【作者】 李静；

【导师】 张风河；

【作者基本信息】 山东大学 ， 口腔临床医学， 2016， 硕士

【摘要】 目的用酶联免疫吸附试验(ELISA法)检测口腔颌面外科围手术期患者血浆中脑源性神经营养因子BDNF的浓度并探讨其临床意义。探讨口腔颌面部鳞状细胞癌患者(OSCC组)、癌前病变患者(OPL组)与正常对照组(NC组)血浆中脑源性神经营养因子(BDNF)、血管内皮生长因子(VEGF)、白细胞介17A(IL-17A)及白细胞介素17F(IL-17F)水平有无差别,并研究其相关性,观察其表达水平与口腔癌患者临床病理特征的关系。材料和方法该实验取得山东大学口腔医学院伦理学会的同意,选择在山东省口腔医院行全麻手术治疗的患者(选择符合纳入标准的患者共118例,其中男63例,女55例,年龄21-77岁,平均年龄46.71±8.53岁,平均受教育年限8.75±3.94年),采用酶联免疫吸附试验(ELISA)法,分别于:入院后清晨(T1)、气管插管成功且手术开始之前(T2)、缝合后(T3)、拔管后20min(T4)、术后第一日清晨(T5)取外周静脉血检测血浆中BDNF浓度,并采用汉密尔顿焦虑量表(Hamilton anxiety scale,HAMA,14项版)来评定患者围手术期的焦虑程度,通过GraphPad Prism 5.0软件进行统计分析,采用非参数检验的曼-惠特尼U检验(Mann-Whitney U test)法来比较患者围手术期血浆中BDNF浓度变化,相关性分析采用spearman等级相关分析(spearman rank correlation analyze)法,以p<0.05 为有统计学意义。在住院患者中选择符合纳入要求的口腔颌面部鳞状细胞癌患者(OSCC组)、癌前病变患者(OPL组)分别入组,并于社会募集正常对照组(NC组),所有研究对象均于清晨空腹留外周静脉血样约3ml,采用酶联免疫吸附试验(ELISA)法测定血浆中脑源性神经营养因子(BDNF)、血管内皮生长因子(VEGF)、白细胞介素17A(IL-17A)及白细胞介素17F(IL-17F)含量,通过GraphPad Prism 5.0软件进行统计分析,相关性分析采用spearman等级相关分析(spearman rank correlation analyze)法,三组均数之间的比较采用K-W检验(Kruskal-Wallis statistic),两两比较采用非参数检验的曼-惠特尼U检验(Mann-Whitney U test),以p<0.05为差异具有统计学意义。结果1.患者一般状况包括年龄、性别、文化程度、经济状况、诊断时间、手术时间、手术持续时间,与其手术前(T1)及手术后(T2)血浆中BDNF的浓度均无明显相关关系,其差异无统计学意义(p＞0.05)。2.围手术期患者血浆中 BDNF 浓度:T1(626.5±283.7pg/ml)、T2(315.5±205.2pg/ml)、T3(710.7±478.5pg/ml)、T4(663.8 ± 533.4pg/ml)、T5(249.2±11O.1pg/ml)。以T1为对照,T2患者血浆中BDNF浓度明显降低,差异有显著统计学意义(p=0.0017);在T3与T4测得血浆中BDNF浓度之间无明显差异(p=0.616),且与T1相比均没有明显差别(p=0.91,p=0.0673);T3患者血浆中BDNF浓度较T2明显升高,差异有显著统计学意义(p<0.01);T5患者血浆中BDNF浓度明显低于T1,差异有显著统计学意义(p<0.01)。3.T3时刻测得血浆中BDNF浓度与手术持续时间长短之间呈正相关关系(rs=0.2289,p=0.0139),此时刻测得血浆中 BDNF 浓度值浮动最大(55pg/ml-1920pg/ml),而术后第一日清晨(T5)血浆中BDNF浓度与手术持续时间长短之间无明显相关关系(rs=0.1539,p=0.1006)。4.患者术后的焦虑程度较术前明显加重,且差异具有统计学意义(p=0.011)。且患者术后的焦虑程度与其血浆中BDNF浓度呈负相关(rs=-0.308,p=0.0008)。5.口腔癌患者测得的血浆中BDNF浓度与癌前病变患者及正常对照之间比较无统计学差异(p=0.6227)。OSCC患者血中BDNF浓度与VEGF浓度之间无明显相关关系(rs=-0.1982,p=0.1508)。OSCC患者血中BDNF浓度与其临床病理学特征之间均无统计学意义。6.OSCC 组血浆中 VEGF(172.4±76.25pg/ml)、IL-17A(33.19±12.6pg/ml)、IL-17F(115.3±51.52pg/ml);OPL 组血浆中 VEGF(130.3±45.24pg/ml)、IL-17A(24.67±11.62pg/ml)、IL-17F(152.9±70.08pg/ml);NC组血浆中 VEGF(103.5±36.17pg/ml)、IL-17A(18.74±6.337pg/ml)、IL-17F(230.9± 161.8pg/ml)。自 OSCC组、OPL组至NC组,其血浆中VEGF和IL-17A的水平依次降低,而IL-17F的水平则为NC组最高,OSCC组最低,且差异均具有统计学意义。OSCC患者血浆中IL-17A与IL-17F的水平均与VEGF水平密切相关,且VEGF水平与IL-17A呈正相关(rs=0.3062,p=0.0243),与 IL-17F 呈负相关(rs=-0.27,p=0.0484),而 IL-17A与 IL-17F 之间无明显相关性(rs=0.0456,p=0.7435)。7.OSCC患者血中VEGF浓度与肿瘤直径(p=0.0181)、TNM分期(p=0.0169)、淋巴结转移(p=0.0249)及分化程度(p=0.008)具有明显相关关系。IL-17A浓度与吸烟(p=0.014)及TNM分期(p=0.0343)、淋巴结转移(p=0.0403)具有明显相关关系。IL-17F浓度与饮酒(p=0.0469)、吸烟(p=0.0272)及肿瘤直径(p=0.0124)、TNM分期(p=0.0334)及淋巴结转移(p=0.0465)具有明显相关关系。8.通过拟合 ROC 曲线(receiver operating characteristic curve)检测 VEGF、IL-17A与IL-17F的诊断效能,VEGF、IL-17A与IL-17F分别可能作为OSCC患者血中诊断标记物并与肿瘤进展相关,而IL-17F/VEGF联合可作为OSCC患者相对有效的肿瘤诊断标志物。结论1.在围手术期,患者血浆中的BDNF浓度有特定的变化趋势且与麻醉及手术持续时间有关;全身麻醉可能会加重患者围手术期焦虑程度,其机制可能与血浆中BDNF水平降低有关。2.OSCC患者血中BDNF与VEGF之间无相关关系且肿瘤的进展无直接相关。3.OSCC患者血中VEGF、IL-17A、IL-17F的水平可能与肿瘤的进展相关,并且可能联合作为相对有效的肿瘤诊断标志物。更多还原

【Abstract】 Objective:To explore the variation of perioperative plasma concentration of BDNF and its clinical significance in patients undergoing perioperative period.To dectect the levels of brain derived neurotrophic factor(BDNF),vascular endothelial growth factor(VEGF),interleukin-17A(IL-17A),interleukin-17F(IL-17F)by enzyme-links immunosorbent assays(ELISA)in patients with oral squamous cell carcinoma,oral precancerous lesions and healthy people.To study the correlation of the four and investigate the relevance of it to the clinicopathologic characteristics of carcinoma patients.Materials and Methods:Totally 118 patients were enrolled in this study(63 boys and 55 girls,mean age of 46.71±8.53).BDNF plasma concentrations were determined in blood samples using enzyme-links immunosorbent assays at five time points:T1(baseline),T2(after intubation and before the surgery was started),T3(end of surgery),T4(20minutes after extubation),T5(24 hours after the surgery).Hamilton anxiety scale(HAMA)was used to determine their status of anxiety.The results were statistically analyzed by GraphPad Prism5.0 software.Mann-Whitney U test was used to compare changes of BDNF plasma concentrations.The relationship was analyzed by spearman rank correlation analyze.p<0.05 was considered statistically significant.We selected patients compliance with our requirement,divided their into groups of Oral squamous cell carcinoma(OSCC),Oral precancerous lesions(OPL).We selected normal control group(NC)in society.All the subject investigated need their peripheral venous blood samples,detected the concentration of BDNF,VEGF,IL-17A,IL-17F by enzyme-links immunosorbent assays(ELISA).The results were statistically analyzed by GraphPad Prism5.0 software.Kruskal-Wallis statistic was used in the comparison of BDNF,VEGF,IL-17A,IL-17F in the three groups.Mann-Whitney U test was used to compare the two groups.The relationship was analyzed by spearman rank correlation analyze.p<0.05 was considered statistically significant.Results:1.Before and after surgery,the concentrations of plasma BDNF and patients basic characteristic have no statistically significant correlation(p>0.05).2.The concentrations of plasma BDNF in patients undergoing perioperative period:T1(626.5 ± 283.7pg/ml),T2(315.5 ± 205.2pg/ml),T3(710.7 ±478.5pg/ml),T4(663.8±533.4pg/ml),T5(249.2 ± 110.1pg/ml).T1 as baseline,concentrations of plasma BDNF at T2 were significantly reduced(p=0.0017).Concentrations of plasma BDNF at T3 and T4 have no statistically significant(p=0.616).BDNF plasma concentrations at T3 were higher than T2(p<0.01).At T5,BDNF plasma concentrations decreased below baseline,have statistically significant(p<0.05).3.There was a statistically significant positive correlation between the duration of surgery and BDNF values at end of surgery(rs=0.2289,p=0.0139),at T3,the variability in BDNF plasma concentrations ranged from 55pg/ml to 1920pg/ml.There was no correction between the BDNF plasma concentrations of T5 and the duration of surgery(rs=0.1539,p=0.1006).4.Combine with Hamilton anxiety scale(HAMA),the degree of anxiety was negatively related to BDNF plasma concentrations,and have statistically significant(rs=-0.308,p=0.0008).After operation,the degree of anxiety was significantly increase(p=0.011).5.The concentrations of plasma BDNF in groups of Oral squamous cell carcinoma group(OSCC),Oral precancerous lesions group(OPL),normal control group(NC):(618± 447.7pg/ml)(555.6±454.1pg/ml)(547.5±434.9pg/ml).The concentrations of plasma BDNF of the three groups have no statistically significant(p=0.6227).The concentrations of plasma BDNF and VEGF of OSCC groups have no statistically correlation with each other(rs=-0.1982,p=0.1508).The concentrations of plasma BDNF have no statistically with patients clinicopathological characteristics.6.The plasma VEGF,IL-17A,IL-17F level of OSCC group were(172.4 ±76.25pg/ml)(33.19 ± 12.6pg/ml)(115.3 ± 51.52pg/ml);The plasma VEGF,IL-17A,IL-17F level of OPL group were(130.3 ±45.24pg/ml)(24.67 ±11.62pg/ml)(152.9 ± 70.08pg/ml);The plasma VEGF,IL-17A,IL-17F level of NC group were(103.5±36.17pg/ml)(18.74±6.337pg/ml)(230.9 ± 161.8pg/ml);In this study showed that plasma concentration of VEGF and IL-17A were remarkably down-regulated from OSCC group,OPL group to normal control group.Conversely,the normal control group had a highest level of IL-17F in the whole groups,and there was a positive correlation between VEGF and IL-17A(rs=0.3062,p=0.0243),a negative correlation between VEGF and IL-17F(rs=-0.27,p=0.0484),no correlation between IL-17F and IL-17A(rs=0.0456,p=0.7435).7.The concentration of VEGF were significantly corrected with tumor size(p=0.0181),tumor-node-metastasis(TNM)stage(p=0.0169),lymph node metastasis(P=0.0249)and differentiation(p=0.008).The concentration of IL-17A were significantly corrected with somking habit(p=0.014),TNM stage(p=0.0343),lymph node metastasis(p=0.0403).The concentration of IL-17F were significantly corrected with drinking(p=0.0469)and somking habit(p=0.0272),tumor size(p=0.0124),TNM stage(p=0.0334),lymph node metastasis(p=0.0465).8.Using ROC curve illustrated the diagnostic value of VEGF、IL-17A 与IL-17F.Results that the ratio of IL-17F/VEGF was the best diagnostic marker of OSCC than other markers.Conclusion:1.These data suggest that patients in perioperative period,BDNF plasma concentrations show a specific pattern that is correlation with the duration of surgery,anesthetic.Anesthesia might be aggravate the anxiety of patients in perioperative period,the mechanism might be related to the decrease of the concentrations of plasma BDNF.2.There have no statistically correlation of the plasma BDNF and VEGF concentrations,further more there have nothing to do with the progression of OSCC.3.The concentrations of plasma VEGF,IL-17A,IL-17F are linked to progression of OSCC,and combined as a good diagnostic marker.更多还原