三維多重訊號人類胰臟組織學方法之建構

Toward 3-D multiplex human pancreas histology in health and disease

簡宏任(Hung-Jen Chien)

指導教授 ： 湯學成

國立清華大學/生命科學院/生物科技研究所/博士(2021年)

摘要

胰臟是兼具內分泌功能與外分泌功能的器官，具有分泌消化酵素與調控血糖恆定的重要功能。獲取胰臟微結構之資訊對於研究胰臟之生理與疾病極具重要意義，胰臟組織學的知識主要建立於常規組織切片觀察(二維平面)。然而胰臟的結構富含血管網路與神經系統等，具三維空間分布特性的組織，難以利用常規組織學技術進行胰臟網路全貌的分析。
為了克服研究胰臟神經組織學時遭遇的技術問題，本研究結合共軛焦顯微鏡技術、免疫螢光染色技術與組織透明化技術，擷取人類胰臟之三維影像，並將正常與疾病狀態下的組織三維影像與常規組織學影像進行交叉評估，以研究胰臟組織中的重塑。
利用三維影像技術，我們建構出人類胰臟於正常與疾病狀態下之傳入和傳出神經的支配模式。由結合胰臟之微結構、脈管系統和神經網路的三維影像資訊，我們發現胰臟內神經節之傳入和傳出神經元具有共定位現象。此外本研究也利用高解析度影像，對人類胰臟中異位性脂肪細胞的神經支配進行分析。並進一步將人類與小鼠的胰臟神經支配模式進行比較。
我們也以三維影像技術分析人類胰臟中局部蘭氏小島重塑與胰臟上皮內瘤樣病變-蘭氏小島複合物(PanIN–islet complex)的關聯性。我們將發生重塑的微環境之三維影像與常規組織學影像相互比對，觀察到發生重塑之胰臟微環境具有蘭氏小島聚集、發炎反應、基質細胞積累和細胞增殖等特徵。同時也觀察到獨特的導管-蘭氏小島細胞簇和蘭氏小島內導管的形成，這些現象表明胰臟上皮內瘤樣病變-蘭氏小島複合物可能是源於胰臟損傷後組織恢復所產生。
綜上所述，本研究結合多維(三維與二維)與多重(螢光、穿透光、H&E與IHC)訊號組織學方法，分析人類胰臟中的神經支配模式與局部病變之重塑，強調了此一新穎的成像策略於研究人類胰臟形態學和組織病理學之潛力。

關鍵字

三維胰臟組織學 ； 胰內神經節 ； 胰臟神經支配 ； 胰臟上皮內瘤樣病變 ； 胰島細胞新生

並列摘要

The pancreas is a vital organ of the exocrine digestive system and a critical regulator of hormone secretion (e.g., insulin and glucagon) to maintain glucose homeostasis in circulation. To study the pancreatic exocrine and endocrine functions in health and disease, histological analysis of the pancreatic microstructure and neurovascular networks is indispensable for evaluating the pancreatic physiology and pathogenesis. To date, investigators routinely use the microtome-based 2-dimensional (2-D) histology to examine the pancreas; yet, the complex nervous and vascular tissues of the organ cannot be easily reconstructed to provide an integrative view of the pancreas tissue networks.
This thesis develops advanced techniques of multiplex confocal imaging with tissue clearing (or optical clearing) to generate transparent pancreatic specimens for 3-dimensional (3-D) imaging. Both the human donor pancreases and the surgical biopsies of pancreatic cancer were analyzed in the investigation. Importantly, we cross-validate the 3-D image data with the classic 2-D H&E and immunohistochemical images to confirm the tissue remodeling in the disease condition. The background information of the human pancreas and the imaging techniques are summarized in Chapter 1.
Using our new 3-D approach, we characterized the human pancreatic afferent and efferent innervation patterns in health and at the tumor boundary. The 3-D image data provide a comprehensive view to analyze the pancreatic microstructure, vasculature, and innervation. The results allow us to identify the co-localization of the afferent and efferent neurons in the intra-pancreatic ganglia and reveal the sympathetic and parasympathetic innervation of the infiltrated adipocytes. The comparison between the mouse and human pancreatic innervation is summarized in Chapter 2.
In Chapter 3, we demonstrate how to use 3-D multiplex histology to study the association between local islet remodeling and the PanIN–islet complex in the human donor pancreas. Our multiplex high-definition images simultaneously reveal the islet aggregation, inflammation, stromal accumulation, cell proliferation, and the likely islet neogenesis, featuring the duct-islet cell cluster and intra-islet ducts, in the peri-lesional microenvironment. The tissue remodeling and the evidence of inflammation and stromal accumulation suggest that the PanIN–islet complex is derived from tissue repair after a local injury.
In summary, this thesis characterizes the human pancreatic innervation patterns and the local pancreatic and islet remodeling with multi-dimensional and multiplex signals (fluorescence, transmitted light, H&E, and IHC signals), highlighting an importance step of using the modern 3-D histology to understand the unknown features of the human pancreas.

並列關鍵字

3-D pancreatic histology ； Intrapancreatic ganglia ； Pancreatic innervation ； Pancreatic intraepithelial neoplasia ； Islet cell neogenesis

參考文獻

1. Huang, H.H., S. Harrington, and L. Stehno-Bittel, The Flaws and Future of Islet Volume Measurements. Cell Transplant, 2018. 27(7): p. 1017-1026.

2. Komatsu, H., et al., Oxygen environment and islet size are the primary limiting factors of isolated pancreatic islet survival. PLoS One, 2017. 12(8): p. e0183780.

3. Peter In’t Veld, S.S., Microscopic Anatomy of the Human Islet of Langerhans., in Islets of Langerhans, M.S. Islam, Editor. 2015, Springer, Dordrecht. p. 19-38.

4. Brissova, M., et al., Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy. J Histochem Cytochem, 2005. 53(9): p. 1087-97.

5. Rahier, J., R.M. Goebbels, and J.C. Henquin, Cellular composition of the human diabetic pancreas. Diabetologia, 1983. 24(5): p. 366-71.