Explore The Pdgf-bb On Vsmcs Within The Tgf-beta <sub> 1 </ Sub> And Its Signaling System And Its Regulation Mechanism, Pcna Expression In Vsmcs

Partâ… Effects of Platelet-derived growth factor-BB on the expressions ofTransforming growth factor-β1 mRNA and Protein and on theexpressions of the downstream proteins of Smad-dependent pathwayin TGF-βsignalling systemObjectivesTo build the cultural method of vascular smooth muscle cells fromadult rat aorta in vitro; to detect the expression of Platelet-derived growthfactor (PDGF) -BB mRNA in male adult rat aorta vascular smoothmuscle cell; to observe the effects of PDGF-BB on the expressions oftransforming growth factor (TGF)-β₁ mRNA and protein in vascularsmooth muscle cells(VSMCs) under serum-free condition and the effectsof PDGF-BB on the expressions of the downstream proteins ofSmad-dependent pathway in TGF-βsignalling system, including TGF-βtypeâ… receptor(ALK-5),Smurf2,pSmad2/3,Smad4,Smad7. The effectsof neutralizing anti-TGF-β₁ antibody on the expressions of these proteinsmodulated by PDGF-BB were also investigated. Methods1. Enzyme digestion of collagenase I was applied for the culture ofnormal smooth muscle cells from male adult rat aorta (weighing100～120g, week age 8～9 weeks).2. RT-PCR analysized the expression of PDGF-BB mRNA in maleadult rat aorta vascular smooth muscle cells.3. The levels of TGF-β₁ mRNA in VSMCs treated with PDGF-BB intime curve and in dose curve were examined by RT-PCR analysis.4. The levels of TGF-β₁ protein secreted by VSMCs treated withPDGF-BB in time curve and in dose curve were examined by ELISAassay.5. Western Blot analysized the expressions of the downstream proteinsof TGF-βsignalling system including TGF-βtypeâ… receptor (ALK-5),Smurf2,pSmad2/3,Smad4,Smad7Results1. VSMCs grew near confluence on the seventh day by Enzymedigestion of collagenase I, showing the typical pattern "Valley-peak".VSMCs were used between passages 5 and 6 for experiments,α-SMAwas abundant in the VSMCs by immunofluorescence staining.2. The expression of PDGF-BB mRNA was not detected in vascularsmooth muscle cell from male adult rat aorta. 3. The expressions of TGF-β₁ mRNA in VSMCs treated withPDGF-BB for 12 hours showed a dose-dependent manner underserum-free conditions.4. The expressions of TGF-β₁ mRNA in VSMCs treated withPDGF-BB (10ng/ml) showed a time-dependent manner underserum-free conditions.5. The expressions of TGF-β₁ proteins in VSMCs treated withPDGF-BB for 24 hours showed a dose-dependent manner underserum-free conditions.6. PDGF-BB (10ng/ml) induced the expressions of TGF-β₁ protein inVSMCs in a time-dependent manner under serum-free conditions.7. PDGF-BB for 48 hours induced the expressions of ALK-5 inVSMCs in a dose-dependent manner under serum-free conditions.8. PDGF-BB for 48 hours induced the expressions of Smurf2 inVSMCs in a dose-dependent manner under serum-free conditions.9. PDGF-BB for 48 hours induced the expressions of pSmad2/3 inVSMCs in a dose-dependent manner under serum-free conditions.10. PDGF-BB for 48 hours induced the expressions of Smad4 inVSMCs in a dose-dependent manner under serum-free conditions.11. PDGF-BB for 48 hours inhibited the expressions of Smad7 inVSMCs in a dose-dependent manner under serum-free conditions.12. These alterations of the downstream proteins of TGF-βsignalling system modulated by PDGF-BB were partially restored by neutralizinganti-TGF-β₁ antibody.Conclusions1. Enzyme digestion of collagenaseâ… is a stable, reliable culturalmethod by which rat VSMCs were cultured. The VSMCs were usedbetween passages 5 and 6 for experiments.2. PDGF-BB mRNA does not express in vascular smooth muscle cellfrom male adult rat aorta.3. PDGF-BB stimulated efficiently the synthesis and secretion ofTGF-β₁ in VSMCs under serum-free condition.4. PDGF-BB upgraded the expressions of ALK-5 in VSMCs underserum-free conditions, which implied that PDGF-BB activated TGF-βsignalling system in VSMCs.5. PDGF-BB upgraded the expressions of pSmad2/3 in VSMCs underserum-free condition, which suggested that PDGF-BB activated theSmad-dependent pathway in TGF-βsignalling system in VSMCs.6. PDGF-BB upgraded the expressions of Smad4 in VSMCs underserum-free conditions, which enhanced the complexes of pSmad2/3 andSmade4 to form.7. PDGF-BB down-regulated the expressions of Smad7 in VSMCsunder serum-free conditions, which enhanced signalling transmission from ALK-5 to pSmad2/3 and Smad4.8.PDGF-BB upregulated the expressions of Smurf2 a in VSMCs underserum-free conditions, which implied negative feedback regulation wasactivated in TGF-βsignalling system in VSMCs.9.PDGF-BB modulated the expressions of the downstream proteins ofSmad-dependent pathway in TGF-βsignalling system partially throughTGF-β₁ overexpression.Partâ…¡Effects of PDGF-BB and PDGF-BB-induced overexpression of TGF-β₁ on the proliferation of VSMCs and the expressions of PCNA inVSMCsObjectivesTo observe the effects of PDGF-BB on the proliferation of VSMCs and the expressions in VSMCs and to observe the effects ofPDGF-BB-induced overexpression of TGF-β₁ on the proliferation ofVSMCs and the expressions of PCNA in VSMCs.Methods1. Effects of PDGF-BB and TGF-β₁ on the proliferation of VSMCs indose curve assayed by Counting Cell Kit (CCK)-8.2. Effects of PDGF-BB on the expression of PCNA in VSMCs in dosecurve detected by Western Blot.3. CCK-8 analysis of the proliferation of VSMCs treated withPDGF-BB and/or neutralizing anti-TGF-β₁ antibody.4. Western Blot analysis of the expression of PCNA in VSMCs treatedwith PDGF-BB and/or neutralizing anti-TGF-β₁ antibody.Results1. PDGF-BB for 48 hours stimulated the proliferation of VSMCs andthe expression of PCNA in VSMCs in dose-dependent manners.2. TGF-β₁ modulated the proliferation of VSMCs in a dose-dependent manner. But the low dose of TGF-β₁ (0.02ng/ml; 0.1ng/ml)inhibited VSMCs proliferation, and the high dose (2.5ng/ml) inducedthe proliferation of VSMCs.3. Neutralizing anti-TGF-β₁ antibody inhibited significantly the proliferation of VSMCs the expression of PCNA in VSMCs induced byPDGF-BB.4. There was a significant correlation between the increments ofVSMCs proliferation by CCK-8 assay and the increments of PCNAexpression in VSMCs by Western Blot in the dose curves (r=0.968,p＜0.01). The increments represent increases in the proliferation ofVSMCs and in expression of PCNA in VSMCs induced by PDGF-BBcompared with controls.ConclusionsPDGF-BB induced the proliferation of VSMCs and the expression ofPCNA in VSMCs partially through the overexpression of TGF-β₁ inducedby PDGF-BB under serum-free condition. The high dose of TGF-β₁stimulated the proliferation of VSMCs.