泪液细胞因子水平在甲状腺相关眼病活动分期中的价值探讨

发布时间：2018-05-15 02:00

本文选题：甲状腺相关眼病 + 泪液细胞因子　；参考：《南方医科大学》2012年硕士论文

【摘要】：研究背景甲状腺相关眼病(Thyroid associated ophthalmopathy, TAO)是一种与甲状腺自身免疫疾病密切相关的眼部疾病,常伴随着Graves'病(Graves'disease,GD)发生,临床上又称为Graves眼病(Graves ophthalmopathy, GO),其眼部表现具有显著的特征：轻者仅有眼部不适、眼睑退缩；重者出现突眼、复视,甚至视力丧失。目前还没有令人满意的治疗方法。 TAO发病机制尚不完全明了,较公认的为“共同抗原学说”,即眼外肌膜成纤维细胞与甲状腺交叉抗原表达,导致眶内活性淋巴细胞浸润,释放各种细胞因子,作用于眼眶组织,引起眼眶的炎症反应,同时刺激成纤维细胞分泌糖胺聚糖(Glycosaminoglycans, GAG),大量亲水性GAG堆积继而导致眼球突出等一系列临床症状,其中TSHR可能作为主要交叉抗原在TAO发病机制中起重要作用。球后成纤维细胞、淋巴细胞及其分泌的细胞因子在TAO的发生、发展中发挥了关键作用,也成为目前较为公认的发病机制所在。之前的研究证实不同分期TAO外周血和球后组织中细胞因子的表达具有差异性,活动期显著升高的细胞因子IL-6、TNF-α等可在一定程度上评判眼病活动分期。作为甲状腺相关眼病中受累组织之一的泪腺,在发病及眼病进展中的作用目前尚不明确。影像学及病理学研究证实,TAO患者泪腺肿大,泪腺组织淋巴细胞浸润和脂肪增生,与球后组织病理改变相似。Eckstein等发现正常泪腺腺泡细胞表面表达促甲状腺素受体(Thyroitropin receptor, TSHR),而TSHR是导致甲状腺和眼眶发病的重要交叉抗原。由此,研究者提出假说,泪腺腺泡细胞可能是TAO发病中的靶细胞之一临床上,TAO患者的眼表症状突出,包括结膜充血水肿、流泪或泪液分泌减少、干涩异物感等,与干眼症相似,亦有研究证实TAO患者泪膜破裂时间缩短,眼表疾病指数升高,干眼症状明显,引起临床医生和学者们的重视。既往的研究认为眼表症状的出现与眼球突出度增加、睑裂增宽,泪液蒸发加速导致泪液渗透压增加相关,但是近来的研究证据显示可能泪腺也参与TAO的干眼症状的发生。泪腺是眼分泌性免疫系统的主要组织,含有多种淋巴细胞和免疫活性成分,对眼表的保护作用和免疫功能的发挥主要依靠分泌泪液实现。泪液中含有复杂的细胞因子成分,可调节多种细胞功能,其浓度变化可引起一系列炎症反应,导致干眼等眼表症状,可作为疾病恢复的信号和干眼症等眼表疾病的标志物。泪腺腺泡细胞与球后成纤维细胞表面同样表达TSHR,并出现与活动期球后组织相似的病理改变,由此,我们提出假设：泪腺是否与球后组织一样存在细胞因子网络的失衡?而作为泪腺分泌物,泪液细胞因子水平是否可以间接反映泪腺炎症反应和眼病活动性?目前国内外此类研究极少,甲状腺相关眼病患者泪液细胞因子表达情况尚不明确。对TAO患者泪液细胞因子谱的研究有助于进一步从非创性角度去初步探讨泪腺在甲状腺相关眼病发病中的作用,对不同活动分期TAO细胞因子浓度的比较,有助于寻求活动分期的可能指标,为临床治疗提供可靠依据。液相芯片是基于美国Luminex公司xMAP(flexible multianalyte profiling)技术的新型芯片技术平台,具有高通量、高灵敏度、高特异性的特点,非常适合于同时检测微量泪液样本中的多个细胞因子。对多个细胞因子水平进行检测与评价比检测单个细胞因子更能反应疾病发生与发展的整体变化。本研究将应用液相芯片技术分析不同活动分期TAO泪液中IL-6、IL-10、TNF-α、IFN-γ、IL-17、IL-8、和IP-10的水平,了解TAO患者泪液细胞因了的表达情况。并随访观察球后放射治疗前后各细胞因子水平的变化,进一步探讨泪腺、泪液炎症因子在TAO发病机制中的可能作用,并寻求甲状腺相关眼病活动分期的可能指标。研究目的利用液相芯片技术检测不同分期TAO泪液中5种细胞因子和2中趋化因子水平,了解TAO患者泪液细胞因子的表达情况,并寻求活动分期的可能指标。研究方法 1、研究对象：连续收集2011.01-2012.02在南方医院内分泌科和南方医科大学第三附属医院内分泌科就诊的近3个月内未行大剂量激素冲击和其他免疫抑制剂治疗的中重度TAO患者47例,按CAS评分标准将其分为活动期27例(CAS≥3)和稳定期20例(CAS3)；同期连续收集正常对照组10例。收集非刺激性泪液,采用LiquiChipTM液相芯片技术检测泪液中IL-6、IL-10、TNF-α、IFN-γ、IL-17、IL-8和IP-10的水平 2、统计方法：采用SPSS13.0统计软件进行数据分析。符合正态分布的计量资料以X+SD表示,偏态分布的计量资料以中位数表示。正态分布的各组间计量资料的分析采用One-way ANOVA,当P≥0.05时,认为方差齐,用LSD法作进一步的多重比较。方差不齐性时,用Welch法校正,并用Dunnett T3法进一步作多重比较。偏态分布的计量资料和等级资料的组间比较采用非参数检验,三组间泪液细胞因子浓度比较采用Kruskal-Wallis H检验,P0.05为差异有统计学意义,进一步组间比较采用Wilcoxon符号秩检验,检验水准采用校正后P值,以P0.017为差异有统计学意义。TAO患者中泪液细胞因子与促甲状腺素受体抗体(thyrotropin receptor antibody,TRAb)和CAS评分的相关性分析用spearman相关分析,P0.05为差异有统计学意义。研究结果 1.TAO与正常人泪液细胞因子表达情况细胞因子IL-6、趋化因子IP10、IL-8在所有57份标本中取得结果(检出率为100%),浓度范围分别为0.32～1583.15pg/ml、58.26～19404.08pg/ml、6098.18-24034pg/ml。TNF-α和IL-10在54份标本中检出(检出率为94.7%),浓度范围分别为0.25～121.23pg/ml、0.33～108.82pg/ml。IL-17仅在1例标本中检出(检出率为1.8%),IFN-γ在所有的标本中均未检出. 2、TAO组与正常人泪液细胞因子浓度比较与正常组相比较,TAO患者泪液IL-6、IL-10、IL-8和IP-10水平显著升高,差异具有统计学差异(P0.001P=0.046P=0.002P=0.026),而TNF-α在TAO和正常组间无显著差异(P=0.664) 3、不同TA0分组间泪液细胞因子浓度比较 IL-6：与正常组比较,活动期TAO组和稳定期TAO组泪液IL-6浓度均明显升高,差异有统计学意义。(P0.001P=0.003),活动期TAO组和稳定期TAO间比较,差异具有统计学意义,活动期TAO组IL-6显著高于稳定期TAO组(P=0.0010.017)。 IL-10：与正常组相比较,活动期TAO泪液IL-10浓度显著升高,差异有统计学意义(P0.001)；活动组与稳定组比较差异具有统计学意义,活动期TAO IL-10浓度显著高于稳定期TAO(P=0.009)；稳定期TAO组与正常组间比较差异无统计学意义(P=0.705) TNF-α：与正常组相比较,活动期TAO组和稳定期TAO组泪液TNF-α浓度无显著差异(P=0.353P=0.555),活动期TAO组与稳定期TAO组比较,差异具有统计学差异(P=0.006) IL-8：与正常组相比较,活动期TAO泪液IL-8浓度显著升高,差异有统计学意义(P0.001)；活动组与稳定组比较差异无统计学意义(P=0.017)稳定期TAO组与正常组间比较差异无统计学意义(P=0.044) IP-10：与正常组相比较,活动期TAO泪液IP10浓度显著升高,差异有统计学意义(P=0.004)；活动组与稳定组比较差异具有统计学意义,活动期TAOIP10浓度显著高于稳定期TAO(P=0.004)；稳定期TAO组与正常组间比较差异无统计学意义(P=0.307) 4、TA0患者泪液细胞因子与CAS、TRAb的相关 TAO患者泪液IL-6、IL-10、TNF-a、IL-8、IP10与活动性评分CAS呈不同程度相关性(r=0.565,0.415,0.453,0.385,0.469；P0.001,0.005,0.002,0.008,0.001) TAO患者泪液IL-6、TNF-α、IL-8、IP10与TRAb浓度呈不同程度相关性(r=0.477,0.325,0.314,0.365；P=0.002,0.032,0.032,0.012),IL-10浓度与TRAb无显著相关(P0.05) 结论 1、甲状腺相关眼病影响泪液细胞因子水平,与正常人比较,TAO患者泪液细胞因子IL-6、IL-10、IL-8和IP10显著浓度升高。 2、不同活动分期TAO泪液细胞因子表达具有差异性,活动期TAO泪液IL-6、IL-10和IP10浓度显著高于稳定期,并与眼病活动性评分CAS呈正相关,泪液IL-6、IL-10和IP10浓度在一定程度上可作为活动分期的评判指标。研究目的纵向观察并比较甲状腺相关眼病患者泪液中5种细胞因子和2种趋化因子水平在球后放射治疗前后的变化,旨在了解其动态变化,进一步明确泪液细胞因子在判断眼病活动分期中的意义。材料与方法 1.研究对象：第一章中活动期TAO组中接受直线加速器球后放射治疗、并完成治疗后3个月随访的14例TAO患者,男2例,女12例,平均年龄(39.00+11.17)岁。治疗后3月收集非刺激性泪液,采用LiquiChipTM液相芯片技术检测泪液中IL-6、IL-10、TNF-α、IFN-γ、IL-8、IP-10和IL-17的水平 2.治疗方法球后放射使用美国VARIAN2100C型直线加速器。分次照射剂量为200cGy,总剂量2000cGy(10次/2周),在模拟机下定位,照射野大小一般为4cmx4cm,机架角度偏转3°～5°,以避开对侧晶体,疗程为2周。治疗开始时同时口服强的松30mg,1/日,治疗结束后逐渐减量至停药。 3.临床相关指标所有入组患者完成治疗前和治疗3月后临床活动评分(CAS),NOSPECS分级、突眼度及睑裂宽度测量,同时抽血进行甲状腺功能和TRAb的检测。CAS、突眼度、睑裂宽度的测定由2名指定的专业人员独立完成,测量结果意见一致。 4.统计方法：采用SPSS13.0统计软件进行数据分析。计量资料以X+SD或者中位数表示。治疗前后CAS、突眼度、睑裂宽度和TRAb浓度比较采用配对t检验,治疗前后泪液细胞因子浓度比较采用两相关样本非参数检验,P0.05为差异有统计学意义。研究结果 1、球后放射治疗前后临床指标比较与球后放射治疗前比较,治疗后3月后CAS评分、TRAb、睑裂宽度和突眼度均显著下降,差异具有统计学意义(P0.001P=0.001P=0.005P=0.009)见表2-1 2、治疗前后泪液细胞因子浓度比较治疗后TAO泪液细胞因子IL-6浓度显著下降,差异具有统计学意义(P=0.041)其余细胞因子IL-10、TNF-α、IL-8和IP10浓度无显著下降(P=0.950P=0.074P=0.096P=0.510) 结论 1、球后放射治疗后TAO泪液IL-6浓度显著下降。机制可能为球后放疗减轻泪腺淋巴细胞浸润和细胞因子IL-6的合成分泌,从而降低泪液IL-6的浓度。 2、球后放射治疗后TAO患者眼病活动性评分、突眼度和睑裂宽度显著下降,证实球后放疗是治疗活动期中重度TAO的有效方法,
[Abstract]:Research background
Thyroid associated ophthalmopathy (TAO) is an ocular disease closely related to the autoimmune disease of the thyroid gland, often accompanied by the occurrence of Graves'disease (Graves'disease, GD), which is also known as Graves ophthalmopathy (Graves ophthalmopathy, GO). Its ocular manifestations are characterized by obvious eye symptoms, only eye discomfort in the light. Eyelid retraction, severe eye exophthalmos, diplopia and even loss of vision. There is no satisfactory treatment at present.
The pathogenesis of TAO is not fully understood. It is generally acknowledged that the "common antigen theory", that is, the expression of the cross antigen of the extraocular myocutaneous fibroblasts and the thyroid gland, leads to the infiltration of active lymphocytes in the orbit, the release of various cytokines, the action of the orbital tissue, the inflammation reaction in the orbit, and the secretion of glycosaminoglycan (Glycos) in the fibroblasts. Aminoglycans, GAG), a large number of hydrophilic GAG accumulation leads to a series of clinical symptoms such as eyeball protrusion, in which TSHR may play an important role in the pathogenesis of TAO. The post ball fibroblast, lymphocyte and its secreted cytokines play a key role in the development of TAO, and have also become more important. Previous studies have shown that the expression of cytokines in the peripheral blood and post - ball tissues of TAO in different stages is different. The cytokine IL-6, TNF- alpha, which are significantly higher in the active stage, can be used to judge the stage of eye disease to a certain extent.
The role of lacrimal gland, one of the affected tissues of the thyroid associated ophthalmopathy, is not clear in the progression of the disease and ophthalmopathy. Imaging and pathological studies have confirmed that the lacrimal gland enlargement, lymphocytic infiltration of lacrimal gland tissue, and fatty hyperplasia in TAO patients are similar to.Eckstein in the pathological changes of the tissue, and the surface of the normal lacrimal gland acinar cell surface is found. Thyroitropin receptor (TSHR), and TSHR is an important cross antigen that causes the thyroid and orbital diseases. Thus, the researchers hypothesized that lacrimal gland acinar cells may be one of the target cells in the pathogenesis of TAO.
Clinically, the ocular surface symptoms of TAO patients are prominent, including conjunctival congestion and edema, tears or tear secretion reduction, dry and foreign body sensation, similar to dry eye. Studies have confirmed that the tear film rupture time of the TAO patients is shortened, the eye surface disease index is increased, and the dry eye symptoms are obvious, which cause the attention of the clinicians and scholars. Appearance and exophthalmos increased, palpebral fissure widened and tear vaporization accelerated to increase lacrimal osmotic pressure, but recent evidence suggests that lacrimal glands may also be involved in the occurrence of dry eye symptoms in TAO.
Lacrimal gland is the main tissue of the secretory immune system of the eye. It contains a variety of lymphocytes and immune active components. The protection and immune function of the eye surface mainly depend on the secretion of tears. The tears contain complex cytokine components, which can regulate a variety of cell functions. The concentration changes can cause a series of inflammatory reactions, leading to a series of inflammatory reactions. Dry eye and other ocular surface symptoms can be used as markers for disease recovery and ocular surface diseases such as dry eye.
The lacrimal gland acinar cells also express TSHR on the surface of the post ball fibroblasts, and appear to be similar to those of the active tissue in the active stage. Therefore, we hypothesized that if the lacrimal gland is like the post ball tissue, there is an imbalance in the cytokine network. As a lacrimal gland secretion, whether the level of lacrimal fluid cytokine can indirectly reflect the mastitis There are few such studies at home and abroad. The expression of tear cytokine in patients with thyroid related ophthalmopathy is not clear. The study of the tear cytokine spectrum of TAO patients is helpful to further explore the role of lacrimal gland in the pathogenesis of thyroid related ophthalmopathy from the noninvasive angle, and to the different stages of TAO The comparison of cytokines concentration can help to find possible indicators of activity staging and provide reliable evidence for clinical treatment.
Liquid chip is a new chip technology platform based on the xMAP (flexible multianalyte profiling) technology of Luminex company in the United States. It is characterized by high throughput, high sensitivity and high specificity. It is very suitable for the simultaneous detection of multiple cytokines in microtear samples. Detection and evaluation of multiple cytokine levels are more than single detection. Cytokines can better reflect the overall changes in disease occurrence and development.
This study will use liquid chip technology to analyze the expression of IL-6, IL-10, TNF- a, IFN-, IL-17, IL-8, and IP-10 in TAO tears of different activity stages, and to investigate the changes in the level of cell factors in the patients with TAO after the post ball radiotherapy, and further explore the pathogenesis of lacrimal gland and tear inflammatory factors in TAO. The possible role of the mechanism and the possible indicators of thyroid related ophthalmopathy activity stages.
research objective
The level of 5 cytokines and chemokines in different stages of TAO tears were detected by liquid chip technology, and the expression of tear cytokine in TAO patients was understood and the possible index of activity stage was sought.
research method
1, subjects: 47 cases of moderate to severe TAO patients who had not been treated with large dose of hormone impact and other immunosuppressive agents in the Department of endocrinology in the Department of endocrinology of southern hospital and the Affiliated Hospital of Southern Medical University for nearly 3 months, were divided into 27 cases (CAS > 3) and 20 cases of stability (CAS > 3) according to the CAS score (CAS). CAS3); 10 cases of normal control group were collected continuously in the same period. Non irritant tears were collected and LiquiChipTM liquid chip technique was used to detect IL-6, IL-10, TNF- alpha, IFN- gamma, IL-17, IL-8 and IP-10 in tears.
2, statistical method: using the SPSS13.0 statistical software to analyze the data. The measurement data conforming to the normal distribution are expressed in the X+SD. The measurement data of the partial distribution are expressed in the median. One-way ANOVA is used for the analysis of the measurement data of the normal distribution. When P is more than 0.05, the variance is homogeneous and the LSD method is used for further multiple comparison. Variance is not the same. At the time of homogeneity, the Welch method was corrected and the Dunnett T3 method was used for multiple comparison. The measurement data of the partial distribution and the grade data were compared with the non parameter test. The concentration of tear cell factors between the three groups was compared with the Kruskal-Wallis H test, and the P0.05 was statistically significant, and the further group was compared with the Wilcoxon sign test. The test level was corrected by the corrected P value, and the correlation analysis of tear cytokine and thyrotropin receptor antibody (thyrotropin receptor antibody, TRAb) and CAS score in.TAO patients with statistically significant difference in P0.017 was statistically significant, and P0.05 was of significant difference.
Research results
Expression of 1.TAO and normal human tear cytokine
The results of cytokine IL-6, chemokine IP10, IL-8 were obtained in all 57 specimens (the detection rate was 100%), the concentration range was 0.32 to 1583.15pg/ml, 58.26 to 19404.08pg/ml, 6098.18-24034pg/ml.TNF- alpha and IL-10 were detected in 54 samples (detection rate was 94.7%), the concentration range was 0.25 to 121.23pg/ml, 0.33 to 108.82pg/ml.IL-17 only. The detection rate was 1.8% in 1 specimens, and IFN- gamma was not detected in all specimens.
2, TAO group and normal human tear cytokine concentration comparison
Compared with the normal group, the levels of IL-6, IL-10, IL-8 and IP-10 in TAO patients were significantly higher, and the difference was statistically significant (P0.001P=0.046P=0.002P=0.026), but there was no significant difference between the TNF- alpha and the normal group (P=0.664).
3, comparison of the concentration of tear cell factors between different TA0 groups
IL-6: compared with the normal group, the tear IL-6 concentration in the active phase TAO group and the stable period TAO group increased significantly, and the difference was statistically significant. (P0.001P=0.003), the difference was statistically significant between the active TAO group and the stable phase TAO, and the IL-6 in the active stage TAO group was significantly higher than that of the stable TAO group (P=0.0010.017).
IL-10: compared with the normal group, the concentration of IL-10 in the active TAO tears was significantly increased, the difference was statistically significant (P0.001), the difference between the active group and the stable group was statistically significant, and the concentration of TAO IL-10 in the active phase was significantly higher than that of the stable TAO (P=0.009), and there was no significant difference between the group of stable TAO and the normal group (P=0.705).
TNF- alpha: compared with the normal group, there was no significant difference in the TNF- alpha concentration between the active TAO group and the stable phase TAO group (P=0.353P=0.555). The difference between the active TAO group and the stable TAO group was statistically significant (P=0.006).
IL-8: compared with the normal group, the concentration of IL-8 in TAO tear was significantly higher in active phase (P0.001), and there was no significant difference between the active group and the stable group (P=0.017), and there was no statistical difference between the TAO group and the normal group (P=0.044).
IP-10: compared with the normal group, the concentration of IP10 in the active TAO tears was significantly increased, the difference was statistically significant (P=0.004). The difference of the activity group and the stable group was statistically significant, and the concentration of TAOIP10 in the active phase was significantly higher than that of the stable phase TAO (P=0.004), and there was no statistical difference between the stable TAO group and the normal group (P=0.307).
4, the correlation between tear cytokine and CAS, TRAb in TA0 patients
The IL-6, IL-10, TNF-a, IL-8, IP10 of TAO patients were related to the activity score (r=0.565,0.415,0.453,0.385,0.469; P0.001,0.005,0.002,0.008,0.001) in varying degrees.
The concentrations of IL-6, TNF-, IL-8, IP10 and TRAb in TAO patients were correlated (r=0.477,0.325,0.314,0.365; P=0.002,0.032,0.032,0.012) in varying degrees (r=0.477,0.325,0.314,0.365; P=0.002,0.032,0.032,0.012). There was no significant correlation between IL-10 concentration and TRAb (P0.05).
conclusion
1, thyroid associated ophthalmopathy affected the level of lacrimal cytokine. Compared with normal subjects, the levels of IL-6, IL-10, IL-8 and IP10 increased significantly in TAO patients.
2, the expression of tear cytokine in TAO with different activity stages was different. The activity stage TAO tear IL-6, IL-10 and IP10 concentration were significantly higher than that in the stable stage, and were positively correlated with the activity score of ophthalmopathy CAS. The concentration of IL-6, IL-10 and IP10 in tear could be used as the evaluation index of the activity stage.
research objective
Longitudinal observation and comparison of the changes of 5 cytokines and 2 chemokine levels in the tears of patients with thyroid related ophthalmopathy before and after the post - ball radiotherapy are designed to understand the dynamic changes and to further clarify the significance of tear cell factors in judging the stage of eye disease.
Materials and methods
1. subjects: in the first chapter, in group TAO, group TAO was treated with linear accelerator post ball radiotherapy, and 14 cases of TAO patients were followed up for 3 months after treatment. 2 men, 12 women, and average age (39.00+11.17) years. After treatment, non irritant tears were collected in March, and IL-6, IL-10, TNF- a, IFN- gamma, IL-8 in tears were detected by LiquiChipTM liquid chip technology. The level of IP-10 and IL-17
2. treatment methods
After the ball, the United States VARIAN2100C linear accelerator was used. The dose of fractional irradiation was 200cGy, the total dose was 2000cGy (10 times /2 weeks). The size of the irradiated field was generally 4cmx4cm, the angle of the frame was 3 to 5 degrees, to avoid the contralateral crystal for 2 weeks. At the beginning of the treatment, the oral prednisone 30mg, 1/ day, gradually after the treatment ended, gradually after the treatment ended. Reduce to withdrawal.
3. clinical related indexes
The clinical activity score (CAS), NOSPECS classification, exophthalmos and palpebral fissure width were measured before and after March, and the measurement of thyroid function and TRAb for.CAS, exophthalmos and palpebral fissure width were determined by 2 designated professionals.
4. statistical method: using SPSS13.0 statistical software for data analysis. The measurement data were expressed in X+SD or median. CAS, exophthalmos, palpebral fissure width and TRAb concentration were compared before and after treatment by paired t test. The concentration of tacrimal cytokine before and after treatment was compared with two related samples, and P0.05 was statistically significant.
Research results
1, comparison of clinical indexes before and after post ball radiotherapy
Compared with post - ball radiotherapy, the CAS score after March after treatment, TRAb, the width of cleft palpebral and the degree of exophthalmos were significantly decreased, and the difference was statistically significant (P0.001P=0.001P=0.005P=0.009) in table 2-1

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R581.1;R771.3

【参考文献】