本文選題：視網(wǎng)膜母細(xì)胞瘤 + RB1基因�。� 參考：《浙江大學(xué)》2011年碩士論文

【摘要】：研究背景 視網(wǎng)膜母細(xì)胞瘤(retinoblastoma, RB)是嬰幼兒最常見的眼科腫瘤,約占兒童惡性腫瘤的4%,也是一類嚴(yán)重的致死性腫瘤(占嬰幼兒死亡率的第一位)。大部分RB患兒在5歲之前發(fā)病。開始發(fā)病時較常見的癥狀為白瞳癥、斜視及視力下降,多因此就診。病情進(jìn)一步發(fā)展則相繼出現(xiàn)青光眼、葡萄膜炎、玻璃體出血,甚至腫瘤轉(zhuǎn)移、死亡等。 RB具有明顯的家系遺傳性,大多數(shù)呈常染色體顯性遺傳方式,外顯率高達(dá)90%。位于13q14.1-q14.2上的RB1基因(OMIM:180200)已被確定為唯一的RB致病基因。RB1基因全長183 kb,包含27個外顯子,屬于較長的基因。RB1基因是人類發(fā)現(xiàn)的第一個抑癌基因,其編碼的pRB蛋白廣泛存在于各種細(xì)胞中。正常的RB1基因表達(dá)對抑制RB的發(fā)生是必不可少的,RB1基因的缺失或變異失活都會導(dǎo)致RB的發(fā)生。 RB在臨床上可表現(xiàn)為單眼發(fā)病和雙眼發(fā)病兩種類型。雙眼患者發(fā)病時間早,多在1歲以內(nèi),90%以上的病例都是遺傳性的；單眼患者發(fā)病較晚,約15%為遺傳性的。92.7%的雙眼患者和14.6%的單眼患者均攜帶高外顯的RB1種系基因突變。因此,RB家系患者和散發(fā)患者的RB1基因的變異組學(xué)研究,對其早期診斷、治療、預(yù)后、遺傳咨詢、產(chǎn)前診斷、植入前遺傳學(xué)診斷以及基因治療等都至關(guān)重要。 研究目的 對1個浙江地區(qū)視網(wǎng)膜母細(xì)胞瘤家系中所有成員的外周血表達(dá)的RBI cDNA序列進(jìn)行測序分析,以期快速獲得基因組水平RB1基因的突變信息,探討RB1基因突變與RB發(fā)病的相關(guān)性。 研究對象 一個居于浙江省杭州地區(qū)的RB家系(包含2例患者),以及10例正常健康對照。 研究方法 (1)抽取所研究家系的所有成員和正常對照個體的外周血(抗凝),用Ambion RiboPure blood kit提純外周血總RNA,用苯酚-氯仿法純化基因組DNA； (2)取所有RNA樣本,反轉(zhuǎn)錄-聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)擴(kuò)增其RBI cDNA全序列并測序； (3)取所有DNA樣本,PCR擴(kuò)增RB1基因外顯子并測序； (4) DNAssist軟件及Chromas 2軟件進(jìn)行測序數(shù)據(jù)分析,并與Ensemb1、NCBI等人類遺傳學(xué)權(quán)威數(shù)據(jù)庫進(jìn)行比對分析； (5)針對所發(fā)現(xiàn)的突變位點,利用堿基錯配引物的等位基因特異性-聚合酶鏈?zhǔn)椒磻?yīng)(AS-PCR法),驗證所發(fā)現(xiàn)的突變,并且篩查家系中的嵌合體患者。 研究結(jié)果 (1)所研究RB家系的2例RB患者(先證者：雙眼；先證者之父：單眼)的外周血RB1基因cDNA均存在第14外顯子的c.1363CT(p.R455X)雜合突變。 (2)RB患者的基因組均存在RB1基因g.76460CT雜合突變。 (3)家系中的2例RB患者的AS-PCR都擴(kuò)增得到特異條帶,其他成員均無條帶。 結(jié)論 (1)RB1基因c.1363CT(p.R455X)突變?yōu)樗芯縍B家系患者的首要致病因素。 (2)先證者的雜合突變遺傳自他的父親。 (3)用RT-PCR/DNA測序相結(jié)合的方法篩查RB1基因突變,是目前RB變異組學(xué)研究中最方便、快捷、廉價的技術(shù)。
[Abstract]:Background retinoblastoma (RBB) is the most common ophthalmic tumor in infants and young children, accounting for about 4% of malignant tumors in children. Most children with RB develop before the age of 5. The more common symptoms of onset are mydriasis, strabismus and decreased visual acuity. There were glaucoma, uveitis, vitreous hemorrhage, tumor metastasis, death and so on. RB had obvious heredity in pedigree, most of them were autosomal dominant inheritance, and the rate of exsertion was as high as 90%. RB1 gene, located on 13q14.1-q14.2, has been identified as the only RB pathogenicity gene. RB1 gene contains 27 exons in 183kb. it is the first oncosuppressor gene found in human. Its encoded PRB protein is widely present in various cells. Normal RB1 gene expression is essential to inhibit the occurrence of RB. Deletion or inactivation of Rb1 gene can lead to RB occurrence. RB can be classified as monocular and binocular. The onset time of binocular patients was early, and 90% of the patients within 1 year of age were hereditary, and 15% of the patients with monocular disease were hereditary, and about 15% of binocular patients and 14.6% of monocular patients were carrying high explicit RB1 gene mutation. Therefore, the mutation study of RB1 gene in RB pedigree and sporadic patients is very important for its early diagnosis, treatment, prognosis, genetic counseling, prenatal diagnosis, preimplantation genetic diagnosis and gene therapy. Objective to sequence and analyze the RBI cDNA sequences expressed in peripheral blood of all members of a family of retinoblastoma in Zhejiang province, in order to obtain the mutation information of RB1 gene at genomic level. To investigate the relationship between RB1 gene mutation and RB. Participants A RB pedigree (including 2 patients and 10 normal controls) living in Hangzhou, Zhejiang Province, was enrolled in the study. Methods 1) the peripheral blood (anticoagulant) was extracted from all the members of the family and the normal individuals. The total RNAs of peripheral blood were purified by Ambion RiboPure blood kit, and the genomic DNAs were purified by phenol-chloroform method. The whole RBI cDNA sequence was amplified and sequenced by reverse transcription-polymerase chain reaction (RT-PCR), and the exon of RB1 gene was amplified and sequenced by PCR from all DNA samples. DNAssist software and Chromas 2 software were sequenced and compared with human genetics authoritative databases such as Ensemb1NCBI. The allele-specific polymerase chain reaction (AS-PCR) method of base mismatch primer was used to verify the mutation and to screen the chimeric patients in the pedigree. Results 1) two RB patients (proband: binocular) from RB pedigree were studied. The RB1 gene cDNA in peripheral blood of proband father: monocular) had heterozygous mutation of exon 14, c. 1363 CTP. R455X. RB1 gene g.76460CT heterozygous mutation was found in the genomes of both patients. Specific bands were obtained from AS-PCR amplification of RB patients. No other members have stripes. Conclusion the mutation of RB1 gene c.1363CTA p.R455X) is the leading pathogenic factor in the RB family studied. The heterozygosity of the proband was inherited from his father. Y3) was sequenced by RT-PCR / DNA sequencing. Methods RB1 gene mutation was screened. It is the most convenient, fast and cheap technique in RB mutation study.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R739.7

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 鄭嵩山;視網(wǎng)膜母細(xì)胞瘤臨床分析及眼內(nèi)化療實驗研究[D];中山大學(xué);2006年

，

本文編號：2015050