當胰小島受損時，其血管與神經網路的改變與重組，影響胰小島在糖尿病發病過程中的生理變化。然而，由於胰臟的不透明性，且胰小島位於胰臟的深處，這雙重的阻礙，使得光學顯微鏡在觀察胰小島血管與神經網路時受到限制。在另一方面，使用傳統切片機所獲得的二維光學顯微影像，只能將視野侷限在某一切面上，無法針對血管與神經網路的三維結構進行觀察。本實驗中我們使用三維影像技術，定性與定量分析神經組織之「膠質細胞網路」 (Schwann cell network)與「血管周細胞」 (pericytes) 在正常胰小島組織的型態，與其在糖尿病小鼠發病過程中的改變與重組。

在胰臟中，Schwann cells主要位於胰小島周邊，區隔內分泌與外分泌組織；pericytes則攀附於血管壁，並隨血管延伸。在中樞神經系統中，當腦與脊髓受損時，Schwann cells與pericytes同時啟動，形成glial scar控制受傷區域。但目前還不清楚在胰小島發炎時 -- 特別是在第1型糖尿病發病初期，當免疫系統開始破壞但未完全摧毀胰小島時 -- 是否有類似的細胞反應幫助胰小島賀爾蒙進入循環系統，維持生理機能運作。在本實驗中我們使用Schwann cells與pericytes的染色方法，並結合三維影像技術，觀察它們在胰小島發炎時於空間中的變化。我們並運用網路組織分析方法，進行第1型糖尿病小鼠模型，包括streptozotocin (STZ) 注射小鼠模型，與nonobese diabetic (NOD) 小鼠模型的胰臟組織影像分析。
從共軛焦顯微鏡的影像，我們觀察到正常小鼠中，膠質細胞網路會從蘭氏小島外圍往核心發展。施打STZ一周後的小鼠，蘭氏小島內微血管的周圍，膠質細胞會增生；同時血管周細胞的密度也會增加。NOD小鼠中，在早期與中期胰島炎，受損區域周圍以及微血管旁會有膠質細胞增生的情形；同時血管周細胞會聚集在受損區域中的喂養微動脈 (feeding arteriole) 上。
從蘭氏小島受損時，這些細胞的反應可以說明他們的可塑性，也說明當實驗性糖尿病對蘭氏小島造成損害時，膠質細胞與血管周細胞會對損傷進行立即性的補救。

目 錄

中文摘要 i
Abstract iii
縮寫對照表 v
圖目錄 vi
1. 文獻回顧 1
1.1 簡介 1
1.2 生物醫學影像技術 2
1.2.1 共軛焦顯微鏡技術 3
1.2.2 生物組織澄清技術 3
1.3 小鼠胰臟內蘭氏小島簡介 4
1.3.1 小鼠胰臟蘭氏小島之血管網路 5
1.3.2 小鼠胰臟蘭氏小島之神經網路 6
1.3.3 小鼠胰臟蘭氏小島之周圍細胞網路 6
1.4 糖尿病簡介 8
1.4.1 Streptozotocin (STZ) 誘發型糖尿病 8
1.4.2 Non-obese diabetic (NOD) mice自發型糖尿病 9
1.5 研究動機 10
2. 實驗材料與方法 12
2.1 實驗動物 12
2.2 實驗材料和儀器 12
2.3 高劑量單次施打Streptozotocin (STZ) 誘發小鼠糖尿病 14
2.4 以染劑灌流進行血管染色 14
2.4.1 染劑配製 14
2.4.2 灌流 14
2.5 樣品處理 15
2.6 組織染色 15
2.6.1 組織螢光免疫染色 15
2.6.2 組織螢光染劑染色 16
2.7 封片 16
2.8 共軛焦顯微鏡 17
2.9 影像投影和分析 17
2.10 統計 17
3. 結果 19
3.1 膠質細胞網路 (Schwann cell network) 在正常胰小島組織的型態 19
3.2 血管周細胞 (pericytes) 在正常胰小島組織的型態 25
3.3 STZ誘發糖尿病小鼠胰小島組織的膠質細胞網路與血管周細胞的變化 29
3.4 淋巴球滲透導致NOD小鼠早期及中期的膠質細胞網路變化 34
3.5 NOD小鼠在胰島炎發生時所引發周細胞在小動脈之變化 41
4. 討論 44
5. 結論與未來展望 46
6. 未來工作 47
7. 參考文獻 48
8. 附錄 54

[1] Merani S. "Optimal implantation site for pancreatic islet transplantation" Br J Surg. 2008 Dec; 95(12):1449-61.

[2] Gromada J, Franklin I, Wollheim CB. "Alpha-cells of the endocrine pancreas: 35 years of research but the enigma remains. " Endocr Rev. 2007 Feb;28(1):84-116. Epub 2007 Jan 16.

[3] Taborsky GJ Jr. "Islets have a lot of nerve! Or do they?"Cell Metab. 2011 Jul 6;14(1):5-6.

[4] Regina E. Burris and Matthias Hebrok"Pancreatic Innervation in Mouse Development and β-cell Regeneration "Neuroscience. 2007 December 12; 150(3): 592–602.

[5]C. WOODS AND DANIEL PORTE, JR"Neural Control of the Endocrine Pancreas "Physiol Rev,1975.54(3)：p.596-619

[6] Katherine Smith "Neurogastroenterology: Improving 3D imaging of the enteric nervous system"Nature Reviews Gastroenterology and Hepatology 8, 600 (November 2011)

[7] Fu, Y. Y. and S. C. Tang (2010). "At the movies: 3-dimensional technology and gastrointestinal histology." Gastroenterology 139(4): 1100-1105, 1105 e1101.

[8] José-Angel Conchello & Jeff W Lichtman"Optical sectioning microscopy"Nature Methods - 2, 920 - 931 (2005)

[9] Fu, Y. Y., C. H. Lu, et al. (2010). "Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution." J Biomed Opt 15(4): 046018.

[10] Jeff W Lichtman & José-Angel Conchello "Fluorescence microscopy"Nature Methods - 2, 910 - 919 (2005)

[11] Wang, R.K. "Investigation of optical clearing of gastric tissue immersed with hyperosmotic agents"IEEE Journal of Selected Topics in Quantum Electronics,2008.13(2):p.021101

[12] Valery V. Tuchin ; Ruikang K. Wang ; Alvin T. Yeh ;"Optical Clearing of Tissues and Cells"J. Biomed. Opt. 13(2), 021101 (April 23, 2008).

[13] Xiangqun Xu and Ruikang K Wang "Synergistic effect of hyperosmotic agents of dimethyl sulfoxide and glycerol on optical clearing of gastric tissue studied with near infrared spectroscopy. " 2004 Phys. Med. Biol. 49 457

[14] Fu, Y. Y. and S. C. Tang (2010). "Optical clearing facilitates integrated 3D visualization of mouse ileal microstructure and vascular network with high definition." Microvasc Res 80(3): 512-521.

[15] Collombat, P., X. Xu, et al. (2010). "Pancreatic beta-cells: from generation to regeneration." Semin Cell Dev Biol 21(8): 838-844.

[16] Lelio Orci a, RogerH Unger b "FUNCTIONAL SUBDIVISION OF ISLETS OF LANGERHANS AND POSSIBLE ROLE OF D CELLS"The Lancet, Volume 306, Issue 7947, Pages 1243 - 1244, 20 December 1975

[17] Cabrera, O., D. M. Berman, et al. (2006). "The unique cytoarchitecture of human pancreatic islets has implications for islet cell function." Proc Natl Acad Sci U S A 103(7): 2334-2339.

[18] Subhadra C. Gunawardana,et al."Imaging beta cell development in real-time using pancreatic explants from mice with green fluorescent protein-labeled pancreatic beta cells"JANUARY AND FEBRUARY 2005, Volume 41, Issue 1-2, pp 7-11

[19] Hideto Kojima , et al."Extrapancreatic insulin-producing cells in multiple organs in diabetes"February 24, 2004 vol. 101 no. 82458-2463

[20] Mary Kay Treutelaar,et al. "Nestin-Lineage Cells Contribute to the Microvasculature but Not Endocrine Cells of the Islet
"Diabetes October 2003 vol. 52 no. 102503-2512

[21] Xianquan Li,et al. "Islet Microvasculature in Islet Hyperplasia and Failure in a Model of Type 2 Diabetes
"Diabetes November 2006 vol. 55 no. 112965-2973

[22] Annika M. Svensson, et al."Age-induced changes in pancreatic islet blood flow: evidence for an impaired regulation in diabetic GK rats"AJP - Endo November 1, 2000 vol. 279 no. 5 E1139-E1144

[23] L. Jansson, P.-O. Carlsson "Graft vascular function after transplantation of pancreatic islets"Diabetologia June 2002, Volume 45, Issue 6, pp 749-763

[24] Marcela Brissova,et al."Pancreatic Islet Production of Vascular Endothelial Growth Factor-A Is Essential for Islet Vascularization, Revascularization, and Function"
Diabetes November 2006 vol. 55 no. 112974-2985

[25] Fu, Y. Y., C. H. Lu, et al. (2010). "Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution." J Biomed Opt 15(4): 046018.

[26] Dosch, H. M., H. Tsui, et al. (2008). "Islet Glia, Neurons, and beta Cells The Neuroimmune Interface in the Pathogenesis of Type 1 Diabetes." Immunology of Diabetes V: From Bench to Bedside 1150: 32-42.

[27] Winer, S., H. Tsui, et al. (2003). "Autoimmune islet destruction in spontaneous type 1 diabetes is not beta-cell exclusive." Nat Med 9(2): 198-205.

[28] Zhang, Y. Q. and N. Sarvetnick (2003). "Development of cell markers for the identification and expansion of islet progenitor cells." Diabetes Metab Res Rev 19(5): 363-374.

[29] Fawcett JW, Asher RA (1999) The glial scar and central nervous system repair. Brain Res Bull 49: 377-391

[30] Pekny M, Nilsson M (2005) Astrocyte activation and reactive gliosis. Glia 50: 427-434


[31] Goritz C, Dias DO, Tomilin N, Barbacid M, Shupliakov O, Frisen J (2011) A pericyte origin of spinal cord scar tissue. Science 333: 238-242

[32] von Boyen GB, Steinkamp M, Reinshagen M, Schafer KH, Adler G, Kirsch J (2004) Proinflammatory cytokines increase glial fibrillary acidic protein expression in enteric glia. Gut 53: 222-228

[33] Cornet A, Savidge TC, Cabarrocas J, et al. (2001) Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn's disease? Proc Natl Acad Sci U S A 98: 13306-13311

[34] Costantini TW, Bansal V, Krzyzaniak M, et al. (2010) Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells. Am J Physiol Gastrointest Liver Physiol 299: G1308-1318

[35] Vasina V, Barbara G, Talamonti L, et al. (2006) Enteric neuroplasticity evoked by inflammation. Auton Neurosci 126-127: 264-272

[36] Kolb, H. (1987). "Mouse models of insulin dependent diabetes: low-dose streptozocin-induced diabetes and nonobese diabetic (NOD) mice." Diabetes Metab Rev 3(3): 751-778.

[37] Donev SR (1984) Ultrastructural evidence for the presence of a glial sheath investing the islets of Langerhans in the pancreas of mammals. Cell Tissue Res 237:343–348

[38] Sunami E, Kanazawa H, Hashizume H, Takeda M, Hatakeyama K, Ushiki T (2001) Morphological characteristics of Schwann cells in the islets of Langerhans of the murine pancreas. Arch Histol Cytol 64:191–201

[39] Hayden MR, Karuparthi PR, Habibi J et al (2008) Ultrastructure of islet microcirculation, pericytes and the islet exocrine interface in the HIP rat model of diabetes. Exp Biol Med (Maywood) 233:1109–1123

[40] Richards OC, Raines SM, Attie AD (2010) The role of blood vessels, endothelial cells, and vascular pericytes in insulin secretion and peripheral insulin action. Endocr Rev 31: 343-363

[41] Winer S, Tsui H, Lau A, et al. (2003) Autoimmune islet destruction in spontaneous type 1 diabetes is not beta-cell exclusive. Nat Med 9: 198-205

[42] Teitelman G, Guz Y, Ivkovic S, Ehrlich M (1998) Islet injury induces neurotrophin expression in pancreatic cells and reactive gliosis of peri-islet Schwann cells. J Neurobiol 34: 304-318
[43] Nakamura M, Kitamura H, Konishi S et al (1995) The endocrine pancreas of spontaneously diabetic db/db mice: microangiopathy as revealed by transmission electronmicroscopy.Diabetes Res Clin Pract 30:89–100

[44] Hayden MR, Karuparthi PR, Habibi J et al (2007) Ultrastructural islet study of early fibrosis in the Ren2 rat model of hypertension.Emerging role of the islet pancreatic pericyte-stellate cell. JOP 8:725–738

[45] Denis MC, Mahmood U, Benoist C, Mathis D,Weissleder R (2004) Imaging inflammation of the pancreatic islets in type 1 diabetes. Proc Natl Acad Sci U S A 101:12634–12639

[46] Chiu YC, Hua TE, Fu YY, Pasricha PJ, Tang SC (2012) 3-D imaging and illustration of the perfusive mouse islet sympathetic innervation and its remodelling in injury. Diabetologia 55: 3252-3261

[47] Bergers G, Song S (2005) The role of pericytes in blood-vessel formation and maintenance. Neuro Oncol 7: 452-464

[48] Sandler S, Jansson L (1985) Vascular permeability of pancreatic islets after administration of streptozotocin. Virchows Arch A Pathol Anat Histopathol 407: 359-367
[49] Papaccio G (1993) Insulitis and islet microvasculature in type 1 diabetes. Histol Histopathol 8: 751-759

[50] Beppu H, Maruta K, Kurner T, Kolb H (1987) Diabetogenic action of streptozotocin: essential role of membrane permeability. Acta Endocrinol (Copenh) 114: 90-95

[51] Enghofer M, Usadel KH, Beck O, Kusterer K (1997) Superoxide dismutase reduces islet microvascular injury induced by streptozotocin in the rat. Am J Physiol 273: E376-382

[52] Papaccio G, Baccari GC, Strate C, Linn T (1994) Pancreatic duct inflammatory infiltration in the nonobese diabetic (NOD) mouse. J Anat 185 ( Pt 3): 465-470