Propofol protects against oxidative-stress-induced COS-7 cell apoptosis by inducing autophagy

BACKGROUND: In oxidative stress, reactive oxygen species (ROS) production contributes to cellular dysfunction and initiates the apoptotic cascade. Autophagy is considered the mechanism that decreases ROS concentration and oxidative damage. Propofol shows antioxidant properties, but the mechanisms underlying the effect of propofol preconditioning (PPC) on oxidative injury remain unclear. Therefore, we investigated whether PPC protects against cell damage from hydrogen peroxide (H₂O₂)-induced oxidative stress and influences cellular autophagy. METHOD: COS-7 cells were randomly divided into the following groups: control, cells were incubated in normoxia (5% CO₂, 21% O₂, and 74% N₂) for 24 h without propofol; H₂O₂, cells were exposed to H₂O₂ (400 µM) for 2 h; PPC + H₂O₂, cells pretreated with propofol were exposed to H₂O₂; and 3-methyladenine (3-MA) + PPC + H₂O₂, cells pretreated with 3-MA (1 mM) for 1 h and propofol were exposed to H₂O₂. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) reduction. Apoptosis was determined using Hoechst 33342 staining and fluorescence microscopy. The relationship between PPC and autophagy was detected using western blot analysis. RESULTS: Cell viability decreased more significantly in the H₂O₂ group than in the control group, but it was improved by PPC (100 µM). Pretreatment with propofol effectively decreased H₂O₂-induced COS-7 cell apoptosis. However, pretreatment with 3-MA inhibited the protective effect of propofol during apoptosis. Western blot analysis showed that the level of autophagy-related proteins was higher in the PPC + H₂O₂ group than that in the H2O2 group. CONCLUSION: PPC has a protective effect on H₂O₂-induced COS-7 cell apoptosis, which is mediated by autophagy activation.

Remifentanil induces autophagy and prevents hydrogen peroxide-induced apoptosis in Cos-7 cells

BACKGROUND: This study investigated the effect of remifentanil pretreatment on Cos-7 cells exposed to oxidative stress, and the influence of remifentanil on intracellular autophagy and apoptotic cell death. METHODS: Cells were divided into 4 groups: (1) Control: non-pretreated cells were incubated in normoxia (5% CO₂, 21% O₂, and 74% N₂). (2) H₂O₂: non-pretreated cells were exposed to H₂O₂ for 24 h. (3) RPC+H₂O₂: cells pretreated with remifentanil were exposed to H₂O₂ for 24 h. (4) 3-MA+RPC+H₂O₂: cells pretreated with 3-Methyladenine (3-MA) and remifentanil were exposed to H₂O₂ for 24 h. We determined the cell viability of each group using an MTT assay. Hoechst staining and FACS analysis of Cos-7 cells were performed to observe the effect of remifentanil on apoptosis. Autophagy activation was determined by fluorescence microscopy, MDC staining, and AO staining. The expression of autophagy-related proteins was observed using western blotting. RESULTS: Remifentanil pretreatment increased the viability of Cos-7 cells exposed to oxidative stress. Hoechst staining and FACS analysis revealed that oxidative stress-dependent apoptosis was suppressed by the pretreatment. Additionally, fluorescence microscopy showed that remifentanil pretreatment led to autophagy-induction in Cos-7 cells, and the expression of autophagy-related proteins was increased in the RPC+H₂O₂ group. CONCLUSIONS: The study showed that remifentanil pretreatment stimulated autophagy and increased viability in an oxidative stress model of Cos-7 cells. Therefore, we suggest that apoptosis was activated upon oxidative stress, and remifentanil preconditioning increased the survival rate of the cells by activating autophagy.

Constructing an expression vector for human lncRNA H19 and the effect of its overexpression on MCF-7 cell proliferation / 中国药理学通报

Aims To construct an expression vector of human lncRNA H 1 9 ,and to determine the effect of H1 9 overexpression on MCF-7 cell proliferation. Methods Total RNA was extracted from MCF-7 cells,and the full-length of H1 9 lncRNA was amplified by RT-PCR and subcloned into pcDNA3.1 (-)ex-pression vector.The constructed H1 9 expression vector was transfected into HEK-293T and COS-7 cells and the H1 9 lncRNA expression was evaluated by real-time PCR.Following the transfection of H1 9 expression vec-tor into MCF-7 cells for 0,24h and 48h and H1 9 siR-NA interference fragment into MCF-7 cells for 24h, MCF-7 cell proliferation was determined by MTS as-say.Results A hH1 9-pcDNA3.1 (-)expression vector was successfully constructed. At Forty-eight hours after the transfection with H1 9 expression vector in to MCF-7 cells,cell proliferation was significantly increased in the transfected group compared to those without transfection and to those transfected with a neg-ative control vector,while twenty-four hours after the transfection with H1 9 siRNA interference fragment into MCF-7 cells,cell proliferation was significantly de-creased in the transfected group compared to those transfected with a negative control vector.Conclusion Ectopic overexpression of H1 9 lncRNA can promote breast cancer MCF-7 cell proliferation.

Construction and expression of a pEGFP-C2-HDAC2 recombinant plamid / 中国药理学通报

Aims HDAC2 gene was cloned into pEGFP-C2 vector to explore the efficiency of the plasmid trans-fection in renal fibroblasts COS-7 cells to identify the expression of both mRNA and protein levels and to ob-serve the distribution of the protein. Methods The HDAC2 cDNA was amlified by PCR and cut with the double enzyme Xho I and BamH I, then inserted into the eukaryotic expression vector pEGFP-C2 with T4 en-zyme. The recombinant vector was verified by PCR, restriction enzymes cut and sequencing identification. Then it was transfected into COS-7 cells and the ex-pression of pEGFP-C2-HDAC2 was monitored by fluo- rescence microscope and PCR. Results Fragments of HDAC2 could be seen after dealt with double diges-tion, and GFP could also be detected in the transfected COS-7 cells. HDAC2 gene expression could be detec-ted by PCR and Western blot. The fusion expression of pEGFP-C2-HDAC2 could be detected by Western blot. Conclusion Eukaryotic expression vector of HDAC2 has been successfully constructed, the fusion expres-sion of HDAC2 and GFP protein can be detected in COS-7 cells.

Effects of Tetrahydroxy-stilbene Glycoside on Over-expression of α-synuclein Protein in Transgenic COS-7 Cells / 中国康复理论与实践

@#Objective To explore the effects of 2,3,5,4'-tetrahydroxy stilbene-2-β-D-glycoside(TSG)on the over-expression and aggregation of α-synuclein in vitro.Methods TSG in different concentrations was incubated with α-synuclein transgenic COS-7 cells for 24 h.The cell viability was measured by MTT method.The expression of α-synuclein protein was determined by immunocytochemistry and Western blotting method.Results Incubation of TSG at the range of 12.5～200.0 μmol/L with α-synuclein transgenic COS-7 cells for 24 h did not influence cell viability,but a dose-dependently inhibition for the over-expression of α-synuclein protein could be observed in the tests of immunocytochemistry and Western blotting.Conclusion TSG can inhibit the over-expression of α-synuclein protein in COS-7 cells in vitro.

Cloning,weukaryotic expremion of the gene encoding glyceraidehydes-3-phosphate dehydrogenase fromperiodic Brugia malayi / 中国地方病学杂志

Objective To clone and express the encoding sequence of glyceraldehydes-3-phosphate dehydrogenase(GAPDH)from periodic Brugia molayi(Bm).Methods Total RNA was extraeted from periodic Brugic malayi.The BmGAPDH gene was amplified by RT-PCR.The PCR product was cloned and then subeloned into pcDNA3.1(+)vector.The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification,and were transformed into COS-7 cell subsequently.The expressed protein was identified by SDS-PAGE.Results BmGAPDH mRNA was highiy expressed in transfected COS-7 cell.The deduced amino acid sequence was identical with that of BmGAPDH.The recombinant pnotein wag about Nr 43 000.Conclusion The recombinant plasmid peDNA3.1(+)-BmGAPDH has been constructed and the protein has been expressed correctly.

Construction of Porcine CCK pDNA and Its Expression in COS-7 Cells / 华中科技大学学报（医学）（英德文版）

CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamine TM2000-mediated transfer method.The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected.And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells,which lays a foundation for further research on the relationship between CCK and tumor.

Construction of recombinant plasmid pIRES2-EGFP/CCK and its expression in vivo and in vitro / 中南大学学报(医学版)

Objective To construct eukaryotic expression plasmid of porcine CCK gene pIRES2-EGFP/CCK and express it in COS-7 cells and hamsters. Methods The aimed segments were obtained from intermediate vector pMD18-T/CCK and were inserted into an eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid pIRES2-EGFP/CCK. The recombinant expression plasmid was transfected into COS-7 cells by liposome-mediated gene transfer method and was observed through fluorescence microscope. The plasmid was injected into the skeletal muscle of hamsters directly to detect the expression of the recombinant plasmid in vivo. Results A recombinant eukaryotic expression plasmid pIRES2-EGFP/CCK was successfully constructed. Green fluorescent protein could be detected in the transfected COS-7 cells 24, 48, and 72 hours after the transfection. On the 4th day postinjection into the skeletal muscle of hamsters, the protein could be detected at the injection site and the fluorescence intensity became much stronger on the 14th day than that on the 4th day. On the 42nd day the protein level increased. The green fluorescence protein was never expressed in the untransfected cells. Conclusion The porcine CCK gene eukaryotic expression plasmid pIRES2-EGFP/CCK is constructed successfully, and is expressed in mammal COS-7 cells and hamsters in vivo. The research paves the way for the cross immunity therapy of hamster pancreatic carcinoma.

Construction of PSA and NCAM differently expressing COS-7 cell line and the effects of PSA on migration and invasion of the diverse COS-7 cell / 中国药理学通报

Aim To construct a polysialic acid(PSA)and neural cell adhesion molecule(NCAM)differently expressing COS-7 cell line,and investigate the effects of PSA on the cell adhesion,migration and invasion,aiming to establish the base for further investigation of the signaling passway of the diverse celluar migration and invasion,and elucidate the molecular mechanism of PSA promoting cancer cell metastasis.Methods A polysialic acid and neural cell adhesion molecule differently expressing COS-7 cell line was constructed by transient cotransfection,and the cotransfection efficiency was determined by Western blot and flow cytometry.Adhesion assay was used to investigate the effect of PSA on cell adhesion ability;transwell assay was used to measure migration and invasion ability.Results The PSA and NCAM differently expressing COS-7 cell line was successfully constructed,which demonstrated PSA inhibited the cell adhesion to basement membrane,and promoted the migration and invasion ability.Conclusions The constructed polysialic acid and neural cell adhesion molecule differently expressing COS-7 cell line can be used to investigate molecular mechanism of promoting cancer cell metastasis induced by PSA in the future.

Cloning and expression of human DcR3 molecule in COS-7 cells / 第三军医大学学报

Objective To clone ORF of DcR3 gene and insert it into eukaryotic expression vector and express it in COS-7 cells. Methods Encoding sequence of human DcR3 gene was cloned by PCR and sequenced. The sequenced ORF was cloned into eukaryotic expression vector pAAV-IRES-hrGFP to construct recombinant plasmid. COS-7 cells were transfected with recombinant plasmid by lipofectamine2000. Expression of recombinant DcR3 gene was verified by Western blotting and confocal microscopy. Results A 1 000-bp gene segment was obtained by PCR and inserted into pAAV-IRES-hrGFP to construct recombinant plasmid. The gene segment was proved to be encoding sequence of human DcR3 gene by sequencing. DcR3 expression in COS-7 cells was verified by Western blotting and confocal microscopy. Conclusion DcR3 gene was successfully cloned and expressed in COS-7 cells.

Construction of pcDNA3.1(+)/hFasL and its expression in COS-7 cells / 重庆医科大学学报

Objective:To construct the recombinant eukaryotic expression vector pcDNA3.1(+)/hFasL and investigate its transfection and transient expression in COS-7 cells.Methods:The full-length cDNA of hFasL was obtained from the plasmid vector pBluescript II KS(+)/hFasL cut with Xba I and then subcloned into eukaryotic expression vector pcDNA3.1(+).The recombinant plasmid pcDNA3.1(+)/hFasL was identified with restriction enzyme digestion and sequence analysis.It was then transfected into the COS-7 cells by Lipofectin Reagent and its expression was determined by immunocytochemical staining.Results:Restriction enzyme digestion analysis with XbaⅠ?DraⅡ?HindⅢ and DNA sequencing indicated the correct construction of the recombinant plasmid;Immunocytochemical staining showed the expession of hFasL on COS-7 cells.Conclusion:The construction and expression of the eukaryotic expression plasmid pcDNA3.1(+)/hFasL have been achieved successfully.

The Effects of Recombinant Human BMP-7, Prepared from a COS-7 Expression System, on the Proliferation and Differentiation of Rat Newborn Calvarial Osteoblasts

A family of proteins, the bone morphogenetic proteins (BMPs), which promote osteoblast differentiation and bone mineralization, have recently been identified. One, BMP-7, has shown the ability to induce cartilage and bone formation processes. In this report, the possibility that other cell lines, to CHO cells, may also be available as host cells for the expression of hBMP-7 was validated. Recombinant human BMP (rhBMP) -7 was produced in COS-7 cells, as a processed mature disulfide-linked homodimer, with an apparent molecular weight of 36, 000. Examination of the expressions of the markers characteristic of osteoblast phenotypes showed that the rhBMP-7 specifically stimulated the inductions of alkaline phosphatase (ALP) (5-fold increase at 100 ng of rhBMP-7/ml), parathyroid hormone (PTH) -mediated intracellular cAMP production (4-fold increase at 100 ng of rhBMP-7/ml) and osteocalcin synthesis (5-fold increase at 100 ng of rhBMP-7/ml). In summary, the in vitro mineralization assay results provide evidence that the rhBMP-7 peptide, produced by COS-7 expression system, possesses intact biological activity. A similar pattern of biological activity was observed for the BMP-7 in COS-7 cells compared to the corresponding CHO cell expression system. Thus, these findings can be experimentally utilized for the production of rhBMPs for in vitro or in vivo studies.

Molecular cloning and sequencing of rat Cdc42 GTPase cDNA

Cdc42 is a member of the Rho family of small GTP-ase and plays an important role in intracellular signaling pathways regulating cell morphology, motility and stimulation of DNA synthesis. We have isolated cDNA encoding Cdc42 from a rat brain cDNA library using PCR-cloning strategy. The sequence of isolated gene revealed an open reading frame of 576 nucleotides with the potential to encode a protein of 191 amino acids with a predicted molecular weight of 21 kD. The resulting sequence was incorporated into the GenBank with accession number, AF205635. Sequence analysis revealed that overall cDNA sequence identity is 96% with human G25K and 52% with rat Chp, a homologue of the GTPase human Cdc42Hs, and having one nucleotide difference from the mouse Cdc42. However, putative protein sequence was identical to the mouse and human brain Cdc42Hs. On expression of the cDNA in COS-7 cells, a protein molecular weight of 21 kD was detected in immunoblotting using anti-human Cdc42 antibodies. Therefore, these results suggest that the cDNA we are reporting is most likely the rat homologue of the GTPase human Cdc42.

The Novel Procedures to Increase the Expression Efficiency of Recombinant Human IL-6 in Eukaryotic Cells / 中国肿瘤生物治疗杂志

In order to improve the expression efficiency of recombinant IL~6 in eukaryotic cells, we constructed a new vector pcDIIL-6 to express IL-6 in eukaryotic cells on the basis of an eukaryotic vector pcDNA3IL-6 by introducin g the first intron of human?-globin gene into the downstream of CMV promoter in the pcDNASIL - 6. The IL-6 level expressed by pcDIIL - 6 is 7. 5 times higher than that of pcDNA3IL - 6. On the other hand , we studied the influence of pAdVAntage vector co - transfection on the expression level of IL-6 in eukaryotic cells and found the level of IL-6 is 3. 6 times higher than that of pcDNA3IL - 6. The results we got are beneficial to express cytokines with high efficiency in eukaryotic cells.

Transient expression of human dentin sialoprotein gene in COS-7 cells / 实用口腔医学杂志

Objective:To express human dentin sialoprotein (hDSP) gene in COS-7 cells. Methods:hDSP gene was subcloned into mammalian expression vector pcDNA3. The recombined plasmids were transfected into COS-7 cells using lipofectamune PLUS TM kit for transient expression. Western blot analysis and immunohistochemical staining were used to examine the gene products. Results:The constructed vectors were confirmed by digestion with restriction enzyme. An immuno-reaction positive band with relative molecular mass of 60 000 was found by Western blot analysis in culture supernatant and cytoplasms of COS-7 cells. Immunohistochemical staining showed strong positive particles in the cytoplasms. Conclution:hDSP gene can be expressed in COS-7 cells.

Construction of CSV-IL-6 and its expression in COS7 cells / 中国免疫学杂志

In this study, two expression vectors for recombinant human interleukin-6(rhIL-6):CSV-IL-6(1) and (2) were successfully constructed by routine gene manipulation, PCR amplification, sitespecific mutagensis, and optimionzation of translation. The constructed expression vectors for rhIL-6 were introduced into a simian cell line COS7 using gene transfer by DEAE-Dextran procedure. The expression level of rhIL-6 was determined by EL-6 ELISA using ELISA KIT for quantification of Human Interleukin-6, and the biological activity of rhIL-6 was assayed by 50% maximum incorporation of ~3H-TdR into 7TD1 cells, an IL-6-dependent hybridoma cell line. At 48h after transfection, the supernatant collected from the culture of COS7 cells transfected with CSV-IL-6 (1) contained rhIL-6 2573pg/ml, and at 72h after transfection it containde 1467pg/ ml; that for CSV-IL-6 (2)were 8261pg/ml and 7101pg/ml, respectively. The biological activity (hybridoma growth factor, HGF) at 48h after transfection from COS-CSV-TL-6 (1) was 10~4U/ ml corresponding to 3.9?10~9U/mg; and that from COS-CSV-IL-6 (2) was 6.3?10~4U/ml mcorresponeding to 7.6?10~9U/mg.

Eukaryotic expression and identification of human DC-SIGN and EGFP fusion protein / 中国免疫学杂志

Objective:To construct the eukaryotic vector of human DC-SIGN and EGFP fusion protein,and to identify the protein in cell line COS7.Methods:RT-PCR,T-vector and pEGFP-C1 vector were used to construct the recombinant expressing plasmid encoding for the fusion protein of DC-SIGN and EGFP.COS7 cells were transfected with the plasmid.Real-time PCR and laser scanning confocal microscope were used to quantificate expression of the fusion protein and cell function of uptaking BCG.Results:DC-SIGN cDNA was successfully cloned into the eukaryotic vector pEGFP-C1.The recombinant vector was transfected into COS7,real-time PCR test showed the amount of mRNA encoding for the fusion protein was 4.52?1011 copies/ml and laser scanning confocal microscope confirmed that the cells could uptake BCG.Conclusion:We have constructed a recombinant vector expressing DC-SIGN and EGFP fusion protein.

Construction of a human Flt3-ligand gene containing eukaryotic expressing vector and its expression in COS-7 cells / 中国免疫学杂志

Objective:To construct an eukaryotic expressing vector pIRES1neo/hFL and express hFL in COS 7 cell.Methods:The cDNA encoding soluble human Flt3 ligand(FL) was obtained by RT PCR from the TF 1 cell lines and was inserted into eukaryotic expressing vector pIRES1neo between EcoR I and BamH I sites after sequencing,and then COS 7 cells were transfected with the recombinant by liposome.The transcription and expression of hFL in the transfected COS 7 cells were assayed by RT PCR,ELISA and the experiment of the human umbilical blood CD34 + cell multiplication,respectively,at 72 hours after transfection.Results:It showed that hFL cDNA cloned in our laboratory was 546 bp in length encoding soluble human FL which was in accordance with the report previously.FL gene was transcripted in transfectants,and FL protein with obvious biological activity was highly expressed with 251 ng/(10 6 cell?d) in the supernatant of the transfectants.Conclusion:Human FL in the recombinant vector is proved to be expressed in COS 7 cells and obvious biological activity of hFL in the supernatant of the transfectants was detected.

Cloning of HLA-A~*0201 and its transient expression on COS-7 cells / 中国免疫学杂志

Objective:To clone HLA A *0201 cDNA and express it transiently on COS 7 cells.Methods:HLA A cDNA isolated from the HLA A *0201 positive lymphocytes by using RT PCR was cloned into pBluescript II SK vector directionally.After the sequence was confirmed,the cDNA was inserted into mammalian expression vector pcDNA3.The recombinant vector was transfected into COS 7 cells by cation liposome.The transient expression on the cells was measured by flow cyteometer.Results:The cDNA was identical with HLA A *0201 cDNA published on Genebank.Determined by flow cytemeter,the expressing rate was recorded for 57%.Conclusion:We have cloned HLA A *0201 successfully and expressed it transiently on COS 7 cell,which would be potentially useful in research on killing tumor restricted by HLA A2.