李文华,李玉香,张新龙,郭宜娇
·论著·
西藏移居内地的藏族和汉族不适症状大学生血清蛋白表达分析
李文华,李玉香*,张新龙,郭宜娇
背景随着世居高原人群往返于高原与平原之间愈发频繁,高原低氧人群对平原富氧环境的适应逐渐引起人们的重视。目的基于血清蛋白组学对移居内地的藏、汉族不适症状大学生的血清蛋白表达进行分析,以期为西藏移居内地的藏、汉族大学生因适应不良而引起的疾病提供简单易行的、精确的早期诊断指标,为其临床治疗提供分子靶标。方法分别选取2013年10—11月、2014年10—11月、2015年10—11月西藏民族大学符合纳入标准的藏、汉族不适症状大学生32例为研究对象。按照研究对象民族,将其分为藏族组和汉族组,各16例。收集研究对象血清样本,采用弱阳离子磁珠(MB-WCX)捕获血清蛋白多肽,采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术进行质谱分析并比较两组血清蛋白表达水平。结果单样本质谱分析结果显示,3次重复取样,藏族组同一个体分子量为1 000~10 000 m/z的血清蛋白多肽曲线基本趋于一致,汉族组得出相似结果;藏族组与汉族组血清蛋白表达水平存在显著差异。整体样本质谱分析结果显示,藏族组与汉族组分子量为1 000~10 000 m/z的血清蛋白表达水平存在显著差异;对其蛋白多肽成分分析,则呈现出相互聚集、较少重合的现象;藏族组与汉族组分子量为905.65、1 062.10、1 078.11、2 138.79、2 313.40、4 094.67、4 172.89、4 213.96、5 910.71 m/z的血清蛋白表达峰值间存在显著差异(P<0.05)。结论西藏移居内地的藏、汉族不适症状大学生血清蛋白表达存在一定差异,其中分子量为4 094.67、2 138.79、905.65 m/z的血清蛋白表达差异较大,为进一步鉴定和定位差异性蛋白的序列和基因奠定了基础。
社会适应;蛋白质组学;质谱分析法;藏族;汉族;学生;西藏移居内地
李文华,李玉香,张新龙,等.西藏移居内地的藏族和汉族不适症状大学生血清蛋白表达分析[J].中国全科医学,2017,20(27):3372-3377.[www.chinagp.net]
LI W H,LI Y X,ZHANG X L,et al.Serum protein expression in Tibetan and Han Chinese college students with unwell symptoms after migration to the inland region of China from Tibet[J].Chinese General Practice,2017,20(27):3372-3377.
我国是一个高原较多的国家,海拔3 000 m以上的高原、高山地区占国土总面积的1/6,高海拔地区低压低氧等环境因素对进入此地区的低海拔人群的机体损害较大,这点已受到广泛关注及研究。随着西藏经济社会的发展和进步、改革开放的深入,西藏地区藏、汉族人群频繁地来往于内地和高原之间,而研究西藏地区藏、汉族人群移居到内地富氧地区的适应问题是摆在研究者面前亟待解决的问题[1]。尤其对移居内地富氧地区3年之内的藏、汉族人群如何平稳地适应富氧环境和血液中哪些血清蛋白的改变是其发生心脑血管疾病的预警因素等的研究具有很好的实际应用前景。因此,本研究基于血清蛋白组学对移居内地的藏、汉族不适症状大学生的血清蛋白表达水平进行分析,以期为西藏移居内地的藏、汉族大学生因适应不良而引起的疾病提供简单易行的、精确的早期诊断指标,为其临床治疗提供分子靶标。
1.1 血清收集 分别选取2013年10—11月、2014年10—11月、2015年10—11月西藏民族大学符合纳入标准的藏、汉族不适症状大学生32例为研究对象。纳入标准:体检新生;有嗜睡、头昏、心悸、乏力、胸闷、心前区隐痛、心律不齐、智力减退、低蛋白血症、心动过缓、心功能下降、肺动脉高压等不适症状。排除标准:体检正常。按照研究对象民族,将其分为藏族组和汉族组,各16例。采用真空采血管采集研究对象晨起空腹静脉全血5 ml,上下方向缓慢地震荡采血管5次,混匀血液中的凝结块,室温(25 ℃)垂直放置30 min,使血液凝结;1 500 r/min离心8 min(离心半径60 cm),取上清液(待检测血清样本)置于-80 ℃冰箱冻存待用。本研究对象均知情同意,本研究经西藏民族大学伦理委员会审批通过。
1.2 血清蛋白多肽捕获 首先,将冻存的血清样本放入冷藏温度为4 ℃的冰箱内解冻,然后运用弱阳离子磁珠(MB-WCX)捕获样本的血清蛋白多肽。具体操作步骤如下:(1)用混匀器完全混匀MB-WCX悬浮液1 min;(2)加10 μl MB-WCX结合液以及10 μl MB-WCX至PCR管,用德国Eppendorf移液枪混匀后加5 μl血清,用移液枪混匀至少5次,静置5 min;(3)将PCR管放入磁珠分离器,使MB-WCX贴壁1 min,液体清澈后弃上清液;(4)加100 μl MB-WCX冲洗液,在磁珠分离器上前后移动PCR管10次,MB-WCX贴壁后弃上清液,重复步骤(3)、(4)2次;(5)加5 μl MB-WCX洗脱液洗涤贴壁的MB-WCX,并反复吹打10次,MB-WCX贴壁2 min,将上清液移入干净的离心管;(6)加5 μl MB-WCX稳定液至离心管并混匀,收集处理好的蛋白样本用于质谱分析。
1.3 质谱分析 首先从藏族组和汉族组中各任意选取1例蛋白样本进行单样本质谱分析,然后选取所有样本进行整体样本质谱分析。将分离得到的蛋白样本与α-氰基-4-羟基肉桂酸混匀,取1 μl点在Anchorchip靶板上,每个样本分别点3个靶点以重复做3次;待室温干燥后将靶板放入基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)仪进行飞行时间质谱分析,采用Flexanalysis软件(一种专业分析蛋白质质谱图的软件系统,能够对单个个体蛋白的指纹进行描图)进行标准品校正后开始检测样本,每个样本要经过总共300次激光打靶(5次点靶,每次打靶2×30次)之后生成质谱图,获得由不同质核比(m/z)组成的蛋白多肽谱图。
1.4 统计学方法 采用ClinProTools 2.1软件进行统计学分析。结合遗传算法等生物统计学和生物信息学方法分析两组血清样本的蛋白多肽谱图。以P<0.05为差异有统计学意义。
2.1 单样本质谱分析结果 3次重复取样,藏族组同一个体分子量为1 000~10 000 m/z的血清蛋白多肽曲线基本趋于一致,汉族组得出相似结果(见图1、2);藏族组与汉族组血清蛋白表达水平存在显著差异(见图3)。
2.2 整体样本质谱分析结果 藏族组与汉族组分子量为1 000~10 000 m/z的血清蛋白表达水平存在显著差异(见图4,本文图4~6彩图见本刊官网www.chinagp.net电子期刊相应文章);对其蛋白多肽成分分析,则呈现出相互聚集、较少重合的现象(见图5、6)。藏族组与汉族组分子量为905.65、1 062.10、1 078.11、2 138.79、2 313.40、4 094.67、4 172.89、4 213.96、5 910.71 m/z的血清蛋白表达峰值间存在显著差异(P<0.05,见表1)。
在“世界屋脊”——青藏高原世代生活的藏族人群经过长期的自然选择已能很好地适应高原环境[2-6],其独特的遗传背景与高原环境适应能力值得进一步探讨[7-9]。目前关于藏族人群移居内地的研究,一方面是由于处于“醉氧状态”的平原藏族人群机体内会产生高活性的氧自由基和一些炎性因子导致机体器官如肝、肾、肺损害,尤其肺损害是脱适应症状表现最早和最明显的器官之一,主要表现为肺动脉干及其分支明显增粗[10-12];另一方面,移居内地高原脱适应人群体内的红细胞生成素相对于高原环境会有不同程度的降低,再加上水钠潴留造成血液稀释,可能与高原脱适应人群产生水肿、头晕等症状相关[13]。藏族人群出现脱适应症状的基因表达改变的研究并不多见,且血清蛋白组学目前主要应用于肿瘤标志物的筛选与诊断[14-18]。西藏移居内地人群机体会发生严重的生理生化变化,多反映在血清蛋白的组分变化上[19-24]。因此,本课题组将血清蛋白组学技术应用于高原医学研究,研究西藏移居内地藏、汉族大学生血清蛋白的组分变化。
图1 藏族组个体移居内地后的血清蛋白分布图
Figure1 Serum protein distribution of individuals migrating to the inland region of China from Tibet in Tibetan group
图2 汉族组个体移居内地后的个体血清蛋白分布图
Figure2 Serum protein distribution of individuals migrating to the inland region of China from Tibet in Han group
图3 藏族组与汉族组个体移居内地后的血清蛋白分布图比较
Figure3 Comparison of serum protein distribution of individuals migrating to the inland region of China from Tibet between Tibetan group and Han group
注:红色为藏族组,蓝色为汉族组
图4 藏族组与汉族组大学生移居内地后的血清蛋白分布图
Figure4 Serum protein distribution of college students migrating to the inland region of China from Tibet in Tibetan group and Han group
注:红色为藏族组,绿色为汉族组
图5 藏族组与汉族组大学生移居内地后的血清蛋白分布散点图
Figure5 Scatter plot of serum protein distribution of college students migrating to the inland region of China from Tibet in Tibetan group and Han group
表1 藏族组与汉族组不同分子量血清蛋白表达峰值比较
Table1 Comparison of the peak value of serum proteins with different molecular weights between Tibetan group and Han group
分子量(m/z)藏族组汉族组倍数P值 905.6511.075.781.92<0.0000011062.1055.6331.241.78<0.0000011078.1139.8721.681.84<0.0000012138.795.042.531.99<0.0000012313.406.203.911.59<0.0000014094.6713.696.542.090.0000214172.892.351.371.720.0006634213.9646.7627.831.680.0005685910.715.823.121.87<0.000001
注:红色为藏族组,绿色为汉族组
图6 藏族组与汉族组大学生移居内地后的血清蛋白分布三维立体图
Figure6 3D stereogram of serum protein distribution of college students migrating to the inland region of China from Tibet in Tibetan group and Han group
本研究单样本质谱分析结果显示,3次重复取样,藏族组同一个体分子量为1 000~10 000 m/z的血清蛋白多肽曲线基本趋于一致,汉族组得出相似结果,表明两组样本中血清蛋白的分布较为稳定,质谱仪器准确;藏族组与汉族组血清蛋白表达水平存在显著差异,说明移居内地后,藏族和汉族大学生的个体血清蛋白表达水平并不相同。整体样本质谱分析结果显示,藏族组与汉族组分子量为1 000~10 000 m/z的血清蛋白表达水平存在显著差异,对其蛋白多肽成分分析,则呈现出相互聚集、较少重合的现象,可知藏族组和汉族组血清蛋白在血清中呈现稳态。藏族组与汉族组分子量为905.65、1 062.10、1 078.11、2 138.79、2 313.40、4 094.67、4 172.89、4 213.96、5 910.71 m/z的血清蛋白表达峰值间存在显著差异,说明西藏藏族和汉族人群移居内地富氧地区后血清蛋白表达不同,生理适应状况不同。这些差异或许与藏族人群独特的基因背景、血液中高红细胞水平、长期的低氧应激以及平原相对较高的氧环境导致醉氧而引发氧自由基、炎性因子释放等有关[25-28],这些均需要进一步研究证实。
本研究的不足之处在于样本量较少,研究群体集中于大学新生人群,希望能够有机会扩大研究对象范围,找到本课题的相关差异蛋白,服务于临床。
综上所述,西藏移居内地的藏、汉族不适症状大学生血清蛋白表达水平存在一定差异,其中分子量为4 094.67、2 138.79、905.65 m/z的血清蛋白表达水平差异较大,为进一步鉴定和定位差异性蛋白的序列和基因奠定了基础。
作者贡献:李文华负责课题设计、数据分析、样本采集、论文修改;李玉香负责课题申报、试验开展、数据收集;张新龙、郭宜娇负责样本收集,进行试验操作,完成试验过程,撰写论文。
本文无利益冲突。
[1]李文华.高原分子医学[M].上海:复旦大学出版社,2011:12. LI W H.Plateau molecular medicine[M].Shanghai:Fudan University Press,2011:12.
[2]SIMONSON T S,MCCLAIN D A,JORDE L B,et al.Genetic determinants of Tibetan high-altitude adaptation[J].Hum Genet,2012,131(4):527-533.DOI:10.1007/s00439-011-1109-3.
[3]MASSCHELEIN E,PUYPE J,BROOS S,et al.A genetic predisposition score associates with reduced aerobic capacity in response to acute normobaric hypoxia in lowlanders[J].High Alt Med Biolm,2015,16(1):34-42.DOI:10.1089/ham.2014.1083.
[4]PENG Y,YANG Z,ZHANG H,et al.Genetic variations in Tibetan populations and high-altitude adaptation at the Himalayas[J].Mol Biol Evol,2011,28(2):1075-1081.DOI:10.1093/molbev/msq290.
[5]YI X,LIANG Y,HUERTA-SANCHEZ E,et al.Sequencing of 50 human exomes reveals adaptation to high altitude[J].Science,2010,329(5987):75-78.DOI:10.1126/science.1190371.
[6]CHEVIRON Z A,BRUMFIELD R T.Genomic insights into adaptation to high-altitude environments[J].Heredity,2012,108(4):354-361.DOI:10.1038/hdy.2011.85.
[7]BUROKER N E,NING X H,ZHOU Z N,et al.EPAS1 and EGLN1 associations with high altitude sickness in Han and Tibetan Chinese at the Qinghai-Tibetan Plateau[J].Blood Cells Mol Dis,2012,49(2):67-73.DOI:10.1016/j.bcmd.2012.04.004.
[8]GE R L,SIMONSON T S,COOKSEY R C,et al.Metabolic insight into mechanisms of high-altitude adaptation in Tibetans[J].Mol Genet Metab,2012,106(2):244-247.
[9]STOBDAN T,ZHOU D,AO-IEONG E,et al.Endothelin receptor B,a candidate gene from human studies at high altitude,improves cardiac tolerance to hypoxia in genetically engineered heterozygote mice[J].Proc Natl Acad Sci U S A,2015,112(33):10425-10430.DOI:10.1073/pnas.1507486112.
[10]BAKER M.Proteomics:the interaction map[J].Nature,2012,484(7393):271-275.DOI:10.1038/484271a.
[11]BENSIMON A,HECK A J,AEBERSOLD R.Mass spectrometry-based proteomics and network biology[J].Annu Rev Biochem,2012,81:379-405.DOI:10.1146/annurev-biochem-072909-100424.
[12]DOERR A.Mass spectrometry-based targeted proteomics[J].Nat Methods,2013,10(1):23.
[13]LI W H,HU Q Y,XU S L,et al.Assessments Hematology of De-adaptation to High Altitude Tibetans in Tibet[C]//2015 International Conference on Medicine and Biopharmaceutical.2016:251-256.DOI:10.1142/9789814719810_0031.
[14]YANG J,HU L,WU Q,et al.A terrified-sound stress induced proteomic changes in adult male rat hippocampus[J].Physiol Behav,2014,128:32-38.DOI:10.1016/j.physbeh.2014.01.038.
[15]YANG J,HU L,SONG T,et al.Proteomic changes in female rat hippocampus following exposure to a terrified sound stress[J].J Mol Neurosci,2014,53(2):158-165.DOI:10.1007/s12031-014-0242-6.
[16]LIU J,JIANG T,JIANG F,et al.Comparative proteomic analysis of serum diagnosis patterns of sputum smear-positive pulmonary tuberculosis based on magnetic bead separation and mass spectrometry analysis[J].Int J Clin Exp Med,2015,8(2):2077-2085.
[17]YANG J,YANG J,GAO Y,et al.Identification of potential serum proteomic biomarkers for clear cell renal cell carcinoma[J].PLoS One,2014,9(11):e111364.DOI:10.1371/journal.pone.0111364.
[18]YANG J,ZHU J,HE K,et al.Proteomic Profiling of Invasive Ductal Carcinoma(IDC) using Magnetic Beads-based Serum Fractionation and MALDI-TOF MS[J].J Clin Lab Anal,2015,29(4):321-327.DOI:10.1002/jcla.21773.
[19]AEBERSOLD R,MANN M.Mass spectrometry-based proteomics[J].Nature,2003,422(6928):198-207.DOI:10.1038/nature01511.
[20]WEBB-ROBERTSON B J,WIBERG H K,MATZKE M M,et al.Review,evaluation,and discussion of the challenges of missing value imputation for mass spectrometry-based label-free global proteomics[J].J Proteome Res,2015,14(5):1993-2001.DOI:10.1021/pr501138h.
[21]OKANO T,SEIKE M,KURIBAYASHI H,et al.Identification of haptoglobin peptide as a novel serum biomarker for lung squamous cell carcinoma by serum proteome and peptidome profiling[J].Int J Oncol,2016,48(3):945-952.DOI:10.3892/ijo.2016.3330.
[22]TSAI T H,SONG E,ZHU R,et al.LC-MS/MS-based serum proteomics for identification of candidate biomarkers for hepatocellular carcinoma[J].Proteomics,2015,15(13):2369-2381.DOI:10.1002/pmic.201400364.
[23]CRUTCHFIELD C A,THOMAS S N,SOKOLL L J,et al.Advances in mass spectrometry-based clinical biomarker discovery[J].Clin Proteomics,2016,13:1.DOI:10.1186/s12014-015-9102-9.
[24]CAMERINI S,MAURI P.The role of protein and peptide separation before mass spectrometry analysis in clinical proteomics[J].J Chromatogr A,2015,1381:1-12.DOI:10.1016/j.chroma.2014.12.035.
[25]VERRASTRO I,PASHA S,JENSEN K T,et al.Mass spectrometry-based methods for identifying oxidized proteins in disease:advances and challenges[J].Biomolecules,2015,5(2):378-411.DOI:10.3390/biom5020378.
[26]CARTER E A,MAYO J R,MACINNIS M J,et al.Individual susceptibility to high altitude and immersion pulmonary edema and pulmonary lymphatics[J].Aviat Space Environ Med,2014,85(1):9-14.
[27]FULCO C S,BEIDLEMAN B A,MUZA S R.Effectiveness of preacclimatization strategies for high-altitude exposure[J].Exercd Sport Sci Rev,2013,41(1):55-63.DOI:10.1097/JES.0b013e31825eaa33.
[28]RICHALET J P.Cardiopulmonary adaptation to high altitude[M]//OSTADAL B,DHALLA N S.Cardiac Adaptations.New York:Springer New York,2013:233-249.DOI:10.1007/978-1-4614-5203-4_12.
(本文编辑:崔丽红)
SerumProteinExpressioninTibetanandHanChineseCollegeStudentswithUnwellSymptomsafterMigrationtotheInlandRegionofChinafromTibet
LIWen-hua,LIYu-xiang*,ZHANGXin-long,GUOYi-jiao
SchoolofMedicine,XizangMinzuUniversity,Xianyang712082,China
*Correspondingauthor:LIYu-xiang,Associateprofessor;E-mail:xzmzxyli@163.com
BackgroundAs the people living in the plateau move back and forth between the plateau and the plain more and more frequently,the adaptation of the plateau hypoxia population to the oxygen enriched environment of the plain has gradually attracted wide attention.ObjectiveTo analyze the serum protein expression in Tibetan and Han Chinese college students with unwell symptoms who migrated to the inland region of China from Tibet based on serum proteomics,in order to provide an accurate and simple method for early diagnosis of the diseases due to maladjustment in such a population,and to offer molecular targets for the related clinical treatment.MethodsThe participants met the inclusion criteria were 32 Tibetan and Han college students〔Tibetan group(n=16) and Han group(n=16)〕 with unwell symptoms who enrolled in Xizang Minzu University during the periods of October to November 2013,October to November 2014 and October to November 2015.Serum samples were collected and the serum protein-derived polypeptide was captured by magnetic bead-based weak cation-exchange chromatography(MB-WCX).The expression of serum proteins was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) technique and compared between the two groups.ResultsMALDI-TOF-MS analysis of single sample found that,the polypeptide curves of serum protein with molecular weight of 1 000-10 000 m/z in the 3 samples from the same individual in the Tibetan group tended to be consistent,and similar results were obtained in the Han group;there were significant differences in serum protein levels between Tibetan group and Han group.The results of the MALDI-TOF-MS analysis of overall sample showed that there were significant differences in expression levels of serum protein with the molecular weight of 1 000-10 000 m/z between the Tibetan group and Han group;the analysis of its protein polypeptide components showed that a phenomenon of mutual aggregation and less overlap;there were significant differences in the peak expression of serum proteins with molecular weights of 905.65,1 062.10,1 078.11,2 138.79,2 313.40,4 094.67,4 172.89,4 213.96,5 910.71 m/z between Tibetan group and Han group(P<0.05).ConclusionThere are some differences in the expression of serum proteins between Tibetan and Han college students with unwell symptoms after migration to the inland region of China from Tibet,and the differences are significant in the expression of serum proteins with molecular weights of 4 094.67,2 138.79 and 905.65 m/z,which can be used as a basis for further identification and localization of the sequences and genes of differential proteins.
Social adjustment;Proteomics;Mass spectrometry;Zang nationality;Han nationality;Students;Migrating to the inland region of China from Tibet
国家自然科学基金资助项目(81360299);西藏自治区自然科学基金资助项目(2016ZR15-17)
R -05 R 394.3
A
10.3969/j.issn.1007-9572.2017.00.037
2017-03-11;
2017-08-04)
712082 陕西省咸阳市,西藏民族大学医学部
*通信作者:李玉香,副教授;E-mail:xzmzxyli@163.com