Study on the Detection of MMP-2,-7,-9,and -12 Enzymatic Activity Using CEACAM1-Derived Fluorescent Peptide Substrate Site 84
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摘要:
目的 探索CEACAM1 源性多肽合成的荧光底物Site 84 对MMP-2,-7,-9,-12 酶活性的检测,并进一步探索荧光多肽底物Site 84在区分同属于MMPs明胶酶谱中MMP-2和MMP-9的应用。 方法 采用荧光酶学法观察荧光多肽底物Site 84 对MMP-2,-7,-9,-12 酶活性的检测;进一步观察底物Site 84对明胶酶谱中MMP-2和MMP-9酶活性的敏感性和特异性;获得底物Site 84与MMP-2的酶促反应的动力学参数Km、Kcat。 结果 以Site 84 为底物,获得MMP-12、-7、-2 的酶活力曲线,但未获得MMP-9的酶活力曲线;此外,以Site 84 为底物,可特异性检测明胶酶谱中MMP-2 的酶活性,其可检测低浓度(0.6 μM)MMP-2 的酶活性,然而对高浓度(6 μM)MMP-9 未见明显酶促反应;最后,MMP-2 和底物 Site 84的酶促反应动力学参数:Km = 315 μM,Kcat/Km = 2565 / MS。结论 以CEACAM1源性底物 Site 84作为新型的荧光多肽底物,可获得MMP-12、-7、-2 的酶活力曲线,且可特异性检测MMPs明胶酶谱中MMP-2的酶活性。 Abstract:Objective To explore the the detection of MMP-2, -7, -9, and -12 enzymatic activity using the CEACAM1-derived fluorescent peptide substrate Site 84, investigating the application of substrate Site 84 to distinguishing between MMP-2 and MMP-9 in the gelatinase spectrum of MMPs. Methods The fluorescent enzymatic method was employed to observe the detection of MMP-2, -7, -9, and -12 enzymatic activity using substrate Site 84; further observations were made on the sensitivity and specificity of substrate Site 84 to enzymatic activity of MMP-2 and MMP-9 within the gelatinase spectrum; the kinetic parameters (Km and Kcat) of the enzymatic reaction between substrate Site 84 and MMP-2 were obtained. Results Using Site 84 as a substrate, enzymic kinetics curves for MMP-12, -7, -2 were obtained, but no enzymatic activity curve for MMP-9 was observed. Furthermore, Site 84 specifically detected the enzymatic activity of MMP-2 within the gelatinase spectrum, capable of detecting low concentration (0.6 μM) of MMP-2 enzymatic activity, but no obvious enzymatic reaction was observed for high concentration (6 μM) of MMP-9; the kinetics parameters for the enzymatic reaction between Site 84 and MMP-2 were Km = 315 μM, Kcat/Km = 2565 /MS.Conclusion The CEACAM1-derived substrate Site 84 serves as a novel fluorescent peptide substrate, enabling the acquisition of enzymatic activity curves for MMP-12, -7 and -2, and specifically detecting the enzymatic activity of MMP-2 within the MMP gelatinase spectrum. -
Key words:
- Site 84 /
- Fluorescent polypeptide substrate /
- Enzymatic activity /
- Gelatinase /
- CEACAM1-derived.
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非糜烂性反流病(non-erosive reflux disease,NERD)指有反酸、烧心、胸骨后疼痛等症状但胃镜检查食管粘膜无充血糜烂表现的一种疾病,临床常见,发病率逐年上升。质子泵抑制剂(proton pump inhibitors,PPIs)是治疗NERD的常用药物,然而有研究[1]显示部分NERD对PPIs治疗的效果并不满意。富马酸伏诺拉生片是1种新型的酸阻滞剂,可抑制胃酸分泌 [2],2019年在我国上市,用于治疗反流性食管炎,目前尚无研究比较富马酸伏诺拉生片与PPIs在治疗NERD中的效果。本研究旨在观察富马酸伏诺拉生片与艾司奥美拉唑镁肠溶片在治疗NERD中的效果及不良反应对比。
1. 资料与方法
1.1 一般资料
将2020 年8月至2022年3月在青岛市第八人民医院治疗的124例NERD患者作为研究对象。本研究通过医院医学伦理委员会审批通过(QBYLL-KY-2023-031),患者知情并签署同意书。采用随机数字表法分成观察组和对照组,各62例。2组的性别、年龄、身体质量指数(body mass index,BMI)、吸烟、饮酒、HP感染率一般资料比较,差异均无统计学意义(均P > 0.05),见表1。纳入标准[3-4]:(1)18~80岁;(2)反酸、烧心或胸骨后疼痛症状超过3个月且排除心血管系统及呼吸系统疾病引起的胸痛;(3)胃镜检查无食管粘膜破损;(4)已完成13C呼气试验,检查结果不限。排除标准[5] :(1)对富马酸伏诺拉生片、艾司奥美拉唑镁肠溶片、青霉素、克拉霉素缓释片、枸橼酸铋钾胶囊任何1种药物过敏者;(2)严重的心肺、肝肾功能不全以及内分泌病患者;(3)生命体征异常患者;(4)心理、精神障碍及不能正常交流患者;(5)胃食管恶性肿瘤及胃食管溃疡患者;(6)1月内服用过抑酸、护胃及促胃肠动力药物患者;(7)孕期或哺乳期患者。
表 1 观察组和对照组一般资料比较[$\bar x \pm s$/n(%)]Table 1. Comparison of general data between treatment group and control group[$ \bar x \pm s $/n(%)]组别 性别
(男/女)年龄
(岁)BMI
(kg/m2)吸烟 饮酒 HP感染 观察组 27/35 57.74±12.81 23.86±2.58 10(16.13) 12(19.35) 34(54.84) 对照组 24/38 56.79±11.29 23.12±2.27 9(14.52) 13(20.97) 32(51.61) t/χ2 0.300 0.439 1.689 0.062 0.050 0.130 P 0.584 0.662 0.094 0.803 0.823 0.719 1.2 方法
观察组给予富马酸伏诺拉生片(takeda pharmaceutical company limited,hikari plant)20 mg/次,口服,1次/d,对照组给予艾司奥美拉唑镁肠溶片(阿斯利康制药公司)20 mg/次,餐前半小时口服,2次/d,疗程均为1月,1月对患者进行随访。对于HP阳性患者给予标准四联根除HP方案,观察组方案(富马酸伏诺拉生片20 mg 2次/d,阿莫西林胶囊(山东鲁抗医药公司)1 g 2次/d,克拉霉素缓释片(江苏恒瑞医药公司)0.5 g 2次/d,枸橼酸铋钾胶囊(丽珠制药厂)0.6 g 2次/d),对照组方案(艾司奥美拉唑镁肠溶片20 mg 2次/d,阿莫西林胶囊1 g 2次/d,克拉霉素缓释片0.5 g 2次/d,枸橼酸铋钾胶囊0.6 g 2次/d),根除HP疗程为14 d,停药至少1月复查13C呼气试验。
1.3 观察指标
(1)2组患者的临床疗效:观察组和对照组患者治疗1月后随访患者反酸、烧心、胸痛等临床症状。显效表示症状基本消失,症状减轻至少75%;有效表示症状较前缓解,症状减轻25%~75%;无效表示症状较前无明显减轻甚至加重,症状减轻小于25%。总有效率=显效率+有效率;(2)2组患者的RDQ评分[6]:治疗前及治疗后对观察组及对照组患者进行RDQ评分:①依症状的频率记分:烧心、胸痛、反酸每周发生频率,依“从未有过、1周 < 1 d、1周1 d、1周2到3 d、1周4到5 d、几乎每天”分别记0、1、2、3、4、5分;②依症状的程度记分:“症状不明显,在医生提示下发现”记1分;“明显,偶尔服药”记3分;“非常明显,长期服药”记5分;“症状1和3分之间”记2分;“3和5分之间”记4分。频率和程度每项最高20分,合计最高40分;(3)2组患者的PSQI评分:治疗前及治疗后分别对2组患者行PSQI评分,评价治疗前后的睡眠质量,此表包含主观睡眠质量、睡眠潜伏期、持续时间等7个因子,按照 0 ~ 3 级计分,总分最高为21分,总分≥8分提示睡眠障碍;(4)2组患者治疗前后食管pH值变化: 治疗前和治疗1月后分别使用pH记录仪(型号:SYS-pH;厂家:辽宁赛亚斯科技有限公司)检测食管pH < 4反流次数、> 5 min反流次数和pH < 4总时间;(5)2组患者的HP根除率:2组患者中HP感染者给予标准四联根除HP方案,根除HP疗程为14d,停药至少1月复查13C呼气试验,复查13C呼气试验阴性表明成功根除,根除率=根除后复查HP转为阴性的例数/根除前HP阳性例数×100%;(6)2组患者的不良反应发生率:统计观察组及对照组患者治疗中及治疗后各种不良反应的例数,计算出总不良反应率。
1.4 统计学处理
应用SPSS22.0对数据进行分析,计量资料以($\bar x \pm s$)表示,采用独立样本t检验。计数资料以 [n(%)]描述,采用χ2检验,P < 0.05为差异有统计学意义。
2. 结果
2.1 2组患者的一般资料比较
2组患者的性别、年龄、BMI、吸烟、饮酒、HP感染率一般资料比较,差异无统计学意义(P > 0.05),见表1。
2.2 2组患者的临床疗效比较
观察组总有效率为93.55%,高于对照组的75.81%,差异有统计学意义(P < 0.05),见表2。
表 2 2组患者治疗后临床疗效[n(%)]Table 2. Clinical efficacy of two groups of patients after treatment [n(%)]组别 n 显效 有效 无效 总有效率 观察组 62 34(54.84) 24(38.71) 4(6.45) 58(93.55) 对照组 62 29(46.77) 18(29.03) 15(24.19) 47(75.81) χ2 / / / / 7.521 P / / / / 0.006* *P < 0.05。 2.3 2组患者的RDQ评分比较
观察组治疗后RDQ评分低于对照组(P < 0.05),见表3。
表 3 2组患者治疗前后RDQ评分[($\bar x \pm s$),分]Table 3. RDQ scores before and after treatment in the two groups [($\bar x \pm s$),points]组别 n 治疗前 治疗后 观察组 62 17.13±6.11 9.03±3.28 对照组 62 16.81±5.59 10.48±2.86 t / 0.307 −2.624 P / 0.760 0.010* *P < 0.05。 2.4 2组患者的PSQI评分比较
观察组治疗后PSQI评分低于对照组(P < 0.05),见表4。
表 4 2组患者治疗前后PSQI评分[($\bar x \pm s$),分]Table 4. PSQI score before and after treatment in the two groups [($\bar x \pm s$),points]组别 n 治疗前 治疗后 观察组 62 7.90±2.51 4.76±1.89 对照组 62 7.85±1.57 5.95±1.74 t / 0.129 −3.660 P / 0.898 < 0.001* *P < 0.05。 2.5 2组患者治疗前后食管pH值变化比较
观察组治疗后pH < 4反流次数、> 5 min反流次数和pH < 4总时间低于对照组(P < 0.05),见表5。
表 5 2组患者治疗前后食管pH检测变化($\bar x \pm s$)Table 5. The changes of esophageal pH before and after treatment in the two groups of patients($\bar x \pm s$)组别 n pH < 4反流次数(次) > 5 min反流次数(次) pH < 4总时间(min) 治疗前 治疗后 治疗前 治疗后 治疗前 治疗后 观察组 62 28.37±8.71 11.16±2.57 5.76±2.41 1.69±0.86 74.84±12.30 16.81±5.02 对照组 62 28.05±8.15 13.02±5.36 5.42±2.27 2.87±1.15 71.77±11.23 23.97±6.17 t / −0.213 2.457 0.804 −6.447 1.449 −7.115 P / 0.832 0.015 0.423 < 0.001* 0.150 < 0.001* *P < 0.05。 2.6 2组患者中HP感染者的根除率比较
观察组HP的根除率为91.2%,高于对照组的62.5%,差异有统计学意义(P < 0.05),见表6。
表 6 2组患者HP感染者根除率(%)Table 6. The eradication rate of HP infection in the two groups of patients (%)组别 n 根除率(%) 根除前HP阳性 根除后HP阴性 观察组 34 31 31/34(91.2) 对照组 32 20 20/32(62.5) χ2 / / 4.030 P / / 0.045* *P < 0.05。 2.7 2组患者的总不良反应发生率比较
观察组出现恶心1例,腹泻1例,腹胀1例,总不良反应发生率4.84%(3/62);对照组出现恶心2例,腹痛1例,头痛2例,总不良反应发生率8.06(5/62);2组总不良反应发生率无明显差异(χ2 = 0.534,P = 0.465)。
3. 讨论
NERD大约占胃食管反流病的50%~70%[7],烧心和反流是该病最常见和最典型的症状,抑制胃酸分泌是治疗NERD最常用的方法[8],伴有睡眠障碍的NERD反酸、烧心症状更明显,这是由于睡眠障碍可降低食管的痛阈,增加食管酸感知的频率及程度 [9]。有研究[10]表明PPIs可明显减轻NERD的症状,然而PPIs治疗NERD的治愈率低,长期服用易出现上腹痛、恶心、呕吐、腹泻、头痛等不良反应,因而寻找1种对NERD治愈率高且不良反应少的抑酸药物对临床十分有益。
富马酸伏诺拉生片是1种新型的抑酸药,可以竞争性阻断H+/K+-ATP酶,从而抑制胃酸分泌。口服吸收迅速,1.5~2.0 h可达到最大血浆浓度,且吸收受食物影响小[11],可迅速进入胃壁细胞并聚集,半衰期长达9 h[1],具有快速抑酸、持久抑酸的特点。在治疗反流性食管炎方面具有较高的黏膜愈合率[12],与PPIs对比,安全性相似[13]。大量研究[14-16]表明富马酸伏诺拉生片治疗难治性反流性食管炎方面疗效优于PPIs,2020年日本的1项研究[17]表明无论在给药后24 h还是夜间抑酸能力,富马酸伏诺拉生片都优于雷贝拉唑。赵莹等[18]研究中报道了富马酸伏诺拉生片与艾司奥美拉唑镁肠溶片在治疗胃食管反流病中的对比,结果提示富马酸伏诺拉生片抑酸作用明显,可提高食管黏膜愈合率,安全性更高。
本研究将在青岛市第八人民医院就诊的124例NERD患者随机分成观察组和对照组,观察富马酸伏诺拉生片与艾司奥美拉唑镁肠溶片在治疗NERD中的效果及不良反应。结果表明,观察组总有效率高于对照组,观察组治疗后RDQ评分低于对照组,考虑主要原因是富马酸伏诺拉生片比艾司奥美拉唑镁肠溶片起效快,半衰期长,抑酸稳定。张泽丹等[19]研究表明胃食管反流病患者睡眠障碍的发生率明显高于无胃食管反流病者,里提甫江·买买提艾力等[20]研究表明NERD患者合并睡眠障碍情况高于反流性食管炎患者。本研究对NERD患者睡眠状况分析,观察组和对照组治疗前都存在睡眠障碍,观察组治疗后PSQI评分低于对照组,这可能由于NERD患者易出现夜间酸反流且食管存在高敏感性,而富马酸伏诺拉生片持久抑酸,可充分抑制夜间的胃酸分泌,从而改善睡眠质量。本研究显示观察组治疗后pH < 4反流次数、> 5 min反流次数和pH < 4总时间低于对照组,表明富马酸伏诺拉生片较艾司奥美拉唑镁肠溶片抑酸作用更强。HP感染可引起胃炎、消化性溃疡、胃恶性肿瘤等疾病[21],根除HP可降低胃恶性肿瘤的发生风险[22]。 2016年日本Hp感染管理指南[23]中提出富马酸伏诺拉生片可作为根除HP的一种治疗药物,多项日本临床研究表明,富马酸伏诺拉生在根除HP中的作用不劣于PPIs,耐受性较好[24],我国建议四联根除HP,本研究将2组患者中HP感染者给予标准四联根除HP方案,结果表明观察组HP根除率高于对照组,提示富马酸伏诺拉生片作为抑酸药在根除HP方面比艾司奥美拉唑镁肠溶片更有优势。本研究对观察组和对照组在治疗中和治疗后出现的不良反应进行比较,在总不良反应率方面无统计学差异,表明富马酸伏诺拉生片作为1种新型抑酸药,并不会增加患者的不良反应,安全性高。
综上所述,在治疗非糜烂性反流病方面,富马酸伏诺拉生片与艾司奥美拉唑镁肠溶片对比,疗效显著,可改善睡眠质量,抑酸更强,提高HP根除率,并不增加不良反应,值得临床推广使用。但是本研究存在一定的局限性,本研究是一项单中心研究,纳入的病例数相对偏少,未来需要多中心、大样本的临床研究,为非糜烂性反流病患者的治疗提供更充分的依据。
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