银屑病患者CD45外显子4和6基因突变的检测
发布时间：2018-10-24 21:40
【摘要】：银屑病是一种慢性炎症性、自身免疫性疾病，其受累器官包括皮肤和关节，T淋巴细胞在银屑病的发生、发展中起重要作用。目前认为遗传因素与银屑病的发病多相关[1]。近年来的实验研究证明，CD45是正常淋巴细胞信号传导的关键[2]，改变CD45的表达对免疫功能有重大影响。由于CD45基因的点突变可使其剪接异构体的构成发生改变，，参与一些自身免疫性疾病的发生[7]。CD45外显子4第59位核苷酸发生C→A突变（C59A）、77位核苷酸发生C→G突变(C77G)，导致第4外显子沉默子的剪接位点失活，使含外显子4的异构体CD45RA的异常增多，导致一些自身免疫性疾病如系统性硬化、多发性硬化等[3，4]；并能增加HIV-1感染的易感性[5]。CD45外显子6第138位核苷酸发生A→G突变（A138G），增强外显子6的剪切，增多活化T细胞的CD45RO异构体，导致IFN-γ的分泌增多[6]，增加部分免疫相关性疾病的易感性。 Lecewicz-Toruń B[8]，Flytlie HA[9]，Alefacept[10]等人的研究也表明了CD45RO与银屑病的相关性。 目的：本实验采用DHPLC及ARMS-PCR方法检测115例寻常型银屑病患者CD45外显子4、外显子6是否存在基因突变，明确寻常型银屑病患者CD45外显子4、外显子6的基因多态性，丰富银屑病的遗传学发病机制内容。 方法：1.采集银屑病患者外周血液，提取基因组DNA。2.设计特异性引物扩增CD45外显子4的基因片段。3.将PCR扩增产物送至北京表观生物技术有限公司进行DHPLC检测。4.设计ARMS-PCR特异性引物，扩增CD45外显子6的基因片段，根据电泳结果将扩增产物分型，并对每种类型的扩增产物进行测序验证。 结果：1.银屑病患者CD45基因外显子4基因突变的检测：（1）以银屑病患者的血液DNA为模板对CD45基因外显子4进行PCR扩增，得到预期大小的特异性片段（171bp）；（2）银屑病患者CD45外显子4的PCR扩增产物的DHPLC检测结果：在所检测的115例患者均为单一峰值，未发现异常洗脱峰，提示银屑病患者在CD45基因外显子4处无基因突变。2.银屑病患者CD45基因外显子6基因突变的检测：（1）ARMS-PCR电泳结果：仅用野生型引物能扩增出预期片段的DNA为A138A型，仅用突变型引物能扩增出预期片段的DNA为G138G型，如果野生型引物和突变型引物都能扩增出预期片段的DNA为A138G型；（2）ARMS-PCR产物的序列测定：A138A型的第138位碱基为A, G138G型的第138位碱基为G, A138G型的第138位碱基为A/G；（3）CD45外显子6第138位基因多态性的分型比例及突变率：统计分析结果分为三种类型，分别为A138A型31例、G138G型2例及A138G型82例，其中突变率G=G/A+G%；（4）银屑病患者CD45外显子6基因多态性的三种类型与性别，疾病严重程度，遗传之间的关系：1）CD45外显子6第138的突变率在男性及女性人群中、在点滴型银屑病和斑块型银屑病中、在初次发病早发组及晚发组中无明显差异；2）在有遗传家族史的患者发生突变的比率明显高于无遗传家族史的患者，提示银屑病患者CD45外显子6的138位A→G与患者的遗传史密切相关；轻度患者的CD45外显子6的第138位基因突变率明显高于中重度患者。 结论：1.所试寻常型银屑病患者CD45外显子4无基因突变位点。2.115例寻常银屑病患者的CD45外显子6第138位存在A→G突变，其中A138A型为31例，A138G型为82例，G138G型为2例，突变率达38.2%。3. CD45外显子6的第138位A→G突变在有家族遗传史的寻常型银屑病患者中多见。
[Abstract]:Psoriasis is a kind of chronic inflammatory, autoimmune disease, its involved organs include skin and joints, T lymphocytes play an important role in the pathogenesis and development of psoriasis. Genetic factors are currently considered to be related to the pathogenesis of psoriasis[1]. Recent studies have shown that CD45 is the key[2] of normal lymphocyte signal transduction, and the change of CD45 expression has a significant effect on the immune function. Because the point mutation of the CD45 gene can change the composition of its splice isomer, it is involved in the occurrence of some autoimmune diseases[7]. In exon 59 of exon 4 of CD45, C/ A mutation (C59A) occurred, and C/ G mutation (C77G) occurred in 77 sites, resulting in the mutation of splice site of exon 4 silence, resulting in abnormal increase of CD45RA containing exon 4, which caused some autoimmune diseases such as systemic sclerosis. Multiple sclerosis et al.[3, 4]; and increased susceptibility to HIV-1 infection[5]. In exon 6 of exon 6 of CD45, A/ G mutation (A138G) was found to enhance the cleavage of exon 6 and increase the CD45RO isomer of activated T cell, resulting in the increase of IFN-jun secretion[6], and to increase the susceptibility of partial immune-related diseases. The correlation between CD45RO and psoriasis was also demonstrated in the study of Lecruicz-Toru, B[8], Fltlie HA[9], Alefacer[10], et al. Objective: We used DHPLC and ARMS-PCR to detect the mutation of exon 4 and exon 6 of CD45 in 115 patients with psoriasis vulgaris. Content. Methods: 1. The peripheral blood of patients with psoriasis was collected and the genome was extracted. DNA. 2. Design of a specific primer to amplify the base of exon 4 of CD45 The PCR amplification product was sent to Beijing Apparent Biotechnology Co., Ltd. for DHPL C detecting. 4. designing an ARMS-PCR specific primer, amplifying the gene fragment of exon 6 of CD45, typing the amplified product according to the electrophoresis result, line sequencing verification Results: 1. Detection of exon 4 gene mutation of CD45 gene in psoriasis patients: (1) PCR amplification of CD45 gene exon 4 was carried out by using blood DNA of psoriasis patients as template to obtain specific fragment of expected size. The results of DHPLC in exon 4 of CD45 in psoriasis patients showed that all 115 patients had a single peak and no abnormal peaks were found, suggesting that psoriasis patients had exon 4 of CD45 gene. Detection of mutations in exon 6 of CD45 gene in patients with psoriasis: (1) ARMS-PCR electrophoresis result: Only the wild-type primer can amplify the DNA of the expected fragment as A138 Atype, and only the mutant primer can amplify the DN of the expected fragment. A is a G138G type, if both the wild-type primer and the mutant primer can amplify the DNA of the expected fragment into the A138G type; (2) sequencing of the ARMS-PCR product: the 138-th base of the A138 Atype is A, the 138-th base of the G138G type is G, and the 13th of the A138G type 8-bit bases were A/ G; (3) The typing ratio and mutation rate of exon 6 of CD45 exon 6 were classified into three types: A138 A31, G138G and A138G 82 cases, among which mutation rate G = G/ A + G%; (4) CD45 exon 6 gene polymorphism in psoriasis patients The relationship between three types of sex, severity of disease and inheritance: 1) mutation rate of exon 6 of CD45 exon 6 in male and female population, early onset and late onset of psoriasis and plaque psoriasis There was no significant difference in the hair group; 2) In patients with a family history of family history, the ratio of mutations was significantly higher than those without a family history of genetic history, suggesting 138 patients with psoriasis patients CD45 exon 6 A, G, and patients The genetic history of CD45 exon 6 in mild patients is closely related to the genetic history; significantly higher than medium weight Conclusion: 1. There is no mutation site in exon 4 of CD45 in patients with psoriasis vulgaris. There is a mutation of A/ G mutation in exon 6 of CD45 in 115 patients with psoriasis vulgaris. Among them, there are 31 cases A138 A138, 82 in A138G type and 2 in G138G type. The mutation rate reached 38. 2%. The 138th A/ G mutation of exon 6 of CD45 was in the history of family heredity.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R758.63
本文编号：2292633
[Abstract]:Psoriasis is a kind of chronic inflammatory, autoimmune disease, its involved organs include skin and joints, T lymphocytes play an important role in the pathogenesis and development of psoriasis. Genetic factors are currently considered to be related to the pathogenesis of psoriasis[1]. Recent studies have shown that CD45 is the key[2] of normal lymphocyte signal transduction, and the change of CD45 expression has a significant effect on the immune function. Because the point mutation of the CD45 gene can change the composition of its splice isomer, it is involved in the occurrence of some autoimmune diseases[7]. In exon 59 of exon 4 of CD45, C/ A mutation (C59A) occurred, and C/ G mutation (C77G) occurred in 77 sites, resulting in the mutation of splice site of exon 4 silence, resulting in abnormal increase of CD45RA containing exon 4, which caused some autoimmune diseases such as systemic sclerosis. Multiple sclerosis et al.[3, 4]; and increased susceptibility to HIV-1 infection[5]. In exon 6 of exon 6 of CD45, A/ G mutation (A138G) was found to enhance the cleavage of exon 6 and increase the CD45RO isomer of activated T cell, resulting in the increase of IFN-jun secretion[6], and to increase the susceptibility of partial immune-related diseases. The correlation between CD45RO and psoriasis was also demonstrated in the study of Lecruicz-Toru, B[8], Fltlie HA[9], Alefacer[10], et al. Objective: We used DHPLC and ARMS-PCR to detect the mutation of exon 4 and exon 6 of CD45 in 115 patients with psoriasis vulgaris. Content. Methods: 1. The peripheral blood of patients with psoriasis was collected and the genome was extracted. DNA. 2. Design of a specific primer to amplify the base of exon 4 of CD45 The PCR amplification product was sent to Beijing Apparent Biotechnology Co., Ltd. for DHPL C detecting. 4. designing an ARMS-PCR specific primer, amplifying the gene fragment of exon 6 of CD45, typing the amplified product according to the electrophoresis result, line sequencing verification Results: 1. Detection of exon 4 gene mutation of CD45 gene in psoriasis patients: (1) PCR amplification of CD45 gene exon 4 was carried out by using blood DNA of psoriasis patients as template to obtain specific fragment of expected size. The results of DHPLC in exon 4 of CD45 in psoriasis patients showed that all 115 patients had a single peak and no abnormal peaks were found, suggesting that psoriasis patients had exon 4 of CD45 gene. Detection of mutations in exon 6 of CD45 gene in patients with psoriasis: (1) ARMS-PCR electrophoresis result: Only the wild-type primer can amplify the DNA of the expected fragment as A138 Atype, and only the mutant primer can amplify the DN of the expected fragment. A is a G138G type, if both the wild-type primer and the mutant primer can amplify the DNA of the expected fragment into the A138G type; (2) sequencing of the ARMS-PCR product: the 138-th base of the A138 Atype is A, the 138-th base of the G138G type is G, and the 13th of the A138G type 8-bit bases were A/ G; (3) The typing ratio and mutation rate of exon 6 of CD45 exon 6 were classified into three types: A138 A31, G138G and A138G 82 cases, among which mutation rate G = G/ A + G%; (4) CD45 exon 6 gene polymorphism in psoriasis patients The relationship between three types of sex, severity of disease and inheritance: 1) mutation rate of exon 6 of CD45 exon 6 in male and female population, early onset and late onset of psoriasis and plaque psoriasis There was no significant difference in the hair group; 2) In patients with a family history of family history, the ratio of mutations was significantly higher than those without a family history of genetic history, suggesting 138 patients with psoriasis patients CD45 exon 6 A, G, and patients The genetic history of CD45 exon 6 in mild patients is closely related to the genetic history; significantly higher than medium weight Conclusion: 1. There is no mutation site in exon 4 of CD45 in patients with psoriasis vulgaris. There is a mutation of A/ G mutation in exon 6 of CD45 in 115 patients with psoriasis vulgaris. Among them, there are 31 cases A138 A138, 82 in A138G type and 2 in G138G type. The mutation rate reached 38. 2%. The 138th A/ G mutation of exon 6 of CD45 was in the history of family heredity.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R758.63
【参考文献】
相关期刊论文 前3条
1 李敬来;CD45—淋巴细胞活化的重要调节分子[J];国外医学(免疫学分册);2002年01期
2 许文,毕新岭;CD45分子及其表达与银屑病[J];中国麻风皮肤病杂志;2003年06期
3 ;Severe vulgaris psoriatic patients with acute myelogenous leukaemia and resolution after allogeneic bone marrow transplantation/ peripheral blood stem cell transplantation[J];Chinese Medical Journal;2005年10期
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