Preparation of the first national reference standard for determination of HEK293 cell DNA content / 中国生物制品学杂志

@#Objective To prepare a national reference standard for the quantification of HEK293 cell DNA content,so as to provide a support for the determination of residual DNA in HEK293 cells in the industry.Methods HEK293 cell DNA prepared using Genomic-tip 500/G and genomic DNA purification reagents was used as source materials,and the purity and content were assessed using ultraviolet spectrophotometry and agarose gel electrophoresis.After dilution to approximately 100 ng/μL,the DNA was aliquoted at 160 μL/tube.Five different laboratories were organized for collaborative calibration by using ultraviolet spectrophotometry, and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A_(260)/A_(280) ratios between 1.8 and 2.0 and displayed a single band on electrophoresis,meeting the specified criteria.Collaborative calibration across five laboratories yielded 78 valid data points with an average content of 104.8 ng/μL,a relative standard deviation(RSD) of 4.2%.The 95% confidence interval for the mean was 103.8—105.8 ng/μL,and the 95% reference range for single measurements was 96.0—113.6 ng/μL.The average confidence limit rate was 1.0%,and the recommended storage condition was-80 ℃.Applicability studies were conducted using two different models of fluorescence quantitative PCR instruments.The reference standard exhibited good applicability within the range of 0.3—3 000 pg/reaction,with amplification efficiencies of 101% and 95%,and R~2 values of 0.999 2 and 0.999 5 for the standard curves,respectively.Conclusion This batch of HEK293 cell DNA national reference standard meets all required specifications and can be utilized as a national reference standard for fluorescence quantitative PCR detection,with a certified content of 104.8 ng/μL,assigned batch number 270039-202301.

Effects of manipulating lactate dehydrogenase gene on metabolism of HEK-293 and production of human adenovirus / 生物工程学报

Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured in vitro, the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out ldha gene and overexpression of ldhb and ldhc genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, ldhc gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O2 ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.

Hyperosmotic stress and perfusion culture strategies increase the yield of recombinant adenoviral vector produced by HEK 293 cells / 生物工程学报

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×1010 IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.

The effect of HMS-01 on stably expressed hERG channel currents in HEK293 cells detected with the manualpatch clamp method / 药学实践杂志

Objective To test the cardiac toxicity of new compound HMS-01 and evaluate the safety profile for clinical trials. Methods Manualpatch clamp method was used to measure human Ether-a-go-go-Related Gene (hERG) potassium channel currents with different concentrations of HMS-01. Cisapride was selected as the positive control drug. HMS-01 was diluted to the concentration of 0.3, 1, 3, 10 and 30 µmol/L and applied to the cells. The changes in electrical currents were recorded and the inhibition rate was calculated. Results At the highest concentration of 30µmol/L, the inhibitory rate of HMS-01 on hERG channel was less than 30%. There was no obvious inhibitory effect compared with cisapride. Conclusion Compared with the cisapride, HMS-01 has no obvious inhibitory effect on hERG channel and has no cardiotoxicity.

Testosterona inhibe la actividad de la aldosterona sintasa silvestre y quimérica in vitro

Background: Familial hyperaldosteronism type I is caused by the generation of a chimeric aldosterone synthase enzyme (ASCE) which is regulated by ACTH instead of angiotensin II. We have reported that in vitro, the wild-type (ASWT) and chimeric aldosterone synthase (ASCE) enzymes are inhibited by progesterone and estradiol does not affect their activity. Aim: To explore the direct action of testosterone on ASWT and ASCE enzymes. Material and Methods: HEK-293 cells were transiently transfected with vectors containing the full ASWT or ASCE cDNAs. The effect of testosterone on AS enzyme activities was evaluated incubating HEK-cells transfected with enzyme vectors and adding deoxycorticosterone (DOC) alone or DOC plus increasing doses of testosterone. Aldosterone production was measured by HPLC-MS/MS. Docking of testosterone within the active sites of both enzymes was performed by modelling in silico. Results: In this system, testosterone inhibited ASWT (90% inhibition at five pM, 50% inhibitory concentration (IC50) =1.690 pM) with higher efficacy andpotency than ASCE (80% inhibition at five pM, IC50=3.176 pM). Molecular modelling studies showed different orientation of testosterone in ASWT and ASCE crystal structures. Conclusions: The inhibitory effect of testosterone on ASWT or ASCE enzymes is a novel non-genomic testosterone action, suggesting that further clinical studies are needed to assess the role of testosterone in the screening and diagnosis of primary aldosteronism.

Down-regulation of Protein Phosphatase 2A Catalytic Subunit Involved in Mitochondria Fission/fusion Dynamics Imbalance and Functional Impairment Induced by Human tau / 中国医学科学院学报

To explore whether the downregulation of protein phosphatase 2A catalytic subunit(PP2Ac)involved in the pathogenesis of mitochondria fission/fusion dynamics and functional imbalance induced by human tau accumulation. After cotransfection with mito-dsRed plasmids and pIRES-eGFP-tau40 plasmids 48 hours,the rat primary hippocampal neurons were observed with a laser scanning confocal microscope for their changes in shape and distribution of mitochondria.The expressions of mitochondria fission/fusion protein and PP2Ac and PP2Ab were detected by Western blotting.Furthermore,the shape and distribution of mitochondria of rat primary hippocampal neuron and wild type 293wt cells were assayed 48 hours after co-transfection with siPP2Ac-EGFP plasmids and mito-DsRed plasmids,and the fission/fusion dynamics of 293wt cells was captured with live cell time-lapse imaging after co-transfection with siPP2Ac plasmids and mito-Dendra2 plasmids.After transfection with siPP2Ac plasmids,the relative level of mitochondria fission/fusion protein of 293wt cells was assayed by Western blotting,and mitochondria membrane potential was detected by JC-1 staining,and the cellular viability was measured by CCK8 assay.Finally,the shape and distribution and membrane potential of mitochondria of HEK293 cells with stable transfection of htau40(293htau)were detected after co-transfection with PP2Ac and mito-dsRed plasmids. Human tau40 expression decreased distribution of mitochondria and significantly lowered PP2Ac level in primary hippocampal neuron(=4.814, =0.0086).Down-regulation of PP2Ac caused mitochondria elongation and perinuclear accumulation in primary hippocampal neuron and 293wt cells;in addition,down-regulation of PP2Ac in 293wt cells significantly increased mitochondria fusion rate(=2.857, =0.0074)and the levels of mitochondria fusion protein mitofusin(MFN)1(=6.768, =0.0025),MFN2(=3.121, =0.0035),and optic atrophy 1(=3.775, =0.0199);however,the levels of dynamin-like protein-1 and Fis1 remained unchanged.The down-regulation of PP2Ac in 293wt cells led to the significant decrease in mitochondria membrane potential(=2.300, =0.0270)and cell viability(=6.249, <0.0001).Finally,up-regulation of PP2Ac attenuated the abnormalities in the shape,distribution and function of mitochondria in the 293htau cells. Down-regulation of PP2Ac is involved in the abnormal shape and distribution of mitochondria and its dysfunction induced by human tau40 in rat primary hippocampal neurons and HEK293 cells.

Effects of Daurisoline on L-Type Calcium Channel CaV1.2 Expressed in HEK293 Cells / 中国药师

Objective:To investigate the effects of daurinsoline (DS) on L-type calcium channel Cav1.2 expressed in HEK293 cells.Methods:Cav1.2 was transferred into HEK293 cells using Lipofectamine 2000, and the effects of DS on Cav1.2 currents (ICav1.2) were analyzed by whole-cell patch clamp techniques. Results:DS at 1,3 and 10 μmol·L-1could inhibit the ICav1.2in HEK293 cells in a dose-dependent manner. The inhibitory rate was(14.68 ± 4.02) %,(32.37 ± 6.63) % and(59.63 ± 5.23) %,respectively. The inhibitory rate of DS at 3 μmol·L-1was 40 % of that of 3 μmol·L-1isradipine(a L-type calcium channel blocker). Conclusion:DS can inhibit the ICav1.2in HEK293 cells in a dose-dependent manner and the inhibition of DS is weaker than that of isradipine.

The F309S mutation increases factor VIII secretion in human cell line

OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate) on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIÄB proteins in HEK 293 cells, but the same effect was not seen for FVIIIÄB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

Inhibitory effects of neferine on Nav1.5 channels expressed in HEK293 cells / 华中科技大学学报（医学）（英德文版）

Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition (IC50) being 26.15 μmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 μmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.

CdCl2 induces autophagy and apoptosis in HEK293 cells / 中国药理学与毒理学杂志

OBJECTIVE To evaluate the possibility that CdCl2 induces autophagy and apoptosis in HEK293 cells,and the role of extracellular regulated protein kinases (ERK1/2) and AKT proteins in autophagy. METHODS Green fluorescence protein(GFP)-light chain 3B(LC3B)expression plasmid was transfected into HEK293 cells. After 24 h,HEK293 cells were induced with CdCl2 2,4,8 and 10μmol·L-1 for 12 h. The expression of GFP-LC3B was detected by fluorescent microscopy. HEK293 cells were induced with CdCl2 2,4,8 and 10μmol · L-1 without transfection of GFP-LC3B for 12 h while autophagic vacuoles were observed by transmission electron microscopy. The expression of LC3B-Ⅱ/Ⅰproteins and the phosphorylation levels of ERK1/2 and AKT were analyzed by Western blotting. Apoptosis was detected by flow cytometry microscopy. HEK293 cells were treated with 3-MA 20μmol · L-1+CdCl2 10 μmol · L-1 for 12 h before cleaved caspase 3 protein was detected by Western blotting. RESULTS When HEK293 cells were exposed to CdCl2(≤10μmol · L-1)for 12 h,cytoplasmic GFP-LC3B punctuates were observed under the fluorescence microscope,and autophagic vacuoles were observed under an electron microscope. The expression of LC3B-Ⅱ/Ⅰ,p-ERK1/2 and p-AKT proteins was significantly increased in CdCl2-induced cells(P<0.05,P<0.01). Moreover,apoptosis was observed. The addition of 3-MA 20μmol · L-1+CdCl2 10μmol · L-1 enhanced apoptosis. Cleaved capase 3 protein expression was significantly increased(P<0.01). CONCLUSION CdCl2(≤10μmol·L-1)can induce autophagy in HEK293 cells. ERK1/2 and AKT proteins might be associated with the activation of autophagy that is accompanied by apoptosis,suggesting that autophagy can inhibit apoptosis at certain concentrations of CdCl2.

Effects of extracellular potassium on expression of HERG gene nonsense mutant L539 fs/47 / 中国病理生理杂志

AIM:To study the effects of extracellular potassium on the protein expression of wild-type HERG and its mutant L539fs/47.METHODS:Wild-type HERG (WT) or its mutant HERG-L539fs/47 (MT) were transfected into HEK293 cells for 36 h.The cells were incubated in different media containing 0.8, 4.3 or 10 mmol/L potassium.Af-ter 6 h of incubation, the protein expression of HERG was detected by flow cytometry.After 12 h of incubation, the locali-zation and quantity of the proteins were detected by laser confocal imaging and Western blot.RESULTS: Different from the retention of mutant protein in cytoplasm, wild-type HERG protein was mainly distributed in the cell membrane.The 2 proteins both increased with the changes of extracellular potassium.Flow cytometry showed that the fluorescence in the 2 groups both increased with the changes of extracellular potassium ( P<0.01 ) .The fluorescence in WT group was signifi-cantly higher than that in MT group (P<0.01).Western blot showed that mutant HERG protein included only one 60 kD band, different from the 135 kD and 155 kD bands in wild-type HERG, which were affected by the changes of extracellular potassium (P<0.05).CONCLUSION:The retention of HERG mutant L539fs/47 protein in the cytoplasm is more than wild-type HERG.Chronic high extracellular potassium keeps the stability of wild-type and mutant HERG proteins on the cell membrane.Chronic low potassium reduces the expression of HERG channel proteins in a time-dependent manner.

Establishment and identification of HEK293 cell lines with stable and high expression of NTCP / 中国药理学通报

Abstrate:Aim To construct HEK293 cell line with stable and high expression of sodium taurocholate cotransporting polypeptide (NTCP ) efficiently and rapidly.Method Vector expressing EGFP-NTCP fusion protein was constructed and verified by DNA sequencing.The pEGFP-NTCP expression vector was transfected into HEK293 cells by FuGENE 6 transfection reagent. The transfected cells with high expression of green fluorescent protein were selected using fluorescence microscope for screening of G418 for 14 days to obtain cell lines stably and highly expressing NTCP.NTCP expression was detected by RT-PCR,qRT-PCR, Western-blot and the uptake experiment of taurocholic acid.Re-sult RT-PCR,qRT-PCR,Western-blot and the uptake experi-ment revealed that compared to the control cells,the expression of NTCP was significantly positive (P<0.01)in stable trans-fected cells showing green fluorescence (P<0.05 ).Conclusion The HEK293 cell line with stable and high expression of NTCP has been established efficiently and rapidly,which provides a cellular model for the study of the mechanism of the uptake of bile acid derivatives.

Production of Lentiviral Vectors Encoding Recombinant Factor VIII Expression in Serum-Free Suspension Cultures

ABSTRACT Lentiviral vector-mediated gene transfer offers several advantages over other gene delivery vectors when considering gene and cell therapy applications. However, using these therapies in clinical applications involves large-scale vector production in an efficient and cost-effective manner. Here we describe a high yield production of a lentivirus encoding recombinant factor VIII in a scalable and GMP-compliant culture system, based on serum free suspension cultures and transient transfection with an inexpensive reagent, polyethylenimine (PEI), reaching a total viral yield of 2.48x108 particles.

Establishment and functional analysis of a cell model expressing the C-type lectin receptor Langerin / 中华皮肤科杂志

Objective To establish a cell model expressing the Langerhans cell-specific C type lectin receptor Langerin in vitro.Methods The cDNA of Langerin was obtained by PCR and cloned into a eukauotic green fluorescent protein (EGFP) expression vector pEGFP-C 1 with EGFP located in the N terminal region of the Langerin gene.Then,the recombinant plasmid was transfected into a human embryonic kidney carcinoma cell line HEK293T.Subsequently,laser confocal microscopy was performed to observe the expression of EGFP-Langerin fusion protein,and flow cytometry to measure the expression of Langerin.Laser confocal microscopy was also conducted to visualize the recognition and endocytosis of dust mite antigen (nDer p 2) by Langerin.Results PCR and Western blot confirmed the successful transfection of HEK293T cells with the recombinant plasmid as well as the expression of Langerin in the transfected cells.As flow cytometry revealed,the expression level of Langerin in transfected HEK293T cells was increased by 43％ compared with untransfected cells.Laser confocal microscopy showed that green fluorescein-labeled Langerin was successfully expressed,and could bind to and endocytose the red fluorescein-labeled antigen nDer p 2.Conclusions The fusion protein EGFP-Langerin expressed in HEK293T cells showed the distribution characteristic of cell surface receptors,and could bind to and endocytose allergens.

Construction of expression vector containing rat rCB1 gene and its influence on the apoptosis in human cervical cancer CaSki cell line / 中国实验动物学报

Objective To construct rCB1 gene eukaryotic expression vector, detect its expression in the cell, and explore its influence on apoptosis in human cervical cancer CaSki cells.Methods The total RNA was extracted from rat brains.The rCB1gene was amplified by RT-PCR.The pcDNA3.1(+)-rCB1 was constructed by enzyme digestion, purifi-cation, bind the PCR purification products and pcDNA3.1 (+) DNA.The pcDNA3.1 (+)-rCB1 plasmid was transfect-ed into HEK293 and CaSki cells by liposomes.The expression and localization of rCB1 were detected by Western blot and immunofluorescence combined with confocal laser scanning microscopy.The apoptosis rate of CaSki cells was detected by flow cytometry.The expression of rCB1, Bcl-2, Bax and Bad was detected by Western blot and real-time fluorescence quantitative RT-PCR (qRT-PCR).Results The 5300 bp pcDNA3.1(+) and 1500 bp rCB1 were obtained after diges-ting the pcDNA3.1 ( +)-rCB1.The result of sequencing was in agreement with the sequence of rCB1 gene ( NM_012784.4 ) .The rCB1 expressed in the membrane and cytoplasm when pcDNA3.1 (+)-rCB1 plasmid was transfected into HEK293 cells.The apoptosis rate of rCB1 group was increased compared with the blank group when pcDNA3.1 (+)-rCB1 plasmid was transfected into CaSki cells (P<0.05).Compared with the blank group, rCB1 gene upregulated the expres-sion of Bax and Bad, and suppressed the expression of Bcl-2.The statistical difference was significant ( P <0.05). Conclusions The pCDNA3.1(+)-rCB1 eukaryotic expression vector is constructed successfully.It is found that rCB1 is expressed in membrane and cytoplasm of HEK293 cells.rCB1 can significantly promote the apoptosis in cervical cancer CaSki cells by up-regulating the expression of Bax and Bad, and down-regulating the expression of Bcl-2 as well.

Salvia fruticosa reduces intrinsic cellular and H2O2-induced DNA oxidation in HEK 293 cells; assessment using flow cytometry

<p><b>OBJECTIVE</b>To investigate the role of water-soluble extract of Salvia fruticosa (Greek sage) (S. fruticosa) leaves in reducing both intrinsic cellular and H2O2-induced DNA oxidation in cultured human embryonic kidney 293 cells. S. fruticosa, native to the Eastern-Mediterranean basin, is widely used as a medicinal herb for treatment of various diseases.</p><p><b>METHODS</b>Dried leaves of S. fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations. Each mL of the preparation contained (7.1±1.0) mg of extract. HEK-293 cells were incubated in one set with S. fruticosa extract in the presence of 0.1 mmol/L H2O2, and in the other set with the addition of the extract alone. The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivatization of 8-oxoguanine moieties. The fluorescence was measured using flow cytometry technique.</p><p><b>RESULTS</b>Cells incubated 3 h with 150 µL extract and exposed to 0.1 mmol/L H2O2 showed lower intensity of fluorescence, and thus lower DNA oxidation. Moreover, cells incubated 3 h with 100 µL of the extract showed lower intensity of fluorescence, and thus lower intrinsic cellular DNA oxidation compared to control (without S. fruticosa).</p><p><b>CONCLUSIONS</b>The results from this study suggest that the water-soluble extract of S. fruticosa leaves protects against both H2O2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells.</p>

More than 3 ku proteins in chicken egg extract up-regulate expression of pluripotent genes Oct-3/4 and Nanog / 中国组织工程研究

BACKGROUND:Reprogramming somatic cells to generate pluripotent stem cells has a wide application in biomedical research. OBJECTIVE:To analyze the effect of different molecular weight proteins in chicken egg-white extract to elevate expression of pluripotent genes Oct-3/4 and Nanog in 293T cells. METHODS:The extracts of chicken egg-white were separated into more than 3 ku and less than 3 ku ingredients to be used for co-culture with 293T cells. There were four groups, 1×105 293T cells per wel , total 500μL. In the control group, 500μL culture medium was added;in the other three groups, 500μL chicken egg-white extract, more than 3 ku and less than 3 ku ingredients were respectively added. Quantitative PCR was used to determine the relative expression levels of pluripotent genes Nanog and Oct-3/4 in 293T cells. RESULTS AND CONCLUSION:By using co-culture method, more than 3 ku ingredients have a role to increase the expression of pluripotent genes Oct-3/4 and Nanog, but less than 3 ku ingredients cannot elevate the expression of pluripotent genes. This indicates that the ingredient of chicken egg-white extract to elevate the expression of pluripotent genes is more than 3 ku proteins.

Establishment of a recombinant cell line that stably expresses rat purinergic receptor P2X4 and verification of its function / 中国药理学与毒理学杂志

OBJECTIVE Toestablisharecombinantcelllinethatcanstablyexpressratpurinergic P2receptorP2X4(rP2X4R).METHODS ToconstructgreenfluorescentproteinandrP2X4recombinant plasmid (pEGFP-N1-rP2X4),lipofectamine was used to transfect pEGFP-N1-rP2X4 into human embry-onic kidney (HEK293)cells that were screened with G41 8 (1 g·L-1 ).The quantitative expression of rP2X4 receptor was verified by qRT-PCR and Western blotting analysis.Whole-cell patch clamp record-ing was used to investigate the function of the stably expressed rP2X4 receptor. RESULTS The sequence of plasmid pEGFP-N1-rP2X4 was verified by PubMed Blastn comparison.qRT-PCR and Western blotting analysis demonstrated that the expression of rP2X4 receptor in HEK293-pEGFP-N1-rP2X4 cell lines remained stable after 25 generations (P1 ,P3,P5,P10,P15,P20 and P25).Whole-cell patch clamp recording experiments showed that the rP2X4 receptor agonist,purine-5′-triphosphate (ATP,3.0 μmol·L-1 ),could activate rP2X4 receptors in HEK293-pEGFP-N1-rP2X4 cell lines.Specific activating current could be blocked by non-selective rP2X4 receptor antagonist TNP-ATP (30.0μmol·L-1).CONCLUSION rP2X4receptorisstablyexpressedinHEK293-pEGFP-N1-rP2X4cell line and maintains stable expression and function within 25 continuous generations.The establish ment of HEK293-pEGFP-N1-rP2X4 cell line can contribute to further investigations of the roles of rP2X4 receptors in neuropathic pain.

Construction of Hi FGF2 eukaryotic expression plasmids and its over-expression induced cell apoptosis / 中国药理学通报

Aim To construct eukaryotic expressing plasmid of hi FGF2 ( high molecular weight isoform fi-broblast growth factor-2,hi FGF2) gene and to investi-gate its effect on apoptosis after its overexpression in HEK293 cells. Methods The DNA template primer was designed and synthesized. The pDsRed1-N1 plas-mids were digested by the restriction enzymes of Nhel and Hind III. The hi FGF2 was ligated with linearized pDsRed1-N1 by T4 DNA Ligase. The recombinant plasmid was identified by endonuclease digestion and sequenced. The recombinant hi FGF2 plasmid was transient transfected into HEK293 cells by Lipofectami-neTM 2000 Reagent. The transfection efficiency was de-tected by fluorescence inversion microscope. The cell apoptosis was detected by Annexin V-FITC/PI apopto-sis detection kit with flow cytometry analysis. Results The pDsRed1-N1 eukaryotic expression vector was consistent with the design. The recombinant hi FGF2 plasmid was transfected in HEK293 cells. The trans-fection rate was more than 70%. The FITC/PI dyeing rate in hi-FGF2 over-expression HEK297 cells was a-bout ( 29. 12 ± 2. 81 )%. Conclusions pDsRed1-N1 eukaryotic expression vector is successfully constructed and transfected into HEK293 cells. Over-expression of hi FGF2 induces cell apoptosis.

Salvia fruticosa reduces intrinsic cellular and H2O2-induced DNA oxidation in HEK 293 cells;assessment using flow cytometry

Objective:To investigate the role of water-soluble extract of Salvia fruticosa (Greek sage) (S. fruticosa) leaves in reducing both intrinsic cellular and H2O2-induced DNA oxidation in cultured human embryonic kidney 293 cells. S. fruticosa, native to the Eastern-Mediterranean basin, is widely used as a medicinal herb for treatment of various diseases. Methods: Dried leaves of S. fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations. Each mL of the preparation contained (7.1±1.0) mg of extract. HEK-293 cells were incubated in one set with S. fruticosa extract in the presence of 0.1 mmol/L H2O2, and in the other set with the addition of the extract alone. The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivatization of 8-oxoguanine moieties. The fluorescence was measured using flow cytometry technique. Results:Cells incubated 3 h with 150 μL extract and exposed to 0.1 mmol/L H2O2 showed lower intensity of fluorescence, and thus lower DNA oxidation. Moreover, cells incubated 3 h with 100 μL of the extract showed lower intensity of fluorescence, and thus lower intrinsic cellular DNA oxidation compared to control (without S. fruticosa). Conclusions: The results from this study suggest that the water-soluble extract of S. fruticosa leaves protects against both H2O2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells.