Involvement of Orai1 in tunicamycin-induced endothelial dysfunction

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca²⁺ homeostasis. The store-operated calcium (SOC) channel is the primary Ca²⁺ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca²⁺ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca²⁺ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.

MicroRNA-199a-3p enhances expressions of fibrosis-associated genes through targeting Smad1 in mouse cardiac fibroblasts / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p.</p><p><b>METHODS</b>Cardiac fibroblasts were isolated from C57BL/6 mice and cultured. The miR-199a-3p mimic and Smad1 siRNA were transiently transfected into the cardiac fibroblasts via liposome. Dual luciferase reporter assay was performed to confirm the interaction between miR-199a-3p and the 3'-UTR of Smad1. The expressions of Smad1 and fibrosis-related genes at the mRNA and protein levels in the cells after miR-199a-3p mimic transfection were determined using RT-qPCR and Western blotting, respectively. The expressions of Smad1, Smad3 and fibrosis-related genes at the protein level in cells transfected with miR-199a-3p mimic and Smad1 siRNA were detected using Western blotting.</p><p><b>RESULTS</b>Over-expression of miR-199a-3p significantly increased the expression of cardiac fibrosis-related genes in cultured mouse cardiac fibroblasts. Dual luciferase reporter assay revealed the interaction of miR-199a-3p with the 3'-UTR of Smad1. The results of RT-qPCR and Western blotting confirmed that miR-199a-3p inhibited Smad1 expression at the post- transcriptional level. Transfection with miR-199a-3p mimic and siRNA-mediated Smad1 silencing consistently activated the Smad3 signaling pathway and enhanced the expressions of cardiac fibrosis-related genes in the cardiac fibroblasts.</p><p><b>CONCLUSIONS</b>As the target gene of miR-199a-3p, Smad1 mediates the pro-fibrotic effect of miR-199a-3p by activating the Smad3 signaling in cultured mouse cardiac fibroblasts.</p>

Model construction of rat coronary artery smooth muscle cell endoplas-mic reticulum stress induced by thapsigargin / 中国病理生理杂志

AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress ( ERS) model in CASMCs of SD rats.METHODS:CASMCs were cultured by tissue explant method .The morphological characteristics were observed under optical micro-scope.The marker proteins of CASMCs , including α-SMA and SM-MHC, were identified by immunofluorescence tech-nique.The protein expression levels of BiP and CHOP , the marker molecules of ERS, were determined by Western blot . RESULTS:The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical hill and valleygrowth pattern of CASMCs at 9~10 d.The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed .The results of Western blot showed that the protein expression of BiP and CHOP in TG ( 1 and 2 μmol/L ) treatment groups was increased compared with control group .Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION:CASMCs can be successfully cultured by tissue explant method .ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.

MEF2C mediates inhibitory effect of microRNA-214 on cardiomyocyte hypertrophy / 中国病理生理杂志

AIM:To investigate the effect of microRNA-214 ( miR-214) on cardiomyocyte hypertrophy and the expression of the potential target genes .METHODS:A cell model of hypertrophy was established based on angiotensin-Ⅱ( Ang-Ⅱ)-induced neonatal mouse ventricular cardiomyocytes ( NMVCs) .Dual luciferase reporter assay was performed to verify the interaction between miR-214 and the 3’ UTR of MEF2C.The expression of MEF2C and hypertrophy-related genes at mRNA and protein levels was determined by RT-qPCR and Western blot , respectively .RESULTS:The expression of ANP, ACTA1,β-MHC and miR-214 was markedly increased in Ang-Ⅱ-induced hypertrophic cardiomyocytes .Dual lu-ciferase reporter assay revealed that miR-214 interacted with the 3’ UTR of MEF2C, and miR-214 was verified to inhibit MEF2C expression at the transcriptional level .The protein expression of MEF2C was markedly increased in the hypertro-phic cardiomyocytes .Moreover, miR-214 mimic, in parallel to MEF2C siRNA, inhibited the expression of hypertrophy-re-lated genes in Ang-Ⅱ-induced NMVCs.CONCLUSION:MEF2C is a target gene of miR-214, which mediates the effect of miR-214 on attenuating cardiomyocyte hypertrophy .

Effect of circRNA_000203 on fibrotic phenotypes in mouse cardiac fibro-blasts / 中国病理生理杂志

AIM:To determine circular RNA (circRNA) profiles in the diabetic mouse myocardium , and to investigate the effect of circRNA_000203 on fibrotic phenotypes in cardiac fibroblasts .METHODS:Masson trichrome stai-ning was performed on the myocardium of the diabetic db /db mice and the non diabetic db/m control mice .circRNA ex-pression profile in the diabetic myocardium was detected by circRNAs microarray .The expression of circRNA_000203 was determined by real time fluorescence quantitative PCR ( RT-qPCR ) .Recombinant circRNA_000203 adenovirus was pre-pared for enforced the expression of circRNA_000203 in mouse cardiac fibroblasts.The expression of Col1a2, Col3a1andα-SMA was determined in circRNA_000203-modified cardiac fibroblasts , respectively .RESULTS:Masson trichrome stai-ning showed that fibrosis was increased in the diabetic mouse myocardium .The results of circRNA array detection revealed that circRNAs were dysregulated in the diabetic myocardium .circRNA_000203 was up-regulated in the diabetic myocardi-um.Significant over-expression of circRNA_000203 was achieved in the cardiac fibroblasts after infection with the recombi-nant circRNA_000203 adenovirus.The mRNA and protein expression of Col1a2, Col3a1 and α-SMA was significantly in-creased in the cardiac fibroblasts with over-expression of circRNA_000203.CONCLUSION:circRNA_000203 is up-regu-lated in the diabetic mouse myocardium .It has pro-fibrotic effect on the cardiac fibroblasts .

MEF2C mediates the effect of microRNA-214 on inhibiting cardiomyocyte hypertrophy / 中国病理生理杂志

AIM:To investigate the effect of miR-214 on cardiomyocyte hypertrophy and the expression of the potential target genes . METHODS:A cell model of hypertrophy was established based on angiotensin-Ⅱ( Ang-Ⅱ)-induced neonatal mouse ventricular car-diomyocytes (NMVCs).Dual luciferase reporter assay was performed to verify the interaction between miR-214 and the 3’ UTR of MEF2C.The expression of MEF2C and hypertrophy-related genes at mRNA and protein levels was determined by RT-qPCR and Wes-tern blotting, respectively.RESULTS:The expression of ANP, ACTA1,β-MHC and miR-214 was markedly increased in Ang-Ⅱ-in-duced hypertrophic cardiomyocytes .Dual luciferase reporter assay revealed that miR-214 interacted with the 3’ UTR of MEF2C, and miR-214 was verified to inhibit MEF2C expression at the transcriptional level .The protein expression of MEF2C was markedly in-creased in the hypertrophic cardiomyocytes .Moreover, miR-214 mimic, in parallel to MEF2C siRNA, inhibited the expression of hy-pertrophy-related genes in Ang-Ⅱ-induced NMVCs.CONCLUSION:MEF2C is a target gene of miR-214, which mediates the effect of miR-214 on attenuating cardiomyocyte hypertrophy .

MicroRNA-1 and-16 inhibit cardiomyocyte hypertrophy by targeting cyclins/Rb pathway / 中国病理生理杂志

AIM:MicroRNAs ( miRNAs) were recognized to play significant roles in cardiac hypertrophy .But, it remains unknown whether cyclin/Rb pathway is modulated by miRNAs during cardiac hypertrophy .This study investigates the potential roles of microRNA-1 (miR-1) and microRNA-16 (miR-16) in modulating cyclin/Rb pathway during cardiomyocyte hypertrophy .METHODS:An animal model of hypertrophy was established in a rat with abdominal aortic constriction (AAC).In addition, a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocyte .RESULTS:miR-1 and-16 expression were markedly de-creased in hypertrophic myocardium and hypertrophic cardiomyocytes in rats .Overexpression of miR-1 and -16 suppressed rat cardiac hypertrophy and hypertrophic phenotype of cultured cardiomyocytes .Expression of cyclins D1, D2 and E1, CDK6 and phosphorylated pRb was increased in hypertrophic myocardium and hypertrophic cardiomyocytes , but could be reversed by enforced expression of miR-1 and -16.CDK6 was validated to be modulated post-transcriptionally by miR-1, and cyclins D1, D2 and E1 were further validated to be modulated post-transcriptionally by miR-16.CONCLUSION: Attenuations of miR-1 and -16 provoke cardiomyocyte hypertrophy via derepressing the cyclins D1, D2, E1 and CDK6, and activating cyclin/Rb pathway.

Cx43 is involved in electrical remodeling of atrial myocytes through regu-lating L-type calcium current / 中国病理生理杂志

AIM:To investigate whether the association of connexin 43 ( Cx43 ) and L-type calcium channel involved in the pathogenesis of atrial fibrillation ( AF) .METHODS:The biochemical assays and whole-cell patch-clamp technique were used to study the expression of Cx43 in human atrial tissue.The co-localization of Cx43 and L-type calcium channel, and the regulation of L-type calcium current in atrial myocytes were investigated.RESULTS:The expression of Cx43 at mRNA and protein levels was decreased in human atrial tissues of AF patients.In cultured atrium-derived myocytes ( HL-1 cells) , knockdown of Cx43 significantly inhibited the mRNA expression of L-type calcium channelα1c subunit, as well as L-type calcium current.Co-localization of Cx43 with L-type calcium channel α1c subunit in mouse atrial myocytes was observed.CONCLUSION:The decrease in Cx43 is involved in the pathogenesis of AF, probably through reducing the L-type calcium current in atrial myoctyes by co-localization with L-type calcium channel, thus representing the potential pathogenesis in atrial fibrillation.

Autopsy procedures and pathological observation of spontaneous main organ lesions in Rongshui miniature pig / 中国比较医学杂志

Objective To establish the data including anatomy and histology of main organs in Rongshui miniature pig (RMP).Methods F1 Rongshui miniature pigs with male and female (2 in each group) in 6 month old were used in this experiment.We measured body weights, dissected these pigs after anaesthesia, recorded total blood volume, total plasma volume, number of spine and dental formula, took main organs for photographs, and made histological sections observed and took photographs by microscope.Results We gained the photographs of main organs and histological sections, organ weights,organic coefficients and other basic data.Conclusion Basic anatomy and histology data of main organs in RMP were collected.

Hsa-mir-202 inhibit the proliferation of lung cancer A549 cells by reducing expression of IL-10 / 中国肿瘤临床

Objective:To study the effects of the overexpression of hsa-miR-202 on the proliferation and molecular mechanism of lung cancer A549 cells. Methods:A sequence of hsa-miR-202 with ppG/miR/eGFP/Blasitidin pasmid was directionally connected and a eukaryotic expression vector pmiR-202 of the target hsa-miR-202 gene was constructed. pmiR-202 was transtected to A549 cell with Lipofectamine 2000. The WST assay was used to detect the cell proliferation rate, and RT-PCR was used to detect the relative gene expression levels. Western blot analysis was used to detect the IL-10 protein expression levels. The interaction between miR-202 and IL-10 was examined using a luciferase reporter assay. Results:The design from the DNA sequencing results shows that a eukaryot-ic expression vector of miR-202 was successfully constructed. The proliferation inhibition rates of A549 cells by Pmir-202 were 12%, 38%, and 52%. The differences in the treatment group compared with the blank control and negative control groups were statistically significant. The RT-PCR results showed that the relative expression levels of miR-202 increased after transfection with pmiR-202. Over-expression of miR-202 can downregulate the relative gene and protein expression levels of IL-10, and the relative levels were 25%and 0.75, respectively. Compared with the blank control and the negative control groups, the difference was statistically significant (P<0.05). The fluorescent activity was reduced when transfection was performed with miR-202 mimics, and IL-10-3'UTR plasmid was cloned. Conclusion: pmiR-202 effectively inhibited the proliferation of A549 cells and exhibited a time-effect relationship with miR-202 by targeted combination with IL-10 3'UTR to downregulate IL-10 expression in A549 cells.

Caspase-8 small hairpin RNA attenuates apoptosis of human bone mar-row mesenchymal stem cells under conditions of serum deprivation and hypoxia / 中国病理生理杂志

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 HEK 293 cells for packaging and amplification of the recombi-nant 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 hM-SCs 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 ad-enovirus ( rAd)-Cap8 shRNA successfully .rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 ex-pression in hMSCs .Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the ap-optotic 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 .

Effect of ketamine on transient outward potassium current of isolated human atrial myocytes / 药学学报

The effects of ketamine on transient outward potassium current (I(to)) of isolated human atrial myocytes were investigated to understand the mechanism of part of its effects by whole-cell patch-clamp. Atrial myocytes were enzymatically isolated from specimens of human atrial appendage obtained from patients under going cardiac valve displacing. Ito is recorded in voltage-clamp modes using the patch-clamp technique at room temperature. Currents signals were recorded by an Axopatch 200B amplifier with the Digidata 1322A-pClamp 9.0 data acquisition system. Ketamine decreased I(to) of human atrial myocytes in a dose-dependent manner. The current-voltage curve was significantly lowered, 30, 100, 300, and 1000 micromol x L(-1) ketamine decreased respectively I(to) current density about (13.62 +/- 0.04)%, (38.92 +/- 0.05)%, (72.24 +/- 0.10)% and (83.84 +/- 0.05)% at the potential of 50 mV, with an IC50 of 121 micromol x L(-1). The I(to) activation curve, inactivation curve and the recovery curve were not altered by ketamine. So, ketamine concentration-dependently decreased I(to) of human atrial myocytes.

The relationship between macrophage migration inhibitory factor and chronic hepatitis B and hepatitis B virus-related cirrhosis / 中华传染病杂志

Objective To investigate the level of serum macrophage migration inhibitory factor (MIF) and its correlation with serum precollagen Ⅲ peptide (PⅢP) and tissue inhibitor of metalloproteinase (TIMP)-1 in patients with chronic hepatitis B (CHB) and hepatitis B virus (HBV)-related cirrhosis. Methods Forty-four CHB patients (hepatitis B group), 44 patients with HBV-related cirrhosis (cirrhosis group) and 30 healthy controls (control group) were enrolled in this study. The venous blood was collected and MIF level was detected by enzyme-linked immunosorbent assay (ELISA). Correlations between MIF and PⅢP, TIMP-1 were analyzed in observed groups. Comparison between groups was done using t test. The correlations between MIF level and alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), plasma thromboplastin antecedent (PTA), PⅢP and TIMP-1 were analyzed by rectilinear correlation. Results The levels of serum MIF, PⅢP and TIMP-1 in CHB group and cirrhosis group were all significantly higher than those in control group (t=12.87,5.28, 10.98,t=11.22,14.84,11.17;all P<0.05), while there were no significant differences between CHB group and cirrhosis group (t= -1.05,1.52,--2.07;all P>0.05). There was no correlation between MIF level and ALT, AST, TBil and PTA. MIF level in CHB patients with hepatitis B e antigen (HBeAg) positive and high viral load were both higher than that in patients with HBeAg negative and low viral load. MIF level was both positively correlated with PⅢP level in CHB group and cirrhosis group (r=0. 603, P<0.05 and r=0. 415, P<0. 05, respectively). MIF level was also positively correlated with TIMP-1 level in CHB group (r=0. 458, P<0.05), while not correlated in cirrhosis group (r=0. 210, P>0.05). Levels of PⅢP and T1MP-1 were both correlated in CHB group and cirrhosis group (r=0. 849, P< 0.05 and r=0. 424, P<0.05, respectively). Conclusions The levels of serum MIF are significantly increased both in patients with CHB and cirrhosis. The early production of MIF might be related with viral replication, but not with liver function. MIF participates in formations of hepatitis, liver fibrosis and cirrhosis, which could reflect the degree of liver cirrhosis.

Combined mesenchymal stem cells and allogenic bone marrow transplantation in treatment of MRL/Ipr mice / 中华内科杂志

In order to study the role of the bone marrow-derived mesenchymal stem cells(MSCs)transplanted with or without bone marrow(BM)in the treatment of lupus mice and the effect of MSCs in the onset of systemic lupus erythematosus(SLE).Method Twenty 12-week-old female MRL/lpr mice were randomly divided into four groups,including simple bone marrow transplantation group(SG,BM 1×107),united group-1(UG1,BM 1×107+MSCs 1×104),united group-2(UG2,BM 1×107+MSCs 1×106),the positive control group(PG,no transplantation).BALB/c mice were used as the negative control group(NG,no transplantation).MSCs which were amplified from the bone marrow of male BALB/c mice in vitro were transplanted into the female MRL/lpr mice with or without BM.One month later Y chromosome was detected to confirm if the transplantation was successful or not.The change of weight, white blood cells,urine protein,anti-dsDNA antibody,the pathology and immunofluorescence of renal were observed to evaluate the therapeutic efficacy.Results Y chromosome was detected in all transplanted female mice.Compared with PG,urine protein concentration in SG,UG1 and UG2 significantly decreased 30 days after transplantation(P<0.05).40 days after transplantation,the rite of anti-dsDNA antibodies in sG(0.91±0.27)was still higher than NG,which OD value wag 0.47 s0.10(P<0.05),but there was no statistical difference among UG1(0.76±0.28),uG2(0.73±0.10)and NG(P>0.05).However,50 days after transplantation,there was no marked difference of the tite of anti-dsDNA antibodies in SG(0.55±0.15),UG1(0.57±0.14)and UC2(0.58±0.05)compared with NG(P>0.05).After transplantation there was no vasculitis.no inflammatory cell infiltration in matrix and no obvious intercapillary cells proliferation in the kidney.The immunofluoroscence became negative or weakly positive.Conclusion MSCs transplantation with or without BM Can both improve the pathogenetic condition of MRL/lpr mice.MSCs can accelerate the clearance of anti-dsDNA antibody and promote the restoration of injured organs.We presume that MSCs are important immunological regulation cells in SLE.

Podocyte apoptosis is suppressed by 1,25(OH)2D3 in puromycin aminonudeoside nephropathy rats / 中华肾脏病杂志

ObjectiveTo evaluate the effects of 1,25(OH)zD3 on podocyte apoptosis in kidney of puremyein aminonueleoside nephropathy (PAN) rats. Methods Seventy-two male Sprague-Dawley rats were randomly divided into three groups: PAN model group(PAN), 1,25 (OH)2D3 treated group (T, 0.2 μg·kg-1d-1 by garages) and normal control group (NC). PAN rat model was constructed by a single intravenous injection of 100 mg/kg body weight. Renal function and 24hour urinary protein were measured at day 3, 7, 14, 21 after PAN injection. The renal tissue morphology was observed by light and electron microscope. Podocyte apeptosis was evaluated by TUNEL. Protein expressions of nephrin, TGF-β1 and p-Smad2/3 were examined by immunofluoreacence, immunohistochemistry and Western blot, respectively. Results(1)The levels of serum creafinine, BUN and 24-h urinary protein [(20.26±4.87) mg vs (1.01±0.41) mg at day 7, P ＜0.01] were significantly higher and the number of glomerular pedocyte was significantly lower [(10.9±4.2)/glomerular volume vs (31.9±6.2)/glomerular volume at day 14, P＜0.01] in PAN group compared with NC group. T group rats had less urinary protein excretion [(9.95±3.82) mg/24 h, P＜0.01] and more glomerular podocytes compared with PAN group. (2) Distribution of nephrin expression was changed from linear to granular pattern in PAN rats on day 7, nephrin mRNA and protein expressions were markedly decreased(P＜0.01), while the number of apoptotic podocyte was increased in PAN group(P＜0.01). However, higher nephrin expression and less apoptotic podocytes were found in T group (P＜0.01). (3) Compared with NC group, the mRNA and protein expression of TGF-β1 and p-Smad2/3 were higher in PAN group (P＜0.01), while 1,25 (OH)2D3 treatment abrogated PAN-induced changes in the expression of TGF-β1 and p-Smad2/3 (P＜0.01). Conclusions 1,25 (OH)2D3 can significantly suppress PAN-induced podocyte apoptosis and ameliorate proteinnuria. The beneficial effect of 1,25(OH)2D3 on podocyte may contribute to direct suppression of TGF-β signaling.

Histiocytic necrotizing lymphadenitis / 临床与实验病理学杂志

Purpose　To describe clinicopathological and immunophenotypic features of 10 cases of histiocytic necrotizing lymphadenitis (HNL). Methods　HE sections of 11 lymph node biopsies were re-examined. Immunophenotyping and detection of apoptotic DNA fragments were performed using S-P and TUNEL methods, respectively. Results　Five cases have been diagnosed as non-Hodgkin lymphoma. Histologically variable-sized discrete or confluent nodules were seen in the paracortex, especially in the interfollicular area, which were composed of proliferative pleomorphic histiocytes, transformed lymphocytes, and karyorrhectic debris. Immunohistochemistry revealed CD3+ and CD45RO+ for lymphocytes, Mac387+ and/or CD68+ for histiocytes, and no expression for CD15,CD30 and CD20 in the lesions. Conclusions　The presence of pleomorphic histiocytes, transformed T-cells, and karyorrhectic debris in the biopsy of lymph nodes, together with the absence of neutrophils support the diagnosis of HNL.

Construction of recombinant adenovirus vector carrying human miR-133a and its expression in vitro / 中国病理生理杂志

AIM:To construct recombinant adenovirus vector carrying human miR-133a and study its expression in human mesenchymal stem cells(hMSCs).METHODS:The PCR product containing miR-133a was amplified from human genomic DNA and inserted into the adenoviral shuttle vector pAdTrack-CMV.Then the recombinant shuttle plasmid linearized by pmeⅠwas cotransformed into competent E.coli.BJ5183 with the adenoviral backbone plasmid pAdEasy-1 to generate the recombinant adenovirus vector rAd-mir-133a.rAd-mir-133a was then packaged and amplified in human embryonic kidney 293(HEK293) cells.The purified rAd-miR-133a was used to infect the hMSCs and the expression of miR-133a was detected by non-quantitative RT-PCR and real-time PCR.RESULTS:The recombinant adenovirus shuttle vector pAdTrack-CMV-miR-133a was constructed and verified by restriction endonuclease analysis and DNA sequence analysis.rAd-miR-133a was successfully packaged and amplified in HEK293 cells.The transcriptions of primary miR-133a and mature miR-133a were over-expressed in the hMSCs infected with rAd-miR-133a.CONCLUSION:The recombinant adenovirus vector carrying human miR-133a is successfully constructed,which lay a foundation for miR-133a function study.

Oxidized low density lipoprotein induces the expression of macrophage migration inhibitory factor / 中国病理生理杂志

AIM: To examine expression of macrophage migration inhibitroy factor (MIF) gene and protein in macrophages induced by oxidized low density lipoprotein (ox-LDL). METHODS: Macrophages were incubated with ox-LDL at the concentration of 150 mg/L for time course (0-36 h) and with ox-LDL at the different concentrations (0-300 mg/L) for 24 h, expression of MIF mRNA and protein were detected by RT-PCR and ELISA. RESULTS: The results showed that ox-LDL increased MIF gene and protein expression in macrophages in a dose and time-dependent manner. After the exposure of macrophage to ox-LDL, the expression of MIF mRNA level increased consistently with protein. CONCLUSION: MIF may play an important role in atherosclerosis. [

siRNA suppresses the green fluorescent protein (GFP) gene expression in human umbilical vein endothelial cells / 中国病理生理杂志

AIM: To establish human umbilical vein endothelial cells (HUVECs) to express green fluorescent protein (GFP), and to study the suppression of GFP by siRNA in HUVECs. METHODS: Using lipofectamine 2000 to transform plasmid pN_3-EGFP encoding GFP into HUVECs. The HUVEC containing pN_3-EGFP, named HUVEC-GFP, was screened and selected by antibiotic G418. Using in vitro transcription T7 kit, GFPsiRNA targeting GFP mRNA and control-siRNA used as control were synthesized. The siRNAs were transfected into HUVEC-GFP with oligofectamine. 48 h later, the expression levels of GFP protein and mRNA in HUVEC-GFP were determined. RESULTS: The HUVEC-GFP was screened to express GFP in the presence of G418. The agarose gel electrophoresis analysis showed that the siRNAs prepared were integrated. 48 h after transfection with siRNAs, compared to control group, the level of GFP fluorescence was obviously decreased in the HUVEC-GFP transfected with GFPsiRNA. The results of RT-PCR detection showed that GFP mRNA expression was obviously suppressed by GFPsiRNA at the rate of 40%, and no obvious suppression of GFP mRNA expression was found in the HUVEC-GFP transfected with control siRNA. CONCLUSION: The siRNA targeting GFP mRNA, synthesized in vitro, efficiently suppresses the GFP expression in HUVECs.

Inward rectifier potassium current and mRNA expression of gene Kir2.1 in human atrial fibrillation / 中国病理生理杂志

AIM: To investigate alteration of inward re ctifier potassium current (I K1) in atrial myocytes and mRNA expression of gene Kir2.1 encoding I K1 in atrial myocardial tissue in patients with chronic atrial fibrillation (AF) compared to that with sinus rhythm (SR).METHODS: Single myocytes were isolated by enzymatic dissociation with the chunk method an d the ionic current was recorded using whole cell patch clamp technique. The sem i-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of Kir2.1 in atrial myocardial tissue, and the g ene GAPDH was used as an internal control.RESULTS: (1) The I K1 density was increased in AF group at hyperpolarizing pot entials, at -120 mV the current densities was (-5.71?0.65) pA/pF in AF group (n=28 cells from 7 patients) and (-4.26?1.22) pA/pF in SR group (n=35 cells from 9 patients) (P0.05).CONCLUSIONS: The increase in I K1 at hyperpolarizing potentials may be related to th e atrial electrophysiological remodeling in chronic human AF. The increased I K1 density in atrial myocytes in AF group without alteration of Kir2.1 mRNA expression in atrial tissue suggests that I K1 may be mediated at post-transcriptional levels.