chinese | WPRIM | ID: wpr-781353

ABSTRACT

OBJECTIVE@#To construct a PA28γ overexpression cell line and determine its effects after infecting an oral squa-mous cell carcinoma (OSCC) cell line.@*METHODS@#The PA28γ gene was cloned into the pLOV.CMV.cherry.2A.EF1a.PuroR lentiviral vector by polymerase chain reaction (PCR), and PCR and DNA sequencing alignment analysis were used for identification. Then, 293T cells were used to package viral diseases. Infected OSCC cells were used to construct a cell line with stable PA28γ overexpression. Finally, the level of PA28γ expression in the OSCC cell line was detected through Western blot.@*RESULTS@#The successful construction of PA28γ recombinant lentiviral vectors was confirmed by DNA sequencing. The results of immunofluorescence showed that the PA28γ overexpression lentivirus successfully infected the OSCC cells and showed cherry red fluorescence. The results of Western blot demonstrated that the constructed cells with stable PA28γ overexpression significantly increased the expression of PA28γ.@*CONCLUSIONS@#The PA28γ overexpression lentiviral vector can significantly increase its protein expression in OSCC cells. We provide a stable OSCC cell line for further study on the effect of PA28γ in OSCC.


Subject(s)
Autoantigens , Carcinoma, Squamous Cell , Cell Line, Tumor , Genetic Vectors , Humans , Lentivirus , Mouth Neoplasms , Proteasome Endopeptidase Complex , Transfection
5.
Article in Chinese | WPRIM | ID: wpr-828675

ABSTRACT

OBJECTIVE@#To study the expression and function of long non-coding RNA linc00467 in childhood acute myeloid leukemia (AML).@*METHODS@#Bone marrow samples were collected from 5 children with AML who were diagnosed from May 2016 to June 2018. Normal bone marrow samples based on bone marrow examination were collected from 3 children as controls. Quantitative real-time PCR was used to measure the expression of linc00467 in the two groups. A lentivirus system was used to achieve overexpression of linc00467 in AML cells (HL-60) (linc00467 overexpression group), and empty vector expressing green fluorescent protein (GFP) was transfected into AML cells to establish a GFP control group. A lentivirus system was used to insert an interfering sequence into AML cells (sh-linc00467 interfering group), and a random sequence was inserted to establish an sh-NC control group. Cell proliferation and resistance to doxorubicin were observed for all groups.@*RESULTS@#Compared with the normal control group, the children with AML had a significant increase in linc00467 (P=0.018). Overexpression and interference with linc00467 expression had no significant effect on cell proliferation. Compared with the GFP control group, the linc00467 overexpression group had a significant increase in the viability of HL-60 cells at the adriamycin concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 μg/mL (P<0.05). Compared with the sh-NC control group, the sh-linc00467 interfering group had a significant reduction in the viability of HL-60 cells at the adriamycin concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 μg/mL (P<0.05). Compared with the untreated group, the adriamycin treatment group had a significant increase in the expression of linc00467 in HL-60 cells (P<0.05).@*CONCLUSIONS@#This study reveals the biological function of linc00467 to promote the resistance to adriamycin in AML, which provides a basis for developing new therapeutic drugs for AML.


Subject(s)
Cell Proliferation , Child , Drug Resistance, Neoplasm , Humans , Lentivirus , Leukemia, Myeloid, Acute , Genetics , RNA, Long Noncoding , Genetics
6.
Chinese Journal of Biotechnology ; (12): 1395-1404, 2020.
Article in Chinese | WPRIM | ID: wpr-826837

ABSTRACT

By inserting microRNAs into the intron of EF1α promoter, we constructed a novel lentiviral vector knocking down PD-1 gene via microRNA and applied it to CAR-T cells. Lentiviral transduction efficiency and PD-1-silencing efficiency were detected by flow cytometry. PD-1 expression was detected by Western blotting. Relative expression of microRNA was measured by Q-PCR. Cytotoxicity of CAR-T cells based on this vector was tested by luciferase bioluminescence and flow cytometry. Compared with lentiviral vector with microRNA transcribed by U6 promotor, the transduction efficiency of lentiviral vector with microRNA which was inserted into the intron of EF1α promoter was more significant, and the knockdown rate of PD-1 was more than 90%, which was validated by flow cytometry and Western blotting. And the relative expression level of microRNA in Jurkat cells transduced with this novel lentiviral vector was shown by Q-PCR. Compared with normal CAR-T cells, CAR-T cells based on this vector showed stronger cytotoxicity against PD-L1 positive Raji cells. We successfully constructed a novel lentiviral vector that knocked down PD-1 via microRNA and verified the superiority of its transduction efficiency and knockdown efficiency of PD-1. CAR-T cells based on this vector can exert a more powerful cytotoxicity, thus providing theoretical support for the subsequent treatment of PD-L1 positive tumors.


Subject(s)
Cell Line, Tumor , Gene Knockdown Techniques , Genetic Vectors , Genetics , Humans , Lentivirus , Genetics , MicroRNAs , Metabolism , Programmed Cell Death 1 Receptor , Promoter Regions, Genetic , Genetics
7.
Chinese Journal of Biotechnology ; (12): 1307-1316, 2019.
Article in Chinese | WPRIM | ID: wpr-771798

ABSTRACT

Gene therapy is a rapidly developing field. The most widely used technique for foreign gene transfer is lentiviral-mediated gene therapy. Lentiviral vector has been developed from the first generation to the third generation in terms of safety. The preparation of lentiviruses with high titer remains difficult. In this study, a Fibra-Cel sheet carrier was used as an HEK293T cell carrier matrix, and several sterile cell culture spinners were combined and cultured on a roller bottle machine to scale up the adherent cells. The virus titer was maximized by screening the factors to optimize the lentivirus titer in the third-generation lentivirus packaging process one by one. Fibra-Cel sheet vector was successfully used as the matrix of HEK293T cell adhesion to culture adherent cells at large scale. The optimal conditions for large-scale preparation of the third-generation lentivirus by bottle roller were screened and three batches of lentiviruses were produced on pilot scale. The production time of lentivirus was shortened from 120 hours to 54 hours from plasmid transfection to virus collection; in terms of cost, a rolling bottle machine was used instead of a bioreactor, leading to lower cost and no need for repeated sterilization during the whole process. The safe, effective and low-cost operation of successful production will provide a technical base for the large-scale preparation of lentivirus and thus lay a firm foundation for its clinical application.


Subject(s)
Genetic Vectors , HEK293 Cells , Humans , Lentivirus , Transduction, Genetic , Transfection
8.
Article in Chinese | WPRIM | ID: wpr-774297

ABSTRACT

OBJECTIVE@#To investigate the effect of steadily down-regulating the expression of calreticulin (CALR) on the invasion of natural killer/T-cell lymphoma SNK6 cells, and explore its possible mechanism.@*METHODS@#The sequences of specific short hairpin RNA (shRNA) targeting on human CALR were designed, and were inserted into pLKO.1-puro lentivirus vector, and the reconbinant lentivirus vector was obtained; the lentivirus particles were backed by three-plasmid system and transfected into SNK6 cells, the SNK6 cells stably down-regulating the CALR expression were sercened by puromytain, the CALR-silencing effect was verified by real-time PCR and Western blot. CCK-8 assay was used to evaluate the cell viability, The transwell invasion assays was used to analyse invasion of SNK6 cells. The mRNA expression of Calreticulin, MMP2, MMP9 and VEGF was determined by real time PCR, the protein expression of Calreticulin and GAPDH was analyzed by Western blot.@*RESULTS@#The recombinant lentiviral vector pLKO.1-puro-shCALR was successfully constructed, packed into the lentivirus, then the SNK6 cells stably down-regulating Calreticulin expression was obtained. When Calreticulin was down-rengulated in SNK6 cells, the proliferation rate was reduced and the invasion ability was decreased; the mRNA levels of VEGF and MMP-2/9 also were reduced.@*CONCLUSION@#The stable down-regnlation of CALR expression in SNK6 cells can attenuate the imvasiveness of SNK6 cells, which maybe related with transcriptional decrease of MMP2, MMP9 and VEGF.


Subject(s)
Calreticulin , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Genetic Vectors , Humans , Lentivirus , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 9 , RNA Interference , RNA, Small Interfering , Transfection , Vascular Endothelial Growth Factor A
9.
Article in Chinese | WPRIM | ID: wpr-773820

ABSTRACT

OBJECTIVE@#To explore the effect of lentivirus-mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells and silk fibroin scaffold on osteoblast transformation.@*METHODS@#The lentivirus BMP-2 overexpression vector was constructed, bone marrow mesenchymal stem cells were cultured, and the combined culture system of nuclear scaffolds was constructed. Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic transformation of bone marrow mesenchymal stem cells in vitro. Ten New Zealand white rabbits, weighing 3.2 to 4.5 kg(averaging 3.9 kg), aged (2.89±0.45) years old, were selected to construct the rabbit tibial defect model by drilling a conical tibial defect (5 mm in length, 2 mm in width and 3 mm in depth) with an oral drill. The repair of the tibial defect in the animal model was observed by HE staining. The experimental group was implanted with silk fibroin scaffold + BMP-2 overexpression vector bone marrow mesenchymal stem cell complex, while the negative control group was implanted with silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cell complex.@*RESULTS@#Compared with the control group(silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cells), the number of adherent cells on the surface of the scaffold in the experimental group(silk fibroin scaffold+transfected BMP-2 overexpression vector BMP-2 complex) increased significantly. Compared with the control group, the ECM secretion in the experimental group increased significantly. EDX analysis showed that the content of calcium ion was 0.22% in the control group and 0.86% in the experimental group, which showed that the ability of inducing calcium ion formation in the experimental group was stronger than that in the control group. Alizarin red staining of calcium nodules showed that there was no obvious change in the naked eye of the control group, and a small amount of calcium nodules could be seen under the microscope. In the experimental group, obvious red area staining was observed by naked eye, and a large number of calcium nodules were observed by microscopy. The results of alkaline phosphatase staining showed that there was no obvious change in the naked eye of the control group, and no obvious change in the microscopic observation. In the experimental group, purple area staining was observed by naked eyes, and ALP staining was strongly positive by microscopy. The combined culture system of silk fibroin scaffold and bone marrow mesenchymal stem cells can repair cartilage defects. The repair effect of BMP-2 bone marrow mesenchymal stem cells after transfection is obviously better than that of non-transfection group. HE staining showed that inflammatory cells decreased and scaffolds disappeared slightly in the control group. In the experimental group, inflammatory cells were significantly reduced, scaffolds disappeared and angiogenesis was observed.@*CONCLUSIONS@#Lentivirus-mediated BMP-2 overexpression plasmid can promote BMSC to differentiate into osteocytes and secrete more extracellular matrix containing Ca²⁺ to promote bone defect repair.


Subject(s)
Animals , Bone Marrow Cells , Bone Morphogenetic Protein 2 , Cells, Cultured , Fibroins , Lentivirus , Mesenchymal Stem Cells , Osteoblasts , Osteogenesis , Plasmids , Rabbits , Transfection
10.
Article in English | WPRIM | ID: wpr-773396

ABSTRACT

OBJECTIVE@#To explore the protective effect of NANOG against hydrogen peroxide (H2O2) -induced cell damage in the human hair follicle mesenchymal stem cells (hHF-MSCs).@*METHODS@#NANOG was expressed from a lentiviral vector, pLVX-IRES-ZsGreen. NANOG hHF-MSCs and vector hHF-MSCs were treated with 400 μmol/L hydrogen peroxide (H2O2) for 2 h, the cell survival rate, cell morphology, ROS production, apoptosis and expression of AKT, ERK, and p21 were determined and compared.@*RESULTS@#Our results showed that NANOG could activate AKT and upregulate the expression of p-AKT, but not p-ERK. When treated with 400 μmol/L H2O2, NANOG hHF-MSCs showed higher cell survival rate, lower ROS production and apoptosis, higher expression of p-AKT, higher ratio of p-AKT/AKT.@*CONCLUSION@#Our results suggest that NANOG could protect hHF-MSCs against cell damage caused by H2O2 through activating AKT signaling pathway.


Subject(s)
Cell Survival , Drug Evaluation, Preclinical , Hair Follicle , Cell Biology , Humans , Hydrogen Peroxide , Lentivirus , Mesenchymal Stem Cells , Metabolism , Nanog Homeobox Protein , Metabolism , Pharmacology , Oxidative Stress , Phosphatidylinositol 3-Kinases , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Signal Transduction
11.
Journal of Experimental Hematology ; (6): 1469-1475, 2019.
Article in Chinese | WPRIM | ID: wpr-775697

ABSTRACT

OBJECTIVE@#To investigate the effect of LNK gene silencing and overexpression on the expression of STAT3 gene in human monocytic leukemia cells (THP-1).@*METHODS@#THP-1 cells were cultured, and the lentivirus was used as a vector to silence and overexpres the LNK gene stably. After transfection for 72 hours, the GFP expression levels were observed by inverted fluorescence microscopy. The lentiviral transfection efficiencies were detected by flow cytometry. The effects of LNK silencing and overexpression were confirmed, and the expression of STAT3 mRNA was detected by RT-PCR. The protein levels of LNK and STAT3 were detected by Western blotting.@*RESULTS@#The GFP expression level of THP-1 cells reached more than 85% after transfection with lentivirus for 72 hours, and the transfection efficiency of cells was above 99%. mRNA expressions levels of LNK and STAT3 in LNK silencing group were signifycantly lower than those in control group, while LNK and STAT3 mRNA levels in the LNK overexpression group was significantly higher than those in control group (P<0.05). The protein expression levels of LNK and STAT3 in LNK silencing group were significantly lower than those in control group, while that in LNK overexpression group was significantly higher than that in control group (P<0.05).@*CONCLUSION@#The THP-1 cell line with LNK gene silencing and overexpression has been successfully established. The LNK gene silencing resulted in decrease of STAT3 expression; LNK gene overexpression and leads to inereases of STAT3 expression indicating that LNK participates in the regulation of STAT3.


Subject(s)
Cell Line, Tumor , Gene Silencing , Genetic Vectors , Humans , Lentivirus , Proteins , RNA, Small Interfering , STAT3 Transcription Factor , Metabolism , THP-1 Cells , Transfection
12.
Yonsei Medical Journal ; : 1045-1053, 2019.
Article in English | WPRIM | ID: wpr-762055

ABSTRACT

PURPOSE: To explore the molecular mechanism of the upregulation of multidrug resistance-associated protein 4 (MRP4) in cholestasis. MATERIALS AND METHODS: The mRNA and protein levels of MRP4 in liver samples from cholestatic patients were determined by quantitative real-time PCR and Western blot. In human hepatoma HepG2 cells, electrophoretic mobility shift assay (EMSA) was used to determine the affinity of nuclear factor-E2-related factor (Nrf2) binding to MRP4 promoter. Dual-luciferase reporter assay was used to detect the binding of tumor necrosis factor α (TNFα) to the promotor of E2F1. The bile duct ligation mouse models were established using male C57BL/6 mice. RESULTS: The mRNA and protein levels of MRP4 were significantly increased in cholestatic patients. TNFα treatment induced the expression of MRP4 and Nrf2 and enhanced cell nuclear extract binding activity to MRP4 promoter, as demonstrated by EMSA. Nrf2 knockdown reduced MRP4 mRNA levels in both HepG2 and Hep-3B cells. In addition, TNFα increased Rb phosphorylation and expression of MRP4 and Nrf2 and activated E2F1 and phosphorylated p38 in HepG2 and Hep-3B cells. These effects were markedly inhibited by pretreatment with E2F1 siRNA. Dual-luciferase reporter assay validated that TNFα induces the transcription of E2F1. Furthermore, the expression of MRP4, Nrf2, E2F1, and p-p38 proteins was improved with treatment of TNFα in a mouse model of cholestasis. E2F1 siRNA lentivirus or SB 203580 (p38 inhibitor) inhibited these positive effects. CONCLUSION: Our findings indicated that TNFα induces hepatic MRP4 expression through activation of the p38-E2F1-Nrf2 signaling pathway in human obstructive cholestasis.


Subject(s)
Animals , Bile Ducts , Blotting, Western , Carcinoma, Hepatocellular , Cholestasis , Electrophoretic Mobility Shift Assay , Hep G2 Cells , Humans , Lentivirus , Ligation , Liver , Male , Mice , Multidrug Resistance-Associated Proteins , Phosphorylation , Real-Time Polymerase Chain Reaction , RNA, Messenger , RNA, Small Interfering , Tumor Necrosis Factor-alpha , Up-Regulation
13.
Article in English | WPRIM | ID: wpr-742359

ABSTRACT

PURPOSE: This study investigated the role of natriuretic peptide receptor 2 (NPR2) on cell proliferation and testosterone secretion in mouse Leydig cells. MATERIALS AND METHODS: Mouse testis of different postnatal stages was isolated to detect the expression C-type natriuretic peptide (CNP) and its receptor NPR2 by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Leydig cells isolated from mouse testis were cultured and treated with shNPR2 lentiviruses or CNP. And then the cyclic guanosine monophosphate production, testosterone secretion, cell proliferation, cell cycle and cell apoptosis in mouse Leydig cells were analyzed by ELISA, RT-qPCR, Cell Counting Kit-8, and flow cytometry. Moreover, the expression of NPR2, cell cycle, apoptosis proliferation and cell cycle related gene were detected by RT-qPCR and Western blot. RESULTS: Knockdown of NPR2 by RNAi resulted in S phase cell cycle arrest, cell apoptosis, and decreased testosterone secretion in mouse Leydig cells. CONCLUSIONS: Our study provides more evidences to better understand the function of CNP/NPR2 pathway in male reproduction, which may help us to treat male infertility.


Subject(s)
Animals , Apoptosis , Blotting, Western , Cell Count , Cell Cycle , Cell Cycle Checkpoints , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Germ Cells , Guanosine Monophosphate , Humans , Infertility, Male , Lentivirus , Leydig Cells , Male , Mice , Natriuretic Peptide, C-Type , Polymerase Chain Reaction , Receptors, Peptide , Reproduction , Reverse Transcription , RNA Interference , S Phase , Testicular Diseases , Testis , Testosterone
14.
Journal of Experimental Hematology ; (6): 1744-1748, 2019.
Article in Chinese | WPRIM | ID: wpr-781403

ABSTRACT

OBJECTIVE@#To analyze the effect of down-regulating the CD59 gene expression by RNAi lentivirus as vector on Jurkat cell line of acute T-lineage leukemia.@*METHODS@#The expression of CD59 in Jurkat cell line of acute T-line leukemia was induced to decrease by RNAi lentivirus as vector. The transfection of RNA lentivirus and the localization of CD59 molecule were analyzed by laser confocal technique. The relative expression of CD59 gene in blank control, negative control and RNAi lentivirus transfected group was detected by real-time fluorescence quantitative PCR, and the enzyme-linked immunosorbent assay was used to detect the expression of TNF-β and IL-3 in supernatants of cultured cells in 3 groups. The expression levels of apoptosis-related molecules including Caspase-3, Survivin, BCL-2 and BCL-2-associated X protein (BAX) were measured by Western blot.@*RESULTS@#The transfection efficiency for Jurkat cells was higher than 90%. CD59 was mainly located on the cell membrane. Compared with the blank control group and the negative control group, the expression level of CD59 mRNA and protein in the RNAi lentivirus transfected group significantly decreased (P<0.05). Compared with the blank control group and the negative control group, the expression of TNF-β and IL-3 in the RNAi lentivirus transfected group were significantly higher and lower (P<0.05) respectively. The expression levels of Survivin and BCL-2 in the RNAi lentivirus transfected group were significantly lower than those in the blank control group and the negative control group, while the expression levels of Caspase-3 and BAX in the RNAi lentivirus transfected group were significantly higher than those in the blank control group and the negative control group (P< 0.05).@*CONCLUSION@#The down-regulation of CD59 gene expression induced by RNAi lenti-virus can decrease the expression of proliferation and differentiation-promoting molecule such as IL-3 and increase the expression of TNF-related factor in Jurkat cell line of acute T-lineage leukemia, which also can increase the expression of apoptosis-related proteins such as Caspase-3 and BAX, and decrease the expression of anti-apoptosis-related proteins such as Survivin and BCL-2.


Subject(s)
Apoptosis , CD59 Antigens , Cell Lineage , Cell Proliferation , Down-Regulation , Humans , Jurkat Cells , Lentivirus , Leukemia , RNA Interference , RNA, Small Interfering , Transfection
15.
Article in Chinese | WPRIM | ID: wpr-813102

ABSTRACT

To determine the effect of a recombinant lentivirus containing human stem cell leukemia (SCL) gene on the expression of c-kit protein in damaged interstitial cells of Cajal (ICC) under high glucose condition.
 Methods: After isolation of ICC, the cells were cultured for 24 hours until the cells were adherent. After identification by inverted microscope and immunofluorescence, ICC cells were divided into two groups: A control group and a high glucose group. The control group was added with a medium containing 5 mmol/L of glucose. The high glucose group was added with a medium containing 20 mmol/L of glucose. After 48 h of continuous cultivation, the high glucose group was divided into 3 subgroups: A blank group, an empty lentivirus group, and an experimental group. The blank group, the empty lentivirus group, and the experimental group were added a medium containing PBS solution, empty lentivirus, and a recombinant lentivirus containing the SCL gene with a glucose concentration of 5 mmol/L, respectively. The cultures were incubated for 24 and 48 h. The expression of c-kit protein in ICC in each group was detected by Western blot.
 Results: After 24 or 48 h, the expression of c-kit protein in ICC was significantly lower in the blank group and the lentivirus group than that in the control group, and the expression of c-kit protein in ICC was significantly higher in the experimental group than that in the blank group and the empty lentivirus group, but it was still lower than that in the control group (all P<0.05).
 Conclusion: The recombinant lentivirus of SCL gene can up-regulate the expression of c-kit protein in functionally impaired ICC under high glucose condition.


Subject(s)
Glucose , Humans , Interstitial Cells of Cajal , Lentivirus , Leukemia, Myeloid, Acute , Proto-Oncogene Proteins c-kit
16.
Yonsei Medical Journal ; : 1205-1213, 2018.
Article in English | WPRIM | ID: wpr-718490

ABSTRACT

PURPOSE: Research has shown that sevoflurane-induced toxicity causes neurodegeneration in the developing brain. miR-34a has been found to negatively regulate ketamine-induced hippocampal apoptosis and memory impairment. However, the role of miR-34a in sevoflurane-induced hippocampal neurodegeneration remains largely unclear. MATERIALS AND METHODS: C57/BL6 mice (7-day-old) inhaled 2.3% sevoflurane for 2 h/day over 3 consecutive days. miR-34a expression was reduced through intracerebroventricular injection with miR-34a interference lentivirus vector (LV-anti-miR-34a) into mouse hippocampus after anesthesia on the first day of exposure. Hippocampal apoptosis was detected by TUNEL assay and flow cytometry analysis. Spatial memory ability was evaluated by the Morris water maze test. The interaction between miR-34a and Wnt1 was confirmed by luciferase reporter assay, RNA immunoprecipitation, Western blot, and immunofluorescence staining. The effects of miR-34a on protein levels of B-cell lymphoma 2 (Bcl-2), bcl-2-like protein 4 (Bax), and Wnt/β-catenin pathway-related proteins were evaluated using Western blot analysis. RESULTS: Sevoflurane upregulated hippocampal miR-34a, and miR-34a inhibitor attenuated sevoflurane-induced hippocampal apoptosis and memory impairment. miR-34a negatively regulated Wnt1 expression by targeting miR-34a in hippocampal neurons. Moreover, forced expression of Wnt1 markedly undermined miR-34a-mediated enhancement of sevoflurane-induced apoptosis of hippocampal neurons, while Wnt1 silencing greatly restored anti-miR-34a-mediated repression of sevoflurane-induced apoptosis of hippocampal neurons. Increased expression of miR-34a inhibited the Wnt/β-catenin pathway in hippocampal neurons exposed to sevoflurane, while anti-miR-34a exerted the opposite effects. CONCLUSION: miR-34a inhibitor may effectively protect against sevoflurane-induced hippocampal apoptosis via activation of the Wnt/β-catenin pathway by targeting Wnt1.


Subject(s)
Anesthesia , Animals , Apoptosis , Blotting, Western , Brain , Flow Cytometry , Fluorescent Antibody Technique , Hippocampus , Immunoprecipitation , In Situ Nick-End Labeling , Lentivirus , Luciferases , Lymphoma, B-Cell , Memory , Mice , Neurons , Repression, Psychology , RNA , Spatial Memory , Water
17.
Article in Chinese | WPRIM | ID: wpr-690988

ABSTRACT

<p><b>OBJECTIVE</b>To evaluate biological effects of OCT4A gene on K562 cells and explore the molecular mechanism of K562 cell apoptosis.</p><p><b>METHODS</b>Two recombinant lentiviral vectors were constructed, which could stablely up- regulate and down- regulate OCT4A protein. Recombinant lentivirus was generated by co-transfection of three-plasmids and transfec-ted into K562 cells. The experiments were divided into 5 groups: normal, pLVX-OCT4A-ZsGreen1, pLVX vector control, PLB-OCT4A shRNA and non-specific shRNA groups. Western blot was applied to detect the expression of OCT4A protein, the cell counting kit-8 was applied to evaluate the effect of OCT4A on proliferation of K562 cells. The apoptosis and differentiation of K562 cells were detected by flow cytometry with AnnexinV/7-AAD double staining. The mRNA expressions of caspase-3,BIM,BCL-xL,BAX in K562 cells were determined by real time PCR.</p><p><b>RESULTS</b>The OCT4A fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR), the 2 lentiviral vectors were successfully constructed. In comparson with those in the control group, the expression of OCT4A protein of pLVX-OCT4A-ZsGreen1 group was significantly increased, but decreased in PLB-OCT4A shRNA group. CCK-8 assay showed that the higher the content of OCT4A protein, the faster the cell proliferation. The apoptosis rate was (3.48±0.52)% of pLVX-OCT4A-ZsGreen1 group, which was lower than that of control group, while the apoptosis rate PLB-OCT4A shRNA group was (7.25±0.57)%, which was higher than that of control group (P<0.05), however, the K562 cells differentiation was not influenced(P>0.05). Compared with control group, the gene expression of Caspase-3,BIM and BAX was down-regulated(P>0.05), but a significant up-regulation of BCL-xL gene expression was observed(P<0.05).</p><p><b>CONCLUSION</b>Two lentiviral vectors have been successfully constructed, which can stably up- and down- regulate the expression of OCT4A in K562 cells respectively. OCT4A can promote the K562 cell proliferation and inhibit the apoptosis, the mechanism may be related with up-regulation of BCL-xl expression.</p>


Subject(s)
Apoptosis , Cell Proliferation , Genetic Vectors , Humans , K562 Cells , Lentivirus , Octamer Transcription Factor-3 , Genetics , Transfection
18.
Article in Chinese | WPRIM | ID: wpr-690934

ABSTRACT

<p><b>OBJECTIVE</b>To clone the circular RNA hsa_circ_0000254 and construct its lentiviral over-expression vector.</p><p><b>METHODS</b>The sequence of hsa_circ_0000254 (a total of 524 bp long) was synthesized and cloned by using pGH vector. The vector was cut by EcoR I and BamH I, and artificial hsa_circ_0000254 was obtained, then inserted in pLCDH-ciR to construct the recombinant expression vector pLCDH-circ254(C254), which was confirmed by DNA sequencing. The lentiviral expression vectors pLCDH-circ254(C254) and NC(pLCDH-ciR) were cotransfected into 293T cells by lipofectamine 2000(lipo2000). After transfection for 40 hours, the cells were collected and verified by PCR and sequencing.</p><p><b>RESULTS</b>Restriction analysis and DNA sequencing demonstrated that the lentiviral vector pLCDH-circ254(C254) was constructed successfully, the expression efficiency increased 10000 times after transfection of cells.</p><p><b>CONCLUSION</b>The successful construction of the lentiviral expression vector pLCDH-circ254(C254) results in the production of high-titer lentivirus, so as to facilitate further study of the molecular functions of hsa_circ_0000254.</p>


Subject(s)
Genetic Vectors , Humans , Lentivirus , Leukemia, Myeloid, Acute , RNA, Small Interfering , Transfection
19.
Article in Chinese | WPRIM | ID: wpr-689574

ABSTRACT

<p><b>OBJECTIVE</b>To explore the killing effect of CAR (CD138-CD28-CD3ζ)-NK cells on myeloma cells through construction of CAR(CD138-CD28-CD3)-NK cells.</p><p><b>METHODS</b>The antiCD138scFv-CD28-CD3 zeta plasmid pcDNA3.1 was constructed, which then together with 3 plasmid lentiviral packaging system were transfected into 293T cells, the virus was collected. Furthermore, in order to get the stably transfected cell line, the NK92MI cell line was infected by the virus, then the positive cells were screened by puromycin. The expression of the CARNK cells were verified by RT-PCR and Western blot. At last the ability of secreting cytokine CD107a was detected by flow cytometry, and the statistical analysis was carried out to verify the anti-myeloma effect of CAR-NK cells.</p><p><b>RESULTS</b>Gene fragment of the CAR(antiCD138scFv-CD28-CD3ζ) was constructed successfully by gene engineering technique in vitro, and the gene sequence was verified to be correct by sequencing. By virus packaging technology, the virus expressing the protein of the CAR was obtained. PCR and Western blot verified the expression of CAR fusion protein on the sufurce of NK cells. The cell killing experiment confirmed that the CAR-NK cells possessed the ability to secrete cytokine CD107a superior to control cells and showed the obvious killing effect on multiple myeloma cells.</p><p><b>CONCLUSION</b>The CAR can be constructed in vitro, and express on NK92 cells. The CAR-NK cells can kill the multiple myeloma cells expressing CD138 antigen, thereby plays an antimyeloma effect.</p>


Subject(s)
Cell Line, Tumor , Humans , Killer Cells, Natural , Lentivirus , Multiple Myeloma , Receptors, Antigen , Receptors, Antigen, T-Cell
20.
Arq. bras. med. vet. zootec ; 69(2): 391-397, mar.-abr. 2017. tab
Article in English | ID: biblio-833836

ABSTRACT

With the objective of detecting the presence of caprine lentivirus (CLV) in ewe milk and in ram semen, ten matrixes and four reproducers experimentally infected with CLV were used. Samples of ewe milk were collected during the four months of lactation, five collections per animal, totaling 50 samples. Regarding the rams, eight semen collections were made per animal, during one year of experimentation, totaling 32 samples. The milk and semen samples were submitted to DNA extraction and the nested polymerase chain reaction test (nPCR) to detect CLV proviral DNA. Eight (16%) of the milk samples were positive in nPCR originating from two ewes. Only one (3.12%) semen sample was positive. The amplification products were sequenced, and were confirmed to be a CLV genomic sequence. Thus, the presence of CLV proviral DNA in sheep milk and semen was demonstrated, confirming the feasibility of infection between species, and alerting to the risk of spreading infections.(AU)


Com o objetivo de detectar a presença do lentivírus caprino (LVC) no leite de ovelhas e no sêmen de carneiros, utilizaram-se 10 matrizes e quatro reprodutores infectados experimentalmente com o LVC. Foram coletadas amostras de leite das ovelhas durante os quatro meses de lactação, ocorrendo cinco coletas por animal, totalizando 50 amostras. Quanto aos carneiros, realizaram-se oito coletas de sêmen por animal, durante um ano de experimentação, totalizando 32 amostras. As amostras de leite e de sêmen foram submetidas à extração de DNA e à prova de reação em cadeia da polimerase do tipo nested (nPCR) visando à detecção de DNA proviral do LVC. Oito (16%) amostras de leite foram positivas na nPCR oriundas de duas ovelhas. Apenas uma (3,12%) amostra de sêmen apresentou positividade. Produtos da amplificação foram sequenciados, confirmando-se tratar de sequência genômica do LVC. Dessa forma, demonstrou-se a presença do DNA proviral do LVC em leite e sêmen de ovinos, confirmando a viabilidade da infecção entre espécies e, assim, alertando sobre o risco de que a infecção seja disseminada.(AU)


Subject(s)
Animals , Male , Female , Lentivirus/isolation & purification , Milk/virology , Ruminants/virology , Semen/virology , Disease Transmission, Infectious/veterinary , Polymerase Chain Reaction/veterinary
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