慢性B型肝炎患者免疫遺傳、免疫受體與免疫調節之研究

Studies on Immunogenetics, Immune-Receptors, and Immunomodulation in Chronic Hepatitis B Patients

黄奕文(Yi-Wen Huang)

指導教授 ： 高嘉宏

共同指導教授 ： 許秉寧(Ping-Ning Hsu)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.00062

摘要

目前仍然未知那些宿主免疫遺傳及免疫受體因素影響慢性Ｂ型肝炎急性發作的嚴重度。我們的目的在於探討免疫遺傳因子HLA-DRB1多形性與男性慢性B型肝炎之肝炎嚴重程度之間的相關性。在此前瞻性世代研究中，共納入了204名在門診已追蹤一年以上的Ｂ肝帶原者 (131名男性和73名女性)。50名ALT < 2X正常值上限 (第一組、平均追蹤83.6個月) 與154名ALT ≧ 2X正常值上限 (第二組、平均跟蹤81.3個月)的帶原者進行比較。HLA-DRB1的對偶基因採用聚合酶鏈反應-序列特定寡核苷酸探針雜交法，而病毒基因型以熔解曲線分析鑒定。在男性帶原者中發現第一組之18% 和第二組之8%帶有HLA-DRB1*1101(調整年齡後OR 0.23， p = 0.020)，女性帶原者是4% 相較於 9.4%(p = 0.094)。男性攜帶DRB1*1101者，其B肝病毒基因型的分佈在兩組之間是相似的。HLA-DRB1*1101與臺灣男性慢性B肝帶原者的輕度肝炎有關。
對於慢性感染和癌症，維生素D可通過維生素D受體 (VDR) 發揮免疫調節和抗增殖作用。我們訂定分為6個不同組別的250名臺灣慢性B肝帶原者之三個多形性限制位點的基因型 (BsmI (rs1544410)、ApaI (rs7975232) 及TaqI (rs731236))。調整年齡和性別後，肝炎急性發作患者的維生素D受體B/b、B/a、B/T、B/a/T頻率均低於未發生急性發作者 (7% 相較於20%，p=0.009；1% 相較於9%，p=0.004；3% 相較於10%，p=0.007；1% 相較於9%，p=0.005)；相反的，肝炎急性發作患者的T/t、A/T、A/t、b/A/t頻率分佈均較高 (8% 相較於3%，p=0.003；49% 相較於34%，p=0.027；2% 相較於1%，P=0.004；0.5% 相較於0%，p=0.001)。此外，HBeAg陽性患者的B/b、B/B、T/t、b/A、B/a、B/A、B/T、B/t、A/t、b/A/T、B/a/T、B/A/T、B/A/t、b/A/t頻率較高。對於併發與未併發肝細胞癌的帶原者，其維生素D受體的頻率分佈相似。維生素D受體的基因多形性與臺灣B型肝炎病毒帶原者的不同臨床表型有關，但與產生肝細胞癌無關。
先天免疫類鐸受體-3的基因變異與慢性B型肝炎有關。我們的目的是探討慢性B型肝炎患者週邊血液單核球細胞與肝臟細胞的類鐸受體-3表現量和接受免疫調節藥物長效型干擾素治療時之變化。我們納入127例慢性B型肝炎患者與64例B型肝炎表面抗原陰性及C型肝炎抗體陰性之控制者。獨立於年齡、性別和ALT值之下 (-13.466，95% 信賴區間 -17.202 –- 9.730，p < 0.001)，相較於對照者，慢性B肝病患在週邊血液單核球細胞上有較低的類鐸受體-3平均熒光強度 (14.61 ± 13.49 相較於 9.70 ± 4.61，p < 0.001)。慢性B肝病患的類鐸受體-3免疫組織化學染色主要侷限在庫氏細胞，而控制者的染色散佈在庫氏細胞和肝細胞上。病患的肝臟細胞類鐸受體-3 mRNA表現量也比對照者低 (0.47 ± 0.30倍相較於1倍)。在12例接受長效型干擾素治療的病患中，5例達到持續病毒學反應。使用治療前類鐸受體-3的平均熒光強度為對照，這5例病患之類鐸受體-3的平均熒光強度在治療後立即回升到平均1.5至1.7倍。在七例治療無反應或復發病患中，類鐸受體-3平均熒光強度減少到平均0.5至0.7倍。在10例接受貝樂克治療且具有治療中病毒學反應的患者，在48個星期的治療中，類鐸受體-3平均熒光強度逐漸回升到平均1.2倍。慢性B型肝炎病患之週邊血液單核球細胞上，在獨立的年齡，性別及ALT值之下，以及肝細胞上的類鐸受體-3的表現量降低。長效型干擾素治療而達到持續病毒反應的患者之類鐸受體-3表現的回升較貝樂克治療者更為顯著。
在lamivudine治療中所檢測到的血清HBV RNA是因為RNA的複製中間產物未受藥物治療之影響。我們的目的在於探討慢性B型肝炎患者中免疫調節藥物 (干擾素) 對於血清HBV RNA的抑制效果。血清檢體中的HBV DNA和RNA通過B肝病毒核酸萃取物的逆轉錄和即時PCR而定量之。將對來自以下三組患者之每2週到3個月的檢體進行分析：10名男性患者，接受核苷類似物單一治療44至48週(5名用lamivudine，5名用entecavir)；6名男性患者，接受相繼干擾素和lamivudine合併治療；以及3名男性患者，接受lamivudine單一治療20至24週。接受治療前，沒有任何患者血清中檢測到HBV RNA，但在接受治療期間，有15名患者血清中檢測出HBV RNA。在這三組中，治療前HBV DNA (8.1 ± 2.4 相較於 7.7 ± 1.4相較於5.1 ± 0.3 log10 copies/ml，p = 0.06)以及治療和追蹤期間 (45.5 ± 2.0 相較於 49.7 ± 5.6 相較於 48.7 ± 6.4 週，p = 0.32) 相似。HBV RNA 在所有接受單一治療的患者之治療結束時以及追蹤結束時都可檢測到，但在接受相繼合併治療患者中卻沒有一位被檢測到(100% 相較於 0%，p＜ 0.001)。與lamivudine治療而檢測到血清HBV RNA相比，免疫調節藥物干擾素治療可通過抑制HBV RNA的複製中間產物來降低HBV DNA 的複製，從而使血清HBV RNA消失。

關鍵字

B 型肝炎 ； 免疫的 ； 人類白血球抗原 ； 維生素D受體 ； 類鐸受體3 ； 核醣核酸 ； 干擾素

並列摘要

Which host immunogenetic factors and immune receptors correlate with hepatitis severity in chronic hepatitis B (CHB) patients (pts) remain unclear. We aimed to study association of HLA-DRB1 with hepatitis severity in male HBV carriers. 204 HBV carriers (131 men, 73 women) who were followed-up for >1y were enrolled. 50 HBV carriers (group I) with ALT <2x ULN (mean f/u 83.6m) were compared with 154 CHB patients (group II) with ALT ≥2x ULN (mean f/u 81.3m). HLA-DRB1 alleles were typed by PCR-sequence specific oligonucleotide probe hybridization and HBV genotypes by melting curve analysis. HLA-DRB1*1101 was found in 18% of group I vs. 8% of group II in male (OR 0.23, p=0.020, adjusted for age) and 4% vs. 9.4% in female (p=0.094). In male harboring DRB1*1101, distribution of HBV genotype was comparable between 2 groups. HLA-DRB1*1101 correlates with less severe hepatitis in Taiwanese male carriers of HBV.
Vitamin D receptor (VDR) has immunomodulatory and antiproliferative effects. We genotyped BsmI (rs1544410), ApaI (rs7975232), TaqI (rs731236) of VDR gene in 250 HBV carriers who were categorized into 6 phenotypes. After adjustment for age and sex, frequencies of VDR B/b, B/a, B/T, B/a/T in pts with hepatitis flare(s) were lower than those without (7%vs.20%, p=0.009; 1%vs.9%, p=0.004; 3%vs.10%, p=0.007; 1%vs.9%, p=0.005), in contrast, T/t, A/T, A/t, b/A/t were higher in flare(s) (8% vs. 3%, p=0.003; 49% vs. 34%, p=0.027; 2% vs. 1%, p=0.004; 0.5% vs. 0%, p=0.001). In addition, B/b, B/B, T/t, b/A, B/a, B/A, B/T, B/t, A/t, b/A/T, B/a/T, B/A/T, B/A/t, b/A/t were higher in HBeAg positive pts. Distribution of VDR genotypes was comparable between pts with and without hepatoma. VDR gene polymorphisms are associated with distinct clinical phenotypes but not with hepatoma.
Innate immune receptor toll-like receptor-3 (TLR3) gene variants correlate with CHB. We aimed to investigate TLR3 expression in PBMCs and liver cells of CHB pts and its response to immunomodulation (pegylated-interferon therapy (peg-IFN)). We enrolled 127 CHB pts and 64 HBsAg-negative, anti-HCV negative controls. Compare to controls, pts had a lower TLR3 mean fluorescence intensity (MFI) on PBMCs (14.61 ± 13.49 versus 9.70 ± 4.61, p<0.001), independent of age, gender and ALT (-13.466, 95% CI -17.202 – -9.730, p<0.001). Pts had limited TLR3 stains on Kupffer cells, controls had diffuse stains on Kupffer and hepatocytes. Hepatic TLR3 mRNA was lower in pts than controls (0.47 ± 0.30 versus 1 fold). Using pre-treatment TLR3 MFI as referent, among 5 of 12 peg-IFN treated pts with sustained virological response (SVR), TLR3 MFI restored to a mean of 1.5-1.7 folds immediately after therapy. Among 7 non-responders or relapsers, TLR3 MFI reduced to a mean of 0.5-0.7 fold. Among 10 entecavir (ETV)-treated pts with on-treatment virological response, TLR3 MFI gradually restored to a mean of 1.2 folds during 48-week therapy. CHB pts have reduced TLR3 expressions on PBMCs, independent of age, gender, ALT and on liver cells. Pts with peg-IFN induced SVR have a more significant restoration of TLR3 expression than those under ETV.
Serum HBV RNA is detected during lamivudine therapy as the consequence of unaffected RNA replicative intermediates. We aimed to determine inhibitory effect of immunomodulation (interferon, IFN) on serum HBV RNA in CHB pts. HBV DNA and RNA were quantified by reverse transcription of HBV nucleic acid extract and real-time PCR; every 2 wks to 3m in 10 male treated with nucleoside analogue monotherapy for 44-48wks (5 lamivudine (LMV), 5 ETV), 6 males with sequential IFN and LMV combination, and 3 males with LMV monotherapy for 20-24 wks. HBV RNA was not detectable in all pts before any therapy, but became detectable in 15 during therapy. Among the 3 groups, pretreatment HBV DNA (8.1±2.4 vs. 7.7±1.4 vs. 5.1±0.3 log cp/mL, p=0.06), treatment and follow-up durations (45.5±2.0 vs. 49.7±5.6 vs. 48.7±6.4 wks, p=0.32) were comparable. HBV RNA was detectable at end of therapy or follow-up in all pts with monotherapy, but none of sequential combination (100% vs. 0%, p<0.001). Compared to LMV with detectable serum HBV RNA, immunomodulation of IFN may reduce HBV DNA replication through inhibition of HBV RNA replicative intermediates, resulting in loss of serum HBV RNA.

並列關鍵字

hepatitis B ； immune ； human leukocyte antigen ； vitamin D receptor ； toll-like receptor-3 ； ribonucleic acid ； interferon

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