替比夫定對慢乙肝患者TCR CDR3譜型的影響
發(fā)布時間：2018-08-12 09:22
【摘要】：目的近年來研究表明慢性乙型肝炎(CHB)的進展及肝癌的發(fā)生與乙型肝炎病毒(HBV)的復(fù)制存在明顯相關(guān)性。在此基礎(chǔ)上,抗病毒治療已經(jīng)逐步成為慢乙肝的主要治療手段。替比夫定是新近上市的一種核苷類似物藥物,國內(nèi)外三期臨床試驗結(jié)果表明,除了提示其較強的病毒抑制作用外,同時顯現(xiàn)出該藥有較高的乙肝e抗原(HBeAg)血清轉(zhuǎn)換率,尤其是對于丙氨酸氨基轉(zhuǎn)移酶(ALT)≥2倍的慢性乙型肝炎患者,2年的HBeAg血清學(xué)轉(zhuǎn)換率明顯高于其他核苷類藥物。這一結(jié)果提示替比夫定治療過程中有可能影響到機體免疫系統(tǒng),但確切的機制尚不清晰。有關(guān)動物實驗提示替比夫定不但可增加感染鼠肝炎病毒大鼠的血中干擾素水平,同時可減少白細(xì)胞介素4(IL-4)分泌。但是替比夫定對人體免疫系統(tǒng)的影響機制報道仍較少。本研究試圖利用T細(xì)胞受體譜型分析技術(shù)(即TCR CDR3譜型分析技術(shù)),對替比夫定治療慢性乙型病毒性肝炎患者的細(xì)胞免疫狀況進行探討,以了解替比夫定對免疫系統(tǒng)的影響機制。 方法(1)選取在我院感染內(nèi)科服用替比夫定治療慢性乙肝患者15例作為研究對象,分別采集服藥前、服藥6個月的外周血作研究,以健康獻血員作對照(排除感染性、腫瘤、自身免疫性疾病等)；(2)入選患者同期檢測治療前后乙型肝炎病毒(HBV DNA)學(xué)指標(biāo),檢測出血清中丙氨酸氨基轉(zhuǎn)移酶(ALT)、天門冬氨酸氨基轉(zhuǎn)移酶(AST)等肝功能指標(biāo)；進行乙肝五項定量測定,分析乙肝表面抗原(HBsAg)滴度及乙肝e抗原滴度(HBeAg)等變化情況；對于該指標(biāo)變化幅度與HBV DNA變化進行相關(guān)性分析；(3)各研究對象每次采集外周血6m1,采用密度梯度離心法分離出外周血單核細(xì)胞(PBMC),細(xì)胞分離,采用TRIZOL試劑提取RNA,互補DNA(cDNA)的合成,PCR擴增；(4)GeneScan掃描分析TCR CDR3譜形。 結(jié)果15例慢乙肝患者HBV-DNA定量(經(jīng)對數(shù)轉(zhuǎn)換后)水平治療前、治療6個月分別為：6.67+0.92、2.76±0.39,與治療前相比治療6個月時數(shù)值明顯下降(P0.01)；乙肝表面抗原(HBsAg)滴度治療前后分別為：25181±9835、11917±6453,與治療前相比,治療6個月明顯下降(P0.01)；乙肝e抗原(HBeAg)滴度治療前后分別為：736±422、456±346,與治療前相比,治療6個月明顯下降(P0.01)；肝功能ALT治療前、治療6個月分別為：203.15±177.92、46.06±22.92,與治療前相比,治療6個月明顯下降(P0.01)；肝功能AST治療前、治療6個月分別為：166.54±156.50、40.20±15.96,與治療前相比,治療6個月明顯下降(P0.01)；患者抗病毒治療6個月時血清HBsAg滴度下降幅度與HBV-DNA下降水平之間有明顯直線相關(guān)關(guān)系,經(jīng)Pearson相關(guān)性檢驗得出,r=0.910,P=0.000；抗病毒治療6個月后血清HBeAg滴度下降幅度大的患者,血清HBV-DNA水平下降幅度也較大,呈正相關(guān)關(guān)系,因數(shù)據(jù)不符合正態(tài)分布,采用Spearman相關(guān)分析顯示,兩者之間的相關(guān)系數(shù)r=0.636,P=0.011。15患者中,完成10例T細(xì)胞受體CDR3譜型分析,10個病例治療前后外周血TCRβ鏈V區(qū)CDR3譜型均或多或少出現(xiàn)了異常譜型；統(tǒng)計各病例抗病毒治療前后出現(xiàn)異常譜型的家族總數(shù),10例患者在替比夫定治療前TCR CDR324個家族中出現(xiàn)異常譜型的家族總數(shù)平均值為5.40±2.07,而治療后出現(xiàn)異常譜型的家族總數(shù)平均值為5.20±1.81；治療前后出現(xiàn)異常譜型的家族總數(shù)經(jīng)配對t檢驗分析,結(jié)果顯示差異無統(tǒng)計學(xué)意義,t=0.238,P=0.817,但具體每例患者,24個BV家族中均有明顯的譜型改變現(xiàn)象。 結(jié)論 1.替比夫定的確是一個較強的抗乙肝病毒的藥物,本實驗入組慢乙肝忠者服用替比夫定抗病毒后,血清學(xué)指標(biāo)發(fā)生明顯變化,升高的ALT下降至正常,多數(shù)患者HBV DNA水平降至檢測線之下； 2.同時檢測到HBsAg滴度、HBeAg滴度下降明顯,HBsAg滴度下降幅度與HBV-DNA下降水平之間有明顯直線相關(guān)關(guān)系,HBeAg滴度下降幅度與血清HBV-DNA水平下降幅度呈正相關(guān)關(guān)系。15例患者中3例出現(xiàn)了E抗原學(xué)轉(zhuǎn)換。這些結(jié)果均提示替比夫定對患者抗乙肝的免疫狀況造成了影響； 3.對10例患者在替比夫定治療前后外周血T淋巴細(xì)胞TCR CDR324個家族中異常譜型的分析,進一步提示替比夫定在抑制HBV的同時,直接影響了慢性乙型肝炎患者的細(xì)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.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R512.62
本文編號：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.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R512.62
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