【作者】 陈明；

【导师】 刘新垣； 黄飚；

【作者基本信息】 浙江理工大学 ， 生物学， 2020， 硕士

【摘要】 T细胞免疫球蛋白和粘蛋白结构域3(T-cell immunoglobulin and mucin domain3,Tim3)是一种1型抑制性跨膜受体蛋白,Tim3在与其配体半乳凝素-9(glactin-9)结合后会抑制1型辅助T细胞(Th1)的活化。Tim3根据分子片段的大小可以分为全长片段的膜结合型Tim3和短片段的可溶性Tim3(soluble Tim3, sTim3),已知膜结合型Tim3经过金属蛋白酶剪切得到 sTim3,随之释放到血液中。已有的研究报道了 sTim3在多种类型疾病中升高,如系统性红斑狼疮、Ig A肾病等。因此, sTim3的表达情况和膜结合型Tim3是密切相关的,通过检测患者血清中 sTim3或许有助于监测疾病状况的发展。目前,血清中 sTim3的检测主要采用酶联免疫吸附试验(ELISA)的方法。因为ELISA使用酶标记抗体蛋白,而酶也是大分子物质,所以容易影响抗体蛋白质原有的结构,从而严重影响酶标记化合物的生物活性。此外,由于ELISA的敏感性较低,导致许多假阴性结果。不足的稳定性和检测范围也是这种方法常见的缺点。相比传统的ELISA,时间分辨荧光免疫分析技术(TRFIA)是一种更加灵敏的常用检测方法。这种检测技术是利用镧系列的元素来标记抗原或抗体,无放射性,尽可能保证标记化合物的整体生物活性。TRFIA与其它传统的检测方法相比在稳定性、灵敏度、检测范围等多方面具有优势。因此,我们建立了可溶性Tim3时间分辨荧光免疫分析法( sTim3-TRFIA),并用双抗体夹心法检测了多种类型的疾病患者和健康个体血清中 sTim3的浓度。研究结果显示在加样血清标本只有20μL的情况下,新建立的 sTim3-TRFIA的灵敏度是0.66ng/m L,高于在加样血清标本为100μL时的 sTim3-ELISA试剂盒的灵敏度(1.11ng/m L)。 sTim3-TRFIA的批内变异系数(CV)为1.64%至4.68%,批间变异系数为5.72%至9.32%。白介素6(IL-6)和肾损伤分子1(KIM-1)在实验中的交叉反应分别为0.25%和0.04%。该方法的检测范围是0.66～100ng/m L,平均回收率为105.26%。我们的研究表明新建立的 sTim3-TRFIA不仅提高了检验血清 sTim3的灵敏度,同时也提高了检测的效率。临床应用研究结果显示, sTim3的水平在我们调查的所有类型慢性肾病患者中均显著升高。不同类型慢性肾病患者 sTim3的浓度与肾小球滤过率成反比,这说明 sTim3与肾病的进展有关, sTim3可能与慢性肾病患者免疫反应的调控作用有关。 sTim3还在胃癌患者和胃癌前病变患者中升高,联合检测胃蛋白酶原和 sTim3可以提高胃癌诊断的准确性。在不同类型肝病患者中, sTim3在肝癌、丙肝、乙肝、自身免疫肝病患者中显著升高, sTim3能够反应这类患者的肝功能情况。在部分类型的肿瘤患者中, sTim3也显著升高,如食管癌、直肠癌,Tim3可能是治愈这类肿瘤患者的分子靶点。总之,我们成功建立了TRFIA检测血清中的 sTim3。然后,我们将这种方法应用于检测不同类型的慢性肾病患者和其他疾病的患者,并且证明 sTim3-TRFIA是检测血清中 sTim3的有效诊断工具。更多还原

【Abstract】 T cell immunoglobulin and mucin domain 3(Tim3)is a type 1 inhibitory transmembrane receptor protein.Tim3 can inhibit the activation of type 1 helper T cells(Th1)after binding to its ligand,galactosin-9(glactin-9).Tim3 can be divided into fulllength segment membrane binding Tim3 and short segment soluble Tim3( sTim3)according to the size of molecular fragments.It is known that membrane-bound Tim3 is sheared by metalloproteinase to obtain sTim3,which is then released into the blood.It has been reported that sTim3 increases in many kinds of diseases,such as systemic lupus erythematosus and Ig A nephropathy.Therefore,the expression of sTim3 is closely related to membrane-bound Tim3.The detection of sTim3 in patients’ serum may be helpful to monitor the development of disease.At present,enzyme-linked immunosorbent assay(ELISA)is mainly used to detect sTim3 in serum.Because ELISA uses enzyme to label antibody protein,and enzyme is also a macromolecular substance,it is easy to affect the original structure of antibody protein,thus seriously affecting the biological activity of enzyme labeled compounds.In addition,the low sensitivity of ELISA results in many false negative results.The lack of stability and detection range is also a common disadvantage of this method.Compared with traditional ELISA,time-resolved fluorescence immunoassay(TRFIA)is a more sensitive and commonly used detection method.This detection technology uses lanthanum series elements to label antigens or antibodies without radioactivity,which can ensure the overall biological activity of the labeled compounds as much as possible.Compared with other traditional detection methods,TRFIA has advantages in stability,sensitivity,detection range and other aspects.Therefore,we established a soluble Tim3 time-resolved fluorescence immunoassay( sTim3-TRFIA),and detected the concentrations of sTim3 in sera of patients with various types of diseases and healthy individuals by double antibody sandwich method.The results showed that the sensitivity of the newly established sTim3-TRFIA was 0.66ng/m L when the serum sample was only 20μL,which was higher than that of the sTim3-ELISA kit when the serum sample was 100μL(1.11ng/m L).The coefficient of variation(CV)of sTim3-TRFIA was 1.64% to 4.68%,and the coefficient of variation between batches was 5.72% to 9.32%.The cross reaction of IL-6 and KIM-1 was 0.25% and 0.04%,respectively.The detection range of this method was 0.66-100ng/m L,and the average recovery was 105.26%.Our research showed that the newly established sTim3-TRFIA not only improves the sensitivity of serum sTim3 detection,but also improves the detection efficiency.The results of clinical application study showed that the level of sTim3 was significantly increased in all types of chronic nephropathy patients we investigated.The concentration of sTim3 was inversely proportional to the glomerular filtration rate in different types of chronic kidney disease,which indicated that sTim3 is related to the progression of kidney disease,and sTim3 may be related to the regulation of immune response in patients with chronic kidney disease. sTim3 also increased in patients with gastric cancer and precancerous lesions.Combined detection of pepsinogen and sTim3 can improve the accuracy of gastric cancer diagnosis.In patients with different types of liver disease, sTim3 was significantly increased in patients with liver cancer,hepatitis C,hepatitis B and autoimmune liver disease. sTim3 can reflect the liver function of these patients.In some types of tumor patients, sTim3 was also significantly increased,such as esophageal cancer,rectal cancer,Tim3 may be the molecular target to cure such tumor patients.In conclusion,we have successfully established TRFIA to detect sTim3 in serum.Then,we applied this method to the detection of different types of chronic nephropathy and other diseases,and proved that sTim3-TRFIA is an effective diagnostic tool for the detection of sTim3 in serum.更多还原