嚴重型急性胰臟炎與糖尿病治療之新策略研究:胰臟腺泡細胞的死亡與蘭氏小島細胞的組織工程

Abstract

第一部分: 於體內阻斷介白素6 (Interleukin 6)，可促進胰臟腺泡細胞的凋亡並減緩嚴重型急性胰臟炎 關於介白素6 (Interleukin 6)在嚴重型急性胰臟炎中，是否扮演發炎或抗細胞凋亡之起始角色，目前仍然並不清楚。本實驗嘗試以中和性抗體(neutralizing antibody)來對抗嚴重型急性胰臟炎之小鼠體內的介白素6，觀察是否能引導胰臟腺泡細胞之凋亡並減輕嚴重型急性胰臟炎之嚴重度。 本實驗以caerulein 50 µg/kg，每小時由腹腔注射一劑共6次後，再由腹腔注射10 mg/kg之脂多糖(lipopolysaccharides, LPS)，來引發實驗小鼠之嚴重型急性胰臟炎。治療組的小鼠，分別在注射第一劑caerulein後5分鐘及2小時時，再以腹腔注射中和性單株抗介白素6抗體(monoclonal anti–IL6 antibody)。在將實驗小鼠胰臟組織切片後，以TUNEL (the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labelling method)方法來檢測胰臟腺泡細胞凋亡之情形。 在注射過caerulein及脂多糖的小鼠中，可發現其血清中澱粉酵素(amylase)和介白素6之濃度上升，並且其胰臟和肺臟嚴重發炎，同時胰臟內訊息傳遞者兼轉譯活化物3 (signal transducer and activator of transcription, STAT3)之磷酸化(phosphorylation)反應增加。而相較於引發嚴重型急性胰臟炎之小鼠，加以注射單株抗介白素6抗體，則可明顯地抑制血清中澱粉酵素和介白素6上升，且胰臟內訊息傳遞者兼轉譯活化物3之磷酸化(phosphorylation)反應也有意義地下降。此外在組織學方面，經注射單株抗介白素6抗體的嚴重型急性胰臟炎小鼠，其胰臟腺泡細胞的壞死較少而凋亡較多，並且胰臟和肺臟發炎的組織學記分亦有意義地進步。 因此，阻斷介白素6可以壓制胰臟內訊息傳遞者兼轉譯活化物3之活化，經由促進胰臟腺泡細胞產生較多的凋亡，來降低嚴重型急性胰臟炎之嚴重度。對於嚴重型急性胰臟炎投予中和性介白素6抗體，可望成為治療的一種新方法。 第二部分: 用人類臍帶幹細胞(HUMSCs)來進行共同培養( co-culture)的新方法，可以維持培養胰臟胰島細胞團之生存和功能 一般認為，胰臟胰島細胞團islet-like cell clusters (ICCs)為一產生胰島素的組織來源。而胰臟胰島細胞團移植是第一型糖尿病目前最好、能永久治癒的方法。但由於胰島細胞團移植因供給不足、分離純化過程中胰島細胞團的流失，不易取得足夠大量純化的胰島細胞團，常為胰島細胞團移植失敗的原因之一。因此我們設計一新方法，以維持培養大鼠胰臟胰島細胞團之生存和功能。我們將培養大鼠胰臟胰島細胞團與人類臍帶間葉幹細胞進行共同培養(co-culture)，可以持續3個月地增加大鼠胰臟胰島細胞團之數量及胰島素分泌量。未加入人類臍帶間葉幹細胞所培養之大鼠胰臟胰島細胞團，其細胞則會逐漸毀壞，並在12天之後死亡。 由cytokine protein array我們發現，在共同培養液(co-culture medium)中，包括interleukin-6, tissue inhibitor of metalloproteinases-1, tissue inhibitor of metalloproteinases-2, monocyte chemoattractant protein-1, growth related oncogene, hepatocytes growth factor, insulin-like growth factor binding proteins 4, and interleukin-8等多種細胞激素的濃度均上升2倍以上，顯示這些細胞激素在此共同培養系統中舉足輕重。 我們發現，以人類臍帶間葉幹細胞進行共同培養之技術，應可保護胰臟胰島細胞團免於毀壞，可茲應用作為一新的培養方法，在移植前用以維持胰臟胰島細胞之生存和功能。 第三部分: 將人類臍帶間葉幹細胞轉化成為似胰島細胞團來控制第一型糖尿病 人類臍帶間葉幹細胞是由臍帶之Wharton’s jelly所分離出來，較胚胎或骨髓幹細胞更易於取得和處理。我們將其於神經條件培養液中，經由逐步的培養後，在體外誘導其轉化成為似胰島細胞團，並觀察其分泌人類胰島素及C-peptide之情形。再利用開腹手術將這些似胰島細胞團移植進入streptozotocin-induced糖尿病大鼠之肝臟內，以評估似胰島細胞團於體內之功能性穩定度。 在移植後12週時，檢測葡萄糖耐受度，以及免疫組織化學與電子顯微鏡分析。發現這些似胰島細胞團含有人類C-peptide，可依生理性葡萄糖濃度而釋放胰島素。並以Real-time reverse transcription-polymerase chain reaction方式偵測這些似胰島細胞團內胰島素及其他胰臟ß細胞相關基因例如Pdx1, Hlxb9, Nkx2.2, Nkx6.1, 及Glut-2之表現。 經過異種移植似胰島細胞團到streptozotocin-induced糖尿病大鼠後，在不需要使用免疫抑制劑的情況下，其高血糖及葡萄糖耐受不良情形明顯地改善。這些移植的細胞持續在體內分泌胰島素。此外，在肝臟內除了可見到這些似胰島細胞團，我們也發現了一些特殊的融合細胞，它們呈現人類胰島素和細胞核之陽性染色結果，並且具有分泌小泡。 在這個實驗中，我們成功地將人類臍帶間葉幹細胞分化成為成熟的似胰島細胞團，而這些似胰島細胞團在體外及體內均具有產生胰島素的能力。由於取得方便、快速且產生排斥的風險也低，若欲分化成人類胰島素分泌細胞，這些來自臍帶Wharton's Jelly之人類臍帶間葉幹細胞應是較佳的幹細胞來源。 因此，臍帶Wharton's Jelly中的人類臍帶間葉幹細胞有潛力成為糖尿病胰島細胞移植治療中的一個極佳選擇。

PART 1: Blockade of interleukin 6 accelerates acinar cell apoptosis and attenuates experimental acute pancreatitis in vivo It remains unclear whether interleukin (IL) 6 plays a role in initiating either the inflammatory or antiapoptotic responses in severe acute pancreatitis. This study examined the effect of neutralizing antibody against IL-6 on the induction of pancreatic acinar cell apoptosis and attenuation of the severity of severe acute pancreatitis. Experiments were conducted on laboratory mice with severe acute pancreatitis induced by lipopolysaccharide injection following six injections of caerulein at intervals of 1 h. Neutralizing monoclonal anti-IL-6 antibody was administered either 5 min or 2 h after the first caerulein injection. Apoptosis in pancreatic sections was determined by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labelling method. Administration of caerulein and LPS induced an increase in serum amylase and IL-6 levels, severe acute pancreatitis, pancreatitis-associated lung injury, and phosphorylation of signal transducer and activator of transcription (STAT) 3 in the pancreas. A neutralizing antibody against IL-6 effectively suppressed these responses. Application of IL-6 neutralizing antibody caused the induction of apoptosis in the pancreatic acinar cells of mice with acute pancreatitis. Blocking IL-6 suppresses STAT-3 activation in the pancreas and consequently attenuates the severity of severe acute pancreatitis by promotion of pancreatic acinar cell apoptosis. PART 2: A novel human stem cell-co-culture system that maintains the survival and function of culture islet-like cell clusters Islet-like cell clusters (ICCs) have been suggested to be a source of insulin-producing tissue for xenotransplantation in type I diabetes. We design an approach to maintain the cultured rat pancreatic ICCs survival and function, when it co-cultured with human umbilical cord mesenchymal stem cells (HUMSCs). HUMSCs in co-culture have ability to maintain ICCs survival and function, which number and insulin secretion of ICCs are increasing and lasting for 3 months, while ICCs gradually crash, which results in cell death after a period of 12 days of culture without HUMSCs. Cytokine protein array showed it has more than a 2-fold increase in several cytokines (interleukin-6, tissue inhibitor of metalloproteinases-1, tissue inhibitor of metalloproteinases-2, monocyte chemoattractant protein-1, growth related oncogene, hepatocytes growth factor, insulin-like growth factor binding proteins 4, and interleukin-8) levels on co-culture medium, implying an important role of these cytokines in this co-culture system. These findings suggest that co-culture with HUMSCs may have a significant potential to protect ICCs from damage during culture, and may be employed in a novel culture approach to maintain islet cell survival and function before transplantation. PART 3: Islet-Like Clusters Derived from Mesenchymal Stem Cells in Wharton's Jelly of the Human Umbilical Cord for Transplantation to Control Type 1 Diabetes HUMSCs isolated from Wharton’s jelly of the umbilical cord, which can be easily obtained and processed compared with embryonic and bone marrow stem cells, were induced to transform into islet-like cell clusters in vitro through stepwise culturing in neuron-conditioned medium. Human insulin and C-peptide for islet-like cell clusters were determined. To assess the functional stability of the islet-like cell clusters in vivo, these cell clusters were transplanted into the liver of streptozotocin-induced diabetic rats via laparotomy. Glucose tolerance was measured on week 12 after transplantation accompanied with immunohistochemistry and electron microscopy analysis. These islet-like cell clusters were shown to contain human C-peptide and release human insulin in response to physiological glucose levels. Real-time reverse transcription-polymerase chain reaction detected the expressions of insulin and other pancreatic ß-cell-related genes, such as Pdx1, Hlxb9, Nkx2.2, Nkx6.1, and Glut-2 in these islet-like cell clusters. The hyperglycemia and glucose intolerance in streptozotocin-induced diabetic rats was significantly alleviated after xenotransplantation of islet-like cell clusters, without the use of immunosuppressants. These transplanted cells continued to express insulin in vivo. In addition to the existence of islet-like cell clusters in the liver, some special fused liver cells were also found, which characterized by human insulin and nuclei-positive staining and possessing secretory granules. In this study, we successfully differentiate HUMSCs into mature islet-like cell clusters, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. HUMSCs in Wharton's Jelly of the umbilical cord seem to be the preferential source of stem cells to convert into insulin-producing cells, because of the large potential donor pool, its rapid availability, no risk of discomfort for the donor, and low risk of rejection. Therefore, HUMSCs in Wharton's Jelly of the umbilical cord have the potential for an excellent candidate in β-cell replacement therapy of diabetes mellitus.