单个肝细胞核内鸭乙型肝炎病毒DNA的水平及动态变化

发布时间：2018-06-23 18:00

本文选题：鸭乙型肝炎病毒 + cccDNA　；参考：《南方医科大学》2013年博士论文

【摘要】：研究背景、目的及意义：共价闭合环状DNA (covalently closed circular DNA, cccDNA)是乙型肝炎病毒(Hepatitis B virus, HBV)的复制模板,长期稳定的以微染色体的形式存在于受感染的肝细胞核内,虽其含量远低于HBV存在的一般形式松弛环状DNA(relaxed circular DNA, rcDNA),但对HBV的复制和感染状态的建立具有举足轻重的意义。cccDNA是HBV感染状态的持续、停用抗病毒药物后病情反复及药物耐药的关键因素,因此只有清除或有效降低细胞核内的cccDNA,才能彻底消除乙肝患者病毒携带状态,或使病情趋于稳定。 cccDNA的定量检测对于理解HBV的复制和清除,评估抗病毒药物的疗效和疗程等具有重要的意义；但rcDNA或双链线性DNA (double strand DNA, dsDNA)的非特异性扩增仍然是目前要面临的主要问题。检测方法不成熟和肝组织来源困难在一定程度上阻碍了对乙肝患者cccDNA的直接研究。在鸭乙型肝炎病毒(Duck hepatitis B virus, DHBV)感染的鸭肝细胞,胞核内病毒DNA主要是以cccDNA为主,而胞浆内作为cccDNA前体的rcDNA可能有其量的10倍。因此定量检测核内病毒DNA可较直接反映cccDNA的水平。本研究将在单个肝细胞核的层面上定量检测慢性DHBV感染状态下核内病毒DNA水平及其影响因素；通过恩替卡韦短期抑制病毒复制,观察血清DHBVDNA阴转时及阴转后单个肝细胞核内病毒水平及受感染肝细胞核数的动态变化。我们的研究首次提出了在单个肝细胞核的水平上分析核内病毒DNA,并通过恩替卡韦抑制病毒复制观察核内病毒DNA的清除动力学。完成这一研究会帮助我们更加深入理解嗜肝DNA病毒的生活周期,病毒与宿主细胞间的相互作用,药物作用下单个核内病毒DNA的清除规律,同时也将为下一步研究慢乙肝患者核内HBV DNA水平及抗病毒疗效评估等奠定基础。研究方法：第一部分：建立鸭乙型肝炎病毒后天感染模型筛选1和58日龄广东樱桃谷鸭各20只,在2日及2月龄时病毒阴性动物静脉接种含1×108DHBV DNA病毒颗粒的血清。接种后不同时间点采血,抽提DNA,PCR定性及定量检测外周血病毒DNA水平。第二部分：慢性感染状态下单个肝细胞核内DHBV DNA水平 1.对纳入的11只45日龄慢性DHBV感染鸭行肝活检手术 2.建立单个核内DHBV DNA定量检测方法采用Taqman探针荧光定量PCR法检测单个核内DHBV DNA水平,并验证该方法的敏感性、特异性及批间差异：已知浓度的标准质粒(PBS-DHBV1.2)倍比稀释为1、10、100、1000和2、20、200、2000copies/μl。按照50μl的反应体系分别向反应孔内加入1、10、100、1000和2、20、200、2000拷贝数的PBS-DHBV1.2质粒进行real-time PCR定量扩增,得到扩增曲线和标准曲线,确定方法的敏感性(最低检测下限)；分别用含有DHBV、HBV及HCV全基因组的质粒进行扩增,验证引物和探针的特异性；分四批定量检测两只动物单个核内DHBV DNA水平,计算核内病毒拷贝数的批间差异,用变异系数(CV%)表示。 3.匀浆研磨5mg肝组织、低速离心、匀浆液充分洗涤得到肝细胞核悬液、通过流式细胞仪分选得到单个细胞核；单个细胞核经蛋白酶K消化、EcoRI酶切后,采用荧光定量PCR法检测核内病毒DNA水平。 4.流式细胞仪分选105个肝细胞核至1ml的PBS溶液中、提取总的核内DNA,采用荧光定量PCR法检测总的核内病毒DNA水平。 5.细胞周期检测并分选处在不同周期的单个核 500μ1的肝细胞核悬液(约1×105个核)中加入15μl PI(100μg/ml)染色液,避光室温孵育至少30min;流式细胞仪将根据核内DNA的量确定细胞周期的分布状况,并分选处在不同细胞周期的单个核至96孔板中。 6.明确整合的病毒DNA影响质粒安全ATP依赖的DNA酶(PSAD)可以选择性消化线性DNA(即整合的片段),对cccDNA和rcDNA无影响。单个核内病毒DNA在1个单位PSAD酶的作用下37℃孵育30min,然后70℃、30min灭活该酶;再加入5个单位的EcoR I,37℃孵育30min。采用荧光定量PCR法检测单个核内病毒DNA水平,确定病毒阳性细胞核数,比较PSAD处理组与未处理组间的差异。 7.病毒DNA在肝内的存在形式采用酚-氯仿抽提法提取肝细胞核、细胞质及肝细胞内的总DNA, Southern blot印迹杂交检测病毒DNA的存在形式。第三部分：恩替卡韦治疗对单个核内DHBV DNA的影响实验分组：将11只45日龄慢性DHBV感染鸭随机分两组：恩替卡韦治疗组(n=6)和未治疗组(n=5)。治疗组每只动物每日接受0.5mg恩替卡韦抗病毒,血清DHBV DNA阴转时,行第二次肝活检；阴转后继续给药治疗16周,治疗结束时处死所用动物,取出肝组织标本。观察单个核内DHBV DNA水平及受感染肝细胞核数的动态变化。统计学分析：所有数据采用SPSS16.0进行处理。计数资料组间比较采用Pearson's chi-square检验,对理论频数小于5的四格表资料采用Fisher精确概率法。连续变量之间的相关性应用Pearson方法。计量资料若满足正态分布或方差齐性采用参数检验方法(两独立样本t检验)；计量资料若不满足正态分布及方差不齐则采用非参数检验方法(Mann-Whitney U检验)。计量资料多组间整体比较采用Kruskal-Wallis H检验。因无法观察同一个肝细胞核在不同时间点或酶处理前后的变化,因此不采用配对比较。所有统计采用双侧检验,P0.05认为差异具有统计学意义。研究结果：第一部分：建立鸭乙型肝炎病毒后天感染模型筛选出4只58日龄和2只1日龄血清DHBV DNA阳性鸭,感染率分别为20%和10%。两组血清病毒阴性动物在2月龄或2日龄时静脉接种含1×108DHBV DNA病毒颗粒的血清。2月龄动物在接种后两个月内均未产生可检测的病毒血症,后天感染率为0,提示在成鸭中较难建立鸭乙型肝炎病毒后天感染模型。2日龄动物在接种后第14天,78.6%的动物可检出病毒血症,血清DHBV DNA水平在108copies/ml左右,随后观察的3个时间点,血清病毒载量均维持在107-109copies/ml之间。第二部分：慢性感染状态下单个肝细胞核内DHBV DNA水平成功建立单个核内DHBV DNA定量检测方法将含有DHBV、HBV和HCV全基因组质粒进行荧光定量PCR扩增,发现除了DHBV质粒能成功扩增外,其余质粒均未产生荧光信号,无假阳性扩增,提示引物和探针的特异性好。分别用1、2、10和20拷贝的PBS-DHBV1.2质粒进行荧光定量PCR扩增,发现除了1拷贝的质粒不能被有效扩增外,其余均能检出。两次独立重复试验均取得相一致结果：单个核内DHBV DNA最低定量检测下限(Low Limit of Detection, LLOD)为2拷贝。分四批定量检测两只动物单个核内DHBV DNA水平,批间变异系数(CV%)分别为2.42%和6.92%。单个核内DHBV DNA水平慢性感染状态下,不是所有肝细胞核均受感染(63.3%-93.3%)。核与核之间病毒拷贝数差异较大(2-204)。Pearson相关性分析发现,核内病毒DNA平均拷贝数(7.57-57.67)与核内总的病毒载量(r=0.927,P0.001)和血清病毒水平(r=0.605,P=0.049)呈正相关。核内DHBV DNA水平与细胞周期有关流式细胞仪对3只45日龄动物行细胞周期检测,发现分别有75%、81%、79%的肝细胞核处在G0/1期,15%、11%、10%的核处在S期,10%、8%、11%的核处于G2/M期。细胞周期间病毒阳性核拷贝数整体比较有显著性差异(P0.001),G0/1期最高,其次是G2/M和S期；阳性细胞核比率整体比较也有显著性差异(P0.05),S期阳性细胞核比率最低,约一半的阳性核病毒拷贝数低于10。 PSAD酶处理对核内DHBV DNA水平影响较小 PSAD酶处理组与未处理组相比较,各动物病毒阳性核拷贝数均未见有显著变化：动物编号22(Z=-0.810, P=0.418),51(Z=-0.352, P=0.725),52(Z=-1.837, P=0.066),62(Z=-0.321, P=0.748),65(Z=1.041, P=0.298);各动物病毒阳性核比率也未发生统计学意义改变：动物编号22(χ2=0.098, P=0.075),51(χ2=0.351, P=0.554),52(χ2=0.741, P=0.389),62(χ2=0.480,P=0.488),65(χ2=1.071,P=0.301)。核内DHBV DNA主要是以cccDNA的形式存在 Southern blot结果显示肝细胞核内DHBV DNA主要以cccDNA的形式存在,也可见有少量rcDNA;胞质中病毒DNA以rcDNA, dsDNA和ssDNA的形式存在。第三部分：恩替卡韦治疗对单个核内DHBV DNA的影响恩替卡韦治疗对病毒阳性肝细胞核比率的影响恩替卡韦抗病毒治疗能显著降低病毒阳性肝细胞核比率(P0.001)。阳性率下降主要发生在从基线至血清病毒DNA阴转时,延长治疗过程中阳性率下降仍较明显。从基线至血清病毒DNA阴转时,恩替卡韦治疗组动物阳性肝细胞核比率明显降低(86.1%vs.50.6%,x2=52.580,P0.001)；未治疗组阳性率未发生有统计学意义改变(70.0%vs.83.3%；x2=2.981,P=0.080)。从血清病毒DNA阴转至治疗结束,恩替卡韦治疗组动物阳性肝细胞核比率明显降低(48.3%vs.25.8%；x2=13.019,P=0.001)；未治疗组阳性率未发生有统计学意义改变(83.3%vs.86.7%；x2=1.310,P=0.718)。恩替卡韦治疗对单个核内DHBV DNA水平的影响血清病毒DNA阴转时,恩替卡韦治疗组动物病毒阳性核拷贝数明显低于基线时水平(Z=-7.984,P=0.000)；未治疗组病毒阳性核拷贝数未发生统计学意义改变(t=0.313,P=0.755)。对每只动物进行分析,发现治疗组中除了9号(Z=-1.745,P=0.081),52号(t=1.479,P=0.148)动物病毒阳性核拷贝数无明显变化外,其余均明显降低。从血清病毒DNA阴转至治疗结束,核内病毒DNA平均拷贝数缓慢降低,但个别核内病毒水平仍较高。结论： 1.本研究成功建立单个核内DHBV DNA定量检测方法,该方法具有较理想的敏感性及特异性。 2.慢性感染状态下单个核内DHBV DNA拷贝数相差较大,核内DHBV DNA平均拷贝数与血清病毒水平及核内总的病毒载量正相关。 3.单个核内DHBV DNA水平与细胞周期状态有关。病毒在G0/1期复制活跃,其次是G2/M和S期。 4.抗病毒治疗能有效降低单个核内DHBV DNA水平及受感染肝细胞核数量。
[Abstract]:Research background, purpose and significance:
Covalently closed ring DNA (covalently closed circular DNA, cccDNA) is a replicating template for hepatitis B virus (Hepatitis B virus, HBV), and is long stable in the form of a micro chromosome in the infected liver nucleus, although its content is far below the general form of HBV relaxation annular DNA. The establishment of replication and infection status is of great significance..cccDNA is the key factor for the persistence of HBV infection state, the recurrence of antiviral drugs and drug resistance. Therefore, only the removal or effective reduction of cccDNA in the nucleus can eliminate the virus carrying status of hepatitis B patients, or make the disease stable.
Quantitative detection of cccDNA is of great significance for understanding the replication and removal of HBV and assessing the efficacy and treatment of antiviral drugs, but the non specific amplification of rcDNA or double chain linear DNA (double strand DNA, dsDNA) is still the main problem to be faced at present. The direct study of cccDNA in patients with hepatitis B was hindered.
In duck hepatitis B virus (Duck hepatitis B virus, DHBV) infected duck liver cells, the main viral DNA in the nucleus is cccDNA, and the rcDNA in the cytoplasm as the precursor of cccDNA precursor may have 10 times its amount. Therefore, the quantitative detection of the intra nuclear virus DNA can directly reflect the cccDNA water level.
In this study, the level of viral DNA in the nucleus of chronic DHBV infection and its influencing factors were quantified at the single liver cell nucleus. The dynamic changes of the virus level and the number of infected liver nuclei in the single liver nucleus were observed by the short-term inhibitory virus replication of entecavir.
Our study was the first to propose an analysis of the nuclear virus DNA at the level of a single liver cell, and to observe the clearance kinetics of the viral DNA in the nucleus through an entecavir inhibition virus replication. This study will help us to understand the life cycle of the liver DNA virus, the interaction between the virus and the host cell, the drug effect. The removal of DNA from the single nucleus will also lay the foundation for further research on the level of HBV DNA and the evaluation of antiviral efficacy in patients with chronic hepatitis B.
Research methods:
Part one: establishment of acquired hepatitis B virus acquired infection model.
20 Guangdong Cherry Valley ducks, 1 and 58 days old, were inoculated with 1 x 108DHBV DNA virus particles in the virus negative animals at 2 and 2 month old days. After inoculation, blood samples were collected at different time points, DNA was extracted, and DNA level of peripheral blood virus was qualitatively and quantitatively detected by PCR.
The second part: DHBV DNA level in a single liver cell under chronic infection.
1. liver biopsy was performed on 11 11 day old DHBV infected ducks.
2. the establishment of quantitative detection method for DHBV DNA in single nucleus
Taqman probe fluorescence quantitative PCR was used to detect the level of DHBV DNA in mononuclear cells, and the sensitivity, specificity and difference between the two methods were verified.
The standard plasmid (PBS-DHBV1.2) of known concentration was diluted to 1,101001000 and 2,202002000copies/ Mu L. in the reaction system of 50 mu l to amplify real-time PCR with 1,101001000 and 2,202002000 copies adding 1,101001000 and 2,202002000 copies in the reaction pore respectively. The amplification curve and standard curve were obtained to determine the sensitivity of the method. Minimum detection limit);
The genomic DNA containing DHBV, HBV and HCV were amplified respectively to verify the specificity of primers and probes.
The level of DHBV DNA in the single nucleus of two animals was quantitatively detected in four batches, and the difference of the number of copies of virus in the nucleus was calculated by means of coefficient of variation (CV%).
3. the homogenate was lapping 5mg liver tissue, low-speed centrifugation and homogenate liquid were washed to get the liver nuclear suspension. The single nucleus was obtained by flow cytometry. The single nucleus was digested by the protease K, and after the EcoRI enzyme was cut, the DNA level of the virus was detected by the fluorescence quantitative PCR method.
4. flow cytometry was used to extract the total nuclear DNA from 105 liver nuclei to 1ml PBS solution. The total nuclear DNA level was detected by fluorescence quantitative PCR.
5. cell cycle detection and sorting in a single cycle with different cycles.
500 mu 1 liver nuclear suspension (about 1 * 105 nuclei) was added to 15 mu L PI (100 g/ml) dyed liquid to incubate at least 30min at room temperature. The flow cytometry will determine the distribution of cell cycle according to the amount of DNA in the nucleus, and select the single nucleus in the different cell cycle to 96 orifice plates.
6. clearly integrated virus DNA impact
Plasmid safe ATP dependent DNA enzyme (PSAD) can selectively digest linear DNA (i.e. integrated fragment) and have no effect on cccDNA and rcDNA. The single nucleus virus DNA incubates 30min at 37 degrees centigrade under the action of 1 unit PSAD enzyme, then 70 degrees C, 30min inactivates the enzyme, then adds 5 units of EcoR I, and incubates 37 centigrade by fluorescence quantitative determination to detect mononuclear nuclei The level of virus DNA was determined, and the number of positive nuclei was determined. The difference between PSAD treated group and untreated group was compared.
The existing form of 7. virus DNA in the liver
Phenols chloroform extraction method was used to extract liver nuclei, cytoplasm and total DNA in liver cells, and Southern blot blot hybridization was used to detect the presence of DNA.
The third part: the effect of entecavir treatment on DHBV DNA in single nucleus.
Experiment group: 11 45 day old chronic DHBV infected ducks were randomly divided into two groups: entecavir treatment group (n=6) and untreated group (n=5).
In the treatment group, each animal received an antivirus 0.5mg of entecavir every day. When the serum DHBV DNA was turned negative, second liver biopsies were performed. After 16 weeks, the animals were killed and the specimens of the liver were removed.
The dynamic changes of DHBV DNA level and the number of nuclei of infected liver were observed.
Statistical analysis:
All data are processed by SPSS16.0. Pearson's chi-square test is used among counting data groups, and Fisher precision probability method is used for the data of four grid tables with theoretical frequency less than 5. The correlation of continuous variables is applied to Pearson method. If measurement data satisfies normal distribution or square difference, the method of parameter test (two independent sample) This t test); non parametric test method (Mann-Whitney U test) is used if the measurement data are not satisfied with normal distribution and variance. Kruskal-Wallis H test is used for the overall comparison of multiple groups of measurement data. The same liver nucleus can not be observed at different time points or after the enzyme treatment. The P0.05 test showed that the difference was statistically significant.
The results of the study:
Part one: establishment of acquired hepatitis B virus acquired infection model.
The serum DHBV DNA positive ducks of 4 58 day old and 2 1 day old were selected. The infection rates were 20% and 10%. respectively.
In the two groups of serum virus negative animals at the age of 2 month old or 2 days of age, the serum of.2 month old animals containing 1 x 108DHBV DNA virus particles had no detectable viremia within two months after inoculation, and the acquired infection rate was 0. It was suggested that the day after infection model of duck hepatitis B virus (.2) in adult ducks was more difficult to establish in Fourteenth days after inoculation fourteenth. Viremia can be detected in 78.6% of the animals, the serum DHBV DNA level is around 108copies/ml, and the 3 time points observed, the serum viral load is maintained between 107-109copies/ml.
The second part: DHBV DNA level in a single liver cell under chronic infection.
Successful establishment of a quantitative detection method for DHBV DNA in mononuclear nuclei
The whole genome plasmid containing DHBV, HBV and HCV was amplified by fluorescence quantitative PCR. It was found that except for the successful amplification of DHBV plasmids, the other plasmids did not produce fluorescent signals and no false positive amplification, suggesting that the specificity of primers and probes was good.
1,2,10 and 20 copies of PBS-DHBV1.2 plasmids were used to amplify the fluorescence quantitative PCR. It was found that the other 1 copies of the plasmid could not be effectively amplified, and the rest could be detected. Two independent repeated tests all obtained the same results: the lowest quantitative detection limit of DHBV DNA in mononuclear (Low Limit of Detection, LLOD) was 2 copies.
The levels of DHBV DNA in the single nucleus of two animals were quantified in four batches, and the inter assay coefficient of variation (CV%) was 2.42% and 6.92%. respectively.
DHBV DNA level in mononuclear nuclei
In the state of chronic infection, not all liver nuclei were infected (63.3%-93.3%). The difference in the number of viral copies between nuclei and nuclei (2-204).Pearson correlation analysis showed that the average copy number of DNA in the nuclear virus (7.57-57.67) was positively correlated with the total viral load (r=0.927, P0.001) and serum virus level (r=0.605, P=0.049) in the nucleus.
The level of DHBV DNA in the nucleus is related to the cell cycle
The flow cytometry was used to detect the cell cycle of 3 45 day old animals. It was found that 75%, 81%, 79% liver nuclei were in G0/1 phase, 15%, 11% and 10% were in phase S, 10%, 8%, 11% were in G2/M phase.
There were significant differences in the number of positive nuclear copies during the period of cell cycle (P0.001), the highest in the G0/1 phase, followed by the G2/M and S stages, and the positive nuclear ratio was also significantly different (P0.05), the S positive nucleus ratio was the lowest, and about half of the positive nuclear virus scallop was less than 10.
PSAD enzyme treatment has little effect on the level of DHBV DNA in the nucleus
PSAD enzyme treatment group compared with the untreated group, no significant changes were found in the number of positive nuclear copies of animal viruses: animal number 22 (Z=-0.810, P=0.418), 51 (Z=-0.352, P=0.725), 52 (Z=-1.837, P=0.066), 62 (Z=-0.321, P=0.748), 65 (Z=1.041, P= 0.298); the positive nuclear ratio of each animal virus did not change statistically: Animal compiling. Number 22 (chi 2=0.098, P=0.075), 51 (chi 2=0.351, P=0.554), 52 (chi 2=0.741, P=0.389), 62 (chi 2=0.480, P=0.488), 65 (chi 2=1.071, P=0.301).
The DHBV DNA in the core is mainly in the form of cccDNA
Southern blot results showed that DHBV DNA existed in the nucleus of the liver mainly in the form of cccDNA, and there was a small amount of rcDNA, and the viral DNA in the cytoplasm existed in the form of rcDNA, dsDNA and ssDNA.
The third part: the effect of entecavir treatment on DHBV DNA in single nucleus.
Effect of entecavir therapy on the ratio of virus positive liver cells
Antiviral therapy of entecavir could significantly reduce the ratio of virus positive liver nuclei (P0.001). The decrease of positive rate was mainly in the period from baseline to serovirus DNA, and the positive rate in the prolonged treatment was still more obvious.
From baseline to sera virus DNA negative, the ratio of positive liver nuclei in entecavir treatment group was significantly lower (86.1%vs.50.6%, x2=52.580, P0.001), and the positive rate of untreated group was not statistically significant (70.0%vs.83.3%; x2=2.981, P=0.080).
In the treatment group of entecavir, the ratio of the positive liver nuclei in the treatment group of entecavir was significantly lower (48.3%vs.25.8%; x2=13.019, P=0.001), and the positive rate of the untreated group was not statistically significant (83.3%vs.86.7%; x2=1.310, P=0.718) from the sera virus DNA to the end of the treatment.
Effect of entecavir therapy on the level of DHBV DNA in single nucleus
The number of positive nuclear copies of the animal virus in the entecavir treatment group was significantly lower than that at the baseline (Z=-7.984, P=0.000), and the number of viral positive nuclear copies in the untreated group was not statistically significant (t=0.313, P=0.755) in the untreated group (t=0.313, P=0.755). In the treatment group, there were 9 (Z=-1.745, P=0.081), t=1.479, P= in the present treatment group. 0.148) there was no significant change in the number of nuclear copies of animal viruses but the rest decreased significantly.
The average copy number of virus DNA decreased slowly from DNA to the end of treatment, but the level of virus in individual nuclei was still high.
Conclusion:
1. the quantitative detection of DHBV DNA in single nucleus was successfully established in this study. The method is more sensitive and specific.
2. in the state of chronic infection, the number of DHBV DNA copies in the mononuclear nucleus is quite different. The average copy number of DHBV DNA in the nucleus is positively correlated with the level of serum virus and the total viral load in the nucleus.
3. the level of DHBV DNA in the single nucleus is related to the cell cycle status. The virus replicated in G0/1 phase.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62

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