室间隔缺损潜在血清标志物锌指蛋白41和SPARC的验证研究

发布时间：2018-01-13 20:00

本文关键词：室间隔缺损潜在血清标志物锌指蛋白41和SPARC的验证研究　出处：《广西医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：先天性心脏病(congenital heart disease, CHD)是出生缺陷中最常见的疾病，室间隔缺损(ventricular septal defect, VSD)作为先天性心脏病中最主要的类型，对婴幼儿的存活及生活质量产生重要影响。室间隔缺损的发病原因及机理复杂，要想全面了解，除了传统相关的易感基因、环境相关因素外，更需对调控心脏异常分化的蛋白及信号通路进行研究，从而为疾病发生和诊断提供线索。本研究针对课题组前期应用同位素标记相对和绝对定量(iTRAQ)技术联合液相色谱串联基质辅助激光解析电离飞行时间质谱(LC-MALDI-TOF/TOF MS)方法筛选出一系列室间隔缺损相关的差异蛋白，结合实验结果及文献检索分析，对其中的锌指蛋白41(zinc finger protein41, ZNF41)和富含半胱氨酸的酸性分泌蛋白（secreted protein acidic and richin cysteine, SPARC）进行验证性研究，应用免疫印迹法（Western blot, WB）和酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)分别检测血清样本中锌指蛋白41和SPARC的表达水平，并应用生物信息学技术对锌指蛋白41和SPARC进行蛋白质结构与功能分析。研究分为两个部分，分别探讨锌指蛋白41和SPARC是否可以作为室间隔缺损的潜在血清标志物，为室间隔缺损的早期诊断及发病机制研究提供参考依据。第一部分室间隔缺损潜在血清标志物锌指蛋白41的验证研究目的验证锌指蛋白41是否能作为室间隔缺损的潜在血清标志物，并应用生物信息学进行蛋白结构与功能分析。方法收集0～10岁的单纯性室间隔缺损、房间隔缺损(atrial septaldefect, ASD)、法洛四联症(tetralogy of Fallot, TOF)患者及健康对照者血清样本各20例。应用Western blot和ELISA检测血清样本中锌指蛋白41的表达水平，应用生物信息学技术对锌指蛋白41的结构功能，参与信号通路等进行分析。结果1. VSD组、ASD组、TOF组和健康对照组间年龄、性别、民族状况均衡可比(P0.05)。 2. Western blot检测结果显示，锌指蛋白41在4组血清中均有表达，但VSD组中的相对表达水平均高于其他3组(P0.05)； 3. ELISA检测结果显示，VSD组患者血清中锌指蛋白41浓度为(136.72±56.44) pg/ml， ASD组为(94.54±41.98) pg/ml， TOF组为(100.69±37.08) pg/ml，健康对照组为(82.08±42.46) pg/ml，4组间差异有统计学意义(P0.05)，进一步两两比较，VSD组与其他3组之间差异均有统计学意义(P0.05)，而其他3组两两间差异均无统计学意义(P0.05)。 4.生物信息学分析表明：锌指蛋白41属于C2H2锌指蛋白家族，具有调节与DNA结合的活性，能调控基因表达、蛋白生成，，影响细胞分化及器官发育等功能。结论锌指蛋白41在室间隔缺损患者血清中表达水平较高，可能是室间隔缺损潜在的血清标志物，但仍需后续的大样本临床确认及锌指蛋白41功能的研究。第二部分室间隔缺损潜在血清标志物SPARC的验证研究目的验证SPARC作为室间隔缺损潜在血清标志物的可能性并应用生物信息学分析。方法收集0～10岁的单纯性室间隔缺损、房间隔缺损、法洛四联症患者及健康对照者血清样本各20例。应用Western blot和ELISA技术检测SPARC在血清样本中的表达水平，运用生物信息学进行SPARC结构功能预测分析。结果1.4组间年龄、性别、民族均衡可比(P0.05)。 2. Western blot检测结果显示，SPARC在4组血清中均有表达，但在VSD组的相对表达水平均高于其他3组(P0.05)； 3. ELISA检测结果可知VSD组患者血清中SPARC浓度为(198.03±87.26) pg/ml， ASD组为(137.02±83.89) pg/ml， TOF组为(140.61±79.98) pg/ml，健康对照组为(91.30±55.50) pg/ml，4组间差异有统计学意义(P0.05)，进一步两两比较，VSD组与其他的3组之间差异均有统计学意义(P0.05)，而其他3组两两间差异均无统计学意义(P0.05)。 4.生物信息学结果，SPARC通过其特有的结构域发挥生物学功能，影响细胞增殖和信号转导，参与心脏等器官的发育。结论SPARC在室间隔缺损的血清中表达水平较高，可能是室间隔缺损的潜在血清标志物，但仍需生物标志物第三阶段的大样本临床确认研究，及SPARC蛋白生物学特性的研究。
[Abstract]:Congenital heart disease (congenital heart, disease, CHD) is the most common disease of birth defects, ventricular septal defect (ventricular septal defect, VSD) as the main types of congenital heart disease in infants, survival and quality of life have an important impact. The etiology and mechanism of ventricular septal defect complicated to a comprehensive understanding, in addition to the traditional related susceptibility genes, environment factors, need more protein to regulate cardiac differentiation and abnormal signal pathway were studied, providing clues for diagnosis and disease.
Based on the previous isobaric tags for relative and absolute quantitation (iTRAQ) series of matrix assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography (LC-MALDI-TOF/TOF MS) method to screen a series of differences in ventricular septal defect associated protein, combined with the analysis of the experimental results and literature retrieval, the zinc finger protein 41 (zinc finger protein41, ZNF41) secreted protein acidic and rich in cysteine (secreted protein acidic and richin cysteine, SPARC) test, by immunoblotting (Western blot, WB) and enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) were detected in serum samples of zinc finger protein expression levels of 41 and SPARC the application of bioinformatics, and technology of zinc finger protein 41 and SPARC analysis of protein structure and function. The research is divided into two parts, respectively. To investigate whether zinc finger protein 41 and SPARC can be used as potential serum markers for ventricular septal defect, and provide reference for early diagnosis and pathogenesis of ventricular septal defect.
Study on the validation of zinc finger protein 41, a potential serum marker of ventricular septal defect in the first part
Objective to verify whether zinc finger protein 41 can be used as a potential serum marker for ventricular septal defect and to use bioinformatics to analyze protein structure and function.
Methods isolated ventricular septal defect 0~10, atrial septal defect (atrial, septaldefect, ASD), tetralogy of Fallot (tetralogy of Fallot, TOF) patients and healthy controls serum samples of 20 cases each. The expression level of zinc finger protein 41 serum samples by Western blot and ELISA, the application of bioinformatics technology the structure and function of zinc finger protein 41, signaling pathways involved in the analysis.
Results the age, sex and national status of the 1. VSD group, the ASD group, the TOF group and the healthy control group were comparable (P0.05).
The results of 2. Western blot showed that zinc finger protein 41 was expressed in the 4 groups, but the relative expression level in the VSD group was higher than that of the other 3 groups (P0.05).
3. ELISA test results showed that the serum in patients with VSD zinc finger protein 41 concentration (136.72 + 56.44) pg/ml, ASD group (94.54 + 41.98) pg/ml, TOF group (100.69 + 37.08) pg/ml, healthy control group (82.08 + 42.46) pg/ml, there were statistically significant differences between the 4 groups (P0.05 further more, 22), VSD group and other 3 groups was statistically significant differences (P0.05), and there was no significant difference between the other 3 groups (P0.05 22).
4. bioinformatics analysis showed that zinc finger protein 41 belonged to the C2H2 zinc finger family. It had the activity of regulating DNA binding, regulating gene expression, protein production, affecting cell differentiation and organ development.
Conclusion zinc finger protein 41 is highly expressed in serum of patients with ventricular septal defect, and it may be a potential serum marker for ventricular septal defect. However, follow-up study is needed for large sample clinical confirmation and zinc finger protein 41 function.
Verification of the potential serum marker SPARC of second parts of ventricular septal defect
Objective to verify the possibility of SPARC as a potential serum marker for ventricular septal defect and to use bioinformatics analysis.
Methods the data of 0 ~ 10 years old of isolated ventricular septal defect, atrial septal defect, tetralogy of Fallot patients and healthy controls serum samples of 20 cases each. The expression level of Western blot and application of ELISA technology in detection of SPARC in serum samples, using bioinformatics of SPARC structure and function prediction analysis.
Results the age, sex, and national equilibrium of the 1.4 groups were comparable (P0.05).
The results of 2. Western blot showed that SPARC was expressed in the 4 groups of serum, but the relative expression level in the VSD group was higher than that of the other 3 groups (P0.05).
3. the results of ELISA SPARC VSD in the sera of patients was (198.03 + 87.26) pg/ml, ASD group (137.02 + 83.89) pg/ml, TOF group (140.61 + 79.98) pg/ml, healthy control group (91.30 + 55.50) pg/ml, there were statistically significant differences between the 4 groups (P0.05), a further 22 compared with the VSD group and the other 3 groups was statistically significant differences (P0.05), and there was no significant difference between the other 3 groups (P0.05 22).
4. bioinformatics results, SPARC, through its unique domain, exerts biological function, affects cell proliferation and signal transduction, and participates in the development of cardiac organs.
Conclusion the expression level of SPARC in serum of ventricular septal defect is relatively high. It may be a potential serum marker for ventricular septal defect. However, the large scale clinical confirmation study of biomarker at the third stage and the biological characteristics of SPARC protein are still needed.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R541;R392

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