台灣地區慢性C型肝炎病患與變形成長因子基因多形性之關係

Transforming growth factor-B1 gene polymorphisms in Taiwanese patients with chronic hepatitis C treated with combined interferon and ribavirin therapy

潘文正(Wen-Cheng Pan)

指導教授 ： 莊萬龍

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

TGF-??1(transforming growth factor- ??1)能促進活化肝星狀細胞(Hepatic stellate cells)及製造細胞外間質蛋白質(extracellular matrix proteins)而導致慢性C型肝炎病患的肝臟纖維化，TGF-??1的製造主要在於基因的控制,特別與基因的多形性有關。最近的研究發現在TGF-??1基因上兩個位置，分別是+ 29 (codon 10)和+ 74 (codon 25)，似乎與肝臟纖維化有關，並且可能影響抗病毒藥物的療效。
本研究的目的在於探討慢性C型肝炎病患TGF-??1基因多形性在codon 10和codon 25之分布情形以及與病患體內C型肝炎病毒基因型，病毒濃度，肝臟發炎程度之關係，及其對干擾素合併ribavirin治療慢性C型肝炎療效之影響。共收集145位慢性C型肝炎病患進入此研究(男性90位，女性55位，平均年齡47.1?b 11.3歲) ，全部病患血清anti-HCV及HCV RNA均呈陽性而HBsAg均呈陰性，病理切片根據HAI (Histological activity index)和兩位病理醫師協助下，將肝臟發炎程度予以分類，其中14位呈現肝硬化現象。血清HCV RNA的定性檢查是以Cobas Amplicor HCV test 2.0測試。HCV基因型乃以Okamoto等人所發展之方法測定，此方法可檢測出1a、1b、2a、2b及3a等五種HCV基因亞型。病毒基因型分型為1b型65 位(44.8%)，2a型43位(29.7%)，2b型17位(11.7%)混合型5位(3.5%)及無法分型者15位(10.3%)。血清HCV RNA定量則以Versant HCV RNA 3.0偵測。DNA 製備乃從病人抽取血液以QIAamp DNA blood kit (Qiagen) 萃取DNA，利用PCR Sequence-specific primer typing的技術，細胞中的DNA以特殊序列之引子(Primer)進行PCR增數。若細胞中之DNA含與引子序列互補之序列.則會經PCR而複製，電泳會出現Band。在codon 10其基因型分別為T/T、T/C、C/C，在codon 25其基因型分別為G/G、G/C、C/C。
全部病患中，136位慢性C型肝炎病患接受IFN-?? 6百萬單位皮下注射，每週三次，治療 24週，ribavirin每天口服1000~1200mg，並在治療滿24週之後接受持續六個月以上之追蹤，方完成此一療程。在治療及追蹤期當中，每位病人均常規檢驗血清ALT值(每個月至少一次)及血清HCV RNA(定性RT-PCR，每三個月至少一次)。
病人對IFN-??/ribavirin合併治療之反應定義如下
1) 持續性反應者(sustained responder)
當病人為治療終點反應者，且持續追蹤血清ALT正常，血清HCV RNA呈PCR陰性滿6個月以上，則視為持續性反應者。
2) 非持續性反應者(non-sustained responder)
不具持續性反應之病患
結果發現：
1. TGF-b1基因多形性的codon 10及25其allele、基因型及表現型均與性別、年齡、治療前C型肝炎病毒濃度及血中ALT、AST濃度無關
2. TGF-b1基因多形性在codon 10基因型為T/T則明顯有較高比率感染C型肝炎病毒基因1b型
3. 並無發現任何基因型或表現型與肝臟纖維化或肝臟組織壞死發炎性有明顯相關性。TGF-b1基因多形性在codon 25的分布與肝纖維化傾向在高加索民族有明顯的相關性，但由於在台灣codon 25幾乎百分百為G/G型所以無法進一步分析研究
4. TGF-b1基因多形性在codon 10基因若為T/T型，對干擾素/ribavirin合併治療有較低的持續反應率，乃因T/T型有較高比率感染C型肝炎病毒基因型1b有關

關鍵字

變形成長因子-B 1 ； 慢性C型肝炎 ； 甲型干擾素

並列摘要

Transforming growth factor-??1 (TGF–??1), which contributes to the activation of hepatic stellate cells(HSC) and their production of extracellular matrix proteins, may promote hepatic fibrogenesis in chronic hepatitis C. The production of TGF-β1 is predominantly under genetic control and significantly associated with gene polymorphism. Two of TGF-β1 genetic polymorphisms have been described in the leader sequence at position +29(codon 10: leucine/proline) and +74(codon 25: arginine /proline) and appears to be associated with hepatic fibrogenesis and to affect the response to antiviral therapy.
This study is to determine the frequency of genotypes associated with TGF- ??1 genetic polymorphisms and investigate their association with HCV genotype, viral load, the severity of liver inflammation, and the response to interferon with ribavirin combination therapy in patients with chronic hepatitis C.
One hundred and forty-five patients with chronic hepatitis C were enrolled in the study. These included 90 males and 55 females, aged between 20 and 73 years (mean 47.1?b11.3 years). Second-generation HCV antibody and hepatitis B surface antigen were detected with commercially available enzyme-linked immunosorbent assay kits. All were positive for anti-HCV antibodies and serum HCV RNA, and negative for hepatitis B surface antigen. Liver histology, assessed blindly by 2 pathologists showed chronic hepatitis of different severity in 131 patients and cirrhosis in 14. Disease activity grade and fibrosis stage were quantitatively scored according to the histological activity index (HAI). Detection of serum HCV RNA was performed using a standardized automated qualitative reverse transcription polymerase chain reaction assay. HCV genotypes 1a, 1b, 2a, 2b and 3a were determined by amplification of the core region using genotype-specific primers described by Okamoto et al. Sixty-five patients were infected by HCV type 1b,43 by type 2a, 17 by type 2b, 5 by mixed types, and 15 had an indeterminate HCV type. Serum HCV RNA levels were measured and performed strictly in accordance with the manufacturer’s instructions. Genomic DNA was purified from whole blood using the QIAamp DNA blood kit according to the manufacturer’s instructions. In PCR sequence-specific primer(SSP) typing , oligonucleotide primers are designed to obtain amplification of specific alleles or groups of alleles. Assignment of alleles is then based on the presence or absence of amplified product usually detected by agarose gel electrophoresis and transillumination. Interpretation of PCR results was based on the presence of the internal control band together with the presence or absence of specific amplified fragment. The genotyping pattern were determined T/T, T/C and C/C at codon 10 and, and G/G , G/C and C/C at codon 25. 136 of all patients were treated 24 weeks with IFN-* at a dose of 6 million units , thrice weekly , combined with 1000-1200 mg of ribavirin, per day. The presence of HCV RNA in the serum was assessed every three months. Sustained viral response was defined as clearance of serum HCV RNA at the end of the therapy and 6 months after the cessation of therapy. All other patients were classified as non-responders.
Our results showed that:
1. All of the alleles, genotypes or phenotypes of TGF-??1 gene polymorphisms at codon 10 and 25 did not correlate to age, sex , pretreatment HCV viral loads, and serum levels of aminotransferase.
2. Patients with the T/T genotype at codon 10 had higher chronic infection rate of HCV genotype 1b
3. No correlation was found between genotypes or phenotypes of TGF-??1 gene polymorphisms and hepatic fibrosis or necroinflammatory activity. Polymorphisms at codon 25 may be associated with hepatic fibrosis in Caucasian populations, but the polymorphism is unlikely to be associated with hepatic fibrosis in Taiwanese population.
4. Lower sustained response rate to combined IFN-* and ribavirin therapy in patients with the T/T genotype at codon 10 was associated with higher infection rate of HCV genotype 1b

並列關鍵字

chronic hepatitis ； Transforming growth factor-B1

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