中國(guó)人群MEFV基因突變及其在炎癥反應(yīng)中的作用
發(fā)布時(shí)間：2019-01-30 18:45
【摘要】：目的：檢測(cè)中國(guó)人群MEFV基因突變情況，觀察脂多糖(LPS)對(duì)人外周血白細(xì)胞MEFV基因mRNA表達(dá)的影響，初步探討MEFV基因突變與炎癥反應(yīng)的相關(guān)性以及Pyrin蛋白在炎癥調(diào)節(jié)機(jī)制中作用；構(gòu)建MEFV基因片段的重組質(zhì)粒，為進(jìn)一步研究其在細(xì)胞中的表達(dá)奠定基礎(chǔ)。方法：收集49份健康成人和46份燒傷病人全血標(biāo)本，運(yùn)用反向雜交(reverse-hybridization)技術(shù)檢測(cè)MEFV基因12個(gè)常見(jiàn)突變位點(diǎn)，統(tǒng)計(jì)突變率、突變類(lèi)型，分析燒傷病人的突變類(lèi)型與臨床炎癥反應(yīng)程度的相關(guān)性；采用半定量逆轉(zhuǎn)錄多聚酶鏈反應(yīng)(RT-PCR)的方法檢測(cè)人外周血白細(xì)胞MEFV基因mRNA表達(dá)水平和給予LPS刺激后對(duì)其表達(dá)的影響；將RT-PCR擴(kuò)增產(chǎn)物導(dǎo)入質(zhì)粒載體pMD18-T后轉(zhuǎn)化至大腸桿菌(E.coli)感受態(tài)細(xì)胞JM109進(jìn)行克隆。結(jié)果：1．中國(guó)人群MEFV基因存在多種突變位點(diǎn)，本實(shí)驗(yàn)共發(fā)現(xiàn)九種突變類(lèi)型，分別為E148Q純合型、E148Q雜合型、P369S雜合型、M680I雜合型、E148Q純合+P369S雜合、E148Q雜合+P369S雜合、E148Q雜合+M680I雜合、E148Q純合+P369S雜合+M680I雜合、148Q雜合+M680I雜合+M694I雜合，其中E148Q突變率最高。除E148Q基因型外，其余各種MEFV基因突變類(lèi)型在中國(guó)人群中尚未見(jiàn)報(bào)道。2．卡方檢驗(yàn)結(jié)果顯示，，MEFV基因突變組膿毒癥發(fā)生率與正常組之間的差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)，尚不能認(rèn)為有MEFV基因突變的病人燒傷后的膿毒癥發(fā)生率高；非條件logistic回歸分析表明，年齡和燒傷面積是影響燒傷病人預(yù)后的主要因素，而是否有MEFV基因突變尚不能確定為直接的影響因素。3．未受LPS刺激的正常人外周血白細(xì)胞表達(dá)低水平的MEFV mRNA，給予1μg／mL LPS和10μg／mL LPS刺激2h后，表達(dá)高于正常對(duì)照(P＜0.05)，而100μg／mL LPS刺激2h時(shí)與正常對(duì)照沒(méi)有差別(P＞0.05)。4．RT-PCR擴(kuò)增后的MEFV基因片段成功導(dǎo)入質(zhì)粒載體，并且可以在大腸桿菌中培養(yǎng)生長(zhǎng)。結(jié)論：1．中國(guó)人群MEFV基因有較高的突變率和多種突變類(lèi)型，E148Q突變率最高。2．尚不能肯定中國(guó)人群MEFV基因突變與燒傷后炎癥反應(yīng)有直接相關(guān)性。3．一定時(shí)間內(nèi)低濃度的LPS刺激可以誘導(dǎo)人外周血白細(xì)胞MEFV基因mRNA表達(dá)上調(diào)。4．成功構(gòu)建MEFV基因片段重組質(zhì)粒，為進(jìn)一步研究MEFV基因的功能提供了分子生物學(xué)基礎(chǔ)。
[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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R363
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相關(guān)期刊論文 前2條
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