WO2018129887A1 - 与原发性胆汁性胆管炎关联的白细胞介素21及其应用 - Google Patents

与原发性胆汁性胆管炎关联的白细胞介素21及其应用 Download PDF

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WO2018129887A1
WO2018129887A1 PCT/CN2017/092504 CN2017092504W WO2018129887A1 WO 2018129887 A1 WO2018129887 A1 WO 2018129887A1 CN 2017092504 W CN2017092504 W CN 2017092504W WO 2018129887 A1 WO2018129887 A1 WO 2018129887A1
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pbc
primary biliary
biliary cholangitis
patients
allele
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刘向东
马雄
陈卫昌
史兴娟
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东南大学
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  • the present invention belongs to the field of immunology, and specifically relates to interleukin 21 (IL21) associated with primary biliary cholangitis and its use.
  • IL21 interleukin 21
  • PBC Primary biliary cholangitis
  • AMA PBC-specific anti-mitochondrial antibodies
  • the positive rate of AMA was found to be higher than 0.16% in middle-aged women. Medium is above 0.3% [5].
  • a positive rate of AMA (PDC-E2) was found to be higher than 0.05% in 8126 adults (18-83 years old) in Guangdong, and higher than 0.15% in middle-aged women.
  • a recent AMA screening of 19,012 residents in Shanghai found that 0.40% of men and 0.94% of women were positive for AMA, and 25 of them (0.13%) had PBC. With the aging of China's population, PBC will rapidly develop into a more common disease in China. It may reach 200,000 to 300,000 patients in 2025 and 35 to 500,000 patients by 2050.
  • Ursodeoxycholic acid is the only drug that effectively controls early PBC patients (especially those with no jaundice), but there is no cure, and the only way to save patients with advanced PBC is Liver transplantation is performed. China is a country with high incidence of viral hepatitis.
  • the clinical symptoms of PBC are similar to viral hepatitis.
  • PBC patients are often misdiagnosed as viral hepatitis or drug-induced hepatitis in the early stage, which seriously affects the diagnosis and treatment of patients with PBC. Therefore, early diagnosis and treatment of PBC patients has important social significance.
  • PBC has a strong genetic susceptibility, and the first-generation relatives of patients have a 100-fold higher incidence of PBC than the general population. According to reports from North America, Europe and Japan, the family incidence of PBC is 3.8-9%. In 2005, a large-scale survey in North America showed that the relative odds ratio of immediate family members of PBC patients was as high as 10.7. Analysis of identical twins in patients with PBC found a common incidence of 63%. In the past four years, our team has found three pairs of identical twin PBC patients in the survey of domestic PBC patients. The sister symptoms and onset time of the three pairs of patients are very close. Combined with international data, the identical pairs of PBC patients The common incidence rate of children 72.7%.
  • GWAS genome-wide association analysis
  • the North American PBC Cooperative Group which is a core member of the applicant, used GWAS and specific site high-density gene polymorphism analysis to conduct a cohort analysis of PBC patients and control populations in North America, confirming the genetic susceptibility of PBC and HLA-II antigens.
  • IL12A, IL12RB2, STAT4, IRF5, ch17q12-21, MMEL1 and SPIB are closely related, and it is revealed that abnormalities in IL12 signaling pathway and Toll-like receptor (TLR)/TNF signaling pathway may lead to PBC .
  • TLR Toll-like receptor
  • the first technical problem to be solved by the present invention is to provide an application of IL21 in the preparation of a product for detecting or assisting detection, screening or predicting primary biliary cholangitis.
  • the second technical problem to be solved by the present invention is to provide a series of single nucleotide polymorphism sites (SNPs) which are significantly associated with primary biliary cholangitis, and to find out which is associated with primary biliary cholangitis. Susceptibility correlates with the most prominent representative sites, thereby providing the use of said single nucleotide polymorphic sites (SNPs) for assessing susceptibility to primary biliary cholangitis.
  • SNPs single nucleotide polymorphism sites
  • a third technical problem to be solved by the present invention is to provide an application of IL21 in the preparation of an animal model of PBC.
  • a fourth technical problem to be solved by the present invention is to provide an application of IL21 in the preparation of a therapeutic drug for PBC.
  • the technical solution adopted by the present invention is:
  • the application of IL21 in preparing a product for detecting or assisting detection, screening or predicting primary biliary cholangitis is provided.
  • the genome-wide association analysis showed that the gene polymorphism of IL21 was closely related to the pathogenesis of PBC.
  • the second aspect provides a series of single nucleotide polymorphisms of IL21 that are significantly associated with susceptibility to primary biliary cholangitis.
  • the study was performed on 1122 Chinese Han PBC patients and 4036 normal subjects.
  • the HumanOmniZhongHua-8 (v1.1) scan was used to analyze the genome-wide association data, and the gene polymorphism (SNP) was classified.
  • SNP gene polymorphism
  • PLINK software was used to compare all the polymorphic loci. The frequency of multiple SNPs of the IL21 gene was significantly different between the PBC and the normal population.
  • the single nucleotide polymorphism of IL21 which is significantly associated with susceptibility to primary biliary cholangitis, is: rs17005934, allele is C/T; rs17005953, allele is T/C; rs28517551, allele Because A/G; rs925550, the allele is A/C; rs60318098, the allele is G/A; rs1026157, the allele is G/T; rs6811183, and the allele is T/G.
  • the present invention finds a linkage relationship between a plurality of SNPs and rs925550 and rs17005934 by performing linkage disequilibrium analysis on SNPs at the IL21 site. Sequenom's iPlex method was used to verify the rs925550 and rs17005934 polymorphism sites; and the Impute2 and PLINK software were used to perform the logistic regression principle. The gender was used as the covariate and analyzed by the additive effect model; similarly, significant differences were found. All of the SNPs in Table 1 are genetic polymorphic sites that are significantly different between PBC and control and can be used for risk prediction of PBC.
  • the present invention analyzes and evaluates the relationship between each SNP locus and primary biliary cholangitis by using PLINK software, logistic regression principle, and calculates the relative risk (ORd ratio, OR) of the dangerous allele and 95 The % confidence interval yields a dangerous allele for this series of SNP loci.
  • the risk alleles of the SNP locus of IL21 are: rs17005934 polymorphism is C; s17005953, polymorphism is T; rs28517551 polymorphism is A; rs925550 polymorphism is A; The rs60318098 polymorphism site is G; rs1026157, the polymorphism site is G; the rs6811183 polymorphism site is T.
  • the present invention specifically analyzes the presence of IL21 in 30 patients with PBC, 30 patients with autoimmune hepatitis (AIH), 25 patients with chronic hepatitis B (CHB), and 6 healthy controls (HC).
  • AIH autoimmune hepatitis
  • CHB chronic hepatitis B
  • HC healthy controls
  • the expression of IL21 protein was measured in liver tissue sections. It was found that IL21 was significantly increased in the liver tissue of PBC patients (P ⁇ 0.01). The expression level of IL21 was positively correlated with the degree of inflammation and fibrosis of liver tissue in PBC patients.
  • an animal model established in the present invention introduces IL21 expression to prepare a PBC animal model, and the model is used for screening of a PBC therapeutic drug.
  • the present invention has the following features and advantages:
  • the present invention finds for the first time that multiple polymorphisms of IL21 are significantly associated with the occurrence of PBC, and can be used as one of the indicators for predicting the risk of PBC.
  • IL21 is significantly elevated in the liver tissue of PBC patients and is useful for PBC targeted therapy. Target.
  • FIG. 1 is a correlation diagram of a single nucleotide polymorphism site of the IL21 gene region and PBC determined in Example 1.
  • the abscissa "Position on chr” is a position on a chromosome (unit: Mb);
  • the coordinates are the P value of the single nucleotide polymorphism site associated with PBC (-log10 (p-value)), the right Recombination rate is the recombination rate, the unit (CM/Mb); the representative site rs925550 to ⁇ Said.
  • Figure 2 is a histochemical analysis of IL21 in 30 patients with PBC, 30 patients with autoimmune hepatitis (AIH), 25 patients with chronic hepatitis B (CHB), and 6 healthy controls (HC).
  • Expression in. 2A is the expression of IL21 in a liver tissue section of a patient with PBC in Example 3.
  • 2B is the expression of IL21 in the liver tissue section of a patient with AIH in Example 3.
  • 2C is the expression of IL21 in liver tissue sections of a patient with CHB in Example 3.
  • 2D is the expression of IL21 in a liver tissue section of an HC tissue in Example 3.
  • FIG. 2E is a statistical analysis of the expression of IL21 in liver tissue sections of 30 patients with PBC, 30 patients with AIH, 25 patients with chronic hepatitis B (CHB), and 6 healthy controls (HC) in Example 3 ( ** is P ⁇ 0.01 and * is P ⁇ 0.05).
  • Fig. 2F is a result of analysis of the correlation between the expression of IL21 in liver tissue sections of 30 PBC patients and the degree of inflammation of liver tissue in PBC patients in Example 3.
  • Fig. 2G is a result of analysis of the correlation between the expression of IL21 in liver tissue sections of 30 PBC patients and the degree of liver tissue fibrosis in PBC patients in Example 3.
  • the HumanOmniZhongHua-8 (v1.1) analysis kit manufactured by Illumina Co., Ltd. includes a chip and a reagent.
  • Phosphate buffer 8 g NaCl, 0.2 g KCl, 0.24 g KH 2 PO 4 , 3.628 g Na 2 HPO 4 ⁇ 12H 2 O, double distilled water to 1 L;
  • Tris-HCl buffer pH 8.0: 30.3g Tris base dissolved in 200ml of double distilled water, hydrochloric acid to adjust the pH to 8.0, to a volume of 250ml;
  • nuclear lysis buffer (Nuclei Lysis Buffer, 500ml): 40ml 5M NaCl, 2ml 0.5M EDTA (pH 8.0), 5ml 1M Tris-HCl (pH 8.0), 453ml double distilled water;
  • Proteinase K buffer (Proteinase K buffer, 100ml): 50ml glycerol, 100ul 1M Tris-HCl (pH 7.5), 200 ⁇ l 1M CaCl 2 , 47ml double distilled water;
  • Proteinase K (Proteinase K Solution, 10mg / ml): 100mg proteinase K powder dissolved in 10ml proteinase K buffer;
  • TE buffer 1ml 1M Tris-HCl buffer (pH 8.0), 0.2ml 0.5M EDTA (pH 8.0), double distilled water to 100ml;
  • TAE buffer 242g Tris-base, 57.1ml glacial acetic acid, 200ml 0.5M EDTA (pH 8.0), double distilled water to 1L.
  • the present invention discovers the association of the IL21 gene locus with primary biliary cholangitis by the following methods and procedures.
  • Non-anticoagulant therapy of patients and normal subjects was collected by non-anticoagulative collection tubes from various hospitals.
  • the collection tube was centrifuged to separate serum and blood clots.
  • the serum of the upper layer of the thawed blood sample was gently mixed with a pipette in a clean bench, and each was placed in four 1.5 ml screw cap storage tubes and stored at -80 °C.
  • the HumanOmniZhongHua-8 (v1.1) chip was purchased from ILLUMINA, and the classification was carried out in accordance with the requirements of the product method. The exact steps are based on the experimental steps of the Illumina HumanOmniZhongHua-8 (v1.1) chip. The relevant description can be found in the published literature [Adler, AJ, Wiley, GB, Gaffney, PM Infinium Assay for Large-scale SNP Genotyping Applications .J.Vis.Exp. (81), e50683, doi: 10.3791/50683 (2013).]. Each DNA sample was treated at 200 nanograms (ng) in strict accordance with the experimental procedure described in the Illumina HumanOmniZhongHua-8 (v1.1) chip.
  • the HumanOmniZhongHua-8 (v1.1) chip typing data was processed and output by the BeylStudio software of ILLUMINA, and the associated data analysis was performed according to the published literature [Zuo, X. et al. Whole-exome SNP array identification 15 new susceptibility loci for psoriasis.Nat Commun 6,6793,2015].
  • the classification data output by BeadStudio is analyzed by PLINK, and the duplicate and affinity samples are excluded according to the pairwise identity-by-state. Using smartpca software, the heterogeneous samples were excluded according to the principle component analysis. The results of 1,122 PBC patients and 4,036 normal controls were used for statistical analysis.
  • SNPs with a success rate of less than 98% exclude SNPs with a minimum allele frequency (MAF) of less than 0.01 in the analysis sample, and exclude Hardy–Weinberg equilibrium values of P ⁇ 1 ⁇ 10 -4 in normal control samples. SNP. After exclusion, a total of 776,516 autosomal SNPs were used for comparison.
  • PLINK software SNP correlation analysis uses the principle of linear regression, using gender as the covariate, using the additive allelic effect, assessing the significance of each SNP locus associated with PBC, and calculating the dangerous alleles.
  • the relative risk of the gene (Odds ratio, OR) and the 95% confidence interval, the OR value, confidence interval and P value were obtained directly in the analysis, thereby obtaining the dangerous allele of the site.
  • the significance level (P value) associated with each PBC in the SNP chip was calculated by genome-wide association (GWAS) analysis, and the chromosomal region with potential association with PBC was found according to the significance level (P value less than 0.0001). And obtain a single nucleotide polymorphism site and its allele that are significantly associated with PBC susceptibility.
  • the SNP calculation (Imputation) is based on the results of the Han people in the thousand human genome project (Reference), using IMPUTE2 software, combined with the results of 1,122PBC patients and 4,036 normal controls.
  • the series of single nucleotide polymorphisms and their alleles are: rs17005934, allele is C/T; rs17005953, allele is T/C; rs28517551, allele is A/G; rs925550 The allele is A/C; rs60318098, the allele is G/A; rs1026157, the allele is G/T; rs6811183, the allele is T/G; the dangerous alleles are: rs17005934 polymorphism
  • the sexual site is C; the rs17005953 polymorphism is T; the rs28517551 polymorphism is A; the rs925550 polymorphism is A; the rs60318098 polymorphism is G; the rs1026157 polymorphism is G
  • the rs6811183 polymorphism site is T. Individuals carrying these dangerous alleles had a relative risk of developing PBC of 1.
  • OR (95% CI) indicates the relative risk of developing PBC in individuals carrying disease-related haploids compared to individuals carrying normal haploids.
  • the chromosomal location is referred to Human Genome GRCh37/hg19.
  • PCR amplification primers and single base extension primers for the SNP site to be tested were designed using Sequenom Genothyping Tools and MassARRAY Assay Design software and synthesized by Integrated DNA Technologies.
  • Primer 1 sequence ACGTTGGATGAAAGTGCAGACCAGGAAAAC;
  • Primer 2 sequence ACGTTGGATGAAAGGGTGACTTCAGGGTAG;
  • Primer 1 sequence ACGTTGGATGGGGAGGGTAATTAAAATTAG;
  • Primer 2 sequence ACGTTGGATGGGGCAAATGAAGCCAATATA
  • PCR amplification was performed using a multiplex PCR technique in a 384-well plate with a total volume of 5 ⁇ l per reaction system.
  • a PCR master mix solution was prepared in a new 1.5 ml EP tube.
  • the 384-well plate is a PCR reaction plate. Take out the prepared DNA sample 384-well plate, adjust the sample volume to 1 ⁇ L using a 24-channel sampler, and each template solution contains 20-50 ng of template DNA, Hotstar Taq 0.5U, 0.5 pmol of each amplification primer. , 0.1 ⁇ l of 25 mM dNTPs.
  • the PCR reaction conditions were set on a 384-well compatible PCR machine: 94 ° C for 4 minutes; 94 ° C for 20 seconds, 56 ° C for 30 seconds, 72 ° C for 1 minute, 45 cycles; 72 ° C for 3 minutes; 4 ° C retention.
  • a 384-well PCR reaction plate was placed on a PCR machine to initiate a PCR reaction.
  • the PCR product was treated with SAP (shrimp alkaline phosphatase) to remove free dNTPs from the system.
  • the single base extension reaction system contained 7 ⁇ l of the SAP-treated PCR product and 2 ⁇ l of the EXTEND Mix solution (wherein each of the extension reaction primer mixtures was 0.94 ⁇ l, the iPLEX enzyme was 0.041 ⁇ l, and the extension mixture was 0.2 ⁇ l).
  • the PCR machine was started to perform a single base extension reaction.
  • MassARRAY Nanodispenser RS1000 spotter was started and the resin extended extension was transferred to a 384 well SpectroCHIP chip.
  • the spotted SpectroCHIP chip was analyzed using MALDI-TOF (matrix-assisted laser desorption/ionization–time of fligh), and the results were classified using TYPER 4.0 software (sequenom) and the results were output. . A total of 907 PBC samples and 2127 control samples gave valid results.
  • the results of 907 PBC samples and 2127 control samples were statistically analyzed by gender as the common variable.
  • the disease-related haploid (G) frequency of rs17005934 was significantly higher in the PBC (0.428) than in the control population (0.362), with a p-value of 1.25 ⁇ 10 -6 .
  • the disease-related haploid (A) frequency of rs925550 was significantly higher in the PBC (0.427) than in the control population (0.353), with a p-value of 7.44 ⁇ 10 -8 .
  • the GWAS results of 1,122 PBC patients and 4,036 normal controls were superimposed with the results of 907 PBC samples and 2127 control samples.
  • the principle of fixed-effect model (I2 ⁇ 25%) was used.
  • the statistical analysis included 2029 PBCs. Sample and 6163 control samples.
  • the results showed that the disease-related haploid (G) frequency of rs17005934 was significantly higher in PBC than in the control group, with a p-value of 1.06 ⁇ 10 -11 and an odds ratio (96% CI) of 1.29 (1.21- 1.39);
  • the disease-related haploid (A) frequency of rs925550 was significantly higher in PBC than in the control population, with a p-value of 3.87 ⁇ 10 -13 and a relative risk value (96% CI) of 1.31 (1.21-1.40) (see table 3).
  • Table 3 Results of IL21 polymorphic loci rs17005934 and rs925550 in genome-wide association analysis and validation findings
  • liver tissues including 30 patients with PBC, 30 patients with autoimmune hepatitis (AIH), and 25 patients with chronic hepatitis B (CHB) were ultrasound-guided liver biopsy at Shanghai Renji Hospital.
  • the liver tissue of 6 healthy controls (HC) was from a liver transplant donor. Liver tissues were preserved in formalin and embedded in paraffin. The degree of inflammation and fibrosis of liver tissue was assessed according to the "Scheuer" scoring system.
  • Liver tissue sections were soaked in sodium citrate buffer for 20 minutes;
  • IL21 expression was significantly elevated in liver tissues of patients with PBC compared with hepatitis B, autoimmune hepatitis and normal liver tissue.
  • IL21-positive cells are inflammatory hepatocytes and infiltrating inflammatory lymphocytes surrounding the portal area of the liver.
  • the experimental results show that the number of IL21 positive cells is proportional to the degree of inflammation in the liver and the degree of fibrosis in the liver.

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Abstract

白细胞介素21(IL21)在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎(Primary Biliary Cholangitis,PBC)的产品中的应用。与原发性胆汁性胆管炎易感性显著相关的白细胞介素21的单核苷酸多态位点为：rs17005934、rs17005953、rs28517551、rs925550、rs60318098、rs1026157、rs6811183。IL21的多个基因多态性位点与PBC的发生显著相关，可用作PBC发病风险预测的指标之一。

Description

与原发性胆汁性胆管炎关联的白细胞介素21及其应用 技术领域
本发明属于免疫学领域，具体涉及与原发性胆汁性胆管炎关联的白细胞介素21(IL21)及其应用。
背景技术
原发性胆汁性胆管炎(primary biliary cholangitis,PBC)，旧称原发性胆汁性肝硬化(primary biliary cirrhosis)，是一种累及肝内胆管系统的慢性进展性自身免疫性疾病，主要表现为肝内小胆管进行性破坏伴门脉炎症性改变，最终导致纤维化及肝硬化。本病主要发生于中年以上女性，男性病例仅占10％。随着对PBC疾病认识的不断提高和诊断方法的建立，PBC的发病率和患病率在全球呈上升趋势。2003年，在上海地区对5011个健康体检者的(26-85岁)PBC特异性抗线粒体抗体(AMA)(PDC-E2)进行筛查，发现AMA阳性率高于0.16％，在中年女性中高于0.3％[5]。2006年，在广东地区对8126个成年人(18-83岁)的普查中发现AMA(PDC-E2)的阳性率高于0.05％，在中年女性中高于0.15％。最近在上海地区对19012个居民的AMA筛查结果发现0.40％的男性和0.94％的女性AMA阳性，其中25人(0.13％)患有PBC。随着我国人口老龄化的加剧，PBC在我国将迅速发展成一种较常见的疾病，在2025年可能达到20-30万患者，到2050年可能高达35-50万患者。
早期诊断和治疗是控制PBC疾病发展的最有效手段。目前对PBC的治疗缺少有效的办法，熊去氧胆酸是唯一对早期PBC患者(尤其是黄疸没有出现的患者)进行有效控制的药物，但无治愈作用，而挽救晚期PBC患者的唯一手段是实施肝脏移植。我国是病毒性肝炎高发的国家，PBC的临床症状与病毒性肝炎相似，PBC患者早期常被误诊为病毒性肝炎或药物性肝炎，严重影响了PBC患者的诊疗。因此，对PBC患者进行早期诊断和治疗，具有重要的社会意义。
PBC具有很强的遗传易感性，患者的第一代亲属的PBC发病率比一般人群高100倍。综合北美、欧洲和日本的报道，PBC的家族发病率为3.8-9％。2005年，一项在北美的大规模调查结果显示，PBC病人直系亲属发病的相对危险度(odds ratio)高达10.7。通过PBC患者的同卵双生子进行分析，发现其共同发病率达63％。我们团队在过去四年对国内PBC患者的调查中发现了3对同卵双生的PBC患者，三对患者的姐妹发病症状和发病时间都非常接近，结合国际已有数据，PBC患者的同卵双生子共同发病率高达 72.7％。
自2009年以来，对白人PBC患者全基因组关联分析(GWAS)的一系列结果显示PBC具有很强的遗传易感性。以申请人为核心成员的北美PBC合作组采用GWAS和特定位点高密度基因多态性分析的方法，对北美PBC患者和对照人群进行队列分析，证实了PBC的遗传易感性与HLA-II类抗原、IL12A、IL12RB2、STAT4、IRF5、ch17q12-21、MMEL1和SPIB等位点密切相关，而且揭示了IL12信号传导通路和Toll-like受体(TLR)/TNF信号传导通路的异常可能导致PBC的发生。日本学者的小规模的GWAS研究也发现了TNFSF15位点与PBC密切相关。我国对PBC的遗传学研究已有一定的基础，国内的学者分别就HLA和CTLA4等基因位点与汉族PBC遗传易感性进行了初步研究。
我们启动并完成了对1122例汉族PBC样品的Illumina中华芯片扫描工作，结合4046例对照数据，进行了大规模的队列分析，在国际上首次发现并证实了IL21的基因位点达到了GWAS显著性，并首次发现IL21在PBC患者血液中异常高表达，其表达程度与肝脏组织纤维化程度呈正比。
目前国际上尚无专利文献提及IL21基因位点以及IL21与PBC的关系。
发明内容
发明目的：本发明所要解决的第一个技术问题是提供了IL21在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎的产品中的应用。
本发明所要解决的第二个技术问题是提供了一系列与原发性胆汁性胆管炎显著相关的单核苷酸多态位点(SNP)，并找出其中与原发性胆汁性胆管炎易感性关联最显著的代表性位点，从而提供将所述的单核苷酸多态位点(SNP)用于评估原发性胆汁性胆管炎易感性的用途。
本发明所要解决的第三个技术问题是提供了IL21在制备PBC动物模型中的应用。
本发明所要解决的第四个技术问题是提供了IL21在制备PBC治疗药物方面的应用。
技术方案：为了解决上述技术问题，本发明所采用的技术方案为：第一方面，提供了IL21在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎的产品中的应用。我们启动并完成了对1122例汉族PBC样品的Illumina中华芯片扫描工作，结合4036例对照数据，进行了大规模的队列分析，在国际上首次发现并证实了IL21的基因位点达到了GWAS显著性，并首次发现IL21在PBC患者肝脏组织中表达程度与肝脏组织纤维化程度呈正比；具体通过全基因组关联分析，发现IL21的基因多态性与PBC发病有密切关系。
第二方面提供了一系列与原发性胆汁性胆管炎易感性显著相关的IL21的单核苷酸多态位点，通过对1122例中国汉族PBC患者样品和4036例正常人样品为研究对象，使用了HumanOmniZhongHua-8(v1.1)扫描进行全基因组关联数据分析，进行基因多态性(SNP)的分型，利用芯片得到的数据，采用PLINK软件对所有多态位点的对比分析，发现IL21基因的多个SNP的频数在PBC和正常人群中有显著差异。与原发性胆汁性胆管炎易感性显著相关的IL21的单核苷酸多态位点为：rs17005934，等位基因为C/T；rs17005953，等位基因为T/C；rs28517551，等位基因为A/G；rs925550，等位基因为A/C；rs60318098，等位基因为G/A；rs1026157，等位基因为G/T；rs6811183，等位基因为T/G。
第三方面，本发明通过对IL21位点的SNP进行连锁不平衡分析，发现多个SNP与rs925550，rs17005934的连锁关系。采用Sequenom的iPlex方法，针对其中rs925550，rs17005934多态性位点进行验证试验；并使用Impute2和PLINK软件，逻辑回归原理进行，以性别为共变量，按叠加效应模型分析；同样发现有显著差异；表1中的所有SNP均为在PBC和对照间有显著差别的基因多态位点，可用于对PBC的风险预测。
第四方面，本发明通过使用PLINK软件，逻辑回归原理进行分析评估每个SNP位点与原发性胆汁性胆管炎的关系，计算危险等位基因的相对危险度(Odd ratio，OR)和95％的可信区间，得到这一系列SNP位点的危险等位基因。IL21的SNP位点的危险等位基因分别是：rs17005934多态性位点为C；s17005953，多态性位点为T；rs28517551多态性位点为A；rs925550多态性位点为A；rs60318098多态性位点为G；rs1026157，多态性位点为G；rs6811183多态性位点为T。
第五方面，本发明通过对IL21特异的组织化学分析对30例PBC患者，30例自身免疫肝炎患者(AIH)，25例慢性乙型肝炎患者(CHB)，和6例健康对照(HC)的肝脏组织切片进行IL21蛋白的表达进行测定，发现IL21在PBC患者肝脏组织中显著升高(P<0.01)，IL21的表达水平与PBC患者肝脏组织的炎症程度和纤维化程度呈正相关。
第六方面，本发明建立的动物模型中引入IL21表达制备PBC动物模型，并将此模型用于PBC治疗药物的筛选。
有益效果：本发明具有如下的特色和优点：
1)本发明首次发现IL21的多个基因多态性位点与PBC的发生显著相关，可用于PBC发病风险预测的指标之一。
2)本发明发现IL21在PBC患者肝脏组织中显著升高，是可用于PBC靶向治疗的 靶点。
3)发明的发现有可能通过在动物模型中引入慢性IL21表达制备PBC动物模型，并将此模型用于PBC治疗药物的筛选。
附图说明
图1为实施例1中所确定的IL21基因区域单核苷酸多态性位点与PBC的关联图；图1中：横坐标“Position on chr”为染色体上的位置(单位Mb)；纵坐标为单核苷酸多态性位点与PBC的关联显著性P值(-log10(p-value))，右侧Recombination rate为重组率，单位(CM/Mb)；代表位点rs925550以◆表示。
图2为实施例3中组织化学分析IL21在30例PBC患者，30例自身免疫肝炎(AIH)患者，25例慢性乙型肝炎(CHB)患者，和6例健康对照(HC)的肝脏组织切片中的表达。图2A为实施例3中在1例PBC患者组织肝脏组织切片的IL21的表达。图2B为实施例3中在1例AIH患者组织肝脏组织切片的IL21的表达。图2C为实施例3中在1例CHB患者组织肝脏组织切片的IL21的表达。图2D为实施例3中在1例HC组织肝脏组织切片的IL21的表达。图2E为实施例3中IL21在30例PBC患者，30例AIH患者，25例慢性乙型肝炎(CHB)患者，和6例健康对照(HC)的肝脏组织切片中的表达的统计分析结果(**为P<0.01，*为P<0.05)。图2F为实施例3中IL21在30例PBC患者的肝脏组织切片中的表达与PBC患者的肝脏组织炎症程度相关性的分析结果。图2G为实施例3中IL21在30例PBC患者的肝脏组织切片中的表达与PBC患者的肝脏组织纤维化程度相关性的分析结果。
具体实施方式
下面通过具体的实施例和附图对本发明进一步说明。
本发明实施例中所用实验材料、主要试剂及配方如下：
主要实验材料和主要试剂：
1、原发性胆汁性胆管炎患者和正常对照人群的DNA样品和血清；
2、全基因组关联分析芯片分析试剂盒：由Illumina公司生产的HumanOmniZhongHua-8(v1.1)分析试剂盒包括芯片和试剂。
主要溶液配制：
1、磷酸缓冲液(PBS，1L)：8g NaCl，0.2g KCl，0.24g KH2PO4，3.628g Na2HPO4·12H2O，双蒸水定容至1L；
2、饱和氯化钠溶液(5M NaCl，1L)：292.5g NaCl，双蒸水溶解定容至1L；
3、1M Tris-HCl缓冲液(pH 8.0)：30.3g Tris base溶于200ml双蒸水中，盐酸调节pH值至8.0，定容至250ml；
4、0.5M EDTA(PH 8.0)：36.5g EDTA溶于NaOH溶液中，调节pH至8.0，定容至250ml；
5、核裂解缓冲液(Nuclei Lysis Buffer，500ml)：40ml 5M NaCl，2ml 0.5M EDTA(PH 8.0)，5ml 1M Tris-HCl(pH 8.0)，453ml双蒸水；
6、蛋白酶K缓冲液(Proteinase K buffer，100ml)：50ml glycerol，100ul 1M Tris-HCl(pH 7.5)，200μl 1M CaCl2，47ml双蒸水；
7、蛋白酶K(Proteinase K Solution，10mg/ml)：100mg蛋白酶K粉末溶于10ml蛋白酶K缓冲液中；
8、消化液(Nuclei-SDS-Proteinase K-Master Mix Lysis buffer，504ml)：420ml Nuclei Lysis Buffer，3.5ml Proteinase K(10mg/ml)，17.5ml 10％SDS，0.35ml 0.5M EDTA(PH 8.0)，62.65ml双蒸水；
9、TE缓冲液(PH 8.0)：1ml 1M Tris-HCl缓冲液(pH 8.0)，0.2ml 0.5M EDTA(PH 8.0)，双蒸水定容至100ml；
10、6×DNA点样缓冲液(10ml):88mg EDTA，5mg溴酚蓝，5mg二甲苯青，溶于4ml双蒸水中，再加入3.6ml甘油后使用NaOH调节至PH7.0，定容至10ml；
11、50×TAE缓冲液：242g Tris-base，57.1ml冰乙酸，200ml 0.5M EDTA(PH 8.0)，双蒸水定容至1L。
实施例1全基因组关联分析
本发明通过下述方法和步骤发现IL21基因位点与原发性胆汁性胆管炎的关联。
一、方法
首先在中国汉族人群中选取了1122例中国汉族PBC患者样品和4036例正常人样品为研究对象，进行HumanOmniZhongHua-8(v1.1)扫描，进行基因多态性(SNP)的分型，采用PLINK软件对所有多态位点的对比分析，发现IL21基因的多个SNP的频数在PBC和正常人群中有显著差异。
二、病例和对照样本入选标准
所有纳入研究的PBC患者均符合以下标准：1)、所有患者均为汉族；2)、AMA-M2血清学检测呈阳性；3)、生化指标显示胆汁淤积证据，主要表现为ALP，GGT升高；5)、 各类肝炎病毒感染以及人类免疫缺陷病毒感染指标阴性(乙肝病毒表面抗原阴性、血清中乙肝病毒DNA小于200拷贝数/毫升、血清中丙肝病毒RNA小于500拷贝数/毫升、戊肝抗原阴性、人类免疫缺陷病毒抗体阴性)；6)、无血吸虫感染史；7)病人主诉饮酒量小于100ml/日。
全基因组扫描对照人群样品来自安徽，江苏，山东的正常体检人群，生化指标正常，无临床疾病表征，自述体健。病例是22～85岁之间筛选，平均年龄为54.7岁；对照为15岁到96岁之间筛选，平均年龄为34.8岁。
三、测试人群的基本特征
测试人群为芯片检测样本其基本特征如下：
表1
Figure PCTCN2017092504-appb-000001
四、原发性胆汁性胆管炎(PBC)患者和正常对照人群的基因组DNA样品的制备
1、病人和正常人的非抗凝血由各医院采集在非抗凝血收集管中。将收集管离心，分离血清和血凝块。在超净工作台内将解冻的血样上层的血清用移液器轻轻混匀，均分装入4个1.5ml螺盖保存管，保存于-80℃。
2、用长柄勺取出分离胶，将血块倒入称量舟，用剪刀将血块充分剪碎，再用塑料吸管将碎血块转移至50ml离心管中。
3、用PBS缓冲液冲洗称量舟及采血管，回收清洗液至50ml离心管，用塑料吸管混匀液体，离心，2500rpm，15min，4℃，弃上清。
4、加5ml消化液，混匀，放入水浴恒温振荡器，50℃，200rpm，过夜消化。
5、消化完全后加1.5ml 5M NaCl，混匀；室温静置10min,期间颠倒混匀多次，离心，3800rpm，30min，室温。
6、转移上清于50ml离心管，加2倍体积无水乙醇，充分混匀。
7、用移液器将可见的絮状DNA沉淀转移至含有500μl 70％乙醇的2ml EP管进行清洗。
8、离心，13000rpm，5min，室温，弃上清；再离心，13000rpm，1min，室温，用移液器弃尽残液，晾干，用双蒸水溶解。
五、HumanOmniZhongHua-8(v1.1)芯片扫描分型和全基因组关联数据分析
HumanOmniZhongHua-8(v1.1)芯片购自ILLUMINA公司，分型按照产品方法要求进行。具体操作严格步骤按Illumina公司HumanOmniZhongHua-8(v1.1)芯片的实验步骤进行，相关描述可同样见发表的文献[Adler,A.J.,Wiley,G.B.,Gaffney,P.M.Infinium Assay for Large-scale SNP Genotyping Applications.J.Vis.Exp.(81),e50683,doi:10.3791/50683(2013).]。每个DNA样品采用200纳克(ng)，严格按照Illumina公司HumanOmniZhongHua-8(v1.1)芯片的说明实验步骤进行处理。
HumanOmniZhongHua-8(v1.1)芯片分型数据由ILLUMINA公司的BeadStudio软件处理并输出，关联数据分析按发表的文献同样进行[Zuo,X.et al.Whole-exome SNP array identifies 15 new susceptibility loci for psoriasis.Nat Commun 6,6793，2015]。BeadStudio输出的分型数据采用PLINK分析，根据状态同一配对原理(pairwise identity-by-state)，排除重复样品和亲缘样品。采用smartpca软件，根据主成分分析原理(principal component analysis)，排除群体异质样品，共有1,122PBC病人和4,036正常对照的结果，用于统计分析。排除分型成功率低于98％的SNP，排除在分析样品中频率(minor allele frequency，MAF)低于0.01的SNP，排除在正常对照样品中Hardy–Weinberg equilibrium值P<1×10-4的SNP。经过排除，共有776,516个常染色体的SNP用于比较。采用PLINK软件，SNP相关分析采用线性回归的原理，以性别为共变量(covariate)，采用加性效应原理(additive allelic effect)，评估每个SNP位点与PBC关联的显著性，计算危险等位基因的相对危险度(Odds ratio,OR)和95％的可信区间，分析中直接获得OR值、可信区间和P值，从而得到所述位点的危险等位基因。通过全基因组关联(GWAS)分析计算获得SNP芯片中每个位点与PBC关联显著性水平(P值)，按照显著性水平(P值小于0.0001)找出与PBC之间存在潜在关联的染色体区域，并得到PBC易感性显著关联的单核苷酸多态性位点及其等位基因。SNP的推算(Imputation)是以千人基因组项目中汉族人结果为参考值(Reference)，采用IMPUTE2软件，结合1,122PBC病人和4,036正常对照的结果进行推算。
分析结果参见表2和图1，结果显示在IL21基因区，有7个单核苷酸多态性位点(rs17005934，rs17005953，rs28517551，rs925550，rs60318098，rs1026157，rs6811183)的疾病相关单倍体频率在PBC中显著高于对照人群，p值为p值小于1.07×10-5。这一系列单核苷酸多态性位点及其等位基因是：rs17005934，等位基因为C/T；rs17005953，等位基因为T/C；rs28517551，等位基因为A/G；rs925550，等位基因为A/C；rs60318098，等位基因为G/A；rs1026157，等位基因为G/T；rs6811183，等位基因为T/G；其中危险等位基因分别是：rs17005934多态性位点为C；rs17005953多态性位点为T；rs28517551多态性位点为A；rs925550多态性位点为A；rs60318098多态性位点为G；rs1026157多态性位点为G；rs6811183多态性位点为T。携带这些危险等位基因的个体发生PBC的相对风险是没有携带者的1.24-1.27倍。
IL21基因区域单核苷酸多态性位点序列：
如序列表中的SEQ ID NO.1所示：
rs17005934
Figure PCTCN2017092504-appb-000002
如序列表中的SEQ ID NO.2所示：
rs17005953
Figure PCTCN2017092504-appb-000003
如序列表中的SEQ ID NO.3所示：
rs28517551
Figure PCTCN2017092504-appb-000004
如序列表中的SEQ ID NO.4所示：
rs925550
Figure PCTCN2017092504-appb-000005
如序列表中的SEQ ID NO.5所示：
rs60318098
Figure PCTCN2017092504-appb-000006
如序列表中的SEQ ID NO.6所示：
rs1026157
Figure PCTCN2017092504-appb-000007
如序列表中的SEQ ID NO.7所示：
rs6811183
Figure PCTCN2017092504-appb-000008
表2 与原发性胆汁性胆管炎显著关联的IL21基因区域单核苷酸多态性位点
Figure PCTCN2017092504-appb-000009
其中“OR(95％CI)”，表示与携带正常的单倍体的个体相比，携带疾病相关单倍体的个体发生PBC的相对风险。染色体位置为参照Human Genome GRCh37/hg19.。
实施例2rs17005934，rs925550的验证实验
通过针对rs17005934，rs925550在907个PBC患者和2027个正常对照的验证，同样发现有显著差异。PBC患者验证样品与实施例1中的标准一样；正常对照样品来自东南大学教师体检，生化指标正常，B超检查无肝脾异常，无临床疾病表征，自述体健。验证实验实施中的材料和时间如无说明，均由Sequenom公司提供。
1、验证IL21基因区域单核苷酸多态性位点序列
rs17005934序列
Figure PCTCN2017092504-appb-000010
rs925550序列
Figure PCTCN2017092504-appb-000011
2、引物设计
使用Sequenom公司Genotyping Tools及MassARRAY Assay Design软件设计待测SNP位点的PCR扩增引物及单碱基延伸引物，并交由Integrated DNA Technologies合成。
设计使用DNA引物和探针如下：
rs17005934：
引物1序列，ACGTTGGATGAAAGTGCAGACCAGGAAAAC；
引物2序列，ACGTTGGATGAAAGGGTGACTTCAGGGTAG；
探针序列，GACCAGGAAAACACAGACTC
rs925550：
引物1序列，ACGTTGGATGGGGAGGGTAATTAAAATTAG；
引物2序列，ACGTTGGATGGGGCAAATGAAGCCAATATA；
探针序列，tTGCTACAAAATTAATATCTATCAAA
3、PCR扩增
PCR扩增采用多重PCR技术，在384孔板中进行，每个反应体系总体积为5μl。在一个新的1.5ml EP管中配制PCR master mix溶液。
配制PCR master mix液：
PCR Mix对每个反应，μL
10×PCR Buffer 0.5μL
MgCl2(25mM)0.4μL
dNTP mix(25mM)0.1μL
HotStar Taq(5U/μL)0.1μL
Water 1.9μL
PCR primer mix(引物1和2各0.5pmol)1μL
Total Volume 4μL
使用24通道加样器，调节加样体积为4μL，在384孔板的每个加样孔中加入PCR master mix液。该384孔板即为PCR反应板。取出已经制备好的DNA样品384孔板，使用24通道加样器，调节加样体积为1μL，每个5μlPCR反应体系中包含模板DNA 20-50ng，Hotstar Taq 0.5U，每条扩增引物0.5pmol，0.1μl的25mM dNTPs。在兼容384孔板的PCR仪上设定PCR反应条件为：94℃4分钟；94℃20秒，56℃30秒，72℃1分钟，45个循环；72℃3分钟；4℃保持。将384孔PCR反应板放置于PCR仪上，启动PCR反应。
4、PCR产物碱性磷酸酶处理
在PCR反应结束后，将PCR产物用SAP(shrimp alkaline phosphatase，虾碱性磷酸酶)处理，以去除体系中游离的dNTPs。
(1)配制碱性磷酸酶处理反应液，SAP Mix。
SAP Mix对每个反应，μL
H2O1.53
SAP Buffer(10x)0.17
SAP Enzyme(1.7U/μL)0.3
Total Volume 2
(2)使用24通道加样器，调节加样体积为2μL，将SAP Mix加入384孔PCR反应板。对于每个碱性磷酸酶处理反应孔，反应体系总体积为7μl，其中PCR产物5μl，SAP Mix 2μl。
(3)将384孔板放置在兼容384孔板的PCR仪上，设定PCR反应条件：37℃40分钟；85℃5分钟；4℃维持，启动PCR仪进行碱性磷酸酶处理。
5、单碱基延伸
(1)在碱性磷酸酶处理结束后，进行单碱基延伸反应，反应体系总体积9μl。
(2)配制单碱基延伸反应液，EXTEND Mix。
Figure PCTCN2017092504-appb-000012
Figure PCTCN2017092504-appb-000013
(3)使用24通道加样器，调节加样体积为2μL，将EXTEND Mix对应加入384孔反应板。对于每个反应孔，单碱基延伸反应体系包含SAP处理后PCR产物7μl及EXTEND Mix液2μl(其中各延伸反应引物混合物0.94μl，iPLEX酶0.041μl，延伸混合物0.2μl)。
(4)将384孔板放置在兼容384孔板的PCR仪上，设定PCR反应条件：
I.94℃for 30seconds；
II.94℃for 5seconds；
III.52℃for 5seconds；
IV.80℃for 5seconds；
V.GOTO III，4more times；
VI.GOTO II，39more times；
VII.72℃for 3minutes；
VIII.4℃forever；
启动PCR仪进行单碱基延伸反应。
6、树脂纯化
(1)将Clean Resin树脂平铺到6mg的树脂板中；
(2)加16μl水到延伸产物的对应孔内；
(3)将干燥后的树脂倒入延伸产物板中，封膜，低速垂直旋转30分钟，使树脂与反应物充分接触；
(4)离心使树脂沉入孔底部。
7、芯片点样
启动MassARRAY Nanodispenser RS1000点样仪，将树脂纯化后的延伸产物移至384孔SpectroCHIP芯片上。
8、质谱检测
将点样后的SpectroCHIP芯片使用MALDI-TOF(matrix-assisted laser desorption/ionization–time of fligh，基质辅助激光解吸附电离飞行时间质谱)分析，检测结果使用TYPER 4.0软件(sequenom)分型并输出结果。共907个PBC样品和2127个对照样品得到了有效的结果。
采用PLINK软件，逻辑回归原理进行，以性别为共变量，按叠加效应模型对907个PBC样品和2127个对照样品的结果进行了统计分析。rs17005934的疾病相关单倍体(G)频率在PBC中(0.428)显著高于对照人群(0.362)，p值为1.25×10-6。rs925550的疾病相关单倍体(A)频率在PBC中(0.427)显著高于对照人群(0.353)，p值为7.44×10-8。将1,122PBC病人和4,036正常对照的GWAS结果，与在907个PBC样品和2127个对照样品的结果进行叠加分析，采用fixed-effect model(I2<25％)的原理，统计分析共包括2029个PBC样品和6163个对照样品。结果表明：rs17005934的疾病相关单倍体(G)频率在PBC中显著高于对照人群，p值为1.06×10-11，相对危险度(odds ratio)值(96％CI)为1.29(1.21-1.39)；rs925550的疾病相关单倍体(A)频率在PBC中显著高于对照人群，p值为3.87×10-13，相对危险度值(96％CI)为1.31(1.21-1.40)(见表3)。
表3：IL21多态位点rs17005934和rs925550在全基因组关联分析和验证发现中的结果
Figure PCTCN2017092504-appb-000014
实施例3原发性胆汁性胆管炎患者肝脏组织中IL21的表达分析
所有肝组织，包括30例PBC患者，30例自身免疫肝炎(AIH)患者，25例慢性乙型肝炎(CHB)患者均为在上海仁济医院超声指导的肝活检组织。6例健康对照(HC)的肝脏组织来自肝脏移植供体。肝组织经福尔马林固定和石蜡包埋保存。肝组织的炎症和纤维化程度按照“Scheuer”评分系统评定。
1、肝组织切片在柠檬酸钠缓冲液中浸泡20分钟；
2、加入1：200稀释的抗IL21抗体(ab5978，Abcam，Cambridge，USA)，室温保持20分钟；
3、用磷酸钠缓冲液清洗玻片；
4、加入1：2000稀释的辣根过氧化酶标记的羊抗兔抗体(全自动免疫组化机二抗组合，47101，Leica)，采用Leica全自动免疫组化机进行免疫组化染色操作；
5、对染色的玻片进行分析，在40*10放大的显微镜下随即选取5个镜下区域，记录染色的阳性细胞数，最终结果按平均值加标准差表述。Mann–Whitney U分析原理(Mann–Whitney U-test)用于数据的统计分析，P值小于0.05被认为统计学显著差异。所有分析采用Prism软件系统(Prism software Version 6.0，Graphpad Software,La Jolla,CA,USA).进行处理。
结果表明，相比于乙型肝炎，自身免疫性肝炎和正常肝组织，IL21在PBC患者肝组织中表达显著升高。在PBC患者中，IL21阳性的细胞是在肝脏门管区周边的炎性肝细胞和浸润的炎症淋巴细胞。实验结果表明，IL21阳性细胞的数目与肝脏的炎症程度和肝脏的纤维化程度呈正比。统计分析发现IL21阳性细胞的数目与肝脏的炎症程度(hepatic inflammation degree)正相关(r＝0.45,p<0.05)；与肝脏的的纤维化程度(hepatic fibrosis degree)正相关((r＝0.66,p<0.01)，相关结果见图2。
上述仅为本发明优选的实施例，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出其它不同形式的变化或变动，这些也应属于本发明的保护范围。
Figure PCTCN2017092504-appb-000015
Figure PCTCN2017092504-appb-000016
Figure PCTCN2017092504-appb-000017
Figure PCTCN2017092504-appb-000018

Claims (10)

  1. IL21在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎的产品中的应用。
  2. IL21基因多态位点rs17005934的试剂在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎的产品中的应用，rs17005934序列如序列表中的SEQ ID NO.1所示，其中，单核苷酸n为C的携带者原发性胆汁性胆管炎的风险高于等位基因T。
  3. IL21基因多态位点rs17005953的试剂在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎的产品中的应用，rs17005953序列如序列表中的SEQ ID NO.2所示，其中，单核苷酸n为T的携带者原发性胆汁性胆管炎的风险高于等位基因C。
  4. IL21基因多态位点rs28517551的试剂在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎的产品中的应用，rs28517551序列如序列表中的SEQ ID NO.3所示，其中，单核苷酸n为A的携带者原发性胆汁性胆管炎的风险高于等位基因G。
  5. IL21基因多态位点rs925550的试剂在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎的产品中的应用rs925550序列如序列表中的SEQ ID NO.4所示，其中，单核苷酸n为A的携带者原发性胆汁性胆管炎的风险高于等位基因C。
  6. IL21基因多态位点rs60318098的试剂在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎的产品中的应用，rs60318098序列如序列表中的SEQ ID NO.5所示，其中，单核苷酸n为G的携带者原发性胆汁性胆管炎的风险高于等位基因A。
  7. IL21基因多态位点rs1026157的试剂在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎的产品中的应用，rs1026157序列如序列表中的SEQ ID NO.6所示，其中，单核苷酸n为G的携带者原发性胆汁性胆管炎的风险高于等位基因T。
  8. IL21基因多态位点rs6811183的试剂在制备检测或辅助检测、筛查或预测原发性胆汁性胆管炎的产品中的应用，rs6811183序列如序列表中的SEQ ID NO.7所示，其中，单核苷酸n为T的携带者原发性胆汁性胆管炎的风险高于等位基因G。
  9. IL21在制备PBC动物模型中的应用。
  10. IL21在制备PBC治疗药物方面的应用。
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