Effection And Regulatory Mechanism Of K~+ Channel Kir2.1/KCNJ2 On The Phenotypic Switching Of Rat Vascular Smooth Muscle Cells

Part 1Construction of Kir2.1-shRNA lentiviral vectors and detection of their kock-down effect to KCNJ2 of rat vascular smooth cells Objective: First, to construct Kir2.1-shRNA lentiviral ?Kir2.1-shRNA-Lv? vectors and transfect rat thoracic aorta vascular smooth muscle cells ?RASMCs? for detection of their kock-down effect to KCNJ2.Methods: Four siRNA?siRNAl-4? sequences targeting the Kir2.1 gene and one negative sequence which was used as negative control group were designed and synthesized according to Genbank published ?accession number NM₀17296.1?, and the corresponding five pairs of complementary single strand DNA of shRNA were formed. The lentiviral particles include virion-packaging elements ?pGag/Pol,pRev,pVSV-G? and recombinant shuttle vector. The recombinant lentivirus vector was transfected into 293T cell line after being detected by DNA sequencing. RASMCs were seeded at 5×105cells/mL in 6-well plates, which were divided five groups?shRNAl-4, NC, CON, respectively?.To infect with recombinant lentivirus encoding for shRNA against Kir2.1 at a multiplicity of infection ?MOI? of 100. Five days post-infection, EGFP expression was examined using fluorescent microscopy and Kir2.1 protein expression was analyzed using Western blot to the five groups.Results: ?1? To determine the lentiviral transduction efficiency in RASMCs, EGFP expression was examined by microscopy at MOI of 100 on 5 days after infection. The efficiency of lentiviral vector transduction in RASMCs was approximately 90% at an MOI of 100. ?2? Western blot showed the Kir2.1 protein expression levels were significantly reduced at shRNAl-4 groups compared with CON, the shRNA3 group had the highest inhibition efficiency, however these were no significant difference inKir2.1 protein expression between CON and NC.Conclusion We successful get the shRNA sequences which can knock-down the Kir2.1 gene expression, and construct the lentivirus. The lentivirus which carried sh-RNA3 vectors had high transduction efficiency and Kir2.1 protein inhibition. The lentivirus provided the best choice for the later research.Part 2To study the effect of Kir2.1-shRNA-Lv Knock down Kir2.1 gene on proliferation, migration, and phenotype protein of RASMCs by PDGF-BB inductionObjective: To investigate the effect of PDGF-BB-induced RASMCs proliferation,migration, and phenotype protein by Kir2.1-shRNA-Lv the Kir2.1 gene.Methods: Primary rat thoracic aorta smooth muscle cells culture and treatment. After 3 days of Kir2.1-shRN A-Lv infection, RASMCs were stimulated PDGF-BB?25ng/mL? 24h. To determine proliferation changes of post-intervention,CCK-8 and BrdU were used. Using wound healing assay and transwell chambers, we observed migration changes of post-intervention. To detect expression levels of post-intervention, Western blot was used.Results: ?1? The PDGF-BB-induced significantly increased the proliferation activity compared with NC, however when PDGF-BB induced after knock-down Kir2.1 significantly suppressed the proliferation activity compared with PDGF-BB-induced.?2? The PDGF-BB-induced can significantly promote the migration activity compared with NC, however when PDGF-BB induced after knock-down Kir2.1 can significantly suppresse the migration activity compared with PDGF-BB-induced. ?3?The PDGF-BB-induced increased the expression of synthetic phenotype protein?SM22?, SM?-actin, calponin?, but reduced the level of contractile phenotype protein?osteopontin? compared with NC. when PDGF-BB induced after knock-down Kir2.1 can significantly suppresse the expression of synthetic phenotype protein, but promote the level of contractile phenotype protein compared with PDGF-BB-induced.Conclusion: Knockdown of Kir2.1 could inhibit proliferation, migration, and phenotype switching of RASMCs by PDGF-BB induction.Part 3To investigate effect of Kir2.1-shRNA-Lv specifically knock-down Kir2.1 on neointima formation after rat carotid artery balloon injury Objective: To detect effect of neointima formation after rat carotid artery balloon injury by Kir2.1-shRNA-Lv specifically knock-down Kir2.1 in vivo.Methods: Twenty-four SD male rats were divided into four groups at random which include Sham group, Saline group, Kir2.1-shRNA-Lv knock-dowm group ?KD group?and Lv-NC group?NC group?. 100?L saline, 100?L Kir2.1-shRNA-Lv or 100?L scrambled RNA was injected into the balloon-injured rat arteries through the balloon catheter side aperture and induced 30min. After 3 weeks, the injuryed carotid arteries were collected for fluorescence microscope, hematoxylin and eosin ?H&E? staining or Western blot to evaluate knock-down efficiency of Kir2.1 and intima forms.Results: ?1? In the KD and NC groups, green fluorescence was visible in the neointima and media. ?2? The expression of Kir2.1 was specifically increased Saline and NC groups compared to Sham group. Copared with Saline group, the expression of Kir2.1 specifically decreased in KD group. ?3? Compared with the Sham group,the KD and NC groups significantly increased balloon injury-induced neointimal areas. The KD group significantly decreased balloon injury-induced neointimal areas compared to Saline group. The medial areas of four groups had not significant difference.Conclusion: Kir2.1-shRNA-Lv specifically knocks down Kir2.1 in vivo and prevents rat carotid stenosis after balloon injury.Part 4The study on the mechanism of Kir2.1 in PDGF-BB-induced RASMCs phenotype switchingObjective: To detect the molecular signal mechanisms of regulation of Kir2.1 in PDGF-BB-induced RASMCs phenotype switching by whole-cell patch clamp.Methods: Using inhabitors of PDGF-BBRa or PDGF-BBR?, PI3K/AKt or cAMP-PKA, and agonist of cAMP or antagonist of cAMP intervened in RASMCs,then PDGF-BB-induced 24h and selected a single cell recorded IK1 currents by protocao voltage in whole cell voltage patch clamp pattern.Results: ?1? To record PDGF-BB-induced RASMCs currents of Kir2.1 by patch clamp: Compared with classic Kir2.1 currents waveform ?CON?, the currents waveform could hardly detect by intervention of Kir2.1 channel antagonist 50uM Ba2+, but the Kir2.1 currents waveform significantly increased by induction by PDGF-BB. ?2? To study Kir2.1 currents after intervention of PDGF-BBRa antagonist or PDGF-BBRa antagonist by patch clamp: Compared with PDGF-BB-induced, the Kir2.1 currents of PDGF-BBRa-intervened was no significant difference, however Kir2.1 currents of PDGF-BBR?-intervened was significantly decreased. ?3? To record currents of Kir2.1 after intervention of AKt inhibitor by patch clamp: the Kir2.1 currents with AKt inhibitor was no significant difference compared with PDGF-BB-induced. ?4? To record currents of Kir2.1 after intervention of PKA inhibitor by patch clamp: the Kir2.1 currents with PKA inhibitor was significantly increased compare with PDGF-BB-induce. ?5? To detect currents of Kir2.1 after intervention of cAMP agonist or cAMP antagonist by patch clamp: Compared toPDGF-BB-induced, the Kir2.1 currents of cAMP agonist was significantly increased,however Kir2.1 currents of cAMP antagonist was significantly decreased.Conclusion: PDGF-BB obviously increased currents of Kir2.1 via PDGF-BBR?through the activation of PKA signaling pathway in RASMCs.