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1.
AIM:Tyrosinase gene was transfected into HEK293 cell as a reporter gene, it's property of synthesizing melanin, which can be examined by magnetic resonance imaging(MRI), is used to evaluate the tyrosinase gene's expression. The aim of this study was to search a way to evaluate the results of gene expression by MRI in vitro.METHODS:The plasmid of pcDNA3tyr which carried the full-length cDNA of tyrosinase gene was transfected into HEK293 cell by lipofectin. To observe the MRI signals of expressed melanin, the transfected cells were scanned by MRI sequences of T1WI, T1WI/SPIR and T2WI. On the other hand, fontana stain was used to search for melanin granules in transfected cells, RT-PCR method was used to search for cDNA of tyrosinase gene.RESULTS:(1) Plasmids of pcDNA3tyr could be transfected into HEK293 cells and could synthesize a large amount of melanin. The synthetic melanins of 106 cells, which had been transfected 5μg, 10μg, 20μg plasmids of pcDNA3tyr separately, were all sufficient to be detected by MRI and appeared high signal in MRI T1WI、T1WI/SPIR、T2WI sequences. The signal intensities of MRI imaging were related to the amounts of transfected plasmids positively. (2) The melanin granules could be found in HEK293 cells by Fontana stain. (3) The cDNA fragment of tyrosinase gene could be detected in transfected HEK293 cells by RT-PCR.CONCLUSION:The fact that MRI could detect the synthet ic melanin of HEK293 cells, which controlled by expression of exogenous gene, demonstrates that medical imaging connecting with molecular biology technology can evaluate the result of gene expression in vitro.  相似文献   

2.
AIM: To investigate the effects of P34H gene silencing on the expression of P34H and activity of hyaluronidase (HYD) in mouse sperm.METHODS: The recombinant plasmid series of P34H targeted short hairpin RNA (shRNA) were constructed by GV248 plasmids vector. These recombinant plasmids were transformed into DH5α competent cells, and the plasmids were taken from DNA sequencing analysis. The HEK293T cells were co-transfected with shRNA and lentiviral packaging plasmids. The 3 kinds of recombinant lentiviruses and negative control lentiviruses were used to inject into the mouse epididymis and the expression of P34H at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The location of P34H protein on the mouse spermatozoa was determined by indirect immunofluorescent staining using P34H antibody. The positive rate and activity intensity of HYD was detected by modified sodium hyaluronate-gelatin membrane. RESULTS: DNA sequencing analysis confirmed that the 3 P34H-shRNA sequences were successfully inserted into the lentiviral vectors. P34H expression in epididymis tissue was significantly decreased at both mRNA and protein levels compared with those of the non-transfected and normal control vectors (P<0.05). The GV-P34H-shRNA-1 played a significant role in reducing the percentage of P34H positive rate and the activity of HYD in mouse sperm. The silencing effect did not significantly differ between the non-transfected and normal control vectors. CONCLUSION: Silencing of P34H significantly inhibits the percentage of P34H positive rate and the activity of hyaluronidase in mouse sperm.  相似文献   

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4.
AIM: To investigate the effect of PI3K/Akt pathway on endoplasmic reticulum (ER) stress-mediated glucose-regulated protein 78 (GRP78) induction in human embryonic kidney 293 cells (HEK293) cells.METHODS: PI3K inhibitor LY294002, dominant negative kinase-dead mutant vector for HA-Akt (K179M) and Akt1 siRNAs were used to block the PI3K/Akt pathway under ER stress. Constitutively active expression vectors for Akt (myr-HA-Akt) were used to up-regulate Akt activity under ER stress. The effects of PI3K/Akt on ER stress-mediated GRP78 induction in HEK293 cells were determined by Western blotting and RT-PCR. RESULTS: GRP78 induction was inhibited by LY294002, Akt1 (K179M) and Akt1 siRNA, but was increased by myr-Akt1 in dithiothreitol-and thapsigargin-treated HEK293 cells. However, both myr-Akt2/3 and Akt2/3 siRNA had no effect on GRP78 induction in HEK293 cells under ER stress. Furthermore, the PI3K/Akt pathway didnt regulated GRP78mRNA induction but increased GRP78 protein stability.CONCLUSION: PI3K/Akt promotes GRP78 accumulation through increasing the stability of GRP78 protein in HEK293 cells under ER stress.  相似文献   

5.
LI Wen-yu  ZHANG Yuan  HE Dong-mei 《园艺学报》2003,19(12):1596-1600
AIM:To explore the effect of hTERT antisense phosphorothioate oligodeoxynucleotide (ASODN) on apoptosis induced by chemotherapeutic drugs in Jurkat cell lines. METHODS:Cell viability was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation, DNA gel electrophoresis and flow cytometry analysis. RESULTS:The survival rates of Jurkat cells cultured with daunorubicin, vincristin, and etoposide, respectively were similar with that cultured with those chemotherapic drugs plus hTERT ASODN. The survival rates of Jurkat cells cultured with cis-diamminedichicloroplatinum(DDP) added 24 hours later were higher than that cultured with hTERT ASODN and DDP added 24 hours later. The survival rates of Jurkat cells cultured with DDP were similar with that cultured with hTERT SODN and DDP. In morphological observation of apoptotic cells using Giemsa staining, cells displayed classic apoptotic changes treated with DDP or DDP combined with hTERT ASODN or SODN at 48 hours. Agarose gel electrophoresis of genomic DNA from Jurkat cells treated with ASODN and DDP combination for 48 hours showed typical DNA "ladder". Neither the DNA from Jurkat cells treated with SODN plus DDP nor the DNA from the cells treated with DDP alone showed ladder pattern. Apoptosis rates of Jurkat cells treated with DDP for 48 hours after 24 hours of exposure to ASODN significantly increased. There was significant difference in the percentage of apoptotic Jurkat cells between hTERT ASODN plus DDP and SODN plus DDP or DDP alone, respectively. CONCLUSION:The hTERT ASODN complementary to the translation initiation region of hTERT mRNA enhanced DDP-induced apoptosis in Jurkat cells.  相似文献   

6.
XIAO Jian-min  FU Hui 《园艺学报》2013,29(10):1788-1792
AIM：To investigate the effects of carvedilol and other β-blockers on ryanodine receptor 2(RyR2)-mediated spontaneous calcium oscillation.
METHODS：HEK293 cells, which steadily and inducibly expressed RyR2, were generated using the Flp-In T-REx Core Kit. Single rat ventricular myocyte was isolated by the method of collagenase. The extracellular Ca2+ concentration was increased step by step to induce calcium oscillation. Single cell calcium imaging was used to test the effects of carvedilol and other β-blockers on RyR2-mediated spontaneous cal-cium oscillation in cardiac cells and HEK293 cells. RESULTS：Carvedilol at concentration of 30 μmol/L obviously suppressed the spontaneous cal-cium oscillation in cardiac cells and HEK293 cells expressing RyR2. The inhibitory rate was (6530±230)% and (6908±530)%, respectively. Metoprolol and other β-blockers had no effects on spontaneous calcium oscillation in cardiac cells and HEK293 cells. CONCLUSION：Carvedilol is the only β-blocker that suppresses RyR2-mediated spontaneous calcium oscillation. This may be one of the mechanisms that carvedilol is better than other β-blockers in reducing the mortality of heart failure.  相似文献   

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8.
AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification (TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK, then DH5α E.coli was transformed with the recombinant plasmid. After identified by PCR, digestion with restriction endonuclease and sequencing, the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didnt influence the localization of exogenous PRAK.  相似文献   

9.
AIM: To investigate the effect of homocysteine(Hcy) on the apoptosis of endothelial cells (EC). METHODS: First-passaged human umbilical vein endothelial cells (hUVEC) were cultured with M199 containing 3 mmol/L Hcy. hUVEC apoptosis was detected as follow: demonstration of nuclear changes by Hoechst 33258 staining, agarose gel electrophoresis of DNA fragments, detection of apoptotic cells by flow cytometry following Annexin V-PI doubled stain, Western blot for P53 and Bax protein detection and colorimetry detecting caspase-3 activity. RESULTS: Compared with control, homocysteine induced characteristic apoptotic changes in hUVEC. The chromosomal DNA of hUVEC appeared "DNA ladder" by agarose gel electrophoresis. Apoptotic cells were increased significantly (P<0.01, n=3). Hcy promoted the expression of protein Bax, P53 (P<0.01, n=3) and enhanced the activity of caspase 3 (P<0.05, n=3). CONCLUSION: Homocysteine induces apoptosis in cultured hUVEC.  相似文献   

10.
AIM: To investigate the effects of ErbB2 overexpression on growth and invasiveness in cultured MCF-7 cell line. METHODS: Retrovirus containing ErbB2 gene was transfected into MCF-7 cells and ErbB2 expression was detected by Western blotting. Proliferation and invasive assays were carried out. Cells overexpressed ErbB2 and its control, AP2, were used in the experiment. RESULTS: ErbB2 was overexpressed in MCF-7 cells after transfection. In vitro, cells overexpressed ErbB2 showed highly proliferated and highly invasive characteristics compared to the control cells. CONCLUSION: Overexpression of ErbB2 promotes cell proliferation and enhances invasiveness in MCF-7 cells. Inhibition of signaling induced by ErbB2 might be a novel strategy for the therapeutics of cancer with ErbB2 overexpression.  相似文献   

11.
AIM: To investigate the regulatory mechanisms of microRNA-29a (miR-29a) on the expression of Bcl-2 and Mcl-1 in rat cardiomyocytes (CM cells). METHODS: The CM cells were isolated from the hearts of newborn rats and transfected with miR-29a mimic (100 nmol/L) by Lipofectamine RNAiMAX. The expression of Bcl-2 and Mcl-1 at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and Western blotting. The luciferase assay was performed in HEK293T cells and CM cells, which were co-transfected with plasmid DNA and miRNA using Lipofectamine 2000. RESULTS: Transfection of miR-29a mimics significantly reduced the expression levels of Bcl-2 and Mcl-1 in CM cells as compared with the control cells (P<0.05). In addition, HEK293T cells co-transfected with miR-29a mimic and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid significantly reduced the luciferase activity as compared with control group (P<0.05). While CM cells transfected with miR-29a inhibitor and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid in succession, the luciferase activity was increased inversely (P<0.05). CONCLUSION: miR-29a may regulate apoptosis by targeting the bcl-2 and mcl-1 genes.  相似文献   

12.
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13.
AIM: To observe the effects of Arid2gene on cell proliferation and cell cycle by interference of endogenous Arid2 expression in hepatoma cells. METHODS: Three pairs of shRNA targeting Arid2gene were cloned into a shuttle vector to construct recombinant adenovirus plasmids. HEK293 cells were transfected with the recombinant adenovirus plasmids. After several rounds of the package and amplification, the high-titer adenoviruses AdsiArid2-1~3 were obtained. To verify the inhibitory effects of AdsiArid2 adenoviruses, Western blotting was used to detect the endogenous Arid2 protien expression in SMMC-7721 cells. Cell growth and cell cycle analysis were carried out by MTS assay and flow cytometry. RESULTS: High- titer recombinant adenovirus of siArid2 were successfully obtained, and named AdsiArid2-1~3, among which the AdsiArid2-3 had the best inhibitory effects. MTS assay showed that the absorbance values at 490 nm were increased at 72 h and 96 h after transduction compared with the mock and Adsicontrol groups. These data indicated that knockdown of Arid2 promoted the proliferation rate of SMMC-7721 cells(P<0.05). Moreover, the flow cytometry analysis revealed that the G1-phase distribution at 72 h in AdsiArid2 group was lower than that in mock group and Adsicontrol group. In contrast, the S-phase distribution in AdsiArid2 group was much higher than that in mock group and Adsicontrol group. CONCLUSION: The recombinant plasmids and recombinant adenovirus were successfully constructed. shRNA-mediated knockdown of Arid2 promotes the proliferation and the transition from G1 phase to S phase of hepatoma cells.  相似文献   

14.
AIM: To investigated the characterization and biological function of P53 binding element in the regulation of NOD8 gene. METHODS: Using the method of bioinformatics, we found a completely preserved P53 binding site in NOD8 core promoter both in humans and chimpanzees. NOD8 gene was amplified by PCR from human cDNA and correctly connected into the vector p NOD8 (760 bp)-EGFP-C2/ mp NOD8 (750 bp)-EGFP-C2. The constructed plasmids p NOD8 (760 bp)-EGFP- NOD8 and mp NOD8 (750 bp)-EGFP- NOD8 were transiently transfected into the cell line HEK293 by JetPeiTM and treated with pifithrin alpha (PFT-α,an inhibitor of P53) at different concentrations for 24 h. The mRNA and protein expression levels of NOD8 were detected by RT-PCR and Western blotting. In addition, chromatin immunoprecipitation (ChIP) assay was performed to determine the binding of P53 to the NOD8 promoter after recombinant plasmid p NOD8 (760 bp)-EGFP was transfected into HEK293 cells. RESULTS: The plasmids p NOD8 (760 bp) -EGFP- NOD8 and mp NOD8 (750 bp)-EGFP- NOD8 were successfully constructed and conformed by restriction digestion and sequence analysis. The results of ChIP confirmed that P53 bound to the promoter of NOD8 . The mRNA expression of NOD8 in the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 was stronger than that in the cells transfected with control vector pEGFP-C2 (P<0.05). Furthermore, the mRNA expression of NOD8 was reduced in HEK293 cells transfectnt with the mutant plasmid mp NOD8 (750 bp)-EGFP- NOD8 compared with the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 . Meanwhile, PFT-α inhibited the mRNA expression of NOD8 in the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 in a concentration-dependent manner, and PFT-α at concentration of 90 μmol/L was the most effective in inhibiting NOD8 mRNA expression (P<0.01). As expected, the protein expression of NOD8 in pNOD8 (760 bp)-EGFP- NOD8 group significantly increased compared with that in pNOD8 -C2 group, the protein expression of NOD8 in mp NOD8 (750 bp)-EGFP- NOD8 group or pNOD8 (760 bp)-EGFP- NOD8 + PFT-α group was dramatically decreased compared with that in p NOD8 (760 bp) -EGFP- NOD8 group. CONCLUSION: The results suggest that the P53 binding site is critical for the regulation of NOD8 gene and there is positive feedback regulation between P53 binding site and NOD8 , which may maintain efficient balance between defense and self-inflicted injury in response to the invasion of pathogen.  相似文献   

15.
AIM: To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 (IPO8) gene on its mRNA expression. METHODS: A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS：The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT,CCT/- and -/-, and the gene frequencies were 1837%, 5510% and 2653%, respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote. CONCLUSION：The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8, thus affecting the gene expression.  相似文献   

16.
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17.
AIM: To prepare histones with different methods and to observe their effects on cell viability and cytokine release by macrophages in vitro.
METHODS: Prokaryotic expression plasmids of histone H3 and histone H4 were constructed by cloning methods. The histones containing GST-tag or His-tag (GST-H3, GST-H3, His-H3, His-H4) were expressed and purified. The histones from eukaryotic cells (RAW264.7 and 293F cells) were extracted with the high salt method. RAW264.7 cells were treated with histones (7.5~50 mg/L) for 4 h and the cell vitality was examined by MTT assay and flow cytometry. The concentrations of IL-6 and TNF-α in the culture supernatants of RAW264.7 cells treated with histones were also detected.
RESULTS: His-H3/H4 and the histones from eukaryotic cells significantly reduced the viability of RAW264.7 cells and induced apoptosis. The effects of histones from different sources on the releases of IL-6 and TNF-α were different.
CONCLUSION: His-H3/His-H4 expressed by prokaryotic plasmids and the histones extracted from eukaryotic cells affect the vitality of macrophages as well as induce late apoptosis and necrosis. Histone may involve in the inflammatory process by promoting the release of inflammatory cytokines.  相似文献   

18.
AIM：To investigate the effect of caspase-8 small hairpin RNA (shRNA) on attenuating apoptosis of human mesenchymal stem cells (hMSCs). METHODS：Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed. Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR. The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid, which was linearized and transfected into HEK293 cells for packaging and amplification of the recombinant adenovirus rAd-Cap8 shRNA. The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting. Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hMSCs under the conditions of serum deprivation and hypoxia. The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR. RESULTS：The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR. The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant adenovirus (rAd)-Cap8 shRNA successfully. rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 expression in hMSCs. Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the apoptotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia, with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2. CONCLUSION：Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia.  相似文献   

19.
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 108.3 and 108.1 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.  相似文献   

20.
AIM: To investigate the potential role of Scy homology 2 domain-containing protein tyrosine phosphatase 2(SHP-2) in apoptosis of 293T cells induced by serum deprivation.METHODS: Transfection of plasmids into 293T cells was performed by liposome protocol. The viability of 293T cells was detected by MTT assay. On day 3 after removing serum the morphological changes of 293T cells were observed by using a transmission electron microscope, apoptosis rate was detected by flow cytometry and the expression of caspase-3 was measured by immunohistochemisty.RESULTS: The apoptosis rate in wild type SHP-2 (WT) group was obviously lower than that in the SHP-2C459S catalytically inactivated group. At the same time, expression of caspase-3 showed the similar results. CONCLUSION: SHP-2 may actively participate in the signal transduction pathway of apoptosis and play a positive role in cell survival. The underlying mechanism of apoptotic inhibition may be caspase-3 dependent.  相似文献   

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