以小鼠模式探討年齡因子造成肝臟先天性免疫環境成熟對B型肝炎病毒容忍性之影響

Age-dependent maturation of hepatic innate immunity and HBV tolerance in the hydrodynamic injection mouse model

洪昭皓(Jau-Hau Horng)

指導教授 ： 陳培哲

國立臺灣大學/醫學院/微生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02782

摘要

慢性B型肝炎在全球約有3億的慢性帶原者。根據臨床上的研究發現，慢性B型肝炎的發生與感染時的年齡有很大的關聯性。尤其是在剛出生的幼兒有高達90%的機會成為慢性帶原者，而5歲之後只有5~7%。目前認為這可能是由於幼兒的免疫系統尚未完全成熟所造成，但是其確實的機制目前仍然未知。由於要研究B型肝炎病毒慢性感染的機制無法在人類進行，本實驗室已建立一以高壓流體注射法為基礎的動物模式，使得B型肝炎病毒能在小鼠肝臟細胞內持續表現。我們發現在較年輕的C57BL/6小鼠，B型肝炎病毒有延遲清除的現象，與我們在人類觀察到的現象類似。因此，我們希望能針對小鼠在不同年齡時免疫系統成熟的情形做比較，找出B型肝炎病毒延遲清除的原因。由於先天性免疫力對控制病毒感染以及後天性免疫的活化有重要的影響，本篇論文將聚焦在小鼠先天性免疫力的成熟。
我們建立了一套可靠的分離肝臟細胞以及非實質型細胞的方法，並利用流式細胞儀技術對非實質型細胞及肝臟內淋巴球之族群進行定量。根據我們的結果，利用膠原蛋白酶灌流分離法得到的竇狀內皮細胞與庫式細胞的數量在成鼠高於幼鼠，但利用免疫組織化學染色並沒有觀察到差異。利用膠原蛋白酶灌流法或破碎法得到的肝臟內白血球則是幼鼠高於成鼠，破碎法在幼鼠分離的效率明顯較好，顯示膠原蛋白酶灌流法在幼鼠的分離效果仍需要改進。另外，肝臟白血球的族群組成在不同年齡有很大的變化。第一，典型自然殺手T細胞以及樹突細胞在成鼠的總量明顯高於離乳前的幼鼠，但在每克肝組織的含量則是離乳前的幼鼠明顯較高。第二，B細胞、γδ-T細胞，以及非典型γδ-自然殺手T細胞於離乳前的幼鼠的總量以及在每克肝組織的含量明顯高於成鼠。第三，離乳前的幼鼠含有大量未成熟的前驅B細胞。
綜合以上結果，我們發現到不同年齡的小鼠，肝臟的免疫環境確實有明顯的改變，但要解釋B型肝炎病毒在年輕小鼠延遲清除的現象，並說明幼鼠的免疫力較差，我們還必須進行生物功能性的測試。在未來，我們希望能夠透過在動物的研究，解釋新生兒容易發生B型肝炎病毒慢性感染的機制。

關鍵字

慢性B型肝炎 ； 先天性免疫環境 ； 年齡因子 ； 肝臟非實質型細胞 ； 肝臟內淋巴球

並列摘要

In the world, approximately 300 million people have chronic hepatitis B (CHB). Mechanisms leading to HBV persistence are multiple, but one important factor is the age of HBV infection. Newborns or infants acquire HBV infection have >90% risk of developing CHB. The risk was reduced to 25~30% in 0~5 years old child and 5~7% after 5 years old. Currently higher risk of developing CHB in young age is proposal to the “immune system immaturity” in the infants. However, the actual mechanism of age factor influencing HBV persistence has not been determined or validated. Studying the mechanism of HBV chronic infection in human is unavailable. Our laboratory established a non-transgenic, immunocompetent mouse model for long-term expression of HBV by hydrodynamic injection method. In our previous study in hydrodynamic injection mouse model, we found that HBV clearance was delayed in younger mice. The phenomenon of delayed HBV clearance in young mouse is similar to human cases. Thus, our purpose is to investigate the mechanism of age factor influences HBV clearance in hydrodynamic injection mouse model with the aspect of immune maturation. We focused on innate immunity because its significant role in control virus infection and its ability to prime effective adaptive immune response.
We set up reproducible protocol to isolate hepatocytes and non-parenchymal cells (NPCs) from mouse liver, and perform quantitative analysis of major NPC and intrahepatic leukocyte (IHL) subpopulations. According to our result, the cell numbers per gram liver of isolated liver sinusoidal endothelial cells (LSECs) and kupffer cells (KCs) by collagenase perfusion method is higher in adult mice than young or pre-weaning one. But immunohistochemistry study shows the amount of LSEC and KCs has no significant difference among different ages. Through collagenase perfusion method or minced preparation, the cell numbers per gram liver of intrahepatic leukocyte is significantly higher in pre-weaning mice than adult one. The yield of intrahepatic leukocyte in pre-weaning mice through minced preparation is higher than collagenase perfusion method. The preliminary collagenase perfusion method requires further improvement, especially for isolating NPC form younger mice. The component of IHL subpopulation varies with age. First, the absolute number of αβ NKT cells and dendritic cells is significant higher in adult mice than pre-weaning one, but the cell numbers per gram liver of pre-waning mice is higher than adult one. Second, both absolute number and cell numbers per gram liver of B cells, γδ NKT cells and γδT cells is higher in pre-weaning mice than adult mice. Third, most of the CD45int cells in pre-weaning mice are immature pre-B cells.
In conclusion, the liver immune environment is different among adult, young and pre-weaning mice. To explain the delayed HBV clearance from the aspect of liver innate immune environment, our prospective aim is perform functional assay for liver immune cells and their response to HBV exposure. In the future, we hope to explain the mechanism that newborns are easily to develop chronic hepatitis B through animal study.

並列關鍵字

Chronic hepatitis B ； Age factor ； Innate immune environment ； Liver non-parenchymal cell ； Intrahepatic leukocytes

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