本文選題：埃他卡林 + 人氣道平滑肌細(xì)胞�。� 參考：《南京醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:氣道重塑是慢性氣道疾病,如哮喘和慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)重要的病理改變。氣道平滑肌細(xì)胞(airway smooth muscle cells,ASMCs)的異常增殖和遷移是氣道重塑的主要原因。ATP敏感性鉀(ATP-sensitive potassium,KATP)通道是氣道平滑肌上的一種重要鉀通道。有研究證實(shí)KATP通道開放能緩解氣道重塑,其開放劑對(duì)氣道重塑有潛在的治療作用。埃他卡林(Iptakalim,Ipt)是一種KATP開放劑,體內(nèi)外肺動(dòng)脈高壓實(shí)驗(yàn)研究證實(shí)Ipt能抑制肺動(dòng)脈平滑肌細(xì)胞增殖及緩解肺血管重構(gòu)。但是,目前有關(guān)Ipt對(duì)氣道重塑方面的研究報(bào)道較少。本實(shí)驗(yàn)在體外采用血小板源性生長(zhǎng)因子BB(platelet-derived growth factor-BB,PDGF-BB)構(gòu)建人氣道平滑肌細(xì)胞(humanairway smooth muscle cells,HASMCs)增殖和遷移的模型,探討Ipt對(duì)HASMCs增殖和遷移的影響及可能的相關(guān)機(jī)制。方法:用平滑肌細(xì)胞培養(yǎng)基(Smooth Muscle Cell Medium,SMCM)常規(guī)培養(yǎng)HASMCs。分別用含有10-8、10-7、10-6、10-5和10-4 M終濃度Ipt的無(wú)血清SMCM預(yù)處理細(xì)胞30min,接著加入終濃度為20 ng/ml的PDGF-BB培養(yǎng)細(xì)胞48h。采用CCK-8實(shí)驗(yàn)檢測(cè)各組細(xì)胞增殖情況,并選定合適的Ipt給藥濃度,在接下來(lái)的試驗(yàn)中我們將HASMCs分為4組:對(duì)照(Control)組、PDGF-BB(20 ng/ml)組、PDGF-BB+Ipt(10-5M)、Ipt組。Ed U(5-ethynyl-2’-deoxyuridine)孵育法進(jìn)一步檢測(cè)Ipt對(duì)HASMCs增殖的影響。流式細(xì)胞儀檢測(cè)Ipt對(duì)HASMCs的周期和凋亡的影響。Transwell遷移實(shí)驗(yàn)和細(xì)胞劃痕實(shí)驗(yàn)共同檢測(cè)每組細(xì)胞的遷移情況。Western blot法檢測(cè)Ipt對(duì)PDGF-BB誘導(dǎo)的Ca2+/鈣調(diào)蛋白依賴性蛋白激酶II(Ca2+/calmodulin-dependent kinase II,Ca MKII)、細(xì)胞外調(diào)節(jié)蛋白激酶1/2(extracellular regulated protein kinases 1/2,ERK1/2)、蛋白激酶B(protein kinase B,PKB/Akt)和環(huán)磷腺苷效應(yīng)元件結(jié)合蛋白(cyclic adenosine monophosphate(c AMP)response element binding protein,CREB)這四種蛋白磷酸化的影響。結(jié)果:1.CCK-8實(shí)驗(yàn)表明PDGF-BB(20 ng/ml)顯著地增加細(xì)胞活力,是對(duì)照組的140.74%(P0.05),而10-5 M Ipt處理細(xì)胞后能將PDGF-BB誘導(dǎo)的140.74%±1.57%細(xì)胞活力降低至112.11%±4.65%(P0.05),并且10-5 M Ipt聯(lián)合PDGF-BB作用于細(xì)胞后與對(duì)照組無(wú)明顯差異(P0.05)。因此,我們選擇10-5 M濃度的Ipt進(jìn)行下一步的實(shí)驗(yàn)。2.PDGF-BB組Ed U-陽(yáng)性細(xì)胞比例(30.04%±3.94%)較對(duì)照組(12.17%±2.68%)顯著增加(P0.05),但是PDGF-BB+Ipt組(15.61%±2.69%)的Ed U-陽(yáng)性細(xì)胞比例顯著地低于PDGF-BB組(P0.05),且Ipt(11.77%±2.83%)單獨(dú)作用對(duì)HASMCs增殖無(wú)影響(P0.05,Ipt組vs對(duì)照組)。3.與對(duì)照組相比(13.62%±4.40%,),PDGF-BB單獨(dú)作用于HASMCs顯著地促進(jìn)細(xì)胞周期進(jìn)入S期(24.53%±3.32%,P0.05),但是PDGF-BB的這個(gè)效應(yīng)可被Ipt阻滯(PDGF-BB+Ipt組16.43%±0.50%,P0.05)。流式凋亡結(jié)果顯示PDGF-BB和Ipt對(duì)HASMCs的凋亡都無(wú)影響。4.在transwell遷移實(shí)驗(yàn)中,我們觀察到PDGF-BB誘導(dǎo)的遷移細(xì)胞數(shù)是對(duì)照組的3.29倍(P0.05),此效應(yīng)可被10-5 M Ipt所抑制(PDGF-BB+Ipt組為對(duì)照組的1.58倍,P0.05,vs PDGF-BB組)。劃痕實(shí)驗(yàn)的結(jié)果與遷移實(shí)驗(yàn)一致。遷移實(shí)驗(yàn)和劃痕實(shí)驗(yàn)均表明Ipt單獨(dú)作用于HASMCs亦能抑制細(xì)胞的遷移(Ipt組為0.725倍,P0.05,vs對(duì)照組)。5.我們的實(shí)驗(yàn)數(shù)據(jù)顯示PDGF-BB刺激細(xì)胞10min能顯著地誘導(dǎo)HASMCs的Ca MKII,ERK1/2,Akt和CREB蛋白磷酸化。10-5 M Ipt預(yù)處理細(xì)胞30min能逆轉(zhuǎn)PDGF-BB誘導(dǎo)的Ca MKII,ERK1/2和CREB磷酸化,并能顯著抑制PDGF-BB誘導(dǎo)的Akt磷酸化。10-5 M Ipt單獨(dú)作用于細(xì)胞不影響Ca MKII,和CREB的磷酸化,但是能部分抑制ERK1/2和Akt的磷酸化。結(jié)論:Ipt抑制PDGF-BB誘導(dǎo)的HASMCs增殖和遷移,此效應(yīng)可能與調(diào)節(jié)Ca MKII,ERK1/2,Akt,和CREB信號(hào)通路有關(guān)。我們的結(jié)果表明Ipt可能是治療慢性氣道疾病氣道重塑的一種有效藥物。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R56

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