替诺福韦酯治疗对慢性乙型肝炎疗效及治疗过程中宿主的免疫应答研究
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摘要
乙型肝炎病毒（HBV）感染是严重威胁我国人民健康的重大传染病，并且是慢性肝炎、肝硬化、肝癌的主要原因。慢性乙型肝炎治疗较困难，目前尚无任何药物治疗可以达到痊愈。关于慢性乙型肝炎的治疗，抗病毒治疗是关键。目前有两种主要途径治疗CHB，一种通过刺激免疫的方法，如干扰素（IFN），另一种是通过抑制HBV复制的方法，口服抗病毒药物：核苷（酸）类似物。干扰素能有效抑制HBV复制从而通过刺激免疫系统诱导肝脏疾病缓解。但有限的疗效、流感样不良反应及相对严格的适应症、禁忌症限制了其临床应用。核苷（酸）类似物具有耐受性、安全性良好和服用方便等特点，已广泛应用于临床抗HBV治疗。但其缺点是长期服药容易出现病毒耐药，耐药变异后的病毒反弹会抵消前期的临床受益，因而针对上述情况，寻求更强效抑制病毒复制、低耐药及长期应用安全的药物是目前药物研发研究的热点。一种新的核苷（酸）类似物抗病毒药物-替诺福韦酯（tenofovir disoproxil fumarate，TDF）的出现给患者带来了新的希望，到今天为止，未见有TDF的HBV临床耐药报告。TDF在我国尚未被批准上市，现正处于临床试验研究中。慢性HBV感染及其治疗过程中宿主因素起到重要作用，炎性细胞因子TNF-α、IFN-γ、IL-2、IL-4、IL-6、IL-10和IL-17A均在此过程中发挥了重要的作用。近些年随着基因组学研究的进步，研究发现宿主的单核苷酸基因多态性(single nucleotide polymorphism，SNP)在HBV感染及清除中也起重要作用。但相关HBV感染的免疫学研究尚不明确。
本研究收集吉林大学第一医院门诊及入院CHB患者，签署知情同意书后进行筛选试验，选取24例核苷和核苷（酸）类似物初治CHB患者，包括HBeAg阳性或阴性的病例（HBV DNA≥105拷贝/毫升，间隔14天以上的两次检测ALT升高HBeAg阳性患者≥2ULN（正常值上限）；HBeAg阴性患者>1ULN且≤10ULN），采用随机、双盲、双模拟、活性药对照研究，按1:1随机分入替诺福韦酯300mg/d治疗组或阿德福韦酯10mg/d治疗组，并于治疗基线、治疗后4周、8周、12周、24周后进行肝功能、乙肝病毒标志物、HBV DNA定量检测；比较治疗后生化学及病毒学的应答情况及细胞因子的动态变化情况，分析其相关性；同时测定24例患者的IL-10的SNPs（rs1800896、rs1800871、rs1800872）并分析与抗病毒治疗的情况和细胞因子的动态变化情况的相关性。
本研究使用三色法流式细胞仪采用微阵列流式细胞术（CBA）方法和焦磷酸测序等技术做了以下工作：
（1）针对替诺福韦酯治疗的慢性乙型肝炎患者，观察其治疗4周、8周、12周、24周后生化学及病毒学的应答情况并与阿德福韦酯治疗的情况相比较。
（2）针对替诺福韦酯治疗的慢性乙型肝炎患者，动态观察其治疗4周、8周、12周、24周后血清中细胞因子的浓度，包括细胞因子TNF-α、IFN-γ、IL-2、IL-4、IL-6、IL-10和IL-17A，分析其变化规律；同时与阿德福韦酯治疗的情况相比较。
（3）针对替诺福韦酯、阿德福韦酯抗HBV治疗慢性乙型肝炎患者进行人类IL-10SNPs（rs1800896、rs1800871、rs1800872）位点检测，分析其与抗病毒疗效及细胞因子的变化的相关性。
本实验结果如下：
（1）慢性乙型肝炎患者经替诺福韦酯治疗后，ALT、AST及HBV DNA较基线时呈下降趋势，治疗4周、8周、12周、24周后，HBV DNA水平与基线相比，均明显下降（P<0.05），且治疗4周与8周之间，8周与12周之间，12周与24周之间均存在显著性差异（p<0.05）；与阿德福韦酯治疗未见明显差异。替诺福韦酯治疗的7例HBeAg阳性患者在治疗24周后，有3例出现HBeAg血清学转换；阿德福韦酯治疗的8例HBeAg阳性患者未出现HBeAg血清学转换。
（2）慢性乙型肝炎患者经替诺福韦酯治疗后，血清IL-10水平在治疗12周、24周后时较基线时下降且存在显著性差异，具有统计学意义（p<0.05）。血清IL-10水平与HBV DNA定量下降水平成负相关（p <0.05）。血清IL-17A水平在治疗24周后，较基线相比明显下降（p <0.05）。血清IFN-γ、TNF-α、IL-2、IL-4、IL-6水平治疗4周、8周、12周、24周后其变化不明显，无统计学意义（p＞0.05）。阿德福韦酯治疗组血清IL-10水平的变化情况与替诺福韦酯治疗组相似，其余六种细胞因子未见明显变化。
（3）替诺福韦酯、阿德福韦酯的抗病毒疗效及血清IL-10、IL-17A水平与IL-10SNPs（rs1800896、rs1800871、rs1800872）位点的相关性分析，未见有统计学意义（p＞0.05）。
通过慢性乙型肝炎患者替诺福韦酯抗病毒治疗过程中细胞因子的变化结果，表明替诺福韦酯可能通过免疫调节起到抑制病毒的作用。病毒学应答分析后，提示血清细胞因子IL-10的水平与HBV DNA具有一定的相关性，故考虑血清细胞因子IL-10水平与替诺福韦酯抗病毒治疗后病毒复制情况密切相关。血清细胞因子IL-10、IL-17A水平在抗病毒早期可以作为慢性乙型肝炎患者治疗过程中的一项临床监测指标，提示疾病预后及转归。
本文通过对慢性乙型肝炎患者抗病毒治疗过程中Th1/Th2/Th17细胞分泌的细胞因子的动态监测，阐明在慢性乙型肝炎抗病毒治疗中血清中Th1/Th2/Th17细胞因子水平的变化，对于明确慢性乙型肝炎的发病机制及指导临床治疗有重要意义。
The hepatitis B virus (HBV) infection is a major infectious disease that seriouslythreatened the health of our people, and it is the main reason for chronic hepatitis, cirrhosisand liver cancer. The therapy of Chronic hepatitis B is very difficult, and no drug treatmentcan make it be healed. With regard to the treatment of chronic hepatitis B, antiviral treatmentis the key. There are two main ways to treat CHB, one is by stimulating the immune, such asinterferon (IFN), the other is by inhibiting replication of HBV, taking orally antiviral drugs:nucleoside (acid) analogues. Interferon can effectively suppress replication of HBV,and thusvia stimulating the immune system to induce liver disease’s mitigation. Nevertheless,thelimited efficacy,flu-like adverse reactions and relatively strict indications, contraindicationslimit its clinical application. Nucleoside (acid) analogues has the feature that tolerance,security, and be easy to take and so on, it has been widely used in clinical anti-HBV therapy.But its drawback is that the long-term medication prone to viral resistance, viral reboundafter resistance mutations will offset the pre-clinical benefit, and thus, to seek more potentinhibition of viral replication, low resistance and long-term safety of the drug is focus ofdrug research. A new nucleoside (acid) analogues of antiviral drug-tenofovir disoproxilfumarate (TDF) has brought new hope to patients, to date, there is no clinical resistancereport in HBV of TDF. TDF has not yet been approved for marketing in our country, and isnow in clinical trials. Host factors play an important role in the process of Chronic HBVinfection and therapy. Inflammatory cytokines TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10andIL-17A play important roles in this process. In recent years, with the development ofgenomics research, we find that single nucleotide polymorphism(SNP) of the host also playan important role in HBV infection and removal.But,immunology research related to HBVinfectIino nth iiss nsotut dcyle,awre. collected outpatients and hospitalized patients with CHB of the FirstHospital of Jilin University,and began screening test after signing informed consent，weselected24previously untreated patients with CHB that giving them nucleoside andnucleotide analogues, including HBeAg-positive or-negative cases (HBVDNA in≥105copies/ml, an interval of14days or more than twice elevated ALT of HBeAg positivepatients≥2ULN(upper limit of normal); HBeAg-negative patients>1ULN and≤10ULN),used randomized, double-blind, double-dummy, active drug controlled study, randomly divided into tenofovir disoproxil fumarate300mg/d treatment group, or adefovirdipivoxil10mg/d treatment one according to1:1, and detected liver function, hepatitis Bvirus markers and HBV DNA quantitative in the treatment of baseline and after4weeks,8weeks,12weeks,24weeks treatment; Compared the response of biochemical andvirological and cytokines in the dynamic changes after treatment, analyzed theirrelevance;Simultaneously determined IL-10SNPs (rs1800896, rs1800871, rs1800872) of the24patients, and analyzed the relevance with antiviral treatment and the dynamic changes ofcytokines.
In this study, we used three-color method of flow cytometry,and adopted the method ofmicro-array flow cytometry (CBA) and pyrosequencing technology to do the followingwork:
(1)Observe the response of biochemical and virological for the treatment of chronichepatitis B patients with tenofovir disoproxil fumarate after4weeks,8weeks,12weeks, andcompared with adefovir dipivoxil.
(2) Dynamicly observed the serum concentrations of cytokines for the treatment ofchronic hepatitis B patients with tenofovir disoproxil fumarate after4weeks,8weeks,12weeks,24weeks, including the cytokines TNF-α,IFN-γ, IL-2, IL-4, IL-6, IL-10and IL-17A,to analyze the variation; and compared with adefovir dipivoxil.
(3)In the chronic hepatitis B patients with tenofovir disoproxil and adefovir dipivoxiltreatment, human IL-10SNPs (rs1800896, rs1800871, rs1800872) loci were detected, toanalyzed the correlation of IL-10SNPs and the antiviral efficacy and the relationshipbetween IL-10SNPs and cytokine changes.
The results are as follows:
(1) In patients with chronic hepatitis B with tenofovir disoproxil treatment, ALT, ASTand HBV DNA were markedly improved, and HBV DNA quantity decreased dramaticallyfrom baseline in4weeks,8weeks,12weeks,24weeks treatment(P <0.05).
Significant differences of the levels of HBV DNA exists between0weeks and12weeks,0weeks and24weeks (p <0.05).The above indicators in tenofovir disoproxil treatmentgroup compared with the adefovir dipivoxil group have the same changing tendency.In sevenHBeAg positive patients with24weeks treatment of tenofovir disoproxil, three casesachieve HBeAg seroconversion. However, no HBeAg seroconversion occurred in8patientswith adefovir dipivoxil treatment.
(2) In the chronic hepatitis B patients with tenofovir disoproxil treatment, thedeclination of serum IL-10levels in12weeks,24weeks compared with baseline is statisticalsignificance (p <0.05). The similar significant changes exist between4weeks and12weeks,8weeks and12weeks,12weeks and24weeks(p <0.05).Serum IL-10levels are negativelyrelated with the declination of HBV DNA quantity（p <0.05）.After24weeks treatment, thelevel of IL-17A in serum decreased clearly compared with the baseline（p <0.05）.The levelsof IFN-γ and TNF-α, IL-2, IL-4, IL-6in serum showed no obviously change in the processof treatment. For the other hand, in the patients of chronic hepatitis B with adefovir dipivoxiltreatment,the same declination occurred as the tenofovir disoproxil treatment group; theother six cytokines showed no significant change.
(3) IL-10SNPs（rs1800896、rs1800871、rs1800872）have little correlation with theantiviral efficacies of the two kinds of medicine,as well as the levels of IL-10,IL-17A（p＞0.05）.
Changes of the cytokines level in the tenofovir disoproxil antiviral treatment processsuggest that immunomodulatory may play a role in the inhibition of viral after using themedicine. Virological response analysis showed the association between the levels of serumcytokines IL-10and HBVDNA.Thus, it is considered that the levels of IL-10in serum areclosely related to the viral replication in the process of tenofovir disoproxil therapy. Thelevels of IL-10, IL-17A can be used as clinical indicators for monitoring the course of theearly stages of the treatment, to evaluate prognosis and the disease outcome in patients withchronic hepatitis B.
The fluctuation of cytokines secreted by Th1/Th2/Th17cells were obtained by dynamicmonitoring of the chronic hepatitis B patients with antiviral treatment, which is significant toelucidate the pathogenesis of chronic hepatitis B and to guide clinical treatment.
本研究收集吉林大学第一医院门诊及入院CHB患者，签署知情同意书后进行筛选试验，选取24例核苷和核苷（酸）类似物初治CHB患者，包括HBeAg阳性或阴性的病例（HBV DNA≥105拷贝/毫升，间隔14天以上的两次检测ALT升高HBeAg阳性患者≥2ULN（正常值上限）；HBeAg阴性患者>1ULN且≤10ULN），采用随机、双盲、双模拟、活性药对照研究，按1:1随机分入替诺福韦酯300mg/d治疗组或阿德福韦酯10mg/d治疗组，并于治疗基线、治疗后4周、8周、12周、24周后进行肝功能、乙肝病毒标志物、HBV DNA定量检测；比较治疗后生化学及病毒学的应答情况及细胞因子的动态变化情况，分析其相关性；同时测定24例患者的IL-10的SNPs（rs1800896、rs1800871、rs1800872）并分析与抗病毒治疗的情况和细胞因子的动态变化情况的相关性。
本研究使用三色法流式细胞仪采用微阵列流式细胞术（CBA）方法和焦磷酸测序等技术做了以下工作：
（1）针对替诺福韦酯治疗的慢性乙型肝炎患者，观察其治疗4周、8周、12周、24周后生化学及病毒学的应答情况并与阿德福韦酯治疗的情况相比较。
（2）针对替诺福韦酯治疗的慢性乙型肝炎患者，动态观察其治疗4周、8周、12周、24周后血清中细胞因子的浓度，包括细胞因子TNF-α、IFN-γ、IL-2、IL-4、IL-6、IL-10和IL-17A，分析其变化规律；同时与阿德福韦酯治疗的情况相比较。
（3）针对替诺福韦酯、阿德福韦酯抗HBV治疗慢性乙型肝炎患者进行人类IL-10SNPs（rs1800896、rs1800871、rs1800872）位点检测，分析其与抗病毒疗效及细胞因子的变化的相关性。
本实验结果如下：
（1）慢性乙型肝炎患者经替诺福韦酯治疗后，ALT、AST及HBV DNA较基线时呈下降趋势，治疗4周、8周、12周、24周后，HBV DNA水平与基线相比，均明显下降（P<0.05），且治疗4周与8周之间，8周与12周之间，12周与24周之间均存在显著性差异（p<0.05）；与阿德福韦酯治疗未见明显差异。替诺福韦酯治疗的7例HBeAg阳性患者在治疗24周后，有3例出现HBeAg血清学转换；阿德福韦酯治疗的8例HBeAg阳性患者未出现HBeAg血清学转换。
（2）慢性乙型肝炎患者经替诺福韦酯治疗后，血清IL-10水平在治疗12周、24周后时较基线时下降且存在显著性差异，具有统计学意义（p<0.05）。血清IL-10水平与HBV DNA定量下降水平成负相关（p <0.05）。血清IL-17A水平在治疗24周后，较基线相比明显下降（p <0.05）。血清IFN-γ、TNF-α、IL-2、IL-4、IL-6水平治疗4周、8周、12周、24周后其变化不明显，无统计学意义（p＞0.05）。阿德福韦酯治疗组血清IL-10水平的变化情况与替诺福韦酯治疗组相似，其余六种细胞因子未见明显变化。
（3）替诺福韦酯、阿德福韦酯的抗病毒疗效及血清IL-10、IL-17A水平与IL-10SNPs（rs1800896、rs1800871、rs1800872）位点的相关性分析，未见有统计学意义（p＞0.05）。
通过慢性乙型肝炎患者替诺福韦酯抗病毒治疗过程中细胞因子的变化结果，表明替诺福韦酯可能通过免疫调节起到抑制病毒的作用。病毒学应答分析后，提示血清细胞因子IL-10的水平与HBV DNA具有一定的相关性，故考虑血清细胞因子IL-10水平与替诺福韦酯抗病毒治疗后病毒复制情况密切相关。血清细胞因子IL-10、IL-17A水平在抗病毒早期可以作为慢性乙型肝炎患者治疗过程中的一项临床监测指标，提示疾病预后及转归。
本文通过对慢性乙型肝炎患者抗病毒治疗过程中Th1/Th2/Th17细胞分泌的细胞因子的动态监测，阐明在慢性乙型肝炎抗病毒治疗中血清中Th1/Th2/Th17细胞因子水平的变化，对于明确慢性乙型肝炎的发病机制及指导临床治疗有重要意义。
The hepatitis B virus (HBV) infection is a major infectious disease that seriouslythreatened the health of our people, and it is the main reason for chronic hepatitis, cirrhosisand liver cancer. The therapy of Chronic hepatitis B is very difficult, and no drug treatmentcan make it be healed. With regard to the treatment of chronic hepatitis B, antiviral treatmentis the key. There are two main ways to treat CHB, one is by stimulating the immune, such asinterferon (IFN), the other is by inhibiting replication of HBV, taking orally antiviral drugs:nucleoside (acid) analogues. Interferon can effectively suppress replication of HBV,and thusvia stimulating the immune system to induce liver disease’s mitigation. Nevertheless,thelimited efficacy,flu-like adverse reactions and relatively strict indications, contraindicationslimit its clinical application. Nucleoside (acid) analogues has the feature that tolerance,security, and be easy to take and so on, it has been widely used in clinical anti-HBV therapy.But its drawback is that the long-term medication prone to viral resistance, viral reboundafter resistance mutations will offset the pre-clinical benefit, and thus, to seek more potentinhibition of viral replication, low resistance and long-term safety of the drug is focus ofdrug research. A new nucleoside (acid) analogues of antiviral drug-tenofovir disoproxilfumarate (TDF) has brought new hope to patients, to date, there is no clinical resistancereport in HBV of TDF. TDF has not yet been approved for marketing in our country, and isnow in clinical trials. Host factors play an important role in the process of Chronic HBVinfection and therapy. Inflammatory cytokines TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10andIL-17A play important roles in this process. In recent years, with the development ofgenomics research, we find that single nucleotide polymorphism(SNP) of the host also playan important role in HBV infection and removal.But,immunology research related to HBVinfectIino nth iiss nsotut dcyle,awre. collected outpatients and hospitalized patients with CHB of the FirstHospital of Jilin University,and began screening test after signing informed consent，weselected24previously untreated patients with CHB that giving them nucleoside andnucleotide analogues, including HBeAg-positive or-negative cases (HBVDNA in≥105copies/ml, an interval of14days or more than twice elevated ALT of HBeAg positivepatients≥2ULN(upper limit of normal); HBeAg-negative patients>1ULN and≤10ULN),used randomized, double-blind, double-dummy, active drug controlled study, randomly divided into tenofovir disoproxil fumarate300mg/d treatment group, or adefovirdipivoxil10mg/d treatment one according to1:1, and detected liver function, hepatitis Bvirus markers and HBV DNA quantitative in the treatment of baseline and after4weeks,8weeks,12weeks,24weeks treatment; Compared the response of biochemical andvirological and cytokines in the dynamic changes after treatment, analyzed theirrelevance;Simultaneously determined IL-10SNPs (rs1800896, rs1800871, rs1800872) of the24patients, and analyzed the relevance with antiviral treatment and the dynamic changes ofcytokines.
In this study, we used three-color method of flow cytometry,and adopted the method ofmicro-array flow cytometry (CBA) and pyrosequencing technology to do the followingwork:
(1)Observe the response of biochemical and virological for the treatment of chronichepatitis B patients with tenofovir disoproxil fumarate after4weeks,8weeks,12weeks, andcompared with adefovir dipivoxil.
(2) Dynamicly observed the serum concentrations of cytokines for the treatment ofchronic hepatitis B patients with tenofovir disoproxil fumarate after4weeks,8weeks,12weeks,24weeks, including the cytokines TNF-α,IFN-γ, IL-2, IL-4, IL-6, IL-10and IL-17A,to analyze the variation; and compared with adefovir dipivoxil.
(3)In the chronic hepatitis B patients with tenofovir disoproxil and adefovir dipivoxiltreatment, human IL-10SNPs (rs1800896, rs1800871, rs1800872) loci were detected, toanalyzed the correlation of IL-10SNPs and the antiviral efficacy and the relationshipbetween IL-10SNPs and cytokine changes.
The results are as follows:
(1) In patients with chronic hepatitis B with tenofovir disoproxil treatment, ALT, ASTand HBV DNA were markedly improved, and HBV DNA quantity decreased dramaticallyfrom baseline in4weeks,8weeks,12weeks,24weeks treatment(P <0.05).
Significant differences of the levels of HBV DNA exists between0weeks and12weeks,0weeks and24weeks (p <0.05).The above indicators in tenofovir disoproxil treatmentgroup compared with the adefovir dipivoxil group have the same changing tendency.In sevenHBeAg positive patients with24weeks treatment of tenofovir disoproxil, three casesachieve HBeAg seroconversion. However, no HBeAg seroconversion occurred in8patientswith adefovir dipivoxil treatment.
(2) In the chronic hepatitis B patients with tenofovir disoproxil treatment, thedeclination of serum IL-10levels in12weeks,24weeks compared with baseline is statisticalsignificance (p <0.05). The similar significant changes exist between4weeks and12weeks,8weeks and12weeks,12weeks and24weeks(p <0.05).Serum IL-10levels are negativelyrelated with the declination of HBV DNA quantity（p <0.05）.After24weeks treatment, thelevel of IL-17A in serum decreased clearly compared with the baseline（p <0.05）.The levelsof IFN-γ and TNF-α, IL-2, IL-4, IL-6in serum showed no obviously change in the processof treatment. For the other hand, in the patients of chronic hepatitis B with adefovir dipivoxiltreatment,the same declination occurred as the tenofovir disoproxil treatment group; theother six cytokines showed no significant change.
(3) IL-10SNPs（rs1800896、rs1800871、rs1800872）have little correlation with theantiviral efficacies of the two kinds of medicine,as well as the levels of IL-10,IL-17A（p＞0.05）.
Changes of the cytokines level in the tenofovir disoproxil antiviral treatment processsuggest that immunomodulatory may play a role in the inhibition of viral after using themedicine. Virological response analysis showed the association between the levels of serumcytokines IL-10and HBVDNA.Thus, it is considered that the levels of IL-10in serum areclosely related to the viral replication in the process of tenofovir disoproxil therapy. Thelevels of IL-10, IL-17A can be used as clinical indicators for monitoring the course of theearly stages of the treatment, to evaluate prognosis and the disease outcome in patients withchronic hepatitis B.
The fluctuation of cytokines secreted by Th1/Th2/Th17cells were obtained by dynamicmonitoring of the chronic hepatitis B patients with antiviral treatment, which is significant toelucidate the pathogenesis of chronic hepatitis B and to guide clinical treatment.
引文
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