黑龙江地区人群HLA-A、B、DRB1高分辨等位基因及单体型多态性研究①

李　鑫　丁　镌　王　鑫　侯　玲　刘　颖　刘　杰

(哈尔滨市血液中心，哈尔滨150056)



黑龙江地区人群HLA-A、B、DRB1高分辨等位基因及单体型多态性研究①

李鑫丁镌王鑫侯玲刘颖②刘杰②

(哈尔滨市血液中心，哈尔滨150056)

[摘要]目的：研究中国造血干细胞捐献者资料库黑龙江分库无关捐献者HLA-A、B、DRB1高分辨等位基因和单体型频率分布特征。方法：采用PCR-测序分型技术(SBT)对13 670名随机、无血缘关系、健康的中华骨髓库黑龙江分库造血干细胞捐献者进行HLA-A、B、DRB1高分辨基因分型，利用计数法和最大似然性原理方法分别计算HLA-A、B、DRB1等位基因及单体型频率。结果：共观察到286个HLA等位基因，其中频率>0.1的A、B、DRB1座位特异性分别为A* 02∶01、A*24∶02、A*11∶01、DR*07∶01、DR*09∶01、DR*15∶01。1 087条A-B单体型中，22条单体型的频率高于0.01,267条单体型呈现显著的正连锁不平衡(ALD>0,HF≥1.09×10-4,χ2>3.84)，12条表现为强连锁不平衡(RLD>0.80)；1 329条B-DRB1单体型中，19条单体型的频率高于0.01,357条单体型呈现显著的正连锁不平衡，18条表现为强连锁不平衡(RLD>0.80)；4 428条A-B-DRB1单体型中，1 348条单体型有意义(频率≥1.09×10-4)，17条单体型频率高于0.005。结论：获得黑龙江地区人群HLA-A、B、DRB1高分辨等位基因频率和单体型分布数据及相关遗传参数，等位基因和单体型的分布特征接近北方汉族人群，但具有其自身分布特征。

HLA系统是迄今所知人类基因中多态性最复杂的遗传系统，其等位基因频率及其连锁不平衡特点在不同种族、民族、地域存在明显差异。其多态性及分布差异是自然选择造成的，它保证了种群能产生对各种病原体特异的免疫应答，以维持群体的稳定性。因此，一个群体高分辨水平的HLA多态性的研究对组织器官移植寻找合适供者、群体遗传学、人类学和法医学至关重要。目前有关用骨髓库样本检测HLA基因多态性在中国人群中的报道尽管已有很多，但大部分或是小样本数据调查，或是低分辨分型数据的研究。我们采用SBT分型技术对2009年至2014年13 670例中国干细胞库黑龙江地区自愿捐献者进行HLA-A、B、DRB1高分辨分型，得到了较为可靠、完整的大样本HLA群体分型数据，统计分析了HLA等位基因频率、单体型频率以及连锁不平衡结果等相关遗传参数资料，现报道如下。

1材料与方法

1.1材料

1.1.1标本来源13 670份标本全部来自2009年至2014年黑龙江地区健康、无血缘关系、18~55周岁造血干细胞捐献者。

1.1.2主要试剂和仪器TIANamp Blood DNA Kit血液基因组DNA提取试剂(美国 Tiangen公司)，PCR-SBT 基因分型试剂盒(德国ROSE公司)，9 700 PCR 扩增仪，Bio-Rad PCR 扩增仪，3730xl DNA Analyzer测序仪。

1.2实验方法外周静脉血样(EDTA抗凝)5 ml,应用TIANamp Blood DNA Kit血液基因组DNA提取试剂(美国 Tiangen公司)提取基因组DNA，检测DNA浓度在40~100 ng/μl，OD260/280比值为1.6~1.8。采用PCR-SBT方法对HLA-A、B、DRB1 进行常规测序分型，操作步骤严格按试剂说明书进行。其中对属于HLAⅠ类基因的A、B位点进行第2、3和4外显子双向测序，而对于HLAⅡ类基因的DRB1进行第2外显子双向测序.对无法确定高分辨分型结果的样本，则选择序列特异性测序引物(Sequence specific sequencing primer,SSSP)对该样本再次测序，获得相应的高分辨结果。测序反应纯化后的产物置于3730xl DNA Analyzer测序仪上进行电泳分析，导出的结果采用序列分析软件ATF-loader进行分析。

1.3统计学处理

1.3.1等位基因频率和单体型频率采用直接计数法和最大数学预期值算法(expectation-maximization,EM)通过Arlequin3.1软件分别计算等位基因频率和单体型频率，同时对各基因位点作Hardy-Weinberg平衡检验[1]。

1.3.2最小群体数量的估计在由n个个体组成的群体中，某单体型只出现一次，该单体型频率HP=1/2n，但并非所有频率≥1/2n的单体型都是可靠的。由此在随机人群中，如果要求有P的把握在群体中检测到该单体型，所需要的群体最少数量为N，n= log(1-P)/log(1-2 hP+HP2)。如果把P设置在95%区间，则n=-1.3010/log(1-2 hP+HP2)[2]。

1.3.3连锁不平衡参数计算两座位HLA单体型连锁不平衡参数的计算采用标准表格法，包括绝对△值(ALD)、最大△值(mlD)和相对△值(RLD)，计算公式：△ij=fij-fi×fj;△max=fij(△ij<0),△max=fi×(1-fj)(△ij≥0且fi

1.3.4频率分布的熵值(Entropy)计算在信息学理论中，频率分布的熵值是对包括多个子项的项目所含信息量的度量，具体到HLA领域，是对1个或1组紧密连锁的HLA基因座位多态性高低的度量[3]，计算公式：E=-∑fi×log2fi(fi为HLA基因或单体型的频率)。

2结果

2.1HLA-A、B、DRB1等位基因频率13670份标本共检测到HLA-A、B、DRB1基因座位上286种HLA编码基因(见表1)。HLA-A、B、DRB1基因座分别检出等位基因80、136和70个基因。A、B、DRB1基因座HW吻合度的检验结果均为P>0.05，表明本研究来源群体的基因分布符合Hardy-Weinberg平衡定律。各座位上等位基因频率的分布情况显示，E(B)>E(DRB1)>E(A)，表明HLA基因座位B、DRB1、A的多态性程度依次降低(见表2)。

2.2最小群体数量的估计在调查的群体样本中，有95%可信度的单体型频率≥1.09×10-4认为是可靠的，小于此数值的单体型频率，则需要在更大样本的群体资料中进行估计。

2.3HLA单体型频率分布情况见表3，观察到1087条HLA-A-B、1329条HLA-B-DRB1和4428条HLA-A-B-DRB1单体型，分别占单体型理论总数的9.99%(1 087/10 880)、13.96%(1 329/9 520)、0.58%(4 428/761 600)。4428条A-B-DRB1单体型中“可靠”的(频率≥1.09×10-4)有1 348条，占单体型总数的30.44%(1 348/4 428)，但累计频率高达85.17%，其中频率>0.001有158条，频率最高的10条HLA-A-B、B-DRB1和A-B-DRB1单体型及其频率数值见表4。

2.4连锁不平衡参数本组资料仅选取频率≥1.09×10-4的有意义单体型计算连锁不平衡参数。结果显示，分别有267条HLA-A-B单体型和357条HLA-B-DRB1单体型呈现显著的正连锁不平衡(ALD>0,HF≥1.09×10-4,χ2>3.84),其中A*02∶53N-B*27∶04、A*25∶01-B*18∶01等12条A-B单体型和B*42∶01-DR*13∶02、B*27∶24-DR*04∶05等18条B-DRB1单体型表现为强连锁不平衡(RLD>0.80)。见表5。表1黑龙江地区HLA-A、B、DRB1高分辨等位基因频率

Tab.1High resolution allele frequencies of HLA-A,B,DRB1 in population from Heilongjiang region

HLA-AGeneFrequecyHLA-BGeneFrequecyHLA-BGeneFrequecyHLA-DRB1GeneFrequecy02∶010.16550813∶020.09513527∶250.00021907∶010.14239224∶020.15032946∶010.07092251∶070.00021909∶010.14074611∶010.14312440∶010.05746253∶010.00021915∶010.13668630∶010.08953915∶010.05665756∶030.00021912∶020.07849333∶030.07867651∶010.05665739∶090.00018311∶010.05449902∶060.07183640∶060.05193944∶050.00018308∶030.05193902∶070.05775413∶010.04758655∶120.00014613∶020.04319701∶010.04890358∶010.04718415∶1980.00011003∶010.03939331∶010.04151444∶030.04019815∶680.00011004∶050.03924703∶010.03628452∶010.03675935∶040.00011015∶020.0339826∶010.02673748∶010.03511340∶110.00011014∶050.02717632∶010.01784954∶010.03120042∶020.00011012∶01∶01G0.02633502∶030.01554515∶110.02904251∶060.00011001∶010.01964268∶010.00716935∶01∶01G0.02699307∶100.00007304∶060.01938602∶050.00669340∶020.02509115∶100.00007313∶010.01806929∶010.0066207∶020.02275115∶190.00007316∶020.01572811∶020.00621815∶180.02110539∶310.00007310∶010.01547202∶100.00508457∶010.01799640∶780.00007304∶010.01269203∶020.00292615∶020.01682544∶270.00007304∶030.01225333∶010.00248738∶020.01667951∶220.00007314∶01∶01G0.01152223∶010.00234155∶020.01620351∶360.00007308∶020.00771824∶200.00226837∶010.01525273∶010.00007314∶030.00720624∶080.001539∶010.01422807∶480.00003711∶040.00636430∶040.00131735∶030.01393613∶160.00003714∶040.00625569∶010.00106167∶010.01075315∶090.00003704∶040.00592566∶010.00095150∶010.01027815∶150.00003701∶020.00303668∶020.00087851∶020.01013215∶1520.00003714∶070.00274324∶070.00076808∶010.00991215∶1780.00003704∶100.00259729∶020.00076815∶270.00888815∶340.00003704∶070.00234102∶53N0.00069544∶02∶01G0.00735215∶660.00003704∶080.00201224∶040.00058527∶040.00596215∶860.00003704∶020.00186511∶030.00054938∶010.00585227∶360.00003713∶120.00179226∶030.00051227∶050.00566927∶400.00003711∶060.0013930∶020.00043907∶05∶01G0.00563335∶110.00003715∶040.0013924∶030.00040214∶020.00398735∶120.00003708∶010.00102402∶480.00036615∶070.00369435∶140.00003714∶120.00087824∶100.00036640∶030.00354837∶020.00003716∶010.00084102∶090.00032918∶010.00292639∶040.00003708∶040.00080526∶020.00021915∶170.00281639∶060.00003713∶070.00076834∶010.00021956∶010.00245139∶240.00003713∶030.000695

(转下页)

(续表1)

HLA-AGeneFrequecyHLA-BGeneFrequecyHLA-BGeneFrequecyHLA-DRB1GeneFrequecy02∶110.00018335∶080.00230440∶1250.00003708∶090.00062225∶010.00018349∶010.00215840∶250.00003711∶030.00043902∶020.00014615∶050.00212140∶430.00003714∶020.00040202∶420.00014635∶020.00201240∶480.00003714∶180.00032968∶240.00014627∶070.00190240∶550.00003715∶060.00021902∶900.00011015∶250.00182940∶590.00003714∶060.00018324∶300.00011041∶010.00153640∶840.00003711∶280.00011030∶180.00011039∶050.00135346∶160.00003713∶500.00011074∶01∶01G0.00011045∶010.0012851∶040.00003715∶030.00011002∶170.00007315∶320.00124451∶090.00003703∶270.00007303∶080.00007359∶010.00106152∶110.00003711∶190.00007311∶060.00007348∶030.00102454∶160.00003713∶050.00007323∶260.00007355∶010.00102454∶170.00003714∶100.00007333∶080.00007314∶010.00095156∶150.00003714∶250.00007368∶380.00007315∶580.00084157∶020.00003714∶490.00007374∶030.00007340∶400.00080557∶030.00003703∶350.00003792∶890.00007355∶040.00080554∶160.00003704∶130.00003701∶030.00003735∶050.00076854∶170.00003711∶200.00003702∶040.00003751∶080.00073256∶150.00003711∶570.00003702∶1450.00003715∶460.00065857∶020.00003712∶030.00003702∶200.00003742∶010.00054957∶030.00003712∶050.00003702∶360.00003781∶01∶01G0.00054981∶010.00003712∶160.00003702∶930.00003715∶130.00051212∶200.00003711∶040.00003747∶010.00043913∶060.00003711∶190.00003755∶070.00043913∶130.00003711∶360.00003727∶020.00040213∶190.00003711∶390.00003781∶020.00040214∶220.00003711∶880.00003715∶120.00036614∶290.00003724∶1210.00003715∶350.00036615∶050.00003724∶170.00003715∶080.00032915∶310.00003724∶210.00003727∶240.00032915∶050.00003724∶280.00003715∶030.00029315∶310.00003724∶320.00003715∶290.00025624∶680.00003727∶060.00025626∶080.00003741∶020.00025626∶180.00003756∶040.00025626∶200.00003715∶210.00021929∶100.00003715∶380.00021932∶030.00003718∶020.00021974∶020.00003727∶030.000219

Note：HLA-A*74∶01∶01G includes 74∶01,74∶02；HLA-B*07∶05∶01G includes 07∶05，07∶06；HLA-B*35∶01∶01G includes 35∶01，35∶42；HLA-B*81∶01∶01G includes 81∶01，81∶02；HLA-B*44∶02∶01G includes 44∶02，44∶19N；HLA-DRB1*12∶01∶01G includes 12∶01，12∶06，12∶10，12∶17；HLA-DRB1*14∶01∶01G includes 14∶01，14∶54.

表2黑龙江地区HLA-A、B、DRB1基因频率分布

Tab.2Frequency distribution for the alleles of HLA-A，B，DRB1

Frequencyrange(×10-2)HLA-AnCumfre(%)HLA-BnCumfre(%)HLA-DRnCumfre(%)>10345.900341.981-101048.462789.411751.90.1-1124.57268.95155.37<0.1551.07831.64350.75Total8013670100EntropyE=3.74E=5.05E=4.19

表3黑龙江地区HLA-A-B、B-DRB1、A-B-DRB1单体型频率分布

Tab.3Haplotype frequency distribution of HLA-A,B,DRB1 in population from Heilongjiang region

Frequencyrange(×10-4)HLA-A-BnCumfre(%)HLA-B-DRB1nCumfre(%)HLA-A-B-DRB1nCumfre(%)100-10002241.161938.38614.4410-10013542.4714640.3715235.6<1093016.37116421.25427049.96Notobserved97930819107571720Total108809520761600EntropyE=7.55E=7.84E=9.86

表5高度连锁不平衡的HLA-A-B、HLA-B-DRB1单体型

Tab.5Positive linkage disequilibrium of HLA-A-B，B-DRB1 haplotypes

ABDRHF(×10-6)ALDmlDRLDχ202∶53N27∶040.0001100.0000940.0000941.0018.0125∶0118∶010.0001100.0001000.0001001.0030.5833∶0114∶020.0024510.0024410.0024770.9916537.0730∶0113∶020.0789720.0704540.0810210.8719337.8629∶0245∶010.0006580.0006340.0007440.85473.8230∶0414∶010.0008050.0008040.0009500.8514133.7429∶0107∶05∶01G0.0047170.0046800.0055960.8416254.6102∶4851∶010.0001830.0001830.0002190.8411419.5502∶4867∶010.0001830.0001830.0002190.8411419.5568∶0227∶030.0001830.0001830.0002190.844756.9202∶0550∶010.0055910.0055220.0066240.8312328.1534∶0115∶210.0001830.0001740.0002100.8398.2142∶0113∶020.0005490.0005250.0005251.00332.6427∶2404∶050.0003290.0003160.0003161.00220.2615∶2115∶020.0002190.0002190.0002191.0013635.8635∶0308∶010.0001100.0000950.0000951.0018.3640∶0208∶040.0001100.0001100.0001101.006848.3254∶0110∶010.0001100.0001070.0001071.0093.3915∶1312∶020.0005010.0004610.0004720.98156.8415∶3215∶040.0010970.0010950.0012420.8819017.4014∶0201∶020.002670.0026580.0030240.8816068.7437∶0108∶020.0001610.0001580.0001800.88249.0235∶0211∶040.0017550.0017420.0019990.876535.5415∶4615∶010.0005740.0004840.0005680.8582.5615∶1713∶020.0024060.0022840.0026940.851229.2681∶01∶01G15∶010.0004750.0004000.0004740.8467.5515∶2915∶010.0002190.0001840.0002210.8330.6508∶0103∶010.0082870.0078970.0095220.834590.6315∶3811∶010.0001830.0001710.0002070.8370.9113∶0207∶010.0808780.0673320.0815890.8311790.63

表4黑龙江地区10条最常见HLA-A-B、B-DRB1、A-B-DRB1单体型频率(×10-6)

Tab.4Major 10 HLA-A-B,B-DRB1,A-B-DRB1 haplotypes and their haplotype frequencies(×10-6)

HLA-A-BHFB-DRB1HFA-B-DRB1HFA*30∶01-B*13∶0278972B*13∶02-DR*07∶0180878A*30∶01-B*13∶02-DR*07∶0167302A*02∶07-B*46∶0140870B*46∶01-DR*09∶0134388A*02∶07-B*46∶01-DR*09∶0120284A*33∶03-B*58∶0136686B*13∶01-DR*12∶0230201A*33∶03-B*58∶01-DR*03∶0115901A*33∶03-B*44∶0324780B*52∶01-DR*15∶0221468A*02∶01-B*13∶01-DR*12∶0214796A*02∶01-B*13∶0118857B*40∶06-DR*09∶0120889A*33∶03-B*44∶03-DR*13∶0213491A*11∶01-B*40∶0116878B*58∶01-DR*03∶0120855A*33∶03-B*58∶01-DR*13∶0212696A*02∶01-B*15∶1116026B*15∶01-DR*15∶0117280A*02∶07-B*46∶01-DR*08∶039679A*24∶02-B*40∶0115502B*44∶03-DR*07∶0116892A*01∶01-B*57∶01-DR*07∶019612A*02∶01-B*15∶0115405B*44∶03-DR*13∶0215977A*01∶01-B*37∶01-DR*10∶018017A*24∶02-B*40∶0613122B*46∶01-DR*08∶0315954A*33∶03-B*44∶03-DR*07∶017483

3讨论

本研究在黑龙江地区人群中共检测到286个HLA-A、B、DRB1等位基因，进一步丰富了中国人群HLA等位基因的多态性。其中又以B座位的多态性最为丰富，不仅是因为B座位比A、DRB1座位存在数目更多的等位基因，还因为B座位中常见等位基因并非处于绝对优势地位(表2)，其>0.1的常见等位基因累计频率为0，明显

80个A位点等位基因、136个B位点等位基因和70个DR位点等位基因的编码基因，理论上可组成10880种A-B、9520种B-DRB1、761600种A-B-DRB1单体型，但其中有90%(9793/10880)的A-B、86.04%(8191/9520)的B-DRB1和99.42%(757172/761600)的A-B-DRB1单体型(表3)未观察到或频率<10-6，提示黑龙江地区人群中HLA-A,B,DRB1等位基因间存在连锁不平衡。这与A-B,B-DRB1和A-B-DRB1单体型的实际熵值低于理论熵值的计算结果(7.55<3.74+5.05,7.84<5.05+4.19,9.86<3.74+5.05+4.19)相符合。黑龙江地区人群中，分别有267条HLA-A-B单体型和357条HLA-B-DRB1单体型呈现显著的正连锁不平衡(ALD>0,HF≥1.09×10-4,χ2>3.84),其中A*02∶53N-B*27∶04、A*25∶01-B*18∶01等12条A-B单体型和B*42∶01-DR*13∶02、B*27∶24-DR*04∶05等18条B-DRB1单体型表现为强连锁不平衡(RLD>0.80)。在呈现强连锁不平衡的A-B、B-DRB1单体型中，属于常见单体型的仅为A*30∶01-B*13∶02和B*13∶02-DR*07:01(表5)。由此可见，高频单体型不一定连锁不平衡，频率较低的等位基因间也存在显著的连锁不平衡，如A*02∶53N-B*27∶04、B*42∶01-DR*13∶02等单体型，频率都<0.001，但在统计学角度上表现为强连锁不平衡，这是本研究调查样本量大的结果。

在黑龙江地区人群中观察到158条常见的HLA-A-B-DRB1单体型，其中频率最高的3条HLA-A-B-DRB1单体型是A*30∶01-B*13∶02-DR*07∶01、A*02∶07-B*46∶01-DR*09∶01、A*33∶03-B*58∶01-DR*03∶01，这与黑龙江地区汉族低分辨单体型研究结果一致[4]。黑龙江地区人群HLA-A-B-DRB1单体型与中国北方(北京、天津、石家庄、山东半岛)汉族人、南方(广东、上海、台湾)汉族人相比，北方、南方汉族人常见的单体型在本组中频率均>0.001[5-9]，而本组频率最高的10种HLA-A-B-DRB1单体型中仅A*01∶01-B*57∶01-DR*07∶01单体型在南方汉族人中频率<0.001 ，其余均>0.001，而且与山东半岛汉族人前10种HLA-A-B-DRB1单体型完全相同[6]；与亚洲其他地区相比，在韩国、越南人群中常见的HLA-A-B-DRB1单体型在本组中均有检出[10,11]。在韩国人群频率最高的10种HLA-A-B-DRB1单体型中有5种单体型与本组相同，这可能是同属黄种人且地域较接近的缘故，而其中A*30∶04-B*14∶01-DR*04∶04、A*02∶01-B*27∶05-DR*01∶01在本组中频率则<0.001，说明两者有各自的单体型分布特征。与越南人相比，在越南人群中频率最高的10种HLA-A-B-DRB1单体型中有4条单体型与本组相同，而有4种HLA-A-B-DRB1单体型在本组中频率则<0.001，说明与越南人的单体型分布特征差异增大；与西方人相比，德国人群中常见的A29∶02-B44∶03-DR07∶01、A02∶01-B15∶01-DR13∶01、美国人群中的A29∶02-B44∶03-DR07∶01单体型在本组中均未观察到[12,13]。通过以上可以看出随着地域、人种不同，HLA-A-B-DRB1单体型频率存在明显差异。

黑龙江省地处中国的最北端，频率较高的HLA单体型的分布符合中国北方汉族人群特征，与中国汉族人群有较大的相似性。分析HLA-A,B,DRB1等位基因多态性、单体型频率及连锁不平衡的特征可为估计捐献者资料的例数对患者查询的满足程度、与患者查询的匹配几率以及与移植成活或预后的关系等研究提供依据。

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[收稿2015-04-21]

(编辑许四平)

[关键词]HLA-A/B/DRB1；高分辨基因分型；基因/单体型频率

Polymorphism research of HLA-A,B,DRB1 high resolution alleles and haplotypes in population from Heilongjiang

LIXin,DINGJuan,WANGXin,HOULing,LIUYing，LIUJie.BloodCenterofHaerbin，HeilongjiangProvince,Harbin150056，China

[Abstract]Objective:To research the distribution features of HLA-A,B and DRB1 high resolution alleles and haplotypes in Heilongjiang population.Methods: PCR-SBT methods was applied for HLA-A,B,DRB1 high resolution genotyping of 13 670 unrelated and healthy donors in region of Heilongjiang,and haplotype frequencies were calculated by counting and maximum likelihood method.Results: A total of 286 HLA alleles were observed and the most frequent alleles(>0.1)were A* 02∶01，A*24∶02，A*11∶01，DR*07∶01，DR*09∶01 and DR*15∶01.Among 1 087 kinds of HLA-A-B haplotype ,there were 22 kinds frequency higher than 0.01,and 267 kinds with statistically significant and positive linkage disequilibrium(ALD>0,HF≥1.09×10-4,χ2>3.84).Moreover,among 1 329 kinds of HLA-B-DRB1 haplotype,there were 19 haplotypes frequency higher than 0.01,and 357 kinds with statistically significant and positive linkage disequilibrium.1 348 kinds of A-B-DR Haplotype were informative with frequency ≥1.66×10-4in 4 428 haplotypes,and a total of 17 kinds of A-B-DR haplotype frequency higher than 0.005.Conclusion: Get the distribution features of HLA high resolution allele and haplotype in Heilongjiang population,and associated genetic parameters,Distribution of alleles and haplotypes close to northern Han population,but have their own distribution.

[Key words]HLA-A/B/DRB1；High resolution genotyping；Gene/Haplotype frequency

作者简介：李鑫(1978年-)，女，硕士，主管技师，主要从事人类免疫遗传学研究，E-mail:xin53@163.com。

通讯作者②，E-mail:liuying54609@126.com；liujie1954@126.com。

中图分类号R457.1+1
①本文为黑龙江省卫生厅科研课题(No.2012-082)。

文献标志码A

文章编号1000-484X(2016)01-0083-07

doi:10.3969/j.issn.1000-484X.2016.01.018