本文選題：鴨乙型肝炎病毒 + cccDNA　； 參考：《南方醫(yī)科大學》2013年博士論文

【摘要】：研究背景、目的及意義： 共價閉合環(huán)狀DNA (covalently closed circular DNA, cccDNA)是乙型肝炎病毒(Hepatitis B virus, HBV)的復制模板,長期穩(wěn)定的以微染色體的形式存在于受感染的肝細胞核內,雖其含量遠低于HBV存在的一般形式松弛環(huán)狀DNA(relaxed circular DNA, rcDNA),但對HBV的復制和感染狀態(tài)的建立具有舉足輕重的意義。cccDNA是HBV感染狀態(tài)的持續(xù)、停用抗病毒藥物后病情反復及藥物耐藥的關鍵因素,因此只有清除或有效降低細胞核內的cccDNA,才能徹底消除乙肝患者病毒攜帶狀態(tài),或使病情趨于穩(wěn)定。 cccDNA的定量檢測對于理解HBV的復制和清除,評估抗病毒藥物的療效和療程等具有重要的意義；但rcDNA或雙鏈線性DNA (double strand DNA, dsDNA)的非特異性擴增仍然是目前要面臨的主要問題。檢測方法不成熟和肝組織來源困難在一定程度上阻礙了對乙肝患者cccDNA的直接研究。 在鴨乙型肝炎病毒(Duck hepatitis B virus, DHBV)感染的鴨肝細胞,胞核內病毒DNA主要是以cccDNA為主,而胞漿內作為cccDNA前體的rcDNA可能有其量的10倍。因此定量檢測核內病毒DNA可較直接反映cccDNA的水平。 本研究將在單個肝細胞核的層面上定量檢測慢性DHBV感染狀態(tài)下核內病毒DNA水平及其影響因素；通過恩替卡韋短期抑制病毒復制,觀察血清DHBVDNA陰轉時及陰轉后單個肝細胞核內病毒水平及受感染肝細胞核數的動態(tài)變化。 我們的研究首次提出了在單個肝細胞核的水平上分析核內病毒DNA,并通過恩替卡韋抑制病毒復制觀察核內病毒DNA的清除動力學。完成這一研究會幫助我們更加深入理解嗜肝DNA病毒的生活周期,病毒與宿主細胞間的相互作用,藥物作用下單個核內病毒DNA的清除規(guī)律,同時也將為下一步研究慢乙肝患者核內HBV DNA水平及抗病毒療效評估等奠定基礎。 研究方法： 第一部分：建立鴨乙型肝炎病毒后天感染模型 篩選1和58日齡廣東櫻桃谷鴨各20只,在2日及2月齡時病毒陰性動物靜脈接種含1×108DHBV DNA病毒顆粒的血清。接種后不同時間點采血,抽提DNA,PCR定性及定量檢測外周血病毒DNA水平。 第二部分：慢性感染狀態(tài)下單個肝細胞核內DHBV DNA水平 1.對納入的11只45日齡慢性DHBV感染鴨行肝活檢手術 2.建立單個核內DHBV DNA定量檢測方法 采用Taqman探針熒光定量PCR法檢測單個核內DHBV DNA水平,并驗證該方法的敏感性、特異性及批間差異： 已知濃度的標準質粒(PBS-DHBV1.2)倍比稀釋為1、10、100、1000和2、20、200、2000copies/μl。按照50μl的反應體系分別向反應孔內加入1、10、100、1000和2、20、200、2000拷貝數的PBS-DHBV1.2質粒進行real-time PCR定量擴增,得到擴增曲線和標準曲線,確定方法的敏感性(最低檢測下限)； 分別用含有DHBV、HBV及HCV全基因組的質粒進行擴增,驗證引物和探針的特異性； 分四批定量檢測兩只動物單個核內DHBV DNA水平,計算核內病毒拷貝數的批間差異,用變異系數(CV%)表示。 3.勻漿研磨5mg肝組織、低速離心、勻漿液充分洗滌得到肝細胞核懸液、通過流式細胞儀分選得到單個細胞核；單個細胞核經蛋白酶K消化、EcoRI酶切后,采用熒光定量PCR法檢測核內病毒DNA水平。 4.流式細胞儀分選105個肝細胞核至1ml的PBS溶液中、提取總的核內DNA,采用熒光定量PCR法檢測總的核內病毒DNA水平。 5.細胞周期檢測并分選處在不同周期的單個核 500μ1的肝細胞核懸液(約1×105個核)中加入15μl PI(100μg/ml)染色液,避光室溫孵育至少30min;流式細胞儀將根據核內DNA的量確定細胞周期的分布狀況,并分選處在不同細胞周期的單個核至96孔板中。 6.明確整合的病毒DNA影響 質粒安全ATP依賴的DNA酶(PSAD)可以選擇性消化線性DNA(即整合的片段),對cccDNA和rcDNA無影響。單個核內病毒DNA在1個單位PSAD酶的作用下37℃孵育30min,然后70℃、30min滅活該酶;再加入5個單位的EcoR I,37℃孵育30min。采用熒光定量PCR法檢測單個核內病毒DNA水平,確定病毒陽性細胞核數,比較PSAD處理組與未處理組間的差異。 7.病毒DNA在肝內的存在形式 采用酚-氯仿抽提法提取肝細胞核、細胞質及肝細胞內的總DNA, Southern blot印跡雜交檢測病毒DNA的存在形式。 第三部分：恩替卡韋治療對單個核內DHBV DNA的影響 實驗分組：將11只45日齡慢性DHBV感染鴨隨機分兩組：恩替卡韋治療組(n=6)和未治療組(n=5)。 治療組每只動物每日接受0.5mg恩替卡韋抗病毒,血清DHBV DNA陰轉時,行第二次肝活檢；陰轉后繼續(xù)給藥治療16周,治療結束時處死所用動物,取出肝組織標本。 觀察單個核內DHBV DNA水平及受感染肝細胞核數的動態(tài)變化。 統(tǒng)計學分析： 所有數據采用SPSS16.0進行處理。計數資料組間比較采用Pearson's chi-square檢驗,對理論頻數小于5的四格表資料采用Fisher精確概率法。連續(xù)變量之間的相關性應用Pearson方法。計量資料若滿足正態(tài)分布或方差齊性采用參數檢驗方法(兩獨立樣本t檢驗)；計量資料若不滿足正態(tài)分布及方差不齊則采用非參數檢驗方法(Mann-Whitney U檢驗)。計量資料多組間整體比較采用Kruskal-Wallis H檢驗。因無法觀察同一個肝細胞核在不同時間點或酶處理前后的變化,因此不采用配對比較。所有統(tǒng)計采用雙側檢驗,P0.05認為差異具有統(tǒng)計學意義。 研究結果： 第一部分：建立鴨乙型肝炎病毒后天感染模型 篩選出4只58日齡和2只1日齡血清DHBV DNA陽性鴨,感染率分別為20%和10%。 兩組血清病毒陰性動物在2月齡或2日齡時靜脈接種含1×108DHBV DNA病毒顆粒的血清。2月齡動物在接種后兩個月內均未產生可檢測的病毒血癥,后天感染率為0,提示在成鴨中較難建立鴨乙型肝炎病毒后天感染模型。2日齡動物在接種后第14天,78.6%的動物可檢出病毒血癥,血清DHBV DNA水平在108copies/ml左右,隨后觀察的3個時間點,血清病毒載量均維持在107-109copies/ml之間。 第二部分：慢性感染狀態(tài)下單個肝細胞核內DHBV DNA水平 成功建立單個核內DHBV DNA定量檢測方法 將含有DHBV、HBV和HCV全基因組質粒進行熒光定量PCR擴增,發(fā)現除了DHBV質粒能成功擴增外,其余質粒均未產生熒光信號,無假陽性擴增,提示引物和探針的特異性好。 分別用1、2、10和20拷貝的PBS-DHBV1.2質粒進行熒光定量PCR擴增,發(fā)現除了1拷貝的質粒不能被有效擴增外,其余均能檢出。兩次獨立重復試驗均取得相一致結果：單個核內DHBV DNA最低定量檢測下限(Low Limit of Detection, LLOD)為2拷貝。 分四批定量檢測兩只動物單個核內DHBV DNA水平,批間變異系數(CV%)分別為2.42%和6.92%。 單個核內DHBV DNA水平 慢性感染狀態(tài)下,不是所有肝細胞核均受感染(63.3%-93.3%)。核與核之間病毒拷貝數差異較大(2-204)。Pearson相關性分析發(fā)現,核內病毒DNA平均拷貝數(7.57-57.67)與核內總的病毒載量(r=0.927,P0.001)和血清病毒水平(r=0.605,P=0.049)呈正相關。 核內DHBV DNA水平與細胞周期有關 流式細胞儀對3只45日齡動物行細胞周期檢測,發(fā)現分別有75%、81%、79%的肝細胞核處在G0/1期,15%、11%、10%的核處在S期,10%、8%、11%的核處于G2/M期。 細胞周期間病毒陽性核拷貝數整體比較有顯著性差異(P0.001),G0/1期最高,其次是G2/M和S期；陽性細胞核比率整體比較也有顯著性差異(P0.05),S期陽性細胞核比率最低,約一半的陽性核病毒拷貝數低于10。 PSAD酶處理對核內DHBV DNA水平影響較小 PSAD酶處理組與未處理組相比較,各動物病毒陽性核拷貝數均未見有顯著變化：動物編號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);各動物病毒陽性核比率也未發(fā)生統(tǒng)計學意義改變：動物編號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)。 核內DHBV DNA主要是以cccDNA的形式存在 Southern blot結果顯示肝細胞核內DHBV DNA主要以cccDNA的形式存在,也可見有少量rcDNA;胞質中病毒DNA以rcDNA, dsDNA和ssDNA的形式存在。 第三部分：恩替卡韋治療對單個核內DHBV DNA的影響 恩替卡韋治療對病毒陽性肝細胞核比率的影響 恩替卡韋抗病毒治療能顯著降低病毒陽性肝細胞核比率(P0.001)。陽性率下降主要發(fā)生在從基線至血清病毒DNA陰轉時,延長治療過程中陽性率下降仍較明顯。 從基線至血清病毒DNA陰轉時,恩替卡韋治療組動物陽性肝細胞核比率明顯降低(86.1%vs.50.6%,x2=52.580,P0.001)；未治療組陽性率未發(fā)生有統(tǒng)計學意義改變(70.0%vs.83.3%；x2=2.981,P=0.080)。 從血清病毒DNA陰轉至治療結束,恩替卡韋治療組動物陽性肝細胞核比率明顯降低(48.3%vs.25.8%；x2=13.019,P=0.001)；未治療組陽性率未發(fā)生有統(tǒng)計學意義改變(83.3%vs.86.7%；x2=1.310,P=0.718)。 恩替卡韋治療對單個核內DHBV DNA水平的影響 血清病毒DNA陰轉時,恩替卡韋治療組動物病毒陽性核拷貝數明顯低于基線時水平(Z=-7.984,P=0.000)；未治療組病毒陽性核拷貝數未發(fā)生統(tǒng)計學意義改變(t=0.313,P=0.755)。對每只動物進行分析,發(fā)現治療組中除了9號(Z=-1.745,P=0.081),52號(t=1.479,P=0.148)動物病毒陽性核拷貝數無明顯變化外,其余均明顯降低。 從血清病毒DNA陰轉至治療結束,核內病毒DNA平均拷貝數緩慢降低,但個別核內病毒水平仍較高。 結論： 1.本研究成功建立單個核內DHBV DNA定量檢測方法,該方法具有較理想的敏感性及特異性。 2.慢性感染狀態(tài)下單個核內DHBV DNA拷貝數相差較大,核內DHBV DNA平均拷貝數與血清病毒水平及核內總的病毒載量正相關。 3.單個核內DHBV DNA水平與細胞周期狀態(tài)有關。病毒在G0/1期復制活躍,其次是G2/M和S期。 4.抗病毒治療能有效降低單個核內DHBV DNA水平及受感染肝細胞核數量。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R512.62

【相似文獻】

相關期刊論文 前10條

1 陳至善!200052上海,熊顯華!200052上海;腎癌細胞增殖活性與預后的關系[J];臨床腫瘤學雜志;1998年02期

2 張秀榮,孟如松，

本文編號：2057944