利用維甲酸促進胰島微血管增生及透過肝細胞重新編程產製胰島素分泌細胞團塊來改善糖尿病的細胞治療

Improving cell-replacement therapies for diabetes via enhancing islet vascularization with retinoic acid and generating insulin-producing cell clusters from hepatocyte reprogramming

簡皎芸(Chiao-Yun Chien)

指導教授 ： 李宣書

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2016年)

https://doi.org/10.6342/NTU201603601

摘要

糖尿病是一種代謝異常疾病，主要原因是由於體內胰島素缺乏或胰島素阻抗性而所造成血糖高於標準值。其中第一型糖尿病(胰島素依賴型糖尿病)的病人因為體內胰島素缺乏，所以可透過胰島素注射治療，但是要達到長期有效的調控及維持血糖，可能的治療方法為胰島移植。然而，胰島移植的兩項瓶頸是胰島來源的絕對缺乏，以及移植後的胰島存活及功能不佳，使得此項治療還未能廣泛應用在治療第一型糖尿病的患者。本篇論文的主要目標以胰島移植來治療糖尿病為基礎下探討可能的改善方式，在研究的第一個部分，我們首先分析並發現人體必需的營養素維他命A在胚胎發育時期是重要影響因子。我們進一步發現全反式維甲酸, 維他命A酸代謝活性產物，對於胰臟發育扮演重要角色。因此，我們透過探討母體維生素Ａ缺乏時如何影響胎兒時期的胰島細胞發育，發現全反式維甲酸藉由調控胰島細胞自行分泌血管內皮生長因子促成胰島內微血管網的生成。根據這樣的結果，我們進一步探討是否全反式維甲酸能夠用於改善糖尿病治療，結果顯示全反式維甲酸能夠逐漸透過增加β細胞的數量，以及修復血管層粘連蛋白的表現來改善糖尿病小鼠的血糖控制，主因是全反式維甲酸能藉由調控胰島細胞自行分泌血管內皮生長因子促成糖尿病鼠胰島內微血管網的生成、血管層粘連蛋白的表現和β細胞增生。重要的是發現全反式維甲酸也可以增進移植胰島內微血管網的生成細及胰島素分泌功能，而能夠有效改善糖尿病小鼠的高血糖症狀。而在研究的第二個部分，我們試著在三維的細胞培養系統來重新編程肝臟細胞成為表現Sox9的肝前驅細胞，這些表現Sox9的肝前驅細胞能在聚乙烯醇(Polyvinyl alcohol, PVA)受質上形成球體狀的結構；進一步結合表現Pdx1, Ngn3 與 MafA三個轉錄因子將其重新編程成為製造胰島素的細胞團塊，當這些能產生胰島素的球體細胞團塊移植到糖尿病小鼠，發現也能夠有效改善糖尿病小鼠的高血糖症狀。為了改善胰島來源的絕對缺乏以及移植後的胰島存活及功能不佳的兩項問題，本論文研究發現可透過維甲酸來增加胰島微血管，來改善移植胰島的存活與功能，更進一步可透過肝細胞重新編程產製具備胰島素分泌功能的細胞團塊，以改善胰島來源缺乏的問題。

關鍵字

維甲酸 ； 糖尿病 ； 治療 ； 胰島微血管增生 ； 胰島素分泌細胞團塊

並列摘要

Diabetes mellitus is a metabolic disorder resulted from insulin deficiency or insulin resistance. The primary treatment for patients with type I diabetes (or insulin-dependent diabetes mellitus) that are absolutely deficient in insulin is insulin injection. However, insulin injection as a regimen for treating diabetes is unable to long-termly control blood sugar levels. Instead, islet transplantation is the potential way to sustainably modulate glycemic status. However, the shortage of donor islets and poor islet graft survival and function limit the potential use of islet transplantation to treat patients with type 1 diabetes. The major goal of the PhD thesis is trying to overcome the barrier of application of islet transplantation.
In the first part of the thesis, we analyzed and found that an essential micronutrient, vitamin A, is an important factor in embryogenesis. All-trans retinoic acid (atRA), the active metabolite of vitamin A, plays an essential role in regulating pancreatic development. We initially investigated how maternal vitamin A deficiency may affect fetal islet development and revealed that atRA is involved in regulating vascularized islet formation via modulating vascular endothelial growth factor secretion. Based on the observation, we next evaluated whether treatment with atRA can ameliorate diabetes. We found that administration of atRA could gradually decrease the blood glucose levels of diabetic mice, increase the amount of β-cells, and restore the vascular laminin expression. Furthermore, atRA induced the expression of vascular endothelial growth factor-A from the pancreatic islets, which mediated the restoration of islet vascularity and recovery of β-cell mass. Importantly, we showed atRA treatment significantly improved grafted islet functionality and vascularity and the combination of islet transplantation and atRA administration could rescue hyperglycemia in diabetic mice. The findings suggest vitamin A derivatives can potentially be used as a supplementary treatment to improve diabetes management and glycemic control.
In the second part of the thesis, we tried to reprogram primary hepatocytes to Sox9-expressing progenitor cells in a three dimensional (3D) culture system. We found these Sox9-expressing progenitors could form spheroid on polyvinyl alcohol (PVA) substrates. In combination with overexpressing Pdx1, Ngn3 and MafA, these hepatocytes could further be reprogramed to insulin-producing clusters. Transplantation of insulin-producing clusters into diabetic mice was found to rescue hyperglycemia. In conclusion, these current works discovered that atRA treatment could improve survival and functionality of the grafted islets. Moreover, these works further developed a novel strategy to generate insulin-secreting cell clusters via hepatocyte reprogramming for transplantation.

並列關鍵字

retinoic acid ； diabetes ； therapy ； islet vascularization ； insulin-producing cell clusters

參考文獻

1. Damasceno DC, Netto AO, Iessi IL, Gallego FQ, Corvino SB, Dallaqua B, Sinzato YK, Bueno A, Calderon IM, Rudge MV: Streptozotocin-induced diabetes models: pathophysiological mechanisms and fetal outcomes. Biomed Res Int 2014, 2014:819065.

3. Dor Y, Brown J, Martinez OI, Melton DA: Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004, 429:41-46.

4. Li W, Zhang H, Nie A, Ni Q, Li F, Ning G, Li X, Gu Y, Wang Q: mTORC1 pathway mediates beta cell compensatory proliferation in 60 % partial-pancreatectomy mice. Endocrine 2016, 53:117-128.

5. Wang G, Rajpurohit SK, Delaspre F, Walker SL, White DT, Ceasrine A, Kuruvilla R, Li RJ, Shim JS, Liu JO, et al: First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic beta-cell mass. Elife 2015, 4.

6. Berney T, Johnson PR: Donor pancreata: evolving approaches to organ allocation for whole pancreas versus islet transplantation. Transplantation 2010, 90:238-243.