乙肝病毒是一種嚴(yán)重影響人類健康的病原體，也是引發(fā)慢性乙肝的元兇。根據(jù)國際衛(wèi)生組織2015年估計，全球約有2.4億慢性乙肝患者，大多分布在低收入及中等收入國家，每年約有78萬患者死于慢性乙肝感染所致的肝衰竭、肝硬化和癌。在中國，約有9300萬慢性乙肝感染者，其中，由乙肝病毒引發(fā)的肝硬化和肝癌患者比例分別高達60%和80%。隨著乙肝疫苗接種的推行，我國嬰幼兒的乙肝陽性率有了大幅度的降低。但是，對于其他感染者，依然缺乏徹底治愈的療法，并且給患者家庭帶來了沉重的經(jīng)濟負擔(dān)和健康威脅。
大量臨床與基礎(chǔ)研究表明，慢性乙肝長期遷延不愈的根本原因在于乙肝病毒共價閉合環(huán)狀DNA(cccDNA)在肝臟內(nèi)的長期存在及其造成的病毒表面抗原血癥。由于cccDNA在慢性乙肝患者肝臟中的水平極低，傳統(tǒng)的分子生物學(xué)方法（如Southern印記、cccDNA特異性聚合酶鏈反應(yīng)）不僅檢測困難，而且無法顯示其在組織內(nèi)的分布特點。
鑒于目前對乙肝cccDNA研究存在的問題，來自復(fù)旦大學(xué)上海醫(yī)學(xué)院醫(yī)學(xué)分子病毒學(xué)重點實驗室的Zhangxiaonan等研究者在bDNA信號擴增技術(shù)基礎(chǔ)上進行二次開發(fā)，成功建立了一種能夠在組織水平顯示cccDNA分布的原位雜交技術(shù)。該技術(shù)還能顯示乙肝病毒的其他復(fù)制中間體，如前基因組RNA(pgRNA)、松弛環(huán)狀DNA(rcDNA)在單細胞水平的分布情況。研究者將該原位雜交技術(shù)與乙肝病毒主要抗原的免疫組化染色相結(jié)合，獲得了病毒核酸與蛋白同時存在的圖像。令人意外的是，乙肝病毒抗原與核酸在單細胞水平呈現(xiàn)驚人的“馬賽克狀”分布，即病毒的ccc DNA、RNA與表面抗原均顯示顯著的負相關(guān)關(guān)系。本研究還進一步證實，經(jīng)過一年以上抗病毒治療的患者的肝細胞內(nèi)仍存在乙肝病毒的cccDNA。綜合前期研究，本研究基于大量患者樣本的觀察結(jié)果，提出在單細胞水平乙肝病毒存在“抗原富集期”、“DNA富集期”和“潛伏期”的“三階段”假說，推測患者體內(nèi)肝細胞生理水平和病毒復(fù)制活躍度的變化可以導(dǎo)致這三個階段的相互轉(zhuǎn)換。該假說從單細胞水平描述了病毒生活周期的復(fù)雜變化模式，豐富了學(xué)術(shù)界對乙肝病毒在肝內(nèi)存活的認(rèn)識，為進一步針對不同病情患者清除乙肝病毒cccDNA的新策略提供了理論基礎(chǔ)。
主要實驗結(jié)果如下：
Figure 1. Validation of the specificity of the strand-specific probe sets design. Tissue sections from CHB patients were pretreated with the indicated nuclease and hybridized with the designated probe sets: (A–C) probe set1(original magnification, ×200; insets, ×400); (D–F)probe set 2 (original magnification, ×200; insets, ×400); (G–I) probe set3 (original magnification, ×400; insets, ×600). After hybridization and signal amplification, NBT/BCIP was used for colorization, followed by counterstaining with nuclear fast red.
Figure 2. Subcellular localization of HBV DNA signal is highly related to its replication status. Representative cytoplasmic (A) and nuclear (B) staining pattern of HBV DNA. (C) Serum HBV DNA levels from cytoplasmic- (n = 26) and nuclear-localized (n = 21) groups were compared. The P value was determined by a Mann-Whitney U test. (D) Comparison of the percentage of cytoplasmic- and nuclear-localized samples from HBeAg-positive and -negative patients.
Figure 3. Spatial relationship between intrahepatic viral nucleic acids (HBV DNA, RNA, and cccDNA) and surface antigen. Probe set 2 or 3 was hybridized with HBV DNA (A and C) or cccDNA (B and D), in conjunction with HBsAg IHC using adjacent sections from the same liver specimen. Images in C and D are higher magnifications of the boxed areas in A and B, respectively, which show similar regions in adjacent slices. HBV RNA was visualized with either probe set 1 (E) or probe set 3 (F) in the context of HBsAg expression using adjacent sections from the same liver specimen. Blue-purple, DNA, cccDNA, or RNA; brown, HBsAg. Original magnification, ×100 (A and B), ×400 (C and D), ×600 (C and D insets), ×200 (E and F), ×400 (E and F insets).
Figure 4. Highly complex distribution of HBsAg, HBcAg, and viral DNA. Liver sections from CHB patients were double stained with anti-HBsAg and anti-HBcAg, followed by visualization with DAB and permanent red, respectively. Results from a representative specimen are shown. Original magnification, ×200 (A), ×400 (B).
The image in B is a magnification of the boxed area in A. Double immunofluorescence staining of HBsAg and HBcAg from the same specimen was performed and analyzed with TissueQuest. (C) Scatterplot showing the expression of HBsAg and HBcAg. (D) Colocalization indices from 25 HBsAg and HBcAg double-positive specimens were calculated. (E and F ) ISH of HBV DNA with probe set 2, in conjunction with HBsAg and HBcAg double IHC. Original magnification, ×400 and ×600 (insets).
Figure 5. Relationship between HBV DNA and cccDNA. Whole-tissue scan of HBV DNA (A) and cccDNA (B) ISH from adjacent sections of 5 CHB liver specimens. One pair of representative images is shown. The selected regions were serially magnified (original magnification, ×200 and ×600) in the insets.
Figure 6. Distribution of HBV DNA and cccDNA before and after adeforvir therapy. Serial liver sections from 9 CHB patients before and 48 weeks after adefovir therapy were hybridized with probe set 2 or 3 to visualize HBV DNA (A and B) or cccDNA (C and D). Results from 1 representative patient (no. 3) are shown. Original magnification, ×200.
其中，HBsAg 和 HBcAg的免疫組化及免疫熒光實驗由奧地利TG公司的TissueFAX系統(tǒng)采圖，TissueQuest軟件分析，該研究建立的乙肝病毒cccDNA原位雜交技術(shù)，不僅對基礎(chǔ)研究具有較大意義，同時也具有廣闊的應(yīng)用前景。該方法可以用來監(jiān)測慢性乙肝患者進行抗病毒治療后cccDNA的清除情況，從而為評價抗病毒藥物清除病毒的效果提供了重要的技術(shù)平臺。目前，研究者正在對該方法進一步開發(fā)，將其作為乙肝病毒分子病理學(xué)檢測方法進行推廣，為臨床醫(yī)師綜合評價患者肝臟內(nèi)病毒學(xué)狀態(tài)提供參考。