中国人群MEFV基因突变及其在炎症反应中的作用
发布时间：2019-01-30 18:45
【摘要】：目的：检测中国人群MEFV基因突变情况，观察脂多糖(LPS)对人外周血白细胞MEFV基因mRNA表达的影响，初步探讨MEFV基因突变与炎症反应的相关性以及Pyrin蛋白在炎症调节机制中作用；构建MEFV基因片段的重组质粒，为进一步研究其在细胞中的表达奠定基础。方法：收集49份健康成人和46份烧伤病人全血标本，运用反向杂交(reverse-hybridization)技术检测MEFV基因12个常见突变位点，统计突变率、突变类型，分析烧伤病人的突变类型与临床炎症反应程度的相关性；采用半定量逆转录多聚酶链反应(RT-PCR)的方法检测人外周血白细胞MEFV基因mRNA表达水平和给予LPS刺激后对其表达的影响；将RT-PCR扩增产物导入质粒载体pMD18-T后转化至大肠杆菌(E.coli)感受态细胞JM109进行克隆。结果：1．中国人群MEFV基因存在多种突变位点，本实验共发现九种突变类型，分别为E148Q纯合型、E148Q杂合型、P369S杂合型、M680I杂合型、E148Q纯合+P369S杂合、E148Q杂合+P369S杂合、E148Q杂合+M680I杂合、E148Q纯合+P369S杂合+M680I杂合、148Q杂合+M680I杂合+M694I杂合，其中E148Q突变率最高。除E148Q基因型外，其余各种MEFV基因突变类型在中国人群中尚未见报道。2．卡方检验结果显示，，MEFV基因突变组脓毒症发生率与正常组之间的差异无统计学意义(P＞0.05)，尚不能认为有MEFV基因突变的病人烧伤后的脓毒症发生率高；非条件logistic回归分析表明，年龄和烧伤面积是影响烧伤病人预后的主要因素，而是否有MEFV基因突变尚不能确定为直接的影响因素。3．未受LPS刺激的正常人外周血白细胞表达低水平的MEFV mRNA，给予1μg／mL LPS和10μg／mL LPS刺激2h后，表达高于正常对照(P＜0.05)，而100μg／mL LPS刺激2h时与正常对照没有差别(P＞0.05)。4．RT-PCR扩增后的MEFV基因片段成功导入质粒载体，并且可以在大肠杆菌中培养生长。结论：1．中国人群MEFV基因有较高的突变率和多种突变类型，E148Q突变率最高。2．尚不能肯定中国人群MEFV基因突变与烧伤后炎症反应有直接相关性。3．一定时间内低浓度的LPS刺激可以诱导人外周血白细胞MEFV基因mRNA表达上调。4．成功构建MEFV基因片段重组质粒，为进一步研究MEFV基因的功能提供了分子生物学基础。
[Abstract]:Objective: to detect the mutation of MEFV gene in Chinese population, to observe the effect of lipopolysaccharide (LPS) on the expression of MEFV gene mRNA in human peripheral blood leukocytes, and to explore the relationship between MEFV gene mutation and inflammatory response and the role of Pyrin protein in the regulation of inflammation. The recombinant plasmid of MEFV gene fragment was constructed to lay a foundation for further study of its expression in cells. Methods: 49 healthy adults and 46 whole blood samples of burn patients were collected and 12 common mutation sites of MEFV gene were detected by reverse hybridization (reverse-hybridization). To analyze the relationship between the mutation type of burn patients and the degree of clinical inflammatory reaction. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of MEFV gene mRNA in human peripheral blood leukocytes and the effect of LPS stimulation on its expression. The RT-PCR amplification product was introduced into plasmid pMD18-T and transformed into Escherichia coli (E.coli) competent cell JM109 for cloning. Results: 1. There were many mutation loci in MEFV gene in Chinese population. Nine mutation types were found in this study, namely, E148Q homozygote, E148Q heterozygote, P369S heterozygote, M680I heterozygote, E148Q homozygous P369S heterozygote, E148Q heterozygote P369S heterozygote, E148Q heterozygous P369S heterozygote. E148Q heterozygosity M680I, E148Q homozygous P369S heterozygote M680I, 148Q heterozygote M680I M694I heterozygote, E148Q heterozygous M680I heterozygosity was the highest. With the exception of E148Q genotype, none of the other MEFV gene mutations have been reported in Chinese population. 2. The results of chi-square test showed that there was no significant difference between the incidence of sepsis in MEFV gene mutation group and normal group (P > 0. 05). The incidence of sepsis after burn in patients with MEFV gene mutation cannot be considered to be high. Non-conditional logistic regression analysis showed that age and burn area were the main factors influencing the prognosis of burn patients. However, whether there is a mutation of MEFV gene can not be identified as a direct influencing factor. 3. The low level of MEFV mRNA, expression in peripheral blood leukocytes of normal subjects without LPS stimulation was induced by 1 渭 g/mL LPS and 10 渭 g/mL LPS for 2 h. The expression of MEFV gene was higher than that of normal control (P < 0. 05), but there was no difference between 100 渭 g/mL LPS and normal control at 2h. The MEFV gene fragment amplified by 4.RT-PCR was successfully introduced into plasmid vector. And can grow in Escherichia coli. Conclusion: 1. MEFV gene has high mutation rate and multiple mutation types in Chinese population. 2. The mutation rate of E148Q is the highest. 2. It is not certain that the mutation of MEFV gene is directly related to the inflammatory reaction after burn in Chinese population. 3. Low concentration of LPS can induce the expression of MEFV gene mRNA in human peripheral blood leukocytes within a certain period of time. The recombinant plasmid of MEFV gene fragment was successfully constructed. It provides molecular biological basis for further study on the function of MEFV gene.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363
本文编号：2418399
[Abstract]:Objective: to detect the mutation of MEFV gene in Chinese population, to observe the effect of lipopolysaccharide (LPS) on the expression of MEFV gene mRNA in human peripheral blood leukocytes, and to explore the relationship between MEFV gene mutation and inflammatory response and the role of Pyrin protein in the regulation of inflammation. The recombinant plasmid of MEFV gene fragment was constructed to lay a foundation for further study of its expression in cells. Methods: 49 healthy adults and 46 whole blood samples of burn patients were collected and 12 common mutation sites of MEFV gene were detected by reverse hybridization (reverse-hybridization). To analyze the relationship between the mutation type of burn patients and the degree of clinical inflammatory reaction. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of MEFV gene mRNA in human peripheral blood leukocytes and the effect of LPS stimulation on its expression. The RT-PCR amplification product was introduced into plasmid pMD18-T and transformed into Escherichia coli (E.coli) competent cell JM109 for cloning. Results: 1. There were many mutation loci in MEFV gene in Chinese population. Nine mutation types were found in this study, namely, E148Q homozygote, E148Q heterozygote, P369S heterozygote, M680I heterozygote, E148Q homozygous P369S heterozygote, E148Q heterozygote P369S heterozygote, E148Q heterozygous P369S heterozygote. E148Q heterozygosity M680I, E148Q homozygous P369S heterozygote M680I, 148Q heterozygote M680I M694I heterozygote, E148Q heterozygous M680I heterozygosity was the highest. With the exception of E148Q genotype, none of the other MEFV gene mutations have been reported in Chinese population. 2. The results of chi-square test showed that there was no significant difference between the incidence of sepsis in MEFV gene mutation group and normal group (P > 0. 05). The incidence of sepsis after burn in patients with MEFV gene mutation cannot be considered to be high. Non-conditional logistic regression analysis showed that age and burn area were the main factors influencing the prognosis of burn patients. However, whether there is a mutation of MEFV gene can not be identified as a direct influencing factor. 3. The low level of MEFV mRNA, expression in peripheral blood leukocytes of normal subjects without LPS stimulation was induced by 1 渭 g/mL LPS and 10 渭 g/mL LPS for 2 h. The expression of MEFV gene was higher than that of normal control (P < 0. 05), but there was no difference between 100 渭 g/mL LPS and normal control at 2h. The MEFV gene fragment amplified by 4.RT-PCR was successfully introduced into plasmid vector. And can grow in Escherichia coli. Conclusion: 1. MEFV gene has high mutation rate and multiple mutation types in Chinese population. 2. The mutation rate of E148Q is the highest. 2. It is not certain that the mutation of MEFV gene is directly related to the inflammatory reaction after burn in Chinese population. 3. Low concentration of LPS can induce the expression of MEFV gene mRNA in human peripheral blood leukocytes within a certain period of time. The recombinant plasmid of MEFV gene fragment was successfully constructed. It provides molecular biological basis for further study on the function of MEFV gene.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363
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本文编号：2418399
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