埃他卡林抑制PDGF-BB诱导的人气道平滑肌细胞增殖和迁移

发布时间：2018-05-06 20:01

本文选题：埃他卡林 + 人气道平滑肌细胞　；参考：《南京医科大学》2015年硕士论文

【摘要】：目的:气道重塑是慢性气道疾病,如哮喘和慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)重要的病理改变。气道平滑肌细胞(airway smooth muscle cells,ASMCs)的异常增殖和迁移是气道重塑的主要原因。ATP敏感性钾(ATP-sensitive potassium,KATP)通道是气道平滑肌上的一种重要钾通道。有研究证实KATP通道开放能缓解气道重塑,其开放剂对气道重塑有潜在的治疗作用。埃他卡林(Iptakalim,Ipt)是一种KATP开放剂,体内外肺动脉高压实验研究证实Ipt能抑制肺动脉平滑肌细胞增殖及缓解肺血管重构。但是,目前有关Ipt对气道重塑方面的研究报道较少。本实验在体外采用血小板源性生长因子BB(platelet-derived growth factor-BB,PDGF-BB)构建人气道平滑肌细胞(humanairway smooth muscle cells,HASMCs)增殖和迁移的模型,探讨Ipt对HASMCs增殖和迁移的影响及可能的相关机制。方法:用平滑肌细胞培养基(Smooth Muscle Cell Medium,SMCM)常规培养HASMCs。分别用含有10-8、10-7、10-6、10-5和10-4 M终浓度Ipt的无血清SMCM预处理细胞30min,接着加入终浓度为20 ng/ml的PDGF-BB培养细胞48h。采用CCK-8实验检测各组细胞增殖情况,并选定合适的Ipt给药浓度,在接下来的试验中我们将HASMCs分为4组:对照(Control)组、PDGF-BB(20 ng/ml)组、PDGF-BB+Ipt(10-5M)、Ipt组。Ed U(5-ethynyl-2’-deoxyuridine)孵育法进一步检测Ipt对HASMCs增殖的影响。流式细胞仪检测Ipt对HASMCs的周期和凋亡的影响。Transwell迁移实验和细胞划痕实验共同检测每组细胞的迁移情况。Western blot法检测Ipt对PDGF-BB诱导的Ca2+/钙调蛋白依赖性蛋白激酶II(Ca2+/calmodulin-dependent kinase II,Ca MKII)、细胞外调节蛋白激酶1/2(extracellular regulated protein kinases 1/2,ERK1/2)、蛋白激酶B(protein kinase B,PKB/Akt)和环磷腺苷效应元件结合蛋白(cyclic adenosine monophosphate(c AMP)response element binding protein,CREB)这四种蛋白磷酸化的影响。结果:1.CCK-8实验表明PDGF-BB(20 ng/ml)显著地增加细胞活力,是对照组的140.74%(P0.05),而10-5 M Ipt处理细胞后能将PDGF-BB诱导的140.74%±1.57%细胞活力降低至112.11%±4.65%(P0.05),并且10-5 M Ipt联合PDGF-BB作用于细胞后与对照组无明显差异(P0.05)。因此,我们选择10-5 M浓度的Ipt进行下一步的实验。2.PDGF-BB组Ed U-阳性细胞比例(30.04%±3.94%)较对照组(12.17%±2.68%)显著增加(P0.05),但是PDGF-BB+Ipt组(15.61%±2.69%)的Ed U-阳性细胞比例显著地低于PDGF-BB组(P0.05),且Ipt(11.77%±2.83%)单独作用对HASMCs增殖无影响(P0.05,Ipt组vs对照组)。3.与对照组相比(13.62%±4.40%,),PDGF-BB单独作用于HASMCs显著地促进细胞周期进入S期(24.53%±3.32%,P0.05),但是PDGF-BB的这个效应可被Ipt阻滞(PDGF-BB+Ipt组16.43%±0.50%,P0.05)。流式凋亡结果显示PDGF-BB和Ipt对HASMCs的凋亡都无影响。4.在transwell迁移实验中,我们观察到PDGF-BB诱导的迁移细胞数是对照组的3.29倍(P0.05),此效应可被10-5 M Ipt所抑制(PDGF-BB+Ipt组为对照组的1.58倍,P0.05,vs PDGF-BB组)。划痕实验的结果与迁移实验一致。迁移实验和划痕实验均表明Ipt单独作用于HASMCs亦能抑制细胞的迁移(Ipt组为0.725倍,P0.05,vs对照组)。5.我们的实验数据显示PDGF-BB刺激细胞10min能显著地诱导HASMCs的Ca MKII,ERK1/2,Akt和CREB蛋白磷酸化。10-5 M Ipt预处理细胞30min能逆转PDGF-BB诱导的Ca MKII,ERK1/2和CREB磷酸化,并能显著抑制PDGF-BB诱导的Akt磷酸化。10-5 M Ipt单独作用于细胞不影响Ca MKII,和CREB的磷酸化,但是能部分抑制ERK1/2和Akt的磷酸化。结论:Ipt抑制PDGF-BB诱导的HASMCs增殖和迁移,此效应可能与调节Ca MKII,ERK1/2,Akt,和CREB信号通路有关。我们的结果表明Ipt可能是治疗慢性气道疾病气道重塑的一种有效药物。
[Abstract]:Objective: airway remodeling is an important pathological change in chronic airway diseases such as asthma and chronic obstructive pulmonary disease (chronic obstructive pulmonary disease, COPD). The abnormal proliferation and migration of airway smooth muscle cells (airway smooth muscle cells, ASMCs) are the main cause of airway remodeling,.ATP sensitive potassium (ATP-sensitive) Channel is an important potassium channel on the airway smooth muscle. It has been proved that opening of KATP channel can alleviate airway remodeling, and its open agent has a potential therapeutic effect on airway remodeling. Iptakalim (Ipt) is a kind of KATP open agent. The experimental study of pulmonary artery hypertension in vitro and in vivo has confirmed that Ipt can inhibit the proliferation and inhibition of pulmonary artery smooth muscle cells. However, there are few reports about airway remodeling in Ipt. In this experiment, a model of proliferation and migration of human airway smooth muscle cells (humanairway smooth muscle cells, HASMCs) was constructed in vitro by using platelet-derived growth factor-BB (PDGF-BB) in vitro. The proliferation and migration of humanairway smooth muscle cells, HASMCs) was studied. Methods: Smooth Muscle Cell Medium (SMCM) was used for normal culture of HASMCs. with HASMCs. without serum SMCM containing 10-8,10-7,10-6,10-5 and 10-4 M terminal concentration Ipt. The cell proliferation of each group was measured and the appropriate concentration of Ipt was selected. In the next experiment, we divided HASMCs into 4 groups: control (Control) group, PDGF-BB (20 ng/ml) group, PDGF-BB+Ipt (10-5M), Ipt group.Ed U (5-ethynyl-2 '-deoxyuridine) incubation method. The effect of cycle and apoptosis on the migration of cells in each group by.Transwell Migration Experiment and cell scratch test,.Western blot method was used to detect Ipt to PDGF-BB induced Ca2+/ calmodulin dependent protein kinase II (Ca2+/calmodulin-dependent kinase II, Ca MKII), and extracellular regulated protein kinase Kinases 1/2, ERK1/2), protein kinase B (protein kinase B, PKB/Akt) and cyclic phosphorous adenosine effect element binding protein (cyclic adenosine monophosphate) (cyclic adenosine monophosphate) effects. Results: the experimental table clearly increased cell viability (20), which was 140.7 of the control group. 4% (P0.05), and 10-5 M Ipt treated cells could reduce the activity of 140.74% + 1.57% cells induced by PDGF-BB to 112.11% + 4.65% (P0.05), and 10-5 M Ipt combined PDGF-BB acted on the cells without significant difference from the control group (P0.05). Therefore, we chose the Ipt of the 10-5 M concentration for the next step of experimental.2.PDGF-BB group positive cells (30) .04% + 3.94%) was significantly higher than that of the control group (12.17% + 2.68%) (P0.05), but the proportion of Ed U- positive cells in group PDGF-BB+Ipt (15.61% + 2.69%) was significantly lower than that in PDGF-BB group (P0.05), and Ipt (11.77% + 2.83%) alone had no effect on HASMCs proliferation (P0.05, Ipt group vs control group) compared with the control group (13.62% + 4.40%,). Cs significantly promoted the cell cycle into the S phase (24.53% + 3.32%, P0.05), but the effect of PDGF-BB could be blocked by Ipt (group PDGF-BB+Ipt 16.43% + 0.50%, P0.05). Flow apoptosis showed that PDGF-BB and Ipt had no effect on.4. in Transwell migration experiments. 3.29 times (P0.05), this effect could be suppressed by 10-5 M Ipt (group PDGF-BB+Ipt was 1.58 times as the control group, P0.05, vs PDGF-BB group). The results of scratch test were in accordance with the migration experiment. Migration Experiment and scratch test showed that Ipt could inhibit cell migration alone (Ipt group was 0.725 times, P0.05, vs control group). The PDGF-BB stimulated cell 10min can significantly induce the Ca MKII of HASMCs, ERK1/2, Akt and CREB protein phosphorylation.10-5 M Ipt pretreatment cell 30min can reverse the induced phosphorylation, and can significantly inhibit the phosphorylation of the induced phosphorylation. However, it can partially inhibit the phosphorylation of ERK1/2 and Akt. Conclusion: Ipt inhibits PDGF-BB induced HASMCs proliferation and migration, which may be related to the regulation of Ca MKII, ERK1/2, Akt, and CREB signaling pathways. Our results suggest that Ipt may be an effective drug for the treatment of airway remodeling in chronic airway diseases.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R56

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