The regulation and gating mechanism of Cav1.2 current by KCNE1 subunit / 中华急诊医学杂志

Objective To explore the regulation and mechanism of Cav1.2 current by KCNE1. Methods Transient transfection was used to transfer Cav1.2 channel plasmids separately or together with KCNE1 plasmids into HEK293 cells. The experiment was divided into 2 groups (15 cells in each group):Cav1.2 group, Cav1.2+KCNE1 group.The whole-cell patch clamp technique was used to record Cav1.2 current and gating dynamics. Results After co-transfection of KCNE1 with Cav1.2, Cav1.2 current decreased significantly. At 0 mV, peak current density of Cav1.2 was reduced from (-14.8±2.5) pA/pF to (-7.5±1.6) pA/pF (n=15, P<0.01). Based on the gate control mechanism, it is found that the regulation of Cav1.2 current by KCNE1 mainly makes the steady-state inactivation curve of the channel shifted to a more negative direction, thus accelerating the inactivation. Meanwhile, the recovery process of the channel after inactivation is slowed down and the recovery time constant was prolonged. Conclusions The KCNE1 subunit can reduce the current density of Cav1.2 by changing the channel inactivation and recovery process.

Establishment of a reliable method for detecting the residual hek293 host cell protein in recombinant adeno-associated virus vector / 中国药学杂志

OBJECTIVE: To develop a reliable method to measure host cell protein(HCP)content of HEK293 cells in rAAV2-KAL vectors for clinic application. METHODS: The rAAV2-KAL vectors used for this study was purified by CsCl ultracentrifugation twice,then were subjected to by Western blot. The optimal linear range, minimum detection limit,and the accuracy and precision were verified by ELISA. Three batches of rAAV2-KAL were prepared by CsCl method. The change of HCP content during purification was determined to verify the method suitability and reliabilities. RESULTS: The optimal linear range of the method developed was 4-200 ng·mL-1, while the minimum detection limit of 4 ng·mL-1. The recovery rates of HEK293 cell protein at various concentrations were at range of 77.0%-115.0%,with a coefficient of variation of less than 20%. The HCP contents in three batches of rAAV2-KAL were less than 100 ng·mL-1, all data taken together indicated that HCP was effectively removed by CsCl ultracentrifugation. CONCLUSION: A reliable ELISA assay for residual host cell protein of HEK293 cells is successfully developed,which might be used for determination of HCP content in CsCl ultracentrifugation purified rAAV2-KAL for clinical application.

The reverse effects of allitridum on sodium current decrease caused by SCN5A-F1473S mutation / 药学学报

This study was designed to test the allitridum (All) activity in correction of sodium current decrease caused by SCN5A-F1473S mutation in HEK293 cells. The result may provide a theoretical basis for screening of new drugs in the treatment of Brugada syndrome. We transferred SCN5A-F1473S channel plasmids into HEK293 cells in a transient transfection. All was administrated acutely and chronically using extracellular irrigation flow and co-culture model. The concentration of All was 30 μmol·L-1. We used whole cell patch clamp technique in voltage clamp mode to record current and gating kinetics. In order to explore the rescue function of All on decreased sodium peak current, we used confocal microscopy and Western blot to detect the expression of channel proteins in the cell membrane. We found a significant increase in sodium peak current of the 30 μmol·L-1 All HEK293 cells (269.8±16.6 pA/pF, PPV1/2,inact returns to -79.5±2.4 mV, PPPSCN5A-F1473S mutation cells. We consider that the main mechanism may be related to the reduced channel inactivation by the drug with an improvement of the migration barrier of the mutational channel.

Isolation, Cloning and Co-expression of Hepatitis C Virus Envelope Proteins: As Potential HCV Detecting Antigens.

Aim: Approximately 3% of the world population is infected with Hepatitis C virus (HCV) which is the main cause of chronic liver disease. Blood transfusion is thought to be the leading cause of global epidemic of HCV. The envelope proteins E1 and E2 are involved in the early stages of the virus life cycle. These proteins have a major role in binding to receptors on the cell surface, fusion and integration of the virus into the host cell. Considering the potency of E1 and E2 in the development of diagnostic methods, the aim of our present study was co-expression of recombinant envelope proteins in eukaryotic HEK293 (human embryonic kidney) cells. Methods: The viral genomic RNA was used for cDNA (complementary DNA) synthesis. Isolation of HCV envelope proteins coding fragment was performed using cDNA and specific primers. The target gene was cloned into pcDNA3.1 expression vector, and transfected into HEK293 cells, an expression host. Accuracy of the cloning and expression was confirmed using PCR and Western blot analysis. Results: The isolation and cloning of the gene fragment encoding the E1 and E2 proteins was successful. Co-expression of these proteins was confirmed using monoclonal antibodies specific for each protein. Conclusion: This study showed that HEK293 host cell is suitable for the expression of hepatitis C virus E1 and E2 coding gene. These proteins can be used in numerous virological studies and detection of HCV infection.

Efeito dual de FGF2 e PMA em células HEK 293 transformadas por H-rasV12 / Dual effects of FGF2 and PMA on H-rasV12 transformed HEK293 cell line

Sabe-se há décadas que mutações nos genes ras estão presentes em cerca de 20% dos cânceres humanos, mas o desenvolvimento de terapias eficazes para o tratamento de câncer dependente dos oncogenes ras permanece um desafio científico importante. Nesse contexto, o nosso grupo publicou recentemente resultados interessantes mostrando que FGF2 exógeno ou PMA, contrariamente à expectativa geral, inibem a proliferação de células de camundongo malignas dependentes dos oncogenes H- ou K-Ras. Para dar continuidade a estes estudos o projeto desta tese foi planejado para investigar os mecanismos subjacentes a possíveis efeitos citotóxicos de FGF2 e PMA em células humanas transformadas por ras. Para esse fim, a linhagem humana imortalizada HEK 293 foi condicionalmente transformada pela expressão ectópica da construção quimérica de DNA ER:H-rasV12, que codifica a oncoproteína de fusão ER:H-RasV12, cuja atividade é induzível por 4-hidroxi-tamoxifen (4OHT). Essa abordagem nos permitiu verificar os efeitos de FGF2 e PMA em sublinhagens HEK/ER:HrasV12 fenotipicamente "normais" ou transformadas por níveis crescentes da oncoproteína H-RasV12. Os principais resultados mostraram que tanto FGF2 como PMA tem efeito dual promovendo ou inibindo a proliferação das células transformadas em função da concentração intracelular crescente de H-RasV12. Ensaios de crescimento de colônias em suspensão de agarose mostraram que: a) as células parentais HEK293 não desenvolveram colônias mesmo quando tratadas com FGF2 ou PMA, resultados que estão de acordo com seu fenótipo não tumoral; b) mas, as sublinhagens HEK/ER:HrasV12 deram origem a colônias mesmo quando tratadas com concentrações pequenas de 4OHT, que condicionaram níveis intracelulares baixos de ER:HRasV12; nestas condições experimentais, FGF2 foi um forte promotor do crescimento de colônias, condizente com sua reconhecida atividade promotora do crescimento de células tumorais em suspensão; ainda nestas condições, PMA não teve efeito significante sobre o crescimento de colônias; c) coerentemente, concentrações elevadas de 4-OHT levaram aos níveis intracelulares mais altos de ER:HRasV12 e, por conseguinte, a desenvolvimento máximo de colônias de células HEK/ER:HrasV12, no entanto, nestas condições, ambos FGF2 e PMA inibiram completamente o crescimento de colônias. Por outro lado, transformação de HEK293 com um vetor de expressão constitutiva de HrasV12 levou à seleção e isolamento das sublinhagens tumorais HEK/HrasV12, cujo fenótipo se caracterizou por: a) nenhum efeito de FGF2 sobre a sua proliferação e b) forte inibição de sua proliferação por PMA. A ação citotóxica de PMA exclusivamente observada em células HEK 293 transformadas por H-rasV12 se caracterizou por: a) total dependência de PKC, provavelmente mediada pela ativação proteolítica específica de PKC δ; b) envolvimento de níveis elevados e sustentados de ROS com disparo tardio de apoptose

It is known for nearly 20 years that mutated ras oncogenes are found in 20% of human malignancies, however efficacious therapies are not yet available for Ras-driven cancer. Along of these lines, our group recently published provocative results showing, against common belief, that FGF2 and PMA inhibited proliferation of Ras-dependent malignant mouse cells. Aiming to gain insight into this intriguing phenomenon, the present thesis project was planned to investigate the possible cytotoxicity of FGF2 and PMA in human Ras-driven malignant cells. To this end an immortalized non-tumorigenic human cell line (HEK293) was stably transformed with the DNA construction ER:H-rasV12, which encodes the fusion protein ER:H-RasV12, whose activity requires activation by 4-hidroxitamoxifen (4-OHT). This approach allowed us to evaluate FGF2 and PMA effects on HEK/ER:HrasV12 sublines under switching from "normal" to transformed phenotypes upon 4-OHT induction. Our main results have shown that both FGF2 and PMA displayed dual effects promoting or inhibiting proliferation of HEK/ER:HrasV12 cells in function of ER:HRasV12 intracellular levels. Clonogenic assays in agarose suspension have shown: a) parental HEK293 line did not develop colonies under FGF2 and PMA treatment or not, in agreement with its non-tumorigenic nature; b) however, HEK/ER:HrasV12 sublines developed colonies even under low 4-OHT concentrations, which led to low ER:HRasV12 intracellular levels; under these conditions FGF2 strongly promoted colony growth and PMA had no effect; c) furthermore, in HEK/ER:HrasV12 sublines, elevated 4-OHT concentrations led to high ER:HRasV12 intracellular levels and maximal colony growth; but, under these experimental conditions both FGF2 and PMA abolished colony growth. On the other hand, HEK293 transformation with a vector that constitutively express HrasV12 yielded HEK/ER:HrasV12 sublines displaying the following phenotypic traits: a) non FGF2 effects on proliferation and b) severe proliferation inhibition by PMA. PMA toxicity, exclusively observed in HrasV12 -transformed HEK293 cells, was characterized by: a) total dependency on PKC, likely mediated by specific proteolytic activation of PKCδ; b) involvement of high and sustained ROS levels correlated with late apoptosis triggering

Construction and expression of the eukaryotic expression plasmid of Human pEGFP/H2B / 重庆医科大学学报

Objective:To construct the eukaryotic expression plasmid of EGFP/H2B and express it in the Human embryo kidney cells(HEK293).Methods:Part H2B gene was obtained by RT-PCR from cultured HEK293 cell's whole RNA.The sequence encoding H2B was cloned into pEGFP-C1 to construct the eukaryotic expression vector.After confirmed by enzymatic digestion and sequencing,the recombination plasmid was transfected to HEK293 cells by lipofectamine technology for observation of the fusion protein's expression.Results:The recombination plasmid of pEGFP/H2B was constructed successfully.Compared with the dispersing green fluorescence among the whole cells in the control cells transfected with the mock plasmid(pEGFP-C1),the green fluorescence was concentrated in the nuclei of HEK293 cells transfected with pEGFP/H2B and distributed with the chromosomes in the mitotic cells.Conclusions:The successful construction of pEGFP/H2B recombination plasmids establishes an available survey system to study the chromosome segregation mechanism.

Construction of Recombinant Eukaryotic Expression Vector for Human Endostatin and Its Expression in HEK293 Cells / 中国康复理论与实践

@#Objective To construct a cell line with long time secreting endostatin. Methods Human endostatin cDNA containing interleukin-2 (IL-2) secreting peptide was cloned into eukaryotic expression plasmid pSNA2.0 to construct recombinant plasmid pSNA2.0/Endostatin. The plasmid pSNA2.0/Endostatin was transfected into HEK293 cells by cationic liposome. The positive cell clones were selected by G418 and named hED/293. The expression of endostatin protein was analyzed by Western-blot. Results Endostatin could be determined in the supernatant of hED/293 cells. Conclusion The recombinant eukaryotic expression vector is correctly constructed. The human endostatin protein can be expressed and secreted.

Construction of eukaryotic expressing vector of human MCHR1 and establishment of stable transfectant HEK293 cell line / 西安交通大学学报(医学版)

Objective To construct eukaryotic expressing vector of human melanin-concentrating hormone receptor1(MCHR1) and transfect HEK293 cells so as to establish stable HEK293 cell line.Methods The full-length MCHR1 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and was inserted into pcDNA3.1(+).After the identification of digestion and sequencing on the recombinant pcDNA3.1(+)/MCHR1,we transfected the recombinant into HEK293 cell by lipofectamineTM2000.After screening culture by G418,stable transfected HEK293 cell line was established,and the expression of MCHR1 was identified by RT-PCR and Western blotting.Results The eukaryotic expression vector pcDNA3.1(+)/MCHR1 was constructed,stable transfected HEK293 cell line was established,and MCHR1 protein was expressed successfully.Conclusion The construction of the eukaryotic expression vector pcDNA3.1(+)/MCHR1 and the establishment of stable transfected HEK293 cell line have provided solid experiment foundation for further studies on the function of MCHR1.

Establishment of HEK293 cell lines stably expressing m-nAChR / 医学研究生学报

Objective: To establish human embryonic kidney(HEK) 293 cell lines that that can stably express the muscle nicotinic acetylcholine receptor(AChR).Methods: The pcDNA3.1 plasmid was constructed and transfected into HEK293 cells with lipofectin,the stable transfectants screened by G418 and the protein expression identified and analyzed by immunohistochemistry.Results: After G418 screening,14 of the transfected cell lines highly expreesed ?-nAchR and 4 showed an obvious expression of ?-nAChR,as demonstrated by the immunohistochemical analysis.Conclusion: HEK293 cell lines stably expressing m-nAChR were constructed successfully.

Establishment of HEK293 cell line expressing sense and antisense Alu-Sx stably and efficiently / 第三军医大学学报

Objective To establish a HEK293 cell line stably and highly expressing sense and antisense Alu-Sx.Methods According to Alu subfamily Sx sequence,a pair of primers containing the sites for given restrictive endonuclease at both ends were designed and synthesized.PCR of the total DNA extracted from HEK293 cell line was performed,the products of which were cloned into a highly efficient eukaryotic expression vector pcDNA3.1/myc-His A.The recombinants were sequenced and identified by restrictive endonuclease digestion and then transfected into the HEK293 cell line by lipofectamine2000.The stable transfectants were screened by G418.Cell subclones were isolated by gradient dilution.The highly expressing clones were identified by Northern blotting.Results Eukaryotic expressing vectors stably and efficiently expressing sense and antisense Alu-Sx were constructed and cell subclones stably and efficiently expressing sense and antisense Alu-Sx were established.Conclusion Cell subclones stably and efficiently expressing sense and antisense Alu-Sx can used for our further study.

Selective actions of iptakalim on the subtypes of K_(ATP) channels / 中国药理学通报

Aim To establish a cell model heterologously expressing Kir6.x and SUR subunits of K_(ATP)channels and to study the selectivity of iptakalim on different subtypes of K_(ATP) channels.Methods Cell were transfected with the pcDNA vector containing the coding sequenced of Kir6.x and SUR using liposome and the pEGFP-N1 vector encoding for green fluorescent protein was added for easy identification of transfected cells.Using immunocytochemical technique,we detected the expression of Kir6.x and SUR protein in[FL(2K2]transfected cells.The electrophysiological experiment was performed after transfection 48-72 h.In whole cell configuration,the effects of K_(ATP) channel openers iptakalim,pinacidil and diazoxide on the clone currents in transfected HEK-293 cells and the antagonism of K_(ATP) channel inhibitor glibenclamide were evaluated.Results Immunocytochemical study identified the expression of Kir6.x and SUR protein in transfected cells.The electrophysiological experiment showed that in cells with recombinant expression of the Kir6.1/SUR2B channel complex,iptakalim(100 ?m),pinacidil(100 ?m)and diazoxide(200 ?m)significantly increased the clone current from(-88.0?30.8) pA to(-2042.6?127.3) pA,(-1431.9?142.4) pA and(-1104.7?228.9) pA,respectively,at-100 mV,and the actions were inhibited by glibenclamide(10 ?m).In cells expressed with Kir6.2/SUR2A channel,both iptakalim and pinacidil activated the currents,which was sensitive to glibenclamide.While diazoxide had no effects on the clone currents.In contrast,in cells with Kir6.2/SUR1channel,diazoxide increased the activity of recombinant K_(ATP) channels,while iptakalim and pinacidil had little effects.Conclusion From these observations,the effects of K_(ATP) channels openers with different chemical structures on the subtypes of K_(ATP) channels were diverse.Iptakalim showed selective action on Kir6.1/SUR2B and Kir6.2/SUR2A,but not Kir6.2/SUR1 K_(ATP) channels

THE EFFECTS OF BASIC FIBROBLAST GROWTH FACTOR ON PROTEIN KINASE B ACTIVITY / 解剖学报

Objective To study the effects of basic fibroblast growth factor(bFGF) on protein kinase B(PKB) activity. Methods The cytosol and membrane activity of PKB at the indicated time periods was determined by [?-()~(32)P]ATP incorporation assay. Results Both membrane and cytosol activity of PKB in HEK293 cell treated with bFGF(75??g/L) reached to the peak at 10?min.Wortmannin efficiently inhibited the activity of PKB in HEK293 cell.Conclusion bFGF rapidly stimulats both membrane and cytosol activity of PKB via P13K in HEK293 cell.