替比夫定对慢乙肝患者TCR CDR3谱型的影响
发布时间：2018-08-12 09:22
【摘要】：目的近年来研究表明慢性乙型肝炎(CHB)的进展及肝癌的发生与乙型肝炎病毒(HBV)的复制存在明显相关性。在此基础上,抗病毒治疗已经逐步成为慢乙肝的主要治疗手段。替比夫定是新近上市的一种核苷类似物药物,国内外三期临床试验结果表明,除了提示其较强的病毒抑制作用外,同时显现出该药有较高的乙肝e抗原(HBeAg)血清转换率,尤其是对于丙氨酸氨基转移酶(ALT)≥2倍的慢性乙型肝炎患者,2年的HBeAg血清学转换率明显高于其他核苷类药物。这一结果提示替比夫定治疗过程中有可能影响到机体免疫系统,但确切的机制尚不清晰。有关动物实验提示替比夫定不但可增加感染鼠肝炎病毒大鼠的血中干扰素水平,同时可减少白细胞介素4(IL-4)分泌。但是替比夫定对人体免疫系统的影响机制报道仍较少。本研究试图利用T细胞受体谱型分析技术(即TCR CDR3谱型分析技术),对替比夫定治疗慢性乙型病毒性肝炎患者的细胞免疫状况进行探讨,以了解替比夫定对免疫系统的影响机制。 方法(1)选取在我院感染内科服用替比夫定治疗慢性乙肝患者15例作为研究对象,分别采集服药前、服药6个月的外周血作研究,以健康献血员作对照(排除感染性、肿瘤、自身免疫性疾病等)；(2)入选患者同期检测治疗前后乙型肝炎病毒(HBV DNA)学指标,检测出血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)等肝功能指标；进行乙肝五项定量测定,分析乙肝表面抗原(HBsAg)滴度及乙肝e抗原滴度(HBeAg)等变化情况；对于该指标变化幅度与HBV DNA变化进行相关性分析；(3)各研究对象每次采集外周血6m1,采用密度梯度离心法分离出外周血单核细胞(PBMC),细胞分离,采用TRIZOL试剂提取RNA,互补DNA(cDNA)的合成,PCR扩增；(4)GeneScan扫描分析TCR CDR3谱形。 结果15例慢乙肝患者HBV-DNA定量(经对数转换后)水平治疗前、治疗6个月分别为：6.67+0.92、2.76±0.39,与治疗前相比治疗6个月时数值明显下降(P0.01)；乙肝表面抗原(HBsAg)滴度治疗前后分别为：25181±9835、11917±6453,与治疗前相比,治疗6个月明显下降(P0.01)；乙肝e抗原(HBeAg)滴度治疗前后分别为：736±422、456±346,与治疗前相比,治疗6个月明显下降(P0.01)；肝功能ALT治疗前、治疗6个月分别为：203.15±177.92、46.06±22.92,与治疗前相比,治疗6个月明显下降(P0.01)；肝功能AST治疗前、治疗6个月分别为：166.54±156.50、40.20±15.96,与治疗前相比,治疗6个月明显下降(P0.01)；患者抗病毒治疗6个月时血清HBsAg滴度下降幅度与HBV-DNA下降水平之间有明显直线相关关系,经Pearson相关性检验得出,r=0.910,P=0.000；抗病毒治疗6个月后血清HBeAg滴度下降幅度大的患者,血清HBV-DNA水平下降幅度也较大,呈正相关关系,因数据不符合正态分布,采用Spearman相关分析显示,两者之间的相关系数r=0.636,P=0.011。15患者中,完成10例T细胞受体CDR3谱型分析,10个病例治疗前后外周血TCRβ链V区CDR3谱型均或多或少出现了异常谱型；统计各病例抗病毒治疗前后出现异常谱型的家族总数,10例患者在替比夫定治疗前TCR CDR324个家族中出现异常谱型的家族总数平均值为5.40±2.07,而治疗后出现异常谱型的家族总数平均值为5.20±1.81；治疗前后出现异常谱型的家族总数经配对t检验分析,结果显示差异无统计学意义,t=0.238,P=0.817,但具体每例患者,24个BV家族中均有明显的谱型改变现象。 结论 1.替比夫定的确是一个较强的抗乙肝病毒的药物,本实验入组慢乙肝忠者服用替比夫定抗病毒后,血清学指标发生明显变化,升高的ALT下降至正常,多数患者HBV DNA水平降至检测线之下； 2.同时检测到HBsAg滴度、HBeAg滴度下降明显,HBsAg滴度下降幅度与HBV-DNA下降水平之间有明显直线相关关系,HBeAg滴度下降幅度与血清HBV-DNA水平下降幅度呈正相关关系。15例患者中3例出现了E抗原学转换。这些结果均提示替比夫定对患者抗乙肝的免疫状况造成了影响； 3.对10例患者在替比夫定治疗前后外周血T淋巴细胞TCR CDR324个家族中异常谱型的分析,进一步提示替比夫定在抑制HBV的同时,直接影响了慢性乙型肝炎患者的细胞免疫功能。
[Abstract]:OBJECTIVE In recent years, studies have shown that the progress of chronic hepatitis B (CHB) and the occurrence of hepatocellular carcinoma are significantly related to the replication of hepatitis B virus (HBV). On this basis, antiviral therapy has gradually become the main treatment for chronic hepatitis B. The results showed that, in addition to suggesting a strong viral inhibitory effect, the drug also showed a higher seroconversion rate of hepatitis B e antigen (HBeAg), especially for patients with chronic hepatitis B whose ALT was more than 2 times, the seroconversion rate of HBeAg in 2 years was significantly higher than that of other nucleoside drugs. Tibivudine may affect the immune system, but the exact mechanism is not clear. Animal experiments suggest that Tibivudine can not only increase the level of interferon in the blood of rats infected with rat hepatitis virus, but also reduce the secretion of interleukin-4 (IL-4). However, the mechanism of Tibivudine's influence on the human immune system is still relatively reported. In this study, we attempted to explore the cellular immune status of patients with chronic viral hepatitis B treated with telbivudine by T cell receptor profiling (TCR CDR3 profiling), in order to understand the mechanism of telbivudine on the immune system.
Methods (1) Fifteen patients with chronic hepatitis B were selected as the subjects, and their peripheral blood samples were collected for 6 months before and after treatment, and healthy blood donors as controls (excluding infections, tumors, autoimmune diseases, etc.); (2) HBV (hepatitis B virus) was detected in selected patients before and after treatment. DNA indicators, detection of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other liver function indicators; quantitative determination of hepatitis B, analysis of hepatitis B surface antigen (HBsAg) titer and hepatitis B e antigen titer (HBeAg) and other changes in the extent of change in this index and HBV DNA changes were analyzed. (3) Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, RNA was extracted by TRIZOL reagent, complementary DNA (cDNA) was synthesized and amplified by PCR. (4) GeneScan scan was used to analyze the spectrum of TCR CDR3.
Results The levels of HBV-DNA in 15 patients with chronic hepatitis B before and after treatment were 6.67+0.92,2.76+0.39 for 6 months, which were significantly lower than those before treatment (P Hepatitis B e antigen (HBeAg) titer decreased significantly (P 0.01), hepatitis B e antigen (HBeAg) titer decreased significantly (P 0.01) before and after treatment (P 0.01) and 6 months after treatment (P 0.01) as compared with before treatment. There was a significant linear correlation between the decrease of HBsAg titer and the decrease of HBV-DNA level at 6 months after antiviral treatment. Pearson correlation test showed that r = 0.910, P = 0.000; after 6 months of antiviral treatment, serum HBsAg titer decreased significantly (P 0.01). The serum HBV-DNA level decreased significantly in patients with large eAg titer decrease, and was positively correlated. Because the data did not conform to normal distribution, Spearman correlation analysis showed that the correlation coefficient between the two was r=0.636, P=0.011.15. The CDR3 pattern of T cell receptor was analyzed in 10 patients before and after treatment. The C region of TCR beta chain V in 10 patients was analyzed before and after treatment. The total number of families with abnormal patterns of DR3 was more or less recorded before and after antiviral treatment. The average number of families with abnormal patterns of TCR CDR 324 families in 10 patients before and after telbivudine treatment was 5.40 (+ 2.07), while the average number of families with abnormal patterns after treatment was 5.20 (+ 1.81). The total number of families with abnormal spectra before and after treatment showed no significant difference by paired t-test analysis, t = 0.238, P = 0.817, but in each patient, 24 BV families had significant spectral changes.
conclusion
1. Telbivudine is indeed a strong anti-HBV drug. After taking Telbivudine, the serum parameters of the patients with chronic hepatitis B in this study changed significantly. The elevated ALT decreased to normal, and the level of HBV DNA in most patients dropped below the detection line.
2. HBsAg titer was detected at the same time, HBeAg titer decreased significantly. There was a linear correlation between HBsAg titer decrease and HBV-DNA level. There was a positive correlation between HBeAg titer decrease and serum HBV-DNA level decrease. The immune status of hepatitis B has been affected.
3. The analysis of the abnormal spectrum of TCR CDR 324 families in peripheral blood T lymphocytes of 10 patients before and after the treatment with tibivudine further indicated that tibivudine could inhibit HBV and directly affect the cellular immune function of patients with chronic hepatitis B.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.62
本文编号：2178627
[Abstract]:OBJECTIVE In recent years, studies have shown that the progress of chronic hepatitis B (CHB) and the occurrence of hepatocellular carcinoma are significantly related to the replication of hepatitis B virus (HBV). On this basis, antiviral therapy has gradually become the main treatment for chronic hepatitis B. The results showed that, in addition to suggesting a strong viral inhibitory effect, the drug also showed a higher seroconversion rate of hepatitis B e antigen (HBeAg), especially for patients with chronic hepatitis B whose ALT was more than 2 times, the seroconversion rate of HBeAg in 2 years was significantly higher than that of other nucleoside drugs. Tibivudine may affect the immune system, but the exact mechanism is not clear. Animal experiments suggest that Tibivudine can not only increase the level of interferon in the blood of rats infected with rat hepatitis virus, but also reduce the secretion of interleukin-4 (IL-4). However, the mechanism of Tibivudine's influence on the human immune system is still relatively reported. In this study, we attempted to explore the cellular immune status of patients with chronic viral hepatitis B treated with telbivudine by T cell receptor profiling (TCR CDR3 profiling), in order to understand the mechanism of telbivudine on the immune system.
Methods (1) Fifteen patients with chronic hepatitis B were selected as the subjects, and their peripheral blood samples were collected for 6 months before and after treatment, and healthy blood donors as controls (excluding infections, tumors, autoimmune diseases, etc.); (2) HBV (hepatitis B virus) was detected in selected patients before and after treatment. DNA indicators, detection of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other liver function indicators; quantitative determination of hepatitis B, analysis of hepatitis B surface antigen (HBsAg) titer and hepatitis B e antigen titer (HBeAg) and other changes in the extent of change in this index and HBV DNA changes were analyzed. (3) Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, RNA was extracted by TRIZOL reagent, complementary DNA (cDNA) was synthesized and amplified by PCR. (4) GeneScan scan was used to analyze the spectrum of TCR CDR3.
Results The levels of HBV-DNA in 15 patients with chronic hepatitis B before and after treatment were 6.67+0.92,2.76+0.39 for 6 months, which were significantly lower than those before treatment (P Hepatitis B e antigen (HBeAg) titer decreased significantly (P 0.01), hepatitis B e antigen (HBeAg) titer decreased significantly (P 0.01) before and after treatment (P 0.01) and 6 months after treatment (P 0.01) as compared with before treatment. There was a significant linear correlation between the decrease of HBsAg titer and the decrease of HBV-DNA level at 6 months after antiviral treatment. Pearson correlation test showed that r = 0.910, P = 0.000; after 6 months of antiviral treatment, serum HBsAg titer decreased significantly (P 0.01). The serum HBV-DNA level decreased significantly in patients with large eAg titer decrease, and was positively correlated. Because the data did not conform to normal distribution, Spearman correlation analysis showed that the correlation coefficient between the two was r=0.636, P=0.011.15. The CDR3 pattern of T cell receptor was analyzed in 10 patients before and after treatment. The C region of TCR beta chain V in 10 patients was analyzed before and after treatment. The total number of families with abnormal patterns of DR3 was more or less recorded before and after antiviral treatment. The average number of families with abnormal patterns of TCR CDR 324 families in 10 patients before and after telbivudine treatment was 5.40 (+ 2.07), while the average number of families with abnormal patterns after treatment was 5.20 (+ 1.81). The total number of families with abnormal spectra before and after treatment showed no significant difference by paired t-test analysis, t = 0.238, P = 0.817, but in each patient, 24 BV families had significant spectral changes.
conclusion
1. Telbivudine is indeed a strong anti-HBV drug. After taking Telbivudine, the serum parameters of the patients with chronic hepatitis B in this study changed significantly. The elevated ALT decreased to normal, and the level of HBV DNA in most patients dropped below the detection line.
2. HBsAg titer was detected at the same time, HBeAg titer decreased significantly. There was a linear correlation between HBsAg titer decrease and HBV-DNA level. There was a positive correlation between HBeAg titer decrease and serum HBV-DNA level decrease. The immune status of hepatitis B has been affected.
3. The analysis of the abnormal spectrum of TCR CDR 324 families in peripheral blood T lymphocytes of 10 patients before and after the treatment with tibivudine further indicated that tibivudine could inhibit HBV and directly affect the cellular immune function of patients with chronic hepatitis B.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.62
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