銀屑病患者CD45外顯子4和6基因突變的檢測
發(fā)布時(shí)間：2018-10-24 21:40
【摘要】：銀屑病是一種慢性炎癥性、自身免疫性疾病，其受累器官包括皮膚和關(guān)節(jié)，T淋巴細(xì)胞在銀屑病的發(fā)生、發(fā)展中起重要作用。目前認(rèn)為遺傳因素與銀屑病的發(fā)病多相關(guān)[1]。近年來的實(shí)驗(yàn)研究證明，CD45是正常淋巴細(xì)胞信號(hào)傳導(dǎo)的關(guān)鍵[2]，改變CD45的表達(dá)對免疫功能有重大影響。由于CD45基因的點(diǎn)突變可使其剪接異構(gòu)體的構(gòu)成發(fā)生改變，，參與一些自身免疫性疾病的發(fā)生[7]。CD45外顯子4第59位核苷酸發(fā)生C→A突變（C59A）、77位核苷酸發(fā)生C→G突變(C77G)，導(dǎo)致第4外顯子沉默子的剪接位點(diǎn)失活，使含外顯子4的異構(gòu)體CD45RA的異常增多，導(dǎo)致一些自身免疫性疾病如系統(tǒng)性硬化、多發(fā)性硬化等[3，4]；并能增加HIV-1感染的易感性[5]。CD45外顯子6第138位核苷酸發(fā)生A→G突變（A138G），增強(qiáng)外顯子6的剪切，增多活化T細(xì)胞的CD45RO異構(gòu)體，導(dǎo)致IFN-γ的分泌增多[6]，增加部分免疫相關(guān)性疾病的易感性。 Lecewicz-Toruń B[8]，F(xiàn)lytlie HA[9]，Alefacept[10]等人的研究也表明了CD45RO與銀屑病的相關(guān)性。 目的：本實(shí)驗(yàn)采用DHPLC及ARMS-PCR方法檢測115例尋常型銀屑病患者CD45外顯子4、外顯子6是否存在基因突變，明確尋常型銀屑病患者CD45外顯子4、外顯子6的基因多態(tài)性，豐富銀屑病的遺傳學(xué)發(fā)病機(jī)制內(nèi)容。 方法：1.采集銀屑病患者外周血液，提取基因組DNA。2.設(shè)計(jì)特異性引物擴(kuò)增CD45外顯子4的基因片段。3.將PCR擴(kuò)增產(chǎn)物送至北京表觀生物技術(shù)有限公司進(jìn)行DHPLC檢測。4.設(shè)計(jì)ARMS-PCR特異性引物，擴(kuò)增CD45外顯子6的基因片段，根據(jù)電泳結(jié)果將擴(kuò)增產(chǎn)物分型，并對每種類型的擴(kuò)增產(chǎn)物進(jìn)行測序驗(yàn)證。 結(jié)果：1.銀屑病患者CD45基因外顯子4基因突變的檢測：（1）以銀屑病患者的血液DNA為模板對CD45基因外顯子4進(jìn)行PCR擴(kuò)增，得到預(yù)期大小的特異性片段（171bp）；（2）銀屑病患者CD45外顯子4的PCR擴(kuò)增產(chǎn)物的DHPLC檢測結(jié)果：在所檢測的115例患者均為單一峰值，未發(fā)現(xiàn)異常洗脫峰，提示銀屑病患者在CD45基因外顯子4處無基因突變。2.銀屑病患者CD45基因外顯子6基因突變的檢測：（1）ARMS-PCR電泳結(jié)果：僅用野生型引物能擴(kuò)增出預(yù)期片段的DNA為A138A型，僅用突變型引物能擴(kuò)增出預(yù)期片段的DNA為G138G型，如果野生型引物和突變型引物都能擴(kuò)增出預(yù)期片段的DNA為A138G型；（2）ARMS-PCR產(chǎn)物的序列測定：A138A型的第138位堿基為A, G138G型的第138位堿基為G, A138G型的第138位堿基為A/G；（3）CD45外顯子6第138位基因多態(tài)性的分型比例及突變率：統(tǒng)計(jì)分析結(jié)果分為三種類型，分別為A138A型31例、G138G型2例及A138G型82例，其中突變率G=G/A+G%；（4）銀屑病患者CD45外顯子6基因多態(tài)性的三種類型與性別，疾病嚴(yán)重程度，遺傳之間的關(guān)系：1）CD45外顯子6第138的突變率在男性及女性人群中、在點(diǎn)滴型銀屑病和斑塊型銀屑病中、在初次發(fā)病早發(fā)組及晚發(fā)組中無明顯差異；2）在有遺傳家族史的患者發(fā)生突變的比率明顯高于無遺傳家族史的患者，提示銀屑病患者CD45外顯子6的138位A→G與患者的遺傳史密切相關(guān)；輕度患者的CD45外顯子6的第138位基因突變率明顯高于中重度患者。 結(jié)論：1.所試尋常型銀屑病患者CD45外顯子4無基因突變位點(diǎn)。2.115例尋常銀屑病患者的CD45外顯子6第138位存在A→G突變，其中A138A型為31例，A138G型為82例，G138G型為2例，突變率達(dá)38.2%。3. CD45外顯子6的第138位A→G突變在有家族遺傳史的尋常型銀屑病患者中多見。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R758.63
本文編號(hào)：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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R758.63
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 李敬來;CD45—淋巴細(xì)胞活化的重要調(diào)節(jié)分子[J];國外醫(yī)學(xué)(免疫學(xué)分冊);2002年01期
2 許文,畢新嶺;CD45分子及其表達(dá)與銀屑病[J];中國麻風(fēng)皮膚病雜志;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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