本文選題：脫發(fā) + 中國人群　； 參考：《中南大學(xué)》2013年博士論文

【摘要】：研究背景：先天性疾病是一種在出生前或出生后一個(gè)月內(nèi)發(fā)生的疾病。很多先天性疾病都是遺傳物質(zhì)異常所導(dǎo)致的遺傳性疾病。先天性出汗性外胚層發(fā)育不良(Hidrotic ectodermal dysplasia, HED),也稱為Clouston綜合癥,是一種常染色體顯性遺傳疾病,其特點(diǎn)是杵狀甲、脫發(fā)以及手掌和腳底角質(zhì)化。先天性杵狀甲(或者杵狀指)是一類臨床上較少見的遺傳性皮膚病,由于甲床和第三指節(jié)骨部分的連接組織增生,導(dǎo)致指甲板擴(kuò)大和手指及腳趾終端分部。目前對(duì)于杵狀甲還缺少具體可行的治療方法,但是針對(duì)其病理癥狀進(jìn)行治療可能會(huì)減少杵狀甲表型的發(fā)生；如果在疾病發(fā)生早期進(jìn)行干預(yù)還有可能逆轉(zhuǎn)病情,但目前尚缺少有效的手術(shù)治療方法。先天性脫發(fā)的臨床特征為正常頭皮毛發(fā)不可再生性脫落,稀疏的眉毛和睫毛以及缺少腋毛、陰毛或其他體毛。有1-2%的先天性脫發(fā)患者會(huì)出現(xiàn)整個(gè)頭皮甚至全部體表出現(xiàn)無毛特征。脫發(fā)病在有家族病史的家系中更易出現(xiàn),這說明遺傳因素可能是脫發(fā)病發(fā)生的一個(gè)誘因。對(duì)有2個(gè)或2個(gè)以上患者的家系的研究結(jié)果表明,基因異常能提高患病風(fēng)險(xiǎn)。針對(duì)不同脫發(fā)病型可采用不同的治療對(duì)策,如果脫發(fā)病灶較小,一些治療可能使頭發(fā)重生脫發(fā)面積變小；但是對(duì)于嚴(yán)重脫發(fā)的患者,注射一些糖皮質(zhì)激素如氯倍他索,醋酸氟輕松及皮質(zhì)類固醇或者使用藥膏的治療方法對(duì)于脫發(fā)并沒有明顯的效果。類固醇藥物的注射經(jīng)常用來治療一些小面積的脫發(fā)及眉毛缺失,但還不能確定其具體療效。 先天性毛發(fā)和指甲疾病一直以來就是具有未知遺傳基礎(chǔ)的一組罕見疾病,并研究較少。有研究指出稱編碼縫隙連接蛋白connexin30的GJB基因突變能導(dǎo)致此類疾病的發(fā)生。間隙連接蛋白-6(GJB6),或connexin30(Cx30),在人體中是由GJB6基因編碼的蛋白質(zhì),是間隙連接復(fù)合物之一。GJB6基因位于13號(hào)染色體長(zhǎng)臂1區(qū)1號(hào)帶和1區(qū)2號(hào)帶1亞帶(13q11-q12.1)之間。間隙連接蛋白(Connexins)是一組結(jié)構(gòu)相關(guān)的跨膜蛋白家族,可以裝配形成脊椎動(dòng)物的間隙連接。間隙連接由兩個(gè)半通道或連接子構(gòu)成,每個(gè)半通道或連接子由六個(gè)間隙連接蛋白裝配組成。間隙連接在多細(xì)胞生物體中能夠直接傳遞細(xì)胞間信號(hào),它們由成簇的連接子構(gòu)成通道集群,能使相鄰細(xì)胞間的離子、營養(yǎng)物質(zhì)以及小的代謝產(chǎn)物自由通過。外胚層上皮細(xì)胞及內(nèi)耳的上皮細(xì)胞表達(dá)大量的間隙連接蛋白,其在協(xié)調(diào)角質(zhì)細(xì)胞的生長(zhǎng)和表皮細(xì)胞分化中扮演重要的角色；在內(nèi)耳的聽覺上皮細(xì)胞中,它能夠回收聽覺轉(zhuǎn)導(dǎo)過程中流失的鉀離子。間隙連接在多種生化反應(yīng)中有至關(guān)重要的作用,如協(xié)調(diào)心臟肌肉去極化過程,胚胎發(fā)育以及毛細(xì)血管的應(yīng)答�；诖�,間隙連接蛋白基因的突變可能導(dǎo)致機(jī)體功能及發(fā)育的異常。 在本研究中,我們對(duì)一個(gè)來自中國人群的先天性指甲發(fā)育不良以及脫發(fā)和掌跖角化過度家系進(jìn)行GJB6的突變篩查,尋找其遺傳病因。我們有望通過鑒定GJB6基因的突變引入快速的分子診斷和新型藥物的治療策略,在未來,可能有助于找到治療這種罕見遺傳性疾病的方法。 研究方法：一個(gè)患有杵狀甲和脫發(fā)的中國家庭,其包括四個(gè)患者和兩個(gè)正常人,被選為研究對(duì)象并簽訂知情同意書。通過家譜分析有力的證明了該疾病是常染色體顯性遺傳并且所有患病的成員是等位基因雜合突變。肝素化處理收集的外周血,用苯酚-氯仿提取基因組DNA,并十二烷基磺酸鈉-蛋白酶K純化DNA。用瓊脂糖凝膠電泳檢測(cè)DNA的提取質(zhì)量。通過聚合酶鏈?zhǔn)椒磻?yīng)(PCR)分別用引物：(1)5'-AGACTAGCAGGGCAGGGAGT-3'(上游)以及5’-GGAAAAAGATGCTGCTGGTG-3'(下游)；(2)5’-CCTCCAGCTGATCTTCGTCT-3'(上游)以及5’-GGTTGGTATTGCCTTCTGGA-3'(下游),擴(kuò)增人類GJB6基因全長(zhǎng)序列,產(chǎn)生1350bp和1250bp擴(kuò)增片段。擴(kuò)增后用濃度為1%的瓊脂糖凝膠電泳分離PCR產(chǎn)物,純化PCR產(chǎn)物,然后在ABI-PRISM3100自動(dòng)測(cè)序儀上進(jìn)行雙向測(cè)序,所有的測(cè)序結(jié)果進(jìn)行組裝、分析并用SeqMan Ⅱ程序與野生型序列進(jìn)行比對(duì)。 結(jié)果：本研究調(diào)查了一個(gè)五代的有常染色體顯性遺傳性家族史的杵狀指和脫發(fā)家系。所有的患者都有先天性的杵狀指、脫發(fā)和掌跖角化等相似的臨床癥狀�；颊哞茽钪赴Y狀在出生時(shí)即發(fā)現(xiàn),脫發(fā)在6歲時(shí)出現(xiàn)。體檢發(fā)現(xiàn)患者頭發(fā)、睫毛、眉毛及其他體毛出現(xiàn)缺失。沒有發(fā)現(xiàn)患者有聽覺、視覺異常,牙齒異常及精神異常。實(shí)驗(yàn)室各檢測(cè)指標(biāo)正常,患者壽命正常。我們對(duì)GJB6基因進(jìn)行聚合酶鏈?zhǔn)椒磻?yīng)擴(kuò)增后,用瓊脂糖凝膠電泳進(jìn)行分離純化。對(duì)這六個(gè)家系成員的GJB6基因進(jìn)行測(cè)序,發(fā)現(xiàn)了一個(gè)雜合的錯(cuò)義突變c.31GA,這一變異導(dǎo)致了GJB6編碼蛋白的第11位氨基酸從甘氨酸G變成了精氨酸R,這種變異可能使蛋白質(zhì)的功能或結(jié)構(gòu)發(fā)生轉(zhuǎn)變從而引發(fā)了脫發(fā)和杵狀指癥狀。這種在細(xì)胞質(zhì)內(nèi)存在的蛋白質(zhì)CX30的氨基端的G11R的突變?cè)?例患者中都存在。家系里的2個(gè)正常人中均未發(fā)現(xiàn)缺失、插入或錯(cuò)義突變。近年來,已發(fā)現(xiàn)了越來越多的連接蛋白參與的人類疾病的發(fā)生。連接蛋白的突變影響到多個(gè)器官和系統(tǒng)并與多種疾病的發(fā)生有關(guān)。另外,不同的連接蛋白突變能引起相同或相似的疾病�；蚪M研究不斷地揭示與連接蛋白有關(guān)的疾病的突變,包括耳聾、皮膚病、中樞和外周神經(jīng)病變、白內(nèi)障以及心血管功能障礙等。GJB6基因的突變導(dǎo)致連接蛋白的組成和結(jié)構(gòu)發(fā)生變化,它可能導(dǎo)致間隙連接的非正常轉(zhuǎn)運(yùn)活動(dòng)�？p隙連接是細(xì)胞間水、離子及小分子交換的通道。它參與細(xì)胞間短程、快速的信息傳遞。由于這個(gè)位置上的突變?cè)谄渌巳褐幸灿羞^報(bào)道,所以我們沒有進(jìn)行后續(xù)的正常對(duì)照研究。 結(jié)論：總結(jié)本研究的結(jié)果就是：“在GJB6基因(在蛋白質(zhì)水平位G11R)中一個(gè)錯(cuò)義突變31G→A在中國人群的天生性HED患者中普遍存在”。為了對(duì)那些有患該病史的家族提供適當(dāng)?shù)脑\斷、遺傳咨詢和產(chǎn)前診斷,GJB6基因突變尤其是G11R等位基因是家族中患杵狀甲和脫發(fā)所必須考慮的因素。GJB6基因的UTR區(qū)域和啟動(dòng)子區(qū)域也應(yīng)該得到鑒定,研究突變體的調(diào)控效果和功能性分析將毫無疑問有助于了解GJB6基因表型變異性的機(jī)制,并綜合先前對(duì)GJB6基因的研究,這些結(jié)果豐富了對(duì)連接蛋白功能特點(diǎn)的了解。然而,還需要研究突變的類型、突變的位置、組織學(xué)檢查患病個(gè)體的候選基因以及鑒定世界范圍內(nèi)不同區(qū)域更多的患者。通過當(dāng)前對(duì)HED疾病的分子生物學(xué)基礎(chǔ)的了解,疾病起因于GJB6基因的突變,我們將引進(jìn)一個(gè)新穎的對(duì)頭發(fā)和指甲的各種疾病治療的藥物學(xué)或者基于基因的治療方法,這類疾病的基因治療方法在臨床上可能不會(huì)立即實(shí)現(xiàn),但是在此領(lǐng)域有效的研究無疑肯明了在未來能對(duì)該疾病有效管理。 研究背景：孤獨(dú)癥是在一種開始于兒童36個(gè)月之前的通過其行為定義的綜合征。自閉癥的特點(diǎn)是在社會(huì)交往中存在普遍的缺陷,即語言和非語言溝通減少和刻板行為及狹隘的興趣和活動(dòng)。之前的研究中已報(bào)道過孤獨(dú)癥患者存在一些神經(jīng)性病變包括海馬體、杏仁核、小腦和大腦皮質(zhì)的異常等。孤獨(dú)癥是一種遺傳性疾病,但是它可能由多種基因缺陷造成。這些缺陷的基因可能與大腦發(fā)育、信號(hào)傳導(dǎo)、轉(zhuǎn)運(yùn)或者細(xì)胞結(jié)構(gòu)有關(guān)。有可靠的證據(jù)證明,孤獨(dú)癥的缺陷基因中有某些特定的基因能夠造成患者的代謝異常,即酶分子的缺陷會(huì)造成異常的酶促反應(yīng)速率,導(dǎo)致代謝底物或產(chǎn)物的濃度異常。盡管在孤獨(dú)癥病人中代謝異常者所占比例未知,但是已有許多研究證明一些代謝缺陷和孤獨(dú)癥有關(guān),包括苯丙酮尿癥、組氨酸血癥、肌酸缺乏綜合征、腺苷酸琥珀酸裂解酶缺陷癥、5’-核苷酸酶高活性和嘌呤代謝紊亂等。腺苷酸琥珀酸裂解酶缺乏癥是一種常染色體隱性遺傳疾病,嘌呤合成異常導(dǎo)致體液中積累琥珀酰嘌呤；該疾病約有一半的患者具有孤獨(dú)癥的特征,80%伴有癲癇。 磷酸腺苷脫氨酶(Adenosine monophosphate deaminase, AMPD)或肌腺苷酸脫氨酶,是在高等真核生物中的一種復(fù)合變構(gòu)酶。單磷酸腺苷脫氨酶即AMPD1,是AMPD家族中具有肌肉組織特異性的一種酶類。AMPD1基因一共有16個(gè)外顯子,在基因組中約占20kb,除了2號(hào)外顯子是由12個(gè)核苷酸組成的,每個(gè)外顯子大小在101至220個(gè)核苷酸之間。AMPDl基因在1號(hào)染色體短臂(p)1區(qū)3帶至2區(qū)1帶之間(1p13-p21)。AMPD1在小鼠中編碼同工酶A,在人類中編碼異構(gòu)體M。在骨骼肌中,AMPD1能夠催化腺苷單磷酸(AMP)的脫氨基作用,產(chǎn)生肌苷單磷酸(IMP)和氨(NH3)。 AMPD1也在嘌呤核苷酸循環(huán)(PNC)中起重要作用。在骨骼肌中,PNC中產(chǎn)生的富馬酸參與三羧酸循環(huán)循環(huán)(TCA),最終導(dǎo)致ATP水平的上升。在我們的前期研究中,采用全基因組關(guān)聯(lián)研究(GWAS)的方法來研究了孤獨(dú)癥易感的單核苷酸多態(tài)性位點(diǎn)(SNP)。在AMPD1基因相鄰位點(diǎn)發(fā)現(xiàn)了一處易感SNP,這一發(fā)現(xiàn)表明AMPD1基因可能是孤獨(dú)癥易感的基因。在本研究中,我們研究了AMPD1在體細(xì)胞HEK293和神經(jīng)母細(xì)胞瘤細(xì)胞SH-SY5Y中的分布。希望這個(gè)研究能夠?qū)ν蛔兓蛘呱锕こ毯铣傻鞍自趧?dòng)物模型中的表達(dá),以及其他研究中有幫助。 研究方法：使用Trizol-氯仿-異丙醇的方法從成年小鼠肌肉組織中提取總RNA。用Oligo (dT)18引物逆轉(zhuǎn)錄合成cDNA,進(jìn)而進(jìn)行AMPD1基因的RT-PCR。 cDNA為模板進(jìn)行AMPD1真核表達(dá)載體的構(gòu)建。以AMPD1基因全長(zhǎng)cDNA的擴(kuò)增引物為：正向引物5'-ATCCGGAATTCAATGCCTCTATTCAAACTAACAGGTC-3', EcoRI為限制性酶切位點(diǎn)；反向引物5'-ATGCGGGATCCTCATTCTGTTGCTTTAAGACCCTCA-3', BamHI為限制性酶切位點(diǎn)。進(jìn)行PCR之后,對(duì)DNA樣本進(jìn)行1%瓊脂糖凝膠電泳；切下目的片段后用Qiagen膠回收試劑盒純化。使用Qiagen質(zhì)粒mini試劑盒抽p3Xflag載體。將mAMPD1的PCR片段和p3Xflag空載體用EcoRI和BamHI進(jìn)行雙酶切。酶切產(chǎn)物進(jìn)行使用純化,并將酶切后的PCR片段和空載體按照比例連接。連接產(chǎn)物轉(zhuǎn)進(jìn)感受態(tài)大腸桿菌后涂板。挑選單克隆在Amp+LB培養(yǎng)基中進(jìn)行培養(yǎng),堿裂解法抽提細(xì)菌質(zhì)粒后,進(jìn)行EcoRI和BamHI的雙酶切鑒定。陽性克隆進(jìn)行測(cè)序鑒定。重組的質(zhì)粒p3Xflag-mAMPD1使用Lipo2000轉(zhuǎn)染進(jìn)HEK293和SH-SY5Y細(xì)胞系中,用免疫組化的方法使用Flag標(biāo)記和內(nèi)質(zhì)網(wǎng)的標(biāo)記蛋白的抗體對(duì)轉(zhuǎn)染后代細(xì)胞進(jìn)行熒光標(biāo)記,研究AMPD1的表達(dá)的細(xì)胞分布。 結(jié)果：在p3Xflag-mAMPD1質(zhì)粒構(gòu)建中,使用小鼠肌肉組織cDNA為模板進(jìn)行PCR擴(kuò)增mAMPDl片段。PCR產(chǎn)物用1%瓊脂糖凝膠電泳分離,切膠純化。p3Xflag (6.4Kb)轉(zhuǎn)化進(jìn)感受態(tài)細(xì)胞中,然后提取質(zhì)粒。純化的PCR產(chǎn)物(97. Ong/ml)以及提取的質(zhì)粒(p3Xflag,237.7ng/ml)進(jìn)行酶切,使用限制性內(nèi)切酶EcoRI和BamHI。mAMPD1(27.lng/ml)和p3Xflag (23.4ng/ml)的酶切產(chǎn)物進(jìn)行連接,然后轉(zhuǎn)進(jìn)E. coli DH5α感受態(tài)細(xì)胞進(jìn)行擴(kuò)增。隨機(jī)選取單克隆,通過測(cè)序檢查插入片段。 轉(zhuǎn)染進(jìn)質(zhì)粒的HEK293細(xì)胞免疫熒光染色發(fā)現(xiàn)AMPD1均勻的分布于胞漿不在細(xì)胞核分布。另一個(gè)細(xì)胞系,SH-SY5Y的轉(zhuǎn)染及免疫熒光結(jié)果也證明AMPD1的胞漿細(xì)胞分布。 結(jié)論：在本研究中,我們檢測(cè)了AMPD1在HEK293和SH-SY5Y細(xì)胞系中的分布情況,發(fā)現(xiàn)AMPD1特異性地分布在胞漿中,這是我們關(guān)于孤獨(dú)癥相關(guān)基因的表達(dá)研究,動(dòng)物以及臨床研究的初步結(jié)果。
[Abstract]:Background: congenital disease is a kind of a month before birth or after birth in the occurrence of the disease. Many congenital diseases are hereditary disease genetic abnormalities caused by congenital hidrotic ectodermal dysplasia (Hidrotic ectodermal, dysplasia, HED), also known as Clouston syndrome, is a constant autosomal dominant genetic disease, which is characterized by clubbing, hair loss and the palms and soles of the feet horny. Congenital clubbed nail (or clubbing) is a clinically rare hereditary skin disease due to connective tissue hyperplasia and third knuckle bone nail bed portion of the nail plate and fingers and expand the lead terminal segment. The toes clubbed a lack of specific and feasible treatment method, but according to the pathological symptoms of the treatment may reduce the occurrence of a clubbing phenotype; if the disease occurs in early intervention and Can reverse the disease, but there is a lack of effective surgical procedures. The clinical features of congenital alopecia is normal scalp hair not renewable shedding, sparse eyebrows and eyelashes and lack of armpit hair, pubic hair or other body hair. 1-2% congenital alopecia patients may have a whole head skin surface appeared glabrous or all alopecia features. It is easy to appear in a family history of the family, suggesting that genetic factors may be a cause of hair loss disease. On 2 or more than 2 of patients with family. The results showed that the abnormal gene can increase the risk of illness. According to the different type of alopecia can be treated by different strategies, if alopecia lesions are smaller, some treatment may make the hair rebirth alopecia area smaller; but for the patients with severe alopecia, injection of glucocorticoids such as clobetasol, fluocinonide and corticosteroids or. Methods of treatment with ointment for hair loss and no obvious effect. Steroid injections are often used to treat hair loss and eyebrows missing some small area, but also can not determine the specific effect.
A group of rare congenital diseases of hair and nails has always been disease with unknown genetic basis, and less research. Studies have shown that GJB gene that encoding connexin connexin30 mutations can lead to such diseases. The gap junction protein -6 (GJB6), or connexin30 (Cx30), in the human body is composed of GJB6 gene encoding protein is one of the gap junction complex.GJB6 gene is located on the long arm of chromosome 13 in 1 District No. 1 zone and 1 Zone No. 2 with 1 sub zones (13q11-q12.1). The gap junction protein (Connexins) is a transmembrane protein family group structure, the formation of the vertebrate gap assembly connection. Gap junction structure by 2.5 channel or connector, each half channel or connector is composed of six connexin assembly. Gap junctional intercellular signal can directly transfer in multicellular organisms, they consist of clusters even Then a channel cluster, can make ion between adjacent cells, nutrients and small metabolites free passage. Ectodermal epithelial cells and inner ear epithelial cells express large gap junction protein, plays an important role in the coordination of keratinocyte growth and differentiation of epidermal cells; in the inner ear auditory epithelial cells. It can recover loss of potassium ions in the process of auditory transduction. Gap junction plays a crucial role in many biochemical reactions, such as the coordination of the heart muscle depolarization process, embryonic development and capillary responses. Based on this, the mutation of gap junction protein genes may cause the body function and developmental abnormalities.
In this study, we performed mutation screening of GJB6 from a China population of congenital hypoplastic nails and hair loss and palmoplantar hyperkeratosis pedigree, find its genetic etiology. We expected treatment strategy, the introduction of molecular rapid diagnosis and new drug through the identification of GJB6 gene mutation in the future may help find a cure for this rare genetic disease.
Methods: with a clubbed nail and hair loss Chinese family, including four patients and two normal people were selected as the research object and signed informed consent. The pedigree analysis proves that the disease is autosomal dominant and all affected members are alleles of heterozygous mutations. Heparinized peripheral blood was collected. Genomic DNA was extracted by phenol chloroform extraction, and the quality of twelve sodium dodecyl sulfate - protease K DNA. purified by agarose gel electrophoresis. The detection of DNA by polymerase chain reaction (PCR) respectively with primers: (1) 5'-AGACTAGCAGGGCAGGGAGT-3'(upstream) and 5 -GGAAAAAGATGCTGCTGGTG-3' (downstream) (; 2) 5 'and 5' -CCTCCAGCTGATCTTCGTCT-3'(upstream) -GGTTGGTATTGCCTTCTGGA-3' (downstream), amplified the full-length sequence of human GJB6 gene, 1350bp and 1250bp amplified fragment. After amplification with concentration of 1% June The separation of PCR lipid products of sugar gel electrophoresis. The purified PCR products were then sequenced on automatic ABI-PRISM3100 sequencer, all sequencing results were assembled, analyzed and compared with the sequence of SeqMan II program with the wild type.
Results: This study investigated a five generations with autosomal dominant familial history of clubbing and alopecia family. All patients had congenital clubbing, hair loss and clinical symptoms similar to patients with palmoplantar keratoderma. Acropachy symptoms at birth, alopecia in 6 when appear. Physical examination found that patients with hair, eyelashes, eyebrows and hair loss. Other patients have found no abnormal vision, hearing, and mental disorders. The abnormal dental lab index of normal patients, normal life. We were amplified by polymerase chain reaction of GJB6 gene was isolated and purified by agarose gel electrophoresis for sequencing. The GJB6 gene of the six family members, found a heterozygous missense mutation c.31GA, the mutation causing GJB6 encoding proteins of eleventh amino acids into arginine glycine from R G, this variant may The protein function or structure change, causing hair loss and acropachy symptoms. This mutation exists in the cytoplasm of the CX30 protein N-terminal G11R exists in 4 patients. 2 normal people in the Department were not found in deletion, insertion or missense mutation. In recent years, has found more and more connected proteins involved in human disease. Connecting protein mutation affects multiple organs and systems and related to many diseases. In addition, connexin mutations can cause similar disease. Different genomic studies continue to reveal and mutation, including protein related diseases connected deafness, skin disease, central and peripheral neuropathy, cataract and cardiovascular dysfunction such as mutation of.GJB6 gene leads to the composition and structure of connexin changes, it may lead to abnormal gap junction Transport activity. Gap junctions are intercellular water, ions and small molecular exchange channels. It is involved in cellular short-range, rapid transmission of information. Because of this position mutations in other populations have also been reported, so we do not have a normal follow-up study.
Conclusion: the results of this study is: "in the GJB6 gene (at the protein level G11R) mutation in a missense 31G, A exists in Chinese people born in HED patients. For those who have had the history of the family to provide the appropriate diagnosis, genetic counseling and prenatal diagnosis, GJB6 gene especially the G11R mutation allele of UTR region and the promoter region of.GJB6 gene family factors with clubbed nail and hair loss that must be considered should be identified, control effect and functional study of mutant analysis will no doubt help to understand the mechanism of GJB6 gene phenotypic variability, and prior to GJB6 genetic studies, these results enrich the understanding of connexin features. However, still need to study types of mutations, mutation location, candidate gene organization examination and identification of affected individuals worldwide Patients in different regions. The more the molecular basis of the current understanding of the HED disease, the disease causing mutation in the GJB6 gene, we will introduce a novel on the hair and nails in treating various diseases pharmacology or gene based treatment, this kind of disease gene therapy may not be immediately implemented in clinically, but effective research in this field is willing in the future to the effective management of the disease.
Background: autism is in a beginning in the 36 months before the children through syndrome definition. Its behavior characteristic of autism is a common defect in social interaction, namely, language and non language communication and reduce the stereotyped behavior and narrow interests and activities. The research has been reported before in autism there were some nerve lesions including the hippocampus, amygdala, cerebellum and cerebral cortex abnormalities. Autism is a genetic disease, but it may be caused by a variety of genetic defects. These defects of genes may be associated with brain development, signal transduction, cell or the transportation structure. There is reliable evidence of autism gene defects in certain genes can cause patients with metabolic abnormalities, including defects of enzyme molecules will cause abnormal enzymatic reaction, resulting in concentration of metabolic substrate or product as abnormal. In patients with autism tube metabolic abnormalities in the proportion of unknown, but many studies prove that some metabolic defects and autism, including phenylketonuria, histidinemia, creatine deficiency syndrome, adenylosuccinate lyase deficiency, 5 '- nucleotidase activity and high purine metabolism. Adenylosuccinate lyase deficiency disease is an autosomal recessive genetic disease, abnormal accumulation of purine synthesis of succinyl purine in body fluids; about half of the patients with autism characteristics of the disease, 80% with epilepsy.
Adenosine monophosphate deaminase (Adenosine monophosphate, deaminase, AMPD) or myoadenylate deaminase, is a complex allosteric enzyme in higher eukaryotes. Adenosine monophosphate deaminase is an enzyme that is AMPD1,.AMPD1 gene has a muscle specific AMPD in the family of a total of 16 exons in the genome of about for 20KB, in addition to exon 2 was composed of 12 nucleotides, each exon size between 101 to 220 nucleotides of.AMPDl gene on the short arm of chromosome 1 (P) 1 to 2 District 1 District 3 zone between (1p13-p21).AMPD1 code of A isoenzyme in mice and in humans encoding M. isoforms in skeletal muscle, AMPD1 can catalyze adenosine monophosphate (AMP) deamination, produce inosine monophosphate (IMP) and ammonia (NH3). AMPD1 is also in the purine nucleotide cycle (PNC) plays an important role in skeletal muscle, fumaric acid produced in the PNC in the three cm Acid circulation (TCA), resulting in increased levels of ATP. In our previous study, a genome-wide association study using (GWAS) method to study the polymorphism of autism susceptibility (SNP). The AMPD1 gene between adjacent sites found a susceptible SNP, which indicates a the AMPD1 gene may be autism susceptibility genes. In this study, we investigated the distribution of AMPD1 in HEK293 cells and neuroblastoma cells in SH-SY5Y. Hope this research can for mutations or protein synthesis of biotechnology in animal models of expression, as well as other studies in help.
Method: using Trizol- chloroform to extract the total RNA. from the muscle of adult mice with Oligo (dT) 18 primer cDNA was synthesized, and then AMPD1 gene RT-PCR. cDNA AMPD1 eukaryotic expression vector as template. The full-length cDNA of AMPD1 gene amplification primer: forward primer 5'-ATCCGGAATTCAATGCCTCTATTCAAACTAACAGGTC-3', EcoRI as the restriction enzyme site; reverse primers 5'-ATGCGGGATCCTCATTCTGTTGCTTTAAGACCCTCA-3', BamHI restriction sites. After PCR, 1% agarose gel electrophoresis of DNA samples; cut fragment with pure Qiagen Gel Extraction Kit

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R758.71;R730.264

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