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Regulatory effect of substance P on proliferation of airway smooth muscle cells
WU Chang-Gui, MAO Bao-Ling, SUN Bin
Department of Respiratory Disease, Xijing Hospital, the 4th Military Medical University, Xian(710032)
Abstract AIM:To investigate the effect of substance P(SP) on the proliferation of canine airway smooth muscle cells (ASMC).METHODS：The fourth generation of cultured cells were used in the study. Cells were cultured in DMEM. At 48 h post incubation, SP and ［Sar9, Met(O2)11］-SP, a specific agonist of NK-1 receptor, were added into the DMEM in varied concentrations, respectively. ［3H］-TdR incorporation rate was used as an index of cell proliferation. Additionally, we investigated whether the ASMC proliferative responses to SP were modulated by preincubation for 2 h with GR71251, a specific antagonist of NK-1 receptor, and neomycin, a phospholipase C(PLC) inhibitor, and nitrendipine, a blockader of Ca2+ channel, respectively.RESULTS：SP and NK-1 receptor-specific agonist induced dose-dependent proliferative response of the cells, the effect of SP was blockaded by GR71251, neomycin and nitrendipin(inhibition rate: 90.5%, 62.8% and 40.3%, respectively). The effects of neomycin and nitrendipine were not enhanced when their concentrations were increased.CONCLUSION：ASMC proliferative response to SP may be mediate by NK-1 receptor. SP binding NK-1 receptor can activate the PLC and cause increase of the concentration of IP3 and Ca2+ in ASMC.
MeSHSubstance P; Muscle, smooth; Cells; Dogs
P物质(substance P, SP)是主要的速激肽之一，广泛分布于包括人类在内的多种哺乳动物肺部，对气道和肺血管功能起着重要的调节作用。业已证实SP参与神经源性炎症的形成，促进胆碱能神经传递、粘液分泌和气道平滑肌收缩［1］。然而SP与气道平滑肌细胞(airway smooth muscle cells, ASMC)增殖的确切关系，目前尚未阐明。本研究探讨SP对犬ASMC体外增殖的影响及机制，旨在进一步了解哮喘气道重塑的有关原因，为哮喘的综合治疗提供实验依据。
材料与方法
1.材料：DMEM、Ⅱ型胶原酶、SP、［Sar9,Met(O2)11］-SP、GR71251和新霉素(均系美国Sigma公司产品)，尼群地平(本校药理学教研室提供)，［3H］-TdR(中国原子能科学研究院产品)，液闪仪(Beckman 公司产品)。
2. 犬ASMC培养：无菌取出健康犬气管，分离气管背侧膜部平滑肌层，将其切成1 mm×1 mm×1 mm大小组织块，置于含0.1%Ⅱ型胶原酶的DMEM中消化6～8 h。终止消化后过100 目不锈钢滤网，离心弃上清，以Hanks液(pH7.4)洗细胞2遍，以含10%FCS DMEM悬浮细胞，计数后置25 cm2培养瓶内于37℃、5%CO2和完全湿化的环境下培养90 min，利用时差法将贴壁的成纤维细胞与平滑肌细胞分开，以1∶2消化传代。选取第4代细胞(平滑肌细胞纯度约90%)作为实验对象。以培养平滑肌细胞的形态特征和以针对平滑肌特有的α-肌动蛋白的单克隆抗体对培养平滑肌细胞作免疫细胞化学染色加以鉴定。
3.细胞增殖实验研究：将平滑肌细胞以1×103/孔接种在96孔板内，含10% FCS-DMEM培养24 h，吸去上清，以无血清DMEM洗涤2次，加入该培养基培养48 h，同步化之后以无血清培养基继续培养48 h(对照组)或分别加入含SP(10-7～10-5 mol/L)和NK-1受体激动剂［Sar9,Met(O2)11］-SP(10-7～10-5 mol/L)的无血清DMEM培养相同时间。为进一步了解SP诱导增殖作用的受体特异性和信号传导途径，我们以分别含GR71251(NK～1受体阻断剂，10-5mol/L)、新霉素［磷脂酶(PLC)抑制剂，10-6、10-5 mol/L］及尼群地平(钙通道阻断剂，10-6、10-5 mol/L)的无血清DMEM预培养犬ASMC 2 h，然后再加入SP(10-5 mol/L)继续培养48 h。在终止培养前16 h，每孔加入［3H］-TdR 3.7×104 Bq。以胰酶消化增殖细胞并收集于玻璃纤维膜上，烤干后置闪烁液内，液闪仪计数counts.min-1。
4. 统计处理：两组间比较采用两均数比较t检验。
增殖抑制率=(SP刺激后counts.min-1-阻断或干预后counts.min-1)/(SP刺激后counts.min-1-对照组counts.min-1)。
结果
1.SP及［Sar9,Met(O2)11］-SP对犬ASMC体外增殖的调节作用：对照组细胞［3H］-TdR掺入量为(1082.0±77.1) counts.min-1；而分别加入3种不同浓度的SP和［Sar9,Met(O2)11］-SP，［3H］-TdR掺入量均明显高于对照组，且呈剂量依赖性，随着浓度的增加，增殖反应增强(图1)。
Fig 1 Influence of substance P(SP) and agonist for NK-1 receptor, ［Sar9,Met(O2)11］-SP, on the proliferation of airway smooth muscle cells from dogs
▲▲P＜0．01，vs control group
图1不同浓度SP和NK-1受体激动剂［Sar9,Met(O2)11］-SP对犬气道平滑肌细胞增殖的影响
2. NK-1受体阻断剂GR71251 对SP诱导ASMC增殖反应的影响：单纯含GR71251(10-5 mol/L) 的无血清DMEM对ASME增殖反应无影响，其［3H］-TdR掺入量为(1081.7±64.0) counts.min-1与对照组相近。经GR71251(10-5 mol/L)预培养2 h后，SP(10-5 mol/L)对ASMC体外增殖反应明显减弱，［3H］-TdR掺入量由(3748.5±68.2) counts.min-1降至(1334.4±72.5) counts.min-1，增殖抑制率为90.5%(图2)。
Fig 2 Inhibitive effect of antagonist for NK-1 receptor, GR71251, on induced proliferation of airway smooth muscle cells from dogs by substance P(SP)
▲▲P＜0.01, vs control group; **P＜0.01, vs treated group by SP
图2NK-1受体拮抗剂GR71251对SP诱导犬气道平滑肌细胞增殖的抑制作用
3.新霉素和尼群地平对SP诱导气道平滑肌细胞体外增殖反应的影响：新霉素(10-6、10-5 mol/L)预培养2 h后，对SP(10-5 mol/L)的促增殖反应呈现明显的抑制效应，［3H］-TdR掺入量分别为(2094.0±55.7)和(2072.5±57.2) counts.min-1，与单纯SP(10-5 mol/L)处理时的(3748.5±232.2) counts.min-1相比差异非常显著(P＜0.01)，增殖抑制率分别为62.0%和62.8%。新霉素浓度的增加并不改变其对SP诱导作用的抑制效应。尼群地平(10-6 mol/L)也明显抑制SP的促ASMC增殖反应，其［3H］-TdR掺入量为(2694.5±270.0) counts.min-1，抑制率为40.3%，增加其浓度(10-5 mol/L)对SP作用无进一步抑制，［3H］-TdR<
