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研究生：	黃千芳 Huang, Chen-Fang
論文名稱：	利用脾臟萃取之間質幹細胞治療小鼠胰臟炎 The usage of spleen-derived mesenchymal stem cell for the treatment of pancreatitis in mice
指導教授：	沈延盛 Shan, Yan-Shen
學位類別：	碩士 Master
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	47
中文關鍵詞：	胰臟炎、間質幹細胞
外文關鍵詞：	pancreatitis, mesenchymal stem cell
相關次數：	點閱：110 下載：1
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胰臟炎顧名思義是發生在胰臟的發炎疾病。依照其發炎持續的時間分為急性及慢性胰臟炎。而近來的研究指出，幹細胞療法對於胰臟炎的治療可能是一大突破。其中，間質幹細胞更已被許多研究證明，在組織修復再生，以及抑制免疫反應上有很好的能力。因此，在本研究中，我們假設從小鼠脾臟萃取之間質幹細胞，在急性或慢性的胰臟炎中，可能藉由去抑制胰臟發炎之發炎反應，或是分化成部分種類的胰臟細胞，來達到修復受損胰臟組織的功能。我們將使用腹腔注射Cerulean藥物至C57BL/6小鼠體內，頻率為一小時一次，持續注射五小時，來建立急性胰臟炎小鼠模式。而從C57BL/6小鼠脾臟萃取的間質幹細胞在標記上Qtracker之後，則是以尾巴靜脈注射的方式來進行治療。在施打間質幹細胞後，將會對小鼠採集血清進行Lipase和Myeloperoxidase (MPO)的活性測量。而在取得胰臟之後，則會以免疫組織染色分析，去觀察胰臟細胞之腫脹程度，壞死程度以及在胰臟炎中過量分泌的amylase表現量。實驗結果顯示，間質幹細胞確實能夠降低在急性胰臟炎情況下，血清當中代表發炎程度的Lipase和Myeloperoxidase (MPO)活性，而在組織切片當中，則是可以看到間質幹細胞確實會在發炎的胰臟中出現，並改善了胰臟細胞之腫脹及壞死程度，且減少了原本胰臟炎中會過量分泌的amylase表現量。在mRNA的表現上，間質幹細胞降低了在發炎胰臟組織中的發炎細胞激素TNF-α以及IFN-γ的表現，同時也升高了抗發炎細胞激素IL-10的表現。另一方面，我們利用手術方式建立了另一種胰臟炎的小鼠模式，主要是利用血管夾夾住部分的胰臟，造成組織的缺氧並壞死，隨著時間的推移，此模式的小鼠胰臟炎也可能會惡化成慢性胰臟炎。我們在此胰臟炎小鼠模式上同樣進行了間質幹細胞的治療，並進行血糖耐受性(IPGTT)分析，結果證實在手術後的小鼠之血糖耐受性會出現下降趨勢，而間質幹細胞則可以改善此現象。根據以上實驗結果，已證明了間質幹細胞對於急性胰臟炎的治療，有很好的抑制發炎以及組織修復的能力。下一步我們將間質幹細胞應用在手術導致胰臟炎的小鼠疾病模式，希望可以進一步證明間質幹細胞擁有再生胰臟細胞的能力，闡明間質幹細胞治療胰臟炎的機制和價值性。

Pancreatitis is inflammation of the pancreas. It may be acute – beginning suddenly and lasting a few days, or chronic – occurring over many years. In recently years, new stem cell therapies have raised the possibility of improving pancreatitis treatment. And the mesenchymal stem cells (MSCs) are considered to play a role in tissue repair and regeneration, including have remarkable immunosuppressive properties. Otherwise, the possibility of the spleen being a reservoir of islet stem cells is supported by close interrelationships between the spleen and pancreas during development. In this study, we suppose that the mesenchymal stem cells from the spleen could have the ability to repair the damaged pancreatic cells after acute/chronic pancreatitis occurred by the way of differentiating into pancreatic cells or suppressing the inflammation to supply the pancreas function. In our research, we found that the MSCs present several MSCs specific surface markers. Then, in the cerulean-caused acute pancreatitis mouse model, the improvement of lipase and MPO activity level in the serum after the MSCs treatment was demonstrated and the labeled MSCs were also recognized in pancreas. And the edema level, necrosis level and amylase expression of pancreas in acute pancreatitis model were down-regulated after the MSCs treatment. Finally, the inflammatory cytokines TNF-α and IFN-γ mRNA expression were down-regulated and the anti-inflammatory cytokine IL-10 was up-regulated by MSCs treatment. In the other hand, we establish the other pancreatitis mouse model by surgery using the vessel champ to obstruct blood flow in a part of pancreas, and this part of pancreas will be ischemia. In this model, the cell loss in pancreas is supposed. As time goes by, the chronic pancreatitis could also be caused. Here we determined the IPGTT (Intraperitoneal Glucose Tolerance Test) on this surgery-caused pancreatitis model with or without MSCs treatment. The result revealed that the MSCs can successfully improve the IPGTT in pancreatitis. According to the above results, the abilities of MSCs to suppress the inflammation and repair the damaged tissue in acute pancreatitis model and pre-chronic pancreatitis model were already proved. So in the future, we are going to confirm that MSCs have the ability to repair pancreatitis not only by regulating inflammation but also by regenerating the pancreatic cells such as beta-cells and acinar cells.

摘要	I
Abstract	III
Acknowledgment	V
Contents	VII
Abbreviation	X
Introduction	1
Pancreatitis	1
Acute pancreatitis	1
chronic pancreatitis 	2
The treatments of pancreatitis  	3
The stem cell therapy	3
The mesenchymal stem cell 	3
Material and methods	7
Isolation of MSCs from donor mice and culture expansion 	8
Phenotype multipotency of cultured MSCs 	8
Preparation of infusion cells	8
Establishment of acute pancreatitis in mice model 	8
MPO assay 	8
Lipase activity level in serum	9
Immunohistochemistry (IHC) and immunofluorescence (IF) 	10
Protein extraction 	11
Flow cytometric analysis 	11
Histological examination	11
RNA extraction	12
Reverse transcriptase-polymerase chain reaction (RT-PCR) 	12
Intraperitoneal Glucose Tolerance Test 	13
Results	14
The mesenchymal stem cells from the spleen of C57BL/6 mice are characterized with several MSC surface markers  	14
The establishment of pancreatitis mouse model by cerulean injection and the 
The Qtracker-positive cells can be recognized significantly in the cerulean-MSC group	15
In the acute pancreatitis mouse model, the lipase and Myeloperoxidase (MPO) activity levels of serum are down regulated after MSC treatment  	15
In acute pancreatitis mouse model, the edema level, necrosis level, and the amylase expression of the pancreas are improved after MSC treatment. 	16
T-Cell suppression by hcMSCs 	16
Effect of MSCs in inflammation response 	16
The establishment of pancreatitis model by surgery 	17
The improvement of IPGTT after the MSCs treatment in surgery-induced pancreatitis model  	17
Discussion 	19
Figure 	21
Figure 1: The mesenchymal stem cells from the spleen of C57BL/6 mice are characterized with several MSC surface markers  	21
Figure 2: The acute pancreatitis mouse model is established by five hourly intraperitoneal injections of 50 μg/kg of cerulean. The MSCs would be labeled with Qtracker and then to treat the pancreatitis model by tail vain injection.  	23
Figure 3: In the acute pancreatitis mouse model, the lipase and Myeloperoxidase (MPO) activity levels of serum are down regulated after MSC treatment. 	25
Figure 4: The pancreas are obtained after the MSCs treatment in the acute pancreatitis mouse model at the 24th hr and 48th hr and to progress H&E staining. 	27
Figure 5: The level of amylase expression of the pancreas is down-regulated after the MSCs treatment in the acute pancreatitis mouse model . 	31
Figure 6: The T cell infiltration in pancreas is also decreased in the acute pancreatitis mouse model at the 48th hr 	33
Figure 7: Effect of MSCs in inflammation response 	35
Figure 8: Establishment of pancreatitis model by surgery  	40
Figure 9: The improvement of IPGTT after the MSCs treatment in surgery-induced pancreatitis model   	41
References 	42
Appendix	46
                                    

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