HBV相关肝病肝移植受体术后外周免疫细胞及骨髓造血干祖细胞HBV存在状态检测及临床意义分析
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摘要
肝移植(liver transplantation，LT)是目前治疗终末期肝病(end stage liver disease，ESLD)的有效手段。在我国，乙肝病毒(hepatitis B virus，HBV)相关肝病已成为肝移植的主要适应症。但是肝移植术后，即使在使用拉米夫定(Lamivudine，LAM)和乙肝免疫球蛋白的情况下，仍有较高的HBV再感染率和乙肝(hepatitis B，HB)复发率，严重影响了肝移植的中远期效果。本课题从研究乙肝病毒相关肝病肝移植术后的免疫-造血系统乙肝病毒库入手，揭示肝移植术后HBV再感染/HB复发的可能机制。
在第一部分，我们建立了一种灵敏可靠的检测HBV共价闭合环状DNA(covalently closed circular DNA，cccDNA)的实时荧光定量聚合酶链式反应方法，为后继的研究打下了基础。在第二部分，我们利用密度梯度离心和免疫磁珠分离法获得骨髓造血干祖细胞和外周血单个核细胞(peripheral blood mononuclear cell，PBMC)、T细胞、B细胞和单核细胞(monocyte，MNC)，分别检测总HBV DNA和HBV cccDNA，证实了细胞内仍存在HBV，但未发现有复制的证据。在第三部分，我们利用特异性的HBV-Alu-PCR方法检测PBMC和骨髓造血干祖细胞内HBV X基因的整合情况，发现上述细胞内无HBV X基因的整合，证实HBV相关肝病肝移植受体术后的外周免疫细胞和骨髓造血干祖细胞不是HBV整合的适宜场所。
第一部分
酶辅助Taq man探针法实时荧光定量PCR检测HBV共价闭合环状DNA方法的建立
目的：建立灵敏可靠的实时荧光定量PCR方法，进行HBV cccDNA的检测。
方法：利用HBV松弛环状DNA(relaxed circular DNA，rcDNA)和cccDNA结构上的差异，参考现有文献，合成两对针对HBV cccDNA的引物，在有或者无绿豆核酸酶(Mung Bean Nuclease，MBN)辅助的情况下，利用不同浓度的从Dane颗粒中提取的HBV rcDNA和含HBV adr_4亚型全基因组的pBHB4质粒为模板，通过预实验选择最佳引物并确定MBN的最适用量，建立用于检测HBV cccDNA的酶辅助实时荧光定量PCR(real-time fluorescence quantitative PCR，RT-FQ PCR)方法。
结果：1．两对引物均可扩增cccDNA，也可扩增较高浓度的HBV rcDNA，即对HBV cccDNA的扩增仅具有相对特异性；2．MBN可特异性降解HBV rcDNA的单链部分，提高HBV cccDNA引物的特异性，其最适用量为5.0IU；3．跨越HBV rcDNA双链上两个缺口的引物对HBVcccDNA的特异性高于仅跨越一个缺口的引物；4．在MBN辅助下，利用跨越HBV rcDNA双链两个缺口的引物，建立了检测HBV cccDNA的Taq man探针RT-FQ PCR方法，其检测限为5.00×10~2～5.00×10~8copies/ml。
结论：用于HBV cccDNA检测的酶辅助RT-FQ PCR方法成功建立，灵敏度高，特异性好，能满足研究的需要。
第二部分
HBV相关肝病肝移植受体术后外周免疫细胞与骨髓CD_(34)~+细胞总HBV DNA及HBV cccDNA的检测与临床意义分析
目的：了解HBV相关肝病肝移植受体术后，在持续规范拉米夫定联合乙肝免疫球蛋白抗病毒治疗下，外周免疫细胞与骨髓造血干祖细胞内HBV含量及其复制状态，分析其临床意义。
方法：25例肝移植受体(男22，女3)术后均应用拉米夫定联合小剂量乙肝免疫球蛋白抗病毒治疗。单用FK_(506)抗排异者12例，单用CsA抗排异者13例。平均术后时间为33.32±14.81月(11～77月)，平均年龄为45.88±8.71岁(36～64岁)。术前非活跃复制者7例；HBV活跃复制者18例，其血清HBV DNA平均滴度为10~(5.954±1.457)copies/ml(5.23×10~3～8.26×10~7copies/ml)。采集25例HBV相关肝病肝移植受体的外周静脉血及其中23例的骨髓血，以密度梯度离心结合单克隆免疫磁珠分离法获取单个核细胞、单核细胞、T细胞、B细胞和骨髓造血干祖细胞(CD_(34)~+细胞)，提取细胞内DNA，以实时荧光定量PCR分别检测各种细胞内总HBV DNA和HBV cccDNA。同时采集血样检测血清HBV DNA定量、乙肝表面抗体浓度、乙肝标志物、血药浓度。
结果：25例Anti-HBs均为阳性，平均全血表面抗体浓度为119.1±90.7IU/L；所有受体的血清HBV抗原成分均为阴性：Anti-HBe和Anti-HBc都阳性者5例，Anti-HBe和Anti-HBc都阴性者6例；Anti-HBe阳性而Anti-HBc阴性者1例；Anti-HBe阴性而Anti-HBc阳性者13例。所有受体血清HBV DNA阴性。25例受体的PBMC、MNC(第9和第17例除外)、B(第8例除外)、T、CD_(34)~+细胞内总HBV DNA均呈阳性，平均浓度分别为10~(3.3263±0.6956)、10~(2.9425±0.6462)、10~(2.7213±0.6223)、10~(2.9522±0.9319)和10~(3.3373±0.6583)copies/10~6cells。方差分析表明不同细胞内HBV DAN含量有差异(F=3.302，P=0.013)，多因素分析表明主要的外周免疫细胞T细胞、B细胞和MNC内的HBV DNA含量与不完全相同的因素相关。所有细胞内及血清内均未检测到HBV cccDNA。
结论：HBV相关肝病肝移植术后，即使在较长的术后时间以及规范的抗病毒治疗之下，外周免疫细胞及骨髓造血干祖细胞(CD_(34)~+细胞)内均有HBV DNA低浓度存在，且受不全相同的因素影响。虽然未检测到细胞内HBV cccDNA，但外周免疫细胞与骨髓造血干祖细胞内的HBV DNA均持续阳性，不能排除外周免疫细胞和骨髓造血干祖细胞仍存在极低水平的HBV复制。在此情况下，不主张中止术后的抗病毒治疗。免疫细胞内HBV含量与免疫功能的关系也待深入研究。
第三部分
利用HBV-Alu-PCR检测HBV相关肝病肝移植受体术后外周血单个核细胞与骨髓造血干祖细胞(CD_(34)~+细胞)HBV X基因整合状态
目的：了解HBV X基因在HBV相关肝病肝移植受体术后外周免疫细胞和骨髓CD_(34)~+细胞内的整合情况及其对PBMC内HBV长期维持阳性的意义。
方法：病例选择及其基本临床资料同第二部分。获得其PBMC及骨髓CD_(34)~+细胞后，提取细胞DNA。因X基因的整合频率最高，设计HBV X基因的特异引物，利用HBV-Alu-PCR方法进行三轮半巢式PCR，终产物进行电泳并回收、连接载体、筛选扩增后测序，检测有无X基因整合。
结果：经PCR后电泳及测序分析，25例HBV相关肝病肝移植受体术后的PBMC内未检测出HBV X基因的整合，其中采集到骨髓标本的23例CD_(34)~+细胞中亦未检测到HBV X基因的整合。
结论：肝移植术后受体体内HBV微生态的剧烈改变，使HBV整合的基本条件丧失，在此情况下，外周免疫细胞及骨髓造血干祖细胞不是发生HBV整合的适宜场所。
Liver transplantation (LT) is a final and effective treatment for end stage liver diseases (ESLD) patients. In China, hepatitis B virus (HBV) related liver diseases have become the primary indication for LT. Eeven lamivudine (LAM) and hepatitis B immunoglobulin (HBIG) were combined administrated in clinic, the HBV graft reinfection and HB recurrence after LT remain still high, which severely leads a poor clinic outcomem of LT. This study aimes to research the status of a major extrahepatic HBV reservoir, namely Immume-Hematopoietic System, try to reveal the possible mechanisms of HBV reinfection and HB recurrence post-LT.
In part 1, a sensitive and credible real-time fluorescence polymerase chain reaction (RT-FQ-PCR) for HBV covalently closed circular DNA (cccDNA) detection was established. In part 2, peripheral blood mononuclear cells (PBMC), T lymphocytes, B lymphocytes, monocytes and bone marrow (BM) hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC) were isolated respectively by Ficoll-Hypaque density-gradient centrifugation combine magnetic cell sorting and separation (MACS). The intracellular and sera total HBV DNA and HBV cccDNA were detected by RT-FQ-PCR. The serological markers of HBV were detected simultaneously. It proved that no evidences of HBV replication and HBV X gene integration were found, but there are HBV DNA persisting positive in immunocytes, HSC and HPC, even undergoing LAM and 1BIG combined therapy for a long time after LT.
Part 1:
An enzyme assistant Taq man probe real-time fluorescence quantitative PCR detection method of HBV cccDNA
Objective: To establish a sensitive and specific real-time fluorescence quantitative PCR for HBV covalently closed circular DNA (HBV cccDNA) detection.
Methods: According to the differences in construction between HBV relaxed circular DNA (HBV rcDNA) and HBV cccDNA, two couples of primers for HBV cccDNA detection, cccF_1-R_1 and cccF_2-R_2, were synthesized basing on attainable literatures at present. Series dilutions of HBV rcDNA which extracted from Dane particles, and plasmid DNA which extracted from pBHB4 plasmid containing a monomer of the HBV adr_4 subtype genome, were using as the templates respectively. Before and after treated by different dosage single-strand-specific mung bean nuclease (MBN), rcDNA and cccDNA were amplified by two couples of primers for HBV cccDNA respectively. The products of PCR were analyzed by 1% agarose gel electrophoresis. The optimal primers of HBV cccDNA detection and the optimum dosage of MBN were chosen for detection of HBV cccDNA, according to the results of PCR and electrophoresis.
Results: 1. cccF_1-R_1 and cccF_2-R_2 can amplify not only cccDNA, but also HBV rcDNA when the titer was more than 1.92×10~6 copies/ml and 1.92×10~5 copies/ml respectively. In other words, the two couples of primers just possess the relative specificity for HBV cccDNA. 2. The specificity of cccF_1-R_1 excels cccF_2-R_2, because the former flanks the nick in minus strand and the gap in plus strand of HBV rcDNA but the latter only flanks the nick in minus strand. 3. MBN can promote the specificity of cccDNA primers by digesting selectively the single strand DNA present only in HBV rcDNA. The optimum dosage of MBN was 5.0IU. 4. Using ccc F_1-R_1, Taq man probe and MBN, a sensitive and specific quantification method of HBV cccDNA detection was established, with a detective range of 5.00×10~2～5.00×10~8 copies/ml.
Conclusions: A sensitive and specific Taq man probe real-time FQ-PCR for HBV cccDNA quantitative detection, assisted by mung bean nuclcasc, has been established successfully, which is content with the requests of our follow research.
Part 2:
Quantitative detection and clinical significance of total HBV DNA and HBV cccDNA in peripheral immunocytes and bone marrow CD_(34)~+ cells from HBV related liver disease patients after liver transplantation undergoing combined prophylaxis of LAM and HBIG
Objective: The long-term clinical outcomes of liver transplantation (LT) had been reduced by HBV graft reinfection/HB recurrence. Peripheral blood mononuclear cell (PBMC), in which HBV DNA can be persistent positive, were considered the major source of HBV graft reinfection/HB recurrence post-LT. But the half-life time of PBMC is limited in some weeks to months. Undergoing the combined prophylaxis protocol of lamivudine (LAM) and hepatitis B immunoglobulin (HBIG), the HBV infected PBMC would be cleared thoroughly step by step after LT. In order to clarify why HBV DNA can persistent in PBMC for long time post liver transplantation, this study was carried out.
Methods: 25 HBV related LT recipients (22 males and 3 females) were selected, whose postoperative time were ranged from 11 to 77 months (33.32±14.81 months) and age were from 33 to 61 years old (43.32±8.76 years). The combined prophylaxis protocol of LAM and HBIG has been chosen to prevent HBV graft re-infection/HB recurrence. 7 case were negative for serum HBV DNA (＜10~3 copies/ml) prior to operation and 18 were positive with a mean trier of 10~(5.954±1.457) copies/ml. In the study, 20ml peripheral vein blood of every case was collected and 23 samples of 20ml bone marrow were obtained from 23 of 25 cases with written content. PBMC, T, B, monocyte (MNC) and CD_(34)~+ cell were isolated respectively by Ficoll-Hypaque density-gadient centrifugation and magnetic cell sorting and separation (MACS). The samples of serum were collected simultaneously. The quantification of anti-HBs was performed using electrochemi-luminescence immunoassay (ECLIA). The detection of HBV markers was performed using enzyme linked immunosorbent assay (ELISA). The cellular DNA was extracted by DNA isolation and purification kit (Watsonbiot, Shanghai China) following the manufacture's instructions. The titer of HBV DNA and HBV covalently closed circular DNA (cccDNA) in serum, PBMCs, T, B lymphocytes, MNCs and CD_(34)~+ cells, including hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), were detected using real-time fluorescence quantitative polymerase chain reaction (RT-FQ-PCR).
Results: 1.25 cases are all positive for anti-HBs with a mean serum anti-HBs titer of 119.1±90.7IU/L, ranged from 12.37 to 372.6IU/L and the serum HBV antigens of all cases are negative. Anti-HBe and anti-HBc are positive in 5 cases; Anti-HBe and Anti-HBc are negative in 6 cases; Anti-HBe is positive and Anti-HBc is negative in 1 case; Anti-HBe is negative and Anti-HBc is positive in 13 cases. 2. All serum HBV DNA is negative in 25 cases. 3. Not only immunocytes including PBMC, MNCs and B, T lymphocytes, but also CD_(34)~+ cells are all positive for HBV DNA, with geometric mean titer of 10~(3.3263±0.6956), 10~(2.9425±0.6462), 10~(2.7213±0.6223), 10~(2.9522±0.932), 10~(3.3373±0.6583) copies per 10~6 cells respectively. And the distribution of HBV DNA in different subset cells is diversity according to the result of ANOVA. The influence factors of HBV DNA titer in different subset cells are also diversity according to the results of regression and correlation analysis. 4. There is no evidence of HBV replication in CD_(34)~+ cells and immunocytes (no HBV cccDNA were detected in cells).
Conclusions: Even combined prophylaxis of lamivudine and hepatitis B immunoglobulin after LT, the HBV related recipients are still HBV DNA positive in peripheral immunocytes and bone marrow CD_(34)~+ cells influenced by complicated factors. Because there is no HBV cccDNA detcted in immunocytes, HBV DNA positive in CD_(34)~+ cells may be a key factor of HBV DNA persisting in immunocytes, especially to lymphomononuclears, which may lead nonresponse to HBV vaccine and HBV graft reinfection/HB recurrence post operatively. But the mechanism of HBV transmission intercellular and the effect of intracellular HBV DNA titer on immune system have not been clarified. Further studies are needed to gain a better understanding on HBV graft reinfection.
Part 3:
Detection of HBV X gene integration in PBMC and bone marrow CD_(34)~+ cells from HBV related liver disease patients using HBV-Alu-PCR after liver transplantation
Objective: To clarify whether HBV X gene integrate in PBMC and bone marrow CD_(34)~+ cells from HBV related liver disease patients after liver transplantation.
Methods: PBMCs were obtained from 25 HBV related liver disease patients after liver transplantation and bone marrow CD_(34)~+ cells obtained from 23 cases among them. The cellular DNA was extracted by DNA isolation and purification kit (Watsonbiot, Shanghai China) following the manufacture's instructions. Specific primers to HBV X gene and to human Alu repeats were used to amplify the virus integration through a 3-round hemi-nest PCR. The PCR final product was judged by 1.2% agarose electrophoresis, ligated to T vector, proliferated in E. coil 5αand sequenced.
Results: According to agarose electrophoresis and sequencing analysis, there were no HBV X gene integration in PBMCs and bone marrow CD_(34)~+ cells from HBV related liver transplant recipients after surgery.
Conclusions: Because of the radical change of HBV bionomy in HBV related liver transplant recipients after operation, the fundamental condition of HBV integration has been lost, which led peripheral immunocytes and bone marrow CD_(34)~+ cells not suit to HBV integrate to human genome.
在第一部分，我们建立了一种灵敏可靠的检测HBV共价闭合环状DNA(covalently closed circular DNA，cccDNA)的实时荧光定量聚合酶链式反应方法，为后继的研究打下了基础。在第二部分，我们利用密度梯度离心和免疫磁珠分离法获得骨髓造血干祖细胞和外周血单个核细胞(peripheral blood mononuclear cell，PBMC)、T细胞、B细胞和单核细胞(monocyte，MNC)，分别检测总HBV DNA和HBV cccDNA，证实了细胞内仍存在HBV，但未发现有复制的证据。在第三部分，我们利用特异性的HBV-Alu-PCR方法检测PBMC和骨髓造血干祖细胞内HBV X基因的整合情况，发现上述细胞内无HBV X基因的整合，证实HBV相关肝病肝移植受体术后的外周免疫细胞和骨髓造血干祖细胞不是HBV整合的适宜场所。
第一部分
酶辅助Taq man探针法实时荧光定量PCR检测HBV共价闭合环状DNA方法的建立
目的：建立灵敏可靠的实时荧光定量PCR方法，进行HBV cccDNA的检测。
方法：利用HBV松弛环状DNA(relaxed circular DNA，rcDNA)和cccDNA结构上的差异，参考现有文献，合成两对针对HBV cccDNA的引物，在有或者无绿豆核酸酶(Mung Bean Nuclease，MBN)辅助的情况下，利用不同浓度的从Dane颗粒中提取的HBV rcDNA和含HBV adr_4亚型全基因组的pBHB4质粒为模板，通过预实验选择最佳引物并确定MBN的最适用量，建立用于检测HBV cccDNA的酶辅助实时荧光定量PCR(real-time fluorescence quantitative PCR，RT-FQ PCR)方法。
结果：1．两对引物均可扩增cccDNA，也可扩增较高浓度的HBV rcDNA，即对HBV cccDNA的扩增仅具有相对特异性；2．MBN可特异性降解HBV rcDNA的单链部分，提高HBV cccDNA引物的特异性，其最适用量为5.0IU；3．跨越HBV rcDNA双链上两个缺口的引物对HBVcccDNA的特异性高于仅跨越一个缺口的引物；4．在MBN辅助下，利用跨越HBV rcDNA双链两个缺口的引物，建立了检测HBV cccDNA的Taq man探针RT-FQ PCR方法，其检测限为5.00×10~2～5.00×10~8copies/ml。
结论：用于HBV cccDNA检测的酶辅助RT-FQ PCR方法成功建立，灵敏度高，特异性好，能满足研究的需要。
第二部分
HBV相关肝病肝移植受体术后外周免疫细胞与骨髓CD_(34)~+细胞总HBV DNA及HBV cccDNA的检测与临床意义分析
目的：了解HBV相关肝病肝移植受体术后，在持续规范拉米夫定联合乙肝免疫球蛋白抗病毒治疗下，外周免疫细胞与骨髓造血干祖细胞内HBV含量及其复制状态，分析其临床意义。
方法：25例肝移植受体(男22，女3)术后均应用拉米夫定联合小剂量乙肝免疫球蛋白抗病毒治疗。单用FK_(506)抗排异者12例，单用CsA抗排异者13例。平均术后时间为33.32±14.81月(11～77月)，平均年龄为45.88±8.71岁(36～64岁)。术前非活跃复制者7例；HBV活跃复制者18例，其血清HBV DNA平均滴度为10~(5.954±1.457)copies/ml(5.23×10~3～8.26×10~7copies/ml)。采集25例HBV相关肝病肝移植受体的外周静脉血及其中23例的骨髓血，以密度梯度离心结合单克隆免疫磁珠分离法获取单个核细胞、单核细胞、T细胞、B细胞和骨髓造血干祖细胞(CD_(34)~+细胞)，提取细胞内DNA，以实时荧光定量PCR分别检测各种细胞内总HBV DNA和HBV cccDNA。同时采集血样检测血清HBV DNA定量、乙肝表面抗体浓度、乙肝标志物、血药浓度。
结果：25例Anti-HBs均为阳性，平均全血表面抗体浓度为119.1±90.7IU/L；所有受体的血清HBV抗原成分均为阴性：Anti-HBe和Anti-HBc都阳性者5例，Anti-HBe和Anti-HBc都阴性者6例；Anti-HBe阳性而Anti-HBc阴性者1例；Anti-HBe阴性而Anti-HBc阳性者13例。所有受体血清HBV DNA阴性。25例受体的PBMC、MNC(第9和第17例除外)、B(第8例除外)、T、CD_(34)~+细胞内总HBV DNA均呈阳性，平均浓度分别为10~(3.3263±0.6956)、10~(2.9425±0.6462)、10~(2.7213±0.6223)、10~(2.9522±0.9319)和10~(3.3373±0.6583)copies/10~6cells。方差分析表明不同细胞内HBV DAN含量有差异(F=3.302，P=0.013)，多因素分析表明主要的外周免疫细胞T细胞、B细胞和MNC内的HBV DNA含量与不完全相同的因素相关。所有细胞内及血清内均未检测到HBV cccDNA。
结论：HBV相关肝病肝移植术后，即使在较长的术后时间以及规范的抗病毒治疗之下，外周免疫细胞及骨髓造血干祖细胞(CD_(34)~+细胞)内均有HBV DNA低浓度存在，且受不全相同的因素影响。虽然未检测到细胞内HBV cccDNA，但外周免疫细胞与骨髓造血干祖细胞内的HBV DNA均持续阳性，不能排除外周免疫细胞和骨髓造血干祖细胞仍存在极低水平的HBV复制。在此情况下，不主张中止术后的抗病毒治疗。免疫细胞内HBV含量与免疫功能的关系也待深入研究。
第三部分
利用HBV-Alu-PCR检测HBV相关肝病肝移植受体术后外周血单个核细胞与骨髓造血干祖细胞(CD_(34)~+细胞)HBV X基因整合状态
目的：了解HBV X基因在HBV相关肝病肝移植受体术后外周免疫细胞和骨髓CD_(34)~+细胞内的整合情况及其对PBMC内HBV长期维持阳性的意义。
方法：病例选择及其基本临床资料同第二部分。获得其PBMC及骨髓CD_(34)~+细胞后，提取细胞DNA。因X基因的整合频率最高，设计HBV X基因的特异引物，利用HBV-Alu-PCR方法进行三轮半巢式PCR，终产物进行电泳并回收、连接载体、筛选扩增后测序，检测有无X基因整合。
结果：经PCR后电泳及测序分析，25例HBV相关肝病肝移植受体术后的PBMC内未检测出HBV X基因的整合，其中采集到骨髓标本的23例CD_(34)~+细胞中亦未检测到HBV X基因的整合。
结论：肝移植术后受体体内HBV微生态的剧烈改变，使HBV整合的基本条件丧失，在此情况下，外周免疫细胞及骨髓造血干祖细胞不是发生HBV整合的适宜场所。
Liver transplantation (LT) is a final and effective treatment for end stage liver diseases (ESLD) patients. In China, hepatitis B virus (HBV) related liver diseases have become the primary indication for LT. Eeven lamivudine (LAM) and hepatitis B immunoglobulin (HBIG) were combined administrated in clinic, the HBV graft reinfection and HB recurrence after LT remain still high, which severely leads a poor clinic outcomem of LT. This study aimes to research the status of a major extrahepatic HBV reservoir, namely Immume-Hematopoietic System, try to reveal the possible mechanisms of HBV reinfection and HB recurrence post-LT.
In part 1, a sensitive and credible real-time fluorescence polymerase chain reaction (RT-FQ-PCR) for HBV covalently closed circular DNA (cccDNA) detection was established. In part 2, peripheral blood mononuclear cells (PBMC), T lymphocytes, B lymphocytes, monocytes and bone marrow (BM) hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC) were isolated respectively by Ficoll-Hypaque density-gradient centrifugation combine magnetic cell sorting and separation (MACS). The intracellular and sera total HBV DNA and HBV cccDNA were detected by RT-FQ-PCR. The serological markers of HBV were detected simultaneously. It proved that no evidences of HBV replication and HBV X gene integration were found, but there are HBV DNA persisting positive in immunocytes, HSC and HPC, even undergoing LAM and 1BIG combined therapy for a long time after LT.
Part 1:
An enzyme assistant Taq man probe real-time fluorescence quantitative PCR detection method of HBV cccDNA
Objective: To establish a sensitive and specific real-time fluorescence quantitative PCR for HBV covalently closed circular DNA (HBV cccDNA) detection.
Methods: According to the differences in construction between HBV relaxed circular DNA (HBV rcDNA) and HBV cccDNA, two couples of primers for HBV cccDNA detection, cccF_1-R_1 and cccF_2-R_2, were synthesized basing on attainable literatures at present. Series dilutions of HBV rcDNA which extracted from Dane particles, and plasmid DNA which extracted from pBHB4 plasmid containing a monomer of the HBV adr_4 subtype genome, were using as the templates respectively. Before and after treated by different dosage single-strand-specific mung bean nuclease (MBN), rcDNA and cccDNA were amplified by two couples of primers for HBV cccDNA respectively. The products of PCR were analyzed by 1% agarose gel electrophoresis. The optimal primers of HBV cccDNA detection and the optimum dosage of MBN were chosen for detection of HBV cccDNA, according to the results of PCR and electrophoresis.
Results: 1. cccF_1-R_1 and cccF_2-R_2 can amplify not only cccDNA, but also HBV rcDNA when the titer was more than 1.92×10~6 copies/ml and 1.92×10~5 copies/ml respectively. In other words, the two couples of primers just possess the relative specificity for HBV cccDNA. 2. The specificity of cccF_1-R_1 excels cccF_2-R_2, because the former flanks the nick in minus strand and the gap in plus strand of HBV rcDNA but the latter only flanks the nick in minus strand. 3. MBN can promote the specificity of cccDNA primers by digesting selectively the single strand DNA present only in HBV rcDNA. The optimum dosage of MBN was 5.0IU. 4. Using ccc F_1-R_1, Taq man probe and MBN, a sensitive and specific quantification method of HBV cccDNA detection was established, with a detective range of 5.00×10~2～5.00×10~8 copies/ml.
Conclusions: A sensitive and specific Taq man probe real-time FQ-PCR for HBV cccDNA quantitative detection, assisted by mung bean nuclcasc, has been established successfully, which is content with the requests of our follow research.
Part 2:
Quantitative detection and clinical significance of total HBV DNA and HBV cccDNA in peripheral immunocytes and bone marrow CD_(34)~+ cells from HBV related liver disease patients after liver transplantation undergoing combined prophylaxis of LAM and HBIG
Objective: The long-term clinical outcomes of liver transplantation (LT) had been reduced by HBV graft reinfection/HB recurrence. Peripheral blood mononuclear cell (PBMC), in which HBV DNA can be persistent positive, were considered the major source of HBV graft reinfection/HB recurrence post-LT. But the half-life time of PBMC is limited in some weeks to months. Undergoing the combined prophylaxis protocol of lamivudine (LAM) and hepatitis B immunoglobulin (HBIG), the HBV infected PBMC would be cleared thoroughly step by step after LT. In order to clarify why HBV DNA can persistent in PBMC for long time post liver transplantation, this study was carried out.
Methods: 25 HBV related LT recipients (22 males and 3 females) were selected, whose postoperative time were ranged from 11 to 77 months (33.32±14.81 months) and age were from 33 to 61 years old (43.32±8.76 years). The combined prophylaxis protocol of LAM and HBIG has been chosen to prevent HBV graft re-infection/HB recurrence. 7 case were negative for serum HBV DNA (＜10~3 copies/ml) prior to operation and 18 were positive with a mean trier of 10~(5.954±1.457) copies/ml. In the study, 20ml peripheral vein blood of every case was collected and 23 samples of 20ml bone marrow were obtained from 23 of 25 cases with written content. PBMC, T, B, monocyte (MNC) and CD_(34)~+ cell were isolated respectively by Ficoll-Hypaque density-gadient centrifugation and magnetic cell sorting and separation (MACS). The samples of serum were collected simultaneously. The quantification of anti-HBs was performed using electrochemi-luminescence immunoassay (ECLIA). The detection of HBV markers was performed using enzyme linked immunosorbent assay (ELISA). The cellular DNA was extracted by DNA isolation and purification kit (Watsonbiot, Shanghai China) following the manufacture's instructions. The titer of HBV DNA and HBV covalently closed circular DNA (cccDNA) in serum, PBMCs, T, B lymphocytes, MNCs and CD_(34)~+ cells, including hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), were detected using real-time fluorescence quantitative polymerase chain reaction (RT-FQ-PCR).
Results: 1.25 cases are all positive for anti-HBs with a mean serum anti-HBs titer of 119.1±90.7IU/L, ranged from 12.37 to 372.6IU/L and the serum HBV antigens of all cases are negative. Anti-HBe and anti-HBc are positive in 5 cases; Anti-HBe and Anti-HBc are negative in 6 cases; Anti-HBe is positive and Anti-HBc is negative in 1 case; Anti-HBe is negative and Anti-HBc is positive in 13 cases. 2. All serum HBV DNA is negative in 25 cases. 3. Not only immunocytes including PBMC, MNCs and B, T lymphocytes, but also CD_(34)~+ cells are all positive for HBV DNA, with geometric mean titer of 10~(3.3263±0.6956), 10~(2.9425±0.6462), 10~(2.7213±0.6223), 10~(2.9522±0.932), 10~(3.3373±0.6583) copies per 10~6 cells respectively. And the distribution of HBV DNA in different subset cells is diversity according to the result of ANOVA. The influence factors of HBV DNA titer in different subset cells are also diversity according to the results of regression and correlation analysis. 4. There is no evidence of HBV replication in CD_(34)~+ cells and immunocytes (no HBV cccDNA were detected in cells).
Conclusions: Even combined prophylaxis of lamivudine and hepatitis B immunoglobulin after LT, the HBV related recipients are still HBV DNA positive in peripheral immunocytes and bone marrow CD_(34)~+ cells influenced by complicated factors. Because there is no HBV cccDNA detcted in immunocytes, HBV DNA positive in CD_(34)~+ cells may be a key factor of HBV DNA persisting in immunocytes, especially to lymphomononuclears, which may lead nonresponse to HBV vaccine and HBV graft reinfection/HB recurrence post operatively. But the mechanism of HBV transmission intercellular and the effect of intracellular HBV DNA titer on immune system have not been clarified. Further studies are needed to gain a better understanding on HBV graft reinfection.
Part 3:
Detection of HBV X gene integration in PBMC and bone marrow CD_(34)~+ cells from HBV related liver disease patients using HBV-Alu-PCR after liver transplantation
Objective: To clarify whether HBV X gene integrate in PBMC and bone marrow CD_(34)~+ cells from HBV related liver disease patients after liver transplantation.
Methods: PBMCs were obtained from 25 HBV related liver disease patients after liver transplantation and bone marrow CD_(34)~+ cells obtained from 23 cases among them. The cellular DNA was extracted by DNA isolation and purification kit (Watsonbiot, Shanghai China) following the manufacture's instructions. Specific primers to HBV X gene and to human Alu repeats were used to amplify the virus integration through a 3-round hemi-nest PCR. The PCR final product was judged by 1.2% agarose electrophoresis, ligated to T vector, proliferated in E. coil 5αand sequenced.
Results: According to agarose electrophoresis and sequencing analysis, there were no HBV X gene integration in PBMCs and bone marrow CD_(34)~+ cells from HBV related liver transplant recipients after surgery.
Conclusions: Because of the radical change of HBV bionomy in HBV related liver transplant recipients after operation, the fundamental condition of HBV integration has been lost, which led peripheral immunocytes and bone marrow CD_(34)~+ cells not suit to HBV integrate to human genome.
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