【摘要】：目的青藏高原是人類生存具有挑戰(zhàn)性的環(huán)境之一,在青藏高原上生活的藏族人群適應(yīng)了高原低壓低氧環(huán)境而成為高原世居人群。本文將從世居藏族各個(gè)系統(tǒng)的高原適應(yīng)情況進(jìn)行流行病學(xué)調(diào)查,以及選擇與藏族低氧適應(yīng)相關(guān)的候選基因來(lái)探討世居藏族高原低氧適應(yīng)相關(guān)性,找出與世居藏族低氧適應(yīng)相關(guān)候選基因多態(tài)位點(diǎn),為高原醫(yī)學(xué)研究提供理論依據(jù)。方法1.流行病學(xué)調(diào)查:在西藏不同海拔高度拉薩市(3650)、山南市(3600)、林芝市(2900)、日喀則市(4100)四個(gè)市采用偶遇調(diào)查方法,對(duì)當(dāng)?shù)厥谰诱吆鸵凭诱吖?884人(總共發(fā)放4000個(gè)問(wèn)卷表,其去掉無(wú)效問(wèn)卷,最后獲得有效問(wèn)卷有2884個(gè))進(jìn)行調(diào)查,其中有1691個(gè)世居藏族和1193個(gè)移居者(漢族為主)通過(guò)自行設(shè)計(jì)的問(wèn)卷調(diào)查表,以自填的方式進(jìn)行流行病學(xué)調(diào)查。問(wèn)卷內(nèi)容包括各系統(tǒng)(呼吸、神經(jīng)、消化系統(tǒng)和循環(huán)系統(tǒng)等)的變化情況。收集的數(shù)據(jù)進(jìn)行統(tǒng)一錄入,并采用SPSS17.0統(tǒng)計(jì)軟件進(jìn)行分析,其中計(jì)數(shù)資料采用卡方檢驗(yàn)(卡方值進(jìn)行哈迪溫伯格檢驗(yàn))。2.候選基因分型,本課題前期通過(guò)全基因組等位基因分型(genome wide association study,GWADS)方法篩選出的與藏族低氧適應(yīng)相關(guān)的候選基因鈉鈣交換因子(SLC8A1)、蛋白激酶C(PRKCE)、細(xì)胞色素C氧化酶合成的核編碼基因(CMC2)、促血管新生蛋白因子(ANGPT4)、腺苷酸脫氨酶家族成員之一(AMPD3)、驅(qū)動(dòng)蛋白超家族(KIF1B)和基因編碼腎上腺素受體(ADRA1A),本課題選擇了241例世居藏族和Hap MapⅢ期中北方和南方平原漢族(208例)利用Sequenom Mass Arry平臺(tái)對(duì)(ADRA1A基因的rs1177946、rs6557946、rs7842829、rs1048101、rs1442341;位于SLC8A1基因上的rs4952404、rs13031392、rs2192771;PRKCE基因上的rs11898209、rs6544852、rs6756452、rs3768753、rs10205024、rs2278775、rs3820729、rs2594489、rs2594494、rs7571879;KIF1B基因上的rs10492970、rs17034615、rs17034716、rs2297881)共4個(gè)候選基因22個(gè)SNP位點(diǎn)利用Squenom Mass ARRAY檢測(cè)平臺(tái)進(jìn)行擴(kuò)大樣本驗(yàn)證。所獲得數(shù)據(jù)采用SPSS17.0統(tǒng)計(jì)軟件進(jìn)行分析,計(jì)量資料采用T檢驗(yàn),計(jì)數(shù)資料采用卡方檢驗(yàn),進(jìn)行哈迪-溫伯格平衡(Hardy-Weinberg equilibrium)檢驗(yàn),群體表型的比較采用單因素方差分析,用T檢驗(yàn)計(jì)算基因型對(duì)臨床表型的影響,用plink軟件進(jìn)行與表型的關(guān)聯(lián)分析,以P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果1.流行病學(xué)調(diào)查:本次調(diào)查獲得2884個(gè)不同海拔高度的世居者和移居者,其中拉薩市世居者579人,移居者536人;女性526人,男性589人;山南市世居者250人,移居者281人;女性164人,男性367人;林芝市世居者153人,移居者206人;女性152人,男性207人;日喀則市世居者708人,移居者171人;女性472人,男性407人;調(diào)查發(fā)現(xiàn)世居者總的高原不適應(yīng)率為34.9%,而移居者總的高原不適應(yīng)率為52.9%,其世居者和移居者神經(jīng)系統(tǒng)不適應(yīng)率為37.1%和50.7%;循環(huán)系統(tǒng)的不適應(yīng)率為23.6%和48.9%;消化系系統(tǒng)不適應(yīng)率為36.4%和44.3%。在不同海拔高度的四個(gè)地區(qū)的世居者和移居者總的高原不適應(yīng)情況調(diào)查顯示,拉薩世居者和移居者總的不適應(yīng)率為34.5%和48.0%;山南世居者和移居者總的不適應(yīng)率為38.3%和52.3%;日喀則世居者和移居者總的不適應(yīng)率為29.6%和52.2%;林芝世居者和移居者總的不適應(yīng)率為55.9%和67.2%,在4個(gè)不同海拔高度的世居者和移居者各組間有統(tǒng)計(jì)學(xué)差異。2.表型數(shù)據(jù)分析:對(duì)241例世居藏族的臨床生化表型進(jìn)行統(tǒng)計(jì),發(fā)現(xiàn)世居藏族的臨床生化指標(biāo)基本在正常參考值范圍內(nèi),其反應(yīng)心室收縮功能的心功能指標(biāo)EF(射血分?jǐn)?shù)%)較高與正常參考值。在世居藏族中男性是73.77±4.86%;女性是73.63±4.36%。3.候選基因的分型:4個(gè)候選基因的22個(gè)SNP位點(diǎn)在世居藏族和平原漢族之間進(jìn)行兩兩比較,并進(jìn)行與表型的關(guān)聯(lián)分析,發(fā)現(xiàn)4個(gè)候選基因均在不同人群中基因型頻率差異顯著且最小等位基因頻率具有顯著相關(guān)性(P㩳0.05)。首先對(duì)4個(gè)候選基因的22個(gè)SNP位點(diǎn)進(jìn)行基因型頻率比較,發(fā)現(xiàn)KIF1B基因上的4個(gè)(rs10492970、rs17034615、rs17034716、rs2297881)SNP位點(diǎn)的基因型頻率均在世居藏族和平原漢族中各組間比較均有顯著差異(P㩳0.05)。SLC8A1基因上的2個(gè)(rs4952404、rs13031392)SNP位點(diǎn)的基因型頻率在世居藏族和平原漢族中各組間比較均均有顯著差異(P㩳0.05)。ADRA1A基因上的5個(gè)(rs1177946、rs6557946、rs7842829、rs1048101、rs1442341)SNP位點(diǎn)的基因型頻率在世居藏族和平原漢族中各組間比較均有顯著差異(P㩳0.05),PRKCE基因上的10個(gè)(rs11898209、rs6544852、rs6756452、rs3768753、rs10205024、rs2278775、rs3820729、rs2594489、rs2594494、rs7571879)SNP位點(diǎn)的基因型頻率在世居藏族和平原漢族中各組間比較均有顯著差異(P㩳0.05)。我們還對(duì)4個(gè)候選基因的22個(gè)SNP位點(diǎn)在世居藏族和平原漢族中進(jìn)行頻率差異性進(jìn)行比較,發(fā)現(xiàn)KIF1B基因上的4個(gè)SNP位點(diǎn);ADRA1A基因的5個(gè)SNP位點(diǎn);SLC8A1基因上的2個(gè)(rs4952404、rs13031392)SNP位點(diǎn);PRKCE基因上的10個(gè)SNP位點(diǎn)均具有顯著差異性(P㩳0.05)。此外,我們發(fā)現(xiàn)了SLC8A1基因上rs4952404和rs13031392位點(diǎn),PRKCE基因上的rs7571879、rs6756452、rs3820729和rs2278775位點(diǎn),ADRA1A基因上的rs7842829位點(diǎn)的最小等位基因頻率隨著海拔升高而增大,其在安多人群中顯示最小等位基因頻率最大。4.數(shù)量性狀關(guān)聯(lián)分析我們對(duì)具有表型數(shù)據(jù)的世居藏族(241例)通過(guò)數(shù)量性狀關(guān)聯(lián)分析發(fā)現(xiàn)PRKCE基因上的rs3768753位點(diǎn)與每搏輸出量(SV)及射血分?jǐn)?shù)(EF)相關(guān);rs2594489位點(diǎn)與紅細(xì)胞壓積(HCT)、最大肺活量(FVC)、最大呼氣流量(PEF)、時(shí)間肺活量(FEV1)、血氧飽和度(Sa O2)相關(guān);rs2594494位點(diǎn)與FEV1、FVC、PEF、HCT相關(guān);rs6544852位點(diǎn)與Sa O2、主動(dòng)脈內(nèi)徑(Ao)、FEV1、FVC、PEF相關(guān);rs6756452位點(diǎn)與Sa O2相關(guān)。ADRA1A基因上的rs1442341位點(diǎn)與Sa O2相關(guān);rs6557946、rs7842829、rs1048101位點(diǎn)均與A0相關(guān);rs11779546位點(diǎn)與Sa O2、SV相關(guān)。KIF1B基因上的rs17034716位點(diǎn)與Sa O2相關(guān);rs10492970位點(diǎn)與AO相關(guān);rs17034615位點(diǎn)與AO相關(guān);rs2297881位點(diǎn)與AO相關(guān)(P0.05)。SLC8A1基因上的rs4952404位點(diǎn)與Sa O2相關(guān)(P0.05)。結(jié)論1.在高原低壓低氧環(huán)境下長(zhǎng)期生活,對(duì)機(jī)體呼吸系統(tǒng)、神經(jīng)系統(tǒng)、循環(huán)系統(tǒng)和消化系統(tǒng)均受到了一定程度的影響。2.世居藏族的高原適應(yīng)性較移居者好,世居藏族總的高原不適應(yīng)率為34.9%,而移居者總的高原不適應(yīng)率為52.9%。不同海拔高度的4個(gè)地區(qū)高原不適應(yīng)率沒(méi)有隨著海拔的升高而增加。3.進(jìn)一步證明了PRKCE、SLC8A1、ADRA1A和KIF1B基因可能是藏族低氧適應(yīng)相關(guān)的候選基因。4.PRKCE基因6個(gè)SNP位點(diǎn)(rs3768753、rs2594489、rs2594494 rs6544852、rs6756452、rs11898209)在不同海拔高度世居藏族中與表型(SV、EF、HCT、FVC、PEF、FEV1、Sa O2、Ao)有顯著相關(guān)性(P0.05),因此推測(cè)PRKCE基因可能在藏族低氧適應(yīng)機(jī)制中發(fā)揮著不容忽視的作用。5.SLC8A1基因rs4952404和rs13031392位點(diǎn)可能是藏族低氧適應(yīng)相關(guān)的重要候選位點(diǎn)。6.ADRA1A基因rs1442341、rs7842829和rs11779546位點(diǎn)可能是藏族低氧適應(yīng)相關(guān)的重要候選位點(diǎn)。7.KIF1B基因rs17034716位點(diǎn)可能是藏族低氧適應(yīng)的重要候選位點(diǎn)。
[Abstract]:Objective The Qinghai-Tibet Plateau is one of the most challenging environments for human survival. The Tibetan people living on the Qinghai-Tibet Plateau have adapted to the high altitude hypobaric hypoxic environment and become the inhabitants of the world. To explore the correlation of hypoxic adaptation in Tibetan plateau and find out the candidate gene polymorphisms related to hypoxic adaptation in Tibetan plateau. Methods 1. Epidemiological investigation: Four cities of Lhasa (3650), Shannan (3600), Linzhi (2900) and Xigaze (4100) in Tibet were selected by chance. Methods A total of 2884 local residents and migrants (4000 questionnaires were sent out, and 2884 valid questionnaires were obtained after eliminating the invalid questionnaires). Among them, 1691 Tibetans and 1133 migrants (mainly Han Chinese) were investigated by self-designed questionnaires. The contents of the paper include the changes of the respiratory, nervous, digestive and circulatory systems. The collected data were input uniformly and analyzed by SPSS17.0 statistical software. The counting data were analyzed by Chi-square test (Hardy-Weinberg test for Chi-square value). 2. Candidate genotyping, which passed the whole genome allele earlier in this project. Genotyping (GWADS) method screened the candidate genes related to Tibetan hypoxic adaptation, including sodium calcium exchange factor (SLC8A1), protein kinase C (PRKCE), cytochrome C oxidase synthesis nuclear coding gene (CMC2), angiogenic protein factor (ANGPT4), adenylate deaminase family member (AMPD3), driving eggs. The white superfamily (KIF1B) and gene-encoded adrenergic receptor (ADRA1A) were selected from 241 Tibetans and 208 Han (Hap Map Phase III) in the northern and southern plains using Sequenom Mass Arry platform pairs (rs1177946, rs6557946, rs7842829, rs1048101, rs1442341; rs4952404, rs13031392, rs21) in the SLC8A1 gene. 92771; PRKCE gene on the rs11898209, rs6544852, rs6756452, rs3768753, rs10205024, rs2278775, rs3820729, rs2594489, rs2594494, rs7571879; KIF1B gene on the rs10492970, rs17034615, rs17034716, rs2297881) a total of four candidate gene 22 SNP loci using the Squenom s ARY detection platform for extended sample validation. SPSS17.0 statistical software was used to analyze the data. T-test was used to measure the data. Chi-square test was used to test the counting data. Hardy-Weinberg equilibrium test was used to test the population phenotype. One-way ANOVA was used to compare the population phenotype. T-test was used to calculate the influence of genotype on the clinical phenotype. Results: 1. Epidemiological survey: 2884 residents and immigrants at different altitudes were obtained, including 579 residents and 536 immigrants in Lhasa, 526 women and 589 men, 250 in Shannan, 281 immigrants, 164 women and 367 men, 153 in Linzhi. 206 migrants; 152 females, 207 males; 708 inhabitants, 171 migrants; 472 females, 407 males; the survey found that the total altitude maladjustment rate of the world's inhabitants was 34.9%, while the total altitude maladjustment rate of the migrants was 52.9%, and the maladjustment rates of the nervous system of the world's inhabitants and the migrants were 37.1% and 50.7%, respectively. The overall altitude maladjustment rates of the settlers and immigrants in four different altitudes were 34.5% and 48.0%, 38.3% and 52.3% respectively, and that of the Shannan settlers and immigrants was 38.3% and 52.3%, respectively. The total maladjustment rates of the inhabitants and the immigrants were 29.6% and 52.2%; the maladjustment rates of the inhabitants and the immigrants of Linzhi were 55.9% and 67.2%. There were statistical differences between the groups of the inhabitants and the immigrants at four different altitudes. 2. Phenotypic data analysis: 241 cases of the clinical biochemical phenotypes of the inhabitants were statistically analyzed. The biochemical parameters of the bed were basically within the normal reference range, and the EF (ejection fraction%) was higher and normal. Among the Tibetans living in the world, the EF (ejection fraction%) was 73.77 (+ 4.86%) in males and 73.63 (+ 4.36%) in females. The genotype frequencies of the four candidate genes were significantly different in different populations and the minimum allele frequencies were significantly correlated (P?0.05). The genotype frequencies of 22 SNP loci of the four candidate genes were compared and four of them (rs10492970, rs17034615, rs170) were found in KIF1B gene. The genotype frequencies of the two SNP loci (rs4952404, rs13031392) in the SLC8A1 gene were significantly different between the Tibetan and Han populations (P?0.05). There were significant differences in the genotype frequencies of the five ADRA1A loci (rs1177). The genotypic frequencies of the SNP loci in 946, rs6557946, rs7842829, rs1048101, rs1442341 were significantly different between Tibetans and Han Chinese (P? 0.05). The genotypic frequencies of 10 PRKCE loci (rs11898209, rs6544852, rs6756452, rs3768753, rs10205024, rs2278775, rs3820729, rs2594489, rs2594494, rs7571879) were significantly different. There were significant differences in the frequencies of 22 SNP loci of 4 candidate genes between Tibetans and Han people in the plains (P?0.05). We also compared the frequencies of 22 SNP loci of 4 candidate genes between Tibetans and Han people in the plains and found 4 SNP loci in the KIF1B gene, 5 SNP loci in the ADRA1A gene, 2 SNPs in the SLC8A1 gene (rs4952404, rs13031392). In addition, we found that the allele frequencies of rs4952404 and rs13031392 in SLC8A1, rs7571879, rs6756452, rs3820729 and rs2278775 in PRKCE, and rs7842829 in ADRA1A increased with elevation. Quantitative trait association analysis revealed that rs3768753 locus in PRKCE gene was associated with stroke output (SV) and ejection fraction (EF), rs2594489 locus with hematocrit (HCT) and maximal vital capacity (MVC) in 241 Tibetans with phenotypic data. FVC, maximum expiratory flow (PEF), time vital capacity (FEV1), blood oxygen saturation (Sa O2) were correlated; rs2594494 locus was correlated with FEV1, FVC, PEF, HCT; rs6544852 locus was correlated with Sa O2, Ao, FEV1, FVC, PEF; rs6756452 locus was correlated with Sa O2; rs6557946, rs7842829, rs1048101 locus was correlated with ADRA1A; rs6557946, rs7842829, rs1048101 locus was correlated with Sa O2; The rs17034716 locus on the KIF1B gene was associated with Sa O2; the rs10492970 locus was associated with AO; the rs17034615 locus was associated with AO; the rs2297881 locus was associated with AO (P 0.05). The rs4952404 locus on the SLC8A1 gene was associated with Sa O2 (P 0.05). Conclusion 1. The body respiratory system, nervous system, circulatory system and digestive system were all affected to a certain extent. 2. The altitude adaptability of the Tibetans living in the world was better than that of the immigrants. The altitude maladjustment rate of the Tibetans living in the world was 34.9%, while that of the immigrants was 52.9%. PRKCE, SLC8A1, ADRA1A and KIF1B may be candidate genes related to hypoxic adaptation in Tibetans. 4. Six SNP loci (rs3768753, rs2594489, rs2594494, rs6544852, rs6756452, rs11898209) of PRKCE gene were significantly correlated with phenotypes (SV, EF, HCT, FVC, PEF, FEV1, Sa O2, Ao) in Tibetans at different altitudes. Sex (P 0.05), so PRKCE gene may play an important role in Tibetan hypoxic adaptation mechanism. 5. SLC8A1 gene rs4952404 and rs13031392 loci may be important candidate loci related to Tibetan hypoxic adaptation. 6. ADRA1A gene rs1442341, rs7842829 and rs11779546 loci may be important candidate loci related to Tibetan hypoxic adaptation. The rs17034716 locus of locus.7.KIF1B may be an important candidate for hypoxic adaptation in Tibetan.
【學(xué)位授予單位】：西藏大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R594.3

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