Construction and expression of recombinant adenovirus vector encoding human somatostatin receptor type 2

AIM: To construct recombinant adenovirus vector carrying the gene of human somatostatin receptor type 2 （SSTR2） for gene therapy of pancreatic carcinoma.METHODS: SSTR2 cDNA was inserted into adenovirus shuttle plasmid pDC316, named pDC316-SSTR2.pDC316-SSTR2 was cotransfected with rescue plasmid pBHGlox (delta) E1, 3Cre into 293 cells by liposome reagent.Ad-SSTR2 was generated by site-specific recombination and confirmed by PCR.Ad-SSTR2 was propagated in 293 cells and purified.The titer of viral stock was determined by end-point dilution assay.Western blotting was used to determine the expression of SSTR2 protein after human pancreatic carcinoma cell capan-2 was infected with recombinant adenovirus.RESULTS: pDC316-SSTR2 was successfully constructed.Recombinant adenovirus Ad-SSTR2 was acquired by pDC316-SSTR2 and pBHGlox (delta) E1, 3Cre cotransfected into 293 cells.Ad-SSTR2 was characterized by PCR.The virus titer was 6.0×1012 pfu/L.SSTR2 protein was detected after adenovirus infected capan-2 48 h with Western blotting.CONCLUSION: The recombinant adenovirus vector encoding human SSTR2 is successfully constructed and correctly expressed in pancreatic carcinoma cells.This investigation provides the basis for study of gene therapy of pancreatic carcinoma.     

Construction of the vector for fusion protein gene driven by IGF-Ⅱ P3 promoter and its expression in hepatocellular carcinoma cells

AIM:To construct the shuttle plasmid vector for thymidine kinase(tk) and EGFP fusion protein gene driven by IGF-Ⅱ P3 promoter,and investigate the specific killing effect of the HSV-tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro.METHODS:Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening,and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT-PCR. Cell killing after ganciclovir(GCV) application was determined by MTT.RESULTS:Identification of pDC316-tkEGFP-P3 by enzyme digestion and sequencing analysis showed that the length,inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells,but not in HeLa cells. The results of RT-PCR showed that only two bands could be seen in the samples of pDC316-tkEGFP-P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells.CONCLUSION:The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.     

Construction and identification of eukaryotic dicistronic vector expressing TCA-12-2/TCB-7.1

AIM: To construct the recombinant dicistronic eukaryotic expression vector pDC315-TCA-12-2-TCB-7.1, which containing T cell antigen receptor (TCR) genes TCA-12-2 and TCB-7.1, and to transfer this recombinant vector into 293 cells to investigate the expression of TCA-12-2 and TCB-7.1. METHODS: The TCA-12-2 was obtained by RT-PCR from the T cells and the TCB-7.1 was amplified by PCR from plamid pcDNA3.1-TCB-7.1 that we constructed before. TCA-12-2 and TCB-7.1 was cloned into vector pIRES2-AcGFP1 firstly, then subcloned into vector pDC315. The recombinant plasmid pDC315-TCA-12-2-TCB-7.1 was verified by restriction enzyme digestion and sequencing, the positive recombinant plasmid was transferred into 293 cells using Lipofectamine 2000. The expressions of gene TCA-12-2 and TCB-7.1 were identified by RT-PCR and flow cytometry. RESULTS: Both TCA-12-2 and TCB-7.1 genes were constructed into eukaryotic expression vector pDC315 and the expressions of genes in 293 cells were detected successfully with RT-PCR and flow cytometry. CONCLUSION: The dicistronic expression vector pDC315-TCA-12-2-TCB-7.1 is successfully constructed and expressed.     

Conditionally replicating adenovirus activated by CXCR4 promoter in lung cancer

AIM: To construct a conditionally replicating adenovirus vector activated by CXCR4 promoter and to evaluate its ability of lysing the lung cancer cells specifically. METHODS: Human CXCR4-E1A gene amplified by PCR was cloned into the shuttle plasmid pDC316-GFP to construct the recombinant shuttle plasmid pDC316-CXCR4-GFP. The recombinat shuttle plasmid and adenovirus genomic plasmid pBHG-lox-E1, 3Cre were transfected into 293 cells to construct the recombinant adenovirus CRAd-CXCR4-GFP. PCR was used to detect the target gene fragments, and the viral titer was determined. A549 cells with the highest mRNA expression of CXCR4 were screened out from 5 kinds of lung cancer cell lines by real-time PCR. CXCR4 promoter activity and adenovirus replication numbers were detected in A549 cells after transfection of CRAd-CXCR4-GFP and Ad-NULL. CRAd-CXCR4-GFP and Ad-NULL were transfected into A549 cells and 16HBE cells, the apoptotic rates were detected by flow cytometry and the viability was analyzed by CCK-8 assay. RESULTS: The recombinant plasmid pDC316-CXCR4-GFP was constructed successfully. Green fluorescence was observed in 293 cells under fluorescent microscope after co-transfection of pDC316-CXCR4-GFP and pBHG-lox-E1, 3Cre at 11 d. Green fluorescence was observed in 293 cells after infection of amplified 3rd generational adenovirus. PCR showed that the purpose gene was successfully integrated in recombinant adenovirus genome. The virus in the supernatant reached a titer of 1×10¹³ PFU/L. The mRNA expression of E1A and E4 in the A549 cells after transfection of CRAd-CXCR4-GFP was markedly increased compared with Ad-NULL group. Compared with Ad-NULL group and empty control group, the apoptotic rate and the viability of A549 cells in CRAd-CXCR4-GFP group had no significant difference in the first 4 d, the apoptotic rate increased significantly at 5 d, and the cell viability declined significantly at 5 d, but the apoptotic rate and the viability of 16HBE cells in each group had no significant difference within 5 days. CONCLUSION: The conditionally replicating adenovirus vector CRAd-CXCR4-GFP has been successfully constructed, which has the ability of lysing lung cancer cells specifically.     

Construction of siArid2 recombinant adenovirus and its effect on proliferation of hepatocarcinoma cells

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.     

Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene

AIM: To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein ( RFP ) reporter gene with the approach of in vitro recombinant ligation. METHODS: The RFP gene fragment of pTurboRFP-N was digested and ligated into pShuttle transfer vector to construct recombinant vector pShuttle-TurboRFP-N. I- Ceu I/PI- Sce I were used to double digest recombinant vector pShuttle-TurboRFP-N and backbone of vector pH5'040.pkGFP-II. The target fragment was collected and ligated, and recombinant adenovirus vector AdH5'.040.CMV.RFP-N was obtained. After linearization, the vector was transfected into AD293 cells by liposome for virus packaging. The efficiency of virus packaging and RFP expression level in AD293 cells were examined using fluorescent microscope. In addition, the biological activity and titer of the virus were tested. Human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were infected with recombinant adenovirus vector AdH5'.040.CMV.RFP-N and control adenovirus vector AdH5.CMV.EGFP respectively. The infection efficiencies of the 2 vectors to different cell lines were compared by evaluating the expression levels of RFP and enhanced green fluorescent protein (EGFP). RESULTS: The recombinant adenovirus vector AdH5'.040.CMV.RFP-N was correctly constructed and confirmed by enzyme digestion. The virus was packaged by the vector in AD293 cells and had the ability to infect the target cells. The target gene in eukaryotic cells was also expressed. The number of recombinant adenoviruses and the titer of the virus after amplification and purification were 3.6×10¹⁵ vp/L and 1×10¹³ pfu/L,respectively. The infection efficiencies of recombinant adenovirus vector Ad5'.040.CMV.RFP-N to human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were higher than those in control adenovirus vector AdH5.CMV.EGFP (P<0.05). CONCLUSION: We have constructed recombinant virus vector carrying RFP reporter gene and provide an important tool for gene therapy and gene vaccine research. The reporter gene can be highly expressed in AD293 cells and has high infection efficiency to cancer cells. RFP is a good substitution and supplement to green fluorescent protein.     

Caspase-8 small hairpin RNA attenuates apoptosis of human bone marrow mesenchymal stem cells under conditions of serum deprivation and hypoxia

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.     

Recombinant hFGF-10 adenovirus promotes proliferation of kerotinocytes

AIM:To construct the recombinant adenoviral vector containing human fibroblast growth factor 10 (hFGF-10) gene, and to study the effect of the recombinant adenovirus on the proliferation of kerotinocytes. METHODS:HFGF-10 gene was amplified by PCR and ligated with shuttle vector pAdTrack-CMV to get the recombinant plasmid pAdTrack-CMV-hFGF-10, which was linearized with PmeI and transferred into Escherichia coli BJ5183 containing the adenoviral bone plasmid pAdEasy-1 for homologous recombination to obtain the recombinant adenoviral plasmid pAdEasy-hFGF-10. The recombinant adenoviral plasmid was then transfected into HEK-293 cell line to package and amplify the recombinant adenovirus. The expression of hFGF-10 in HaCat cells infected with the recombinant adenovirus was detected by Western blotting. The influence of the recombinant adenovirus on the proliferation of kerotinocytes was checked by MTT. RESULTS:The recombinant adenovirus containing hFGF-10 gene was successfully constructed, which effectively infected HaCat cells. The result of Western blotting showed that a protein in culture media of the infected HaCat cells reacted with hFGF-10 antibody. The recombinant adenovirus stimulated the proliferation of kerotinocytes. CONCLUSION:HaCat cells infected with the recombinant adenovirus expresses and secrets hFGF-10 protein, which promotes the proliferation of HaCat cells.     

Construction of recombinant adenoviruses expressing the helicobacter pylori CagA antigen

AIM: To construct a replication-defective recombinant adenovirus, which expresses the CagA gene. METHODS AND RESULTS: The CagA gene was amplified by PCR. This heterogeneous gene was cloned into shuttle vector pAdTrack-CMV. The recombinant adenovirus DNA was obtained by the homologous recombination between the shuttle vector and adenovirus DNA in E.coli 5183. After linearization， the recombinant adenovirus DNA was transfected into 293 cells and the recombinant adenovirus was obtained. Through this technique, the replication- defective recombinant adenovirus AdEasyCagA was constructed. CONCLUSIONS: The replication-defective recombinant adenoviruses AdEasyCagA was constructed successfully. The work will make a good foundation for studying the effects of the replication-defective recombinant adenovirus on Th1/Th2 balance in asthma and be useful for finding a new pathway to prevent and cure asthma.     

Effect of P53 binding site on regulation of human NOD8 gene

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 JetPei^TM 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.     

Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro

AIM:To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro.METHODS:BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo.RESULTS:We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION:Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.     

PI3K/Akt regulates endoplasmic reticulum stress-mediated GRP78 induction

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.     

Construction of pNTAP-PRAK eukaryotic expression plasmid and its expression in HEK293 cells

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.     

Effects of adiponectin siRNA on the glucose transport in 3T3-L1 adipocytes

AIM: To construct the adenovirus vector with adiponectin (Acrp30) siRNA, and to observe its effect on the Acrp30 expression and glucose transport in 3T3-L1 adipocytes. METHODS: Mouse Acrp30 siRNA fragment was designed, synthesized and cloned into the adenovirus vector. 3T3-L1 cells were infected with the two recombinant adenoviruses, respectively. The mRNA expression and protein levels of Acrp30 in these cells were evaluated by RT-PCR and ELISA. Glucose transport was measured by 2-Deoxy-［3H］-D-glucose incorporation method. RESULTS: The recombinant adenoviruses were successfully constructed. They remarkably downregulated the expression of Acrp30 at both mRNA and protein levels in 3T3-L1 cells, and decreased the glucose transport in 3T3-L1 adipocytes (P<0.05). CONCLUSION: The siRNA expression vectors effectively inhibit the expression of Acrp30 in 3T3-L1 adipocytes, and decrease the glucose transport.     

Morphological study of macrophages infected with constructed recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain

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 10^8.3 and 10^8.1 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.     

Effects of lentivirus-mediated transfection of shRNA targeting Adra1d gene on calcium and calmodulin in rat aortic vascular smooth muscle cells

AIM:To investigate the effects of lentivirus-mediated transfection of shRNA targeting α_1D-adrenergic receptor (Adra1d) gene on calcium ion (Ca²⁺) and calmodulin (CaM) in vascular smooth muscle cells (VSMCs) of rat aorta. METHODS:Single oligonucleotide sequences of shRNA targeting rat Adra1d gene were design and synthesized, and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293T cells together and packed with lentivirus, and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-qPCR and Western blot. The concentration of Ca²⁺ in VSMCs was detected by laser confocal inspection, and the expression of CaM at mRNA and protein levels in the VSMCs was determined by RT-qPCR and Western blot. RESULTS:The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3×10¹¹ TU/L. The mRNA and protein expression levels of Adra1d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1d gene, compared with scrambled group, the Ca²⁺ fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover, the mRNA and protein expression levels of CaM were also increased significantly. CONCLUSION:A lentiviral shRNA expression vector targeting rat Adra1d gene was successfully constructed, which significantly increased Ca²⁺ concentration and CaM expression in rat aortic VSMCs.     

Role of ANX A2 in APL-induced expression of tissue factor in THP-1 cells

AIM: To construct a lentiviral vector harboring RNAi sequence targeting human annexin A2 (ANX A2) and to investigate the functions of ANX A2 in antiphospholipid antibody (APL)-induced tissue factor (TF) expression in monocytes. METHODS: Four different short hairpin RNAs (shRNA) targeting ANX A2 gene were constructed and cloned into the pGCSIL-GFP vector. After identification with PCR and sequencing, the effective interference sequence was further determined by Western blotting analysis. The recombinant lentivirus LV-RNAi-ANX A2 harvested from 293T cells was transferred into THP-1 cells and the ANX A2 mRNA and protein expression on THP-1 cells were examined. The transferred-THP-1 cells were stimulated by APL/β2GPI compound, and the TF mRNA and TF activity was assayed. RESULTS: The RNAi sequences targeting the human ANX A2 gene were successfully inserted into the lentiviral vector and the high-performance RNA interference sequence was sieved out. The recombinant lentivirus was harvested from 293T cells with a viral titer of 3×1012 TU/L. THP-1 cells infected with LV-RNAi-ANX A2 showed almost lockout of ANX A2 both at mRNA and protein levels. The TF expression was also significantly decreased in the transferred-THP-1 cells induced by APL/β2GPI compound. CONCLUSION: The lentiviral vector constructed in the present study blocks the ANX A2 expression in THP-1 cells and further inhibits the TF expression induced by APL/β2GPI compound, which indicates that ANX A2 do play an important role in APL-induced TF expression on monocytes.