臺灣博碩士論文加值系統

腎臟疾病因逐年增加的發病率和治療成本居高不下，目前已在世界上影響到數百萬人，是一個需要大家共同面對的的醫療問題。其中，以缺血後再灌流（Ischemia reperfusion, IR）所造成的急性腎臟損傷（Acute kidney injury, AKI）最為常見。AKI經常發生在不同的臨床情況中，包括腎臟移植、體外循環手術、主動脈搭橋手術、意外創傷、敗血症和腎積水等。IR後的腎臟組織的特徵是在於細胞外基質（Extracellular matrix, ECM）的堆積，正常腎臟實質被纖維化組織所取代，導致腎功能喪失或異常。腎功能異常會積聚代謝廢物、毒素和多餘的水分在腎臟內，會導致腎小管擴張，腎小球硬化症，蛋白尿，或腎間質纖維化，嚴重的話最終會導致腎衰竭。目前亟需新穎有效的治療方法。
本次實驗決定使用骨髓間葉幹細胞（Bone marrow mesenchymal stem cell, BMSC）作為治療材料之一，因為它們是多能性幹細胞之一，具有分化多種細胞類型的能力，包括：成骨細胞、軟骨細胞和脂肪細胞。另外，幹細胞（Stem cell）具有兩種特性，第一是分化能力，第二則是自我更新能力。而且本實驗還使用富含血小板的纖維蛋白（Platelet rich fibrin, PRF）釋放液，是一種天然生長因子的來源，當中含有多種影響組織再生的生長因子，包含：TGFβ-1（Transforming growth factor）、VEGF（Vascular endothelial growth factor）、PDGF（Platelet-derived growth factor）等等。在本次實驗中，我們使用以上兩種生醫材料測試治療因為IR後所造成腎臟損傷之大鼠模型中的恢復情況。
我們將富含血小板纖維蛋白釋放液（PRFr）與大鼠骨髓幹細胞和共同培養，來模擬腎臟組織中的細胞生長實驗，探討釋放液是否能提高幹細胞的生長能力。結果顯示有加入釋放液組別的骨髓幹細胞生長較沒加釋放液的組別快速，細胞存活率也高出許多。因此我們推斷富含血小板纖釋放液對骨髓幹細胞生長有極大幫助。
在完成細胞實驗之後，我們接著利用動物實驗，去探討富含血小板纖釋放液對AKI之治療成效。AKI動物模型建立是利用IR手術大鼠，8周齡大的SD大鼠會隨機分配到非手術組和急性腎臟損傷手術組兩個組別，非手術組大鼠作為正常對照組（Sham組），而IR手術組中大鼠則分別接受PRF釋放液（PRFr組，注射0.8毫升PRF釋放液）、骨髓幹細胞（BMSC組，注射2x106細胞數/0.8毫升PBS）、骨髓幹細胞+PRF釋放液（BMSC+PRFr組，注射2x106細胞數/0.8毫升PRF釋放液）以及不接受任何治療（Control組，注射0.8毫升PBS）。所使用的骨髓間葉幹細胞是從健康同種異體大鼠取得，而富含血小板纖維蛋白釋放液PRFr則是從健康異種異體兔子抽取血液製備而成。治療時將幹細胞或富含血小板纖維蛋白釋放液注射至大鼠的薦尾靜脈中，以調查其用於腎臟功能與組織的治療潛力。
大鼠進行完手術後一天，大鼠置入代謝籠中，進行24小時的尿液收集。第一個月以一週一次的頻率收集，爾後以一月一次的頻率收集總共四個月份的尿液檢體。血液檢驗則在犧牲大鼠的同時採集其血液，接著將所抽取到的血液利用3000 rpm離心10分鐘，以從分離出的血清作分析。每組大鼠於治療後16週犧牲，並取下腎臟組織對半切後以10％中性福馬林固定之。
從尿液檢驗及血清檢驗結果，亦或是長時間收集檢驗結果來觀察，單獨治療組別（BMSC組或是PRFr組）雖然在前四週有治療期間內，可以提供不錯的治療恢復效果，但當治療停止，BUN和Creatinine檢驗數值都有上升的趨勢，腎臟損傷評估方面也未有較好的成績；反觀利用富含血小板纖維蛋白釋放液結合骨髓間葉幹細胞都可以提供較好的治療效果，腎臟切片觀察也最接近的正常的腎臟組織結構。從以上研究的結果表示出，在IR大鼠腎臟受損模型中，富含血小板纖維蛋白釋放液結合骨髓間葉幹細胞，可以達到良好的組織修復能力，並且減緩手術誘導的急性腎臟損傷的傷害，以及可能應用在急性腎臟損傷的早期治療，並且做為一個有效的實驗數據。

Kidney disease affects millions of people in the world because of increasing incidence and prevalence and the high costs of treatment. Renal injury is mainly caused by toxic and ischemic injury. Renal ischemia-reperfusion occurs in different clinical situations including kidney transplantation, cardiopulmonary bypass, aortic bypass surgery, accidental trauma, sepsis, and hydronephrosis. Post ischemia-reperfusion kidney is characterized by the accumulation of extracellular matrix and usually results in a loss of function when normal parenchyma is replaced by fibrotic tissue. Abnormal kidney function will accumulate waste, toxins and useless moisture in the kidney tissue, may cause renal tubular dilation, glomerular sclerosis, proteinuria, or renal interstitial fibrosis, and finally lead to acute kidney injury (AKI).
Stem cells are a kind of undifferentiated cells which may differentiate into other cell types. And they can also divide indefinitely into stem cell line, but may become cancer cells at about the tenth generation. We collect the bone marrow stem cells (BMSCs) for our research.
Platelet-rich fibrin (PRF) is a natural source of growth factors, which contains a large amount of growth factors that can facilitate bone regeneration, Such as: TGFb-1 (Transforming growth factor), PDGF (platelet-derived growth factor) , VEGF (vascular endothelial growth factor) and Matrix proteins (thrombospondin-1, fibronectin, vitronectin). The growth factors secreted from PRF will cause cell transformation by promoting cell proliferation, matrix formation, bone formation and synthesis of collagen.
We co-cultured rat bone marrow stem cells with platelet-rich fibrin releasate (PRFr), to investigate whether the release would improve stem cell proliferation in vitro or not. The results show that proliferation rate of the bone marrow stem cells which co-cultured with releasate were more rapid, and cell viability was much higher. Therefore, we deduced that platelet-rich fiber release solution have significant assistance on the growth and differentiation of bone marrow stem cells.
To evaluate therapeutic efficacy of cell-based therapy in AKI: a bone marrow stem cells (BMSCs) and platelet-rich fibrin releasate (PRFr) were used for the treatment. An AKI was established with a rat model by ischemia reperfusion (IR). Transplantation of BMSCs, PRFr, and BMSCs + PRFr into IR rats for investigating the therapeutic potential for recovery of renal function. IR or sham operations were performed on SD rats at 10-weeks old, which were randomly divided into two parts: SHAM, and IR.6 rats in SHAM group was subjected to sham surgery, and 24 rats in IR group rats will accept four different treatments (1)Control group, injected 0.8ml PBS; (2)PRFr group, injected PRFr 0.8ml; (3)BMSCs group, injected BMSCs 2106 cells combine 0.8 ml PBS; (4)BMSCs + PRFr group, injected BMSCs 2106 cells combine 0.8 ml PRFr once a week for four weeks. At the firth month, urine was collected in one time a week; at the following three months, urine was collected in one time a month. At four months after surgery, the blood was taken for checking the value of blood urea nitrogen (BUN) and creatinine; the kidney was made into a Paraffin section for checking the effectiveness of treatments.
Estimates based on preliminary data, not only the urinalysis (urine test) but also the serum analysis results indicated that BMSCs combined with PRFr group was significant lower than other groups. By three months after injection, the results of BMSCs combined with PRFr group were also measured in normal values, which performed better than those other groups. It was concluded that the promising experimental data are beginning to emerge in support of the use of MSCs combine PRFr for regenerative applications.

論文題目 ······················································· I
口試委員審定書 ················································· II
致謝 ··························································· III
目錄 ··························································· IV
圖目錄 ························································· VII
中文摘要 ······················································· IX
Abstracts ······················································· XI
第一章 前言 ··················································· 1
第二章 文獻探討 ··············································· 3
第一節 急性腎臟損傷的介紹 ································ 3
2.1.1 腎臟簡介 ············································ 3
2.1.2 急性腎臟損傷 ········································ 5
第二節 血小板相關產品之介紹與應用 ························ 7
2.2.1 高濃度血小板血漿及富含血小板纖維蛋白 ··············· 7
2.2.2 富含血小板纖維蛋白釋放液 ··························· 8
第三節 幹細胞之介紹 ······································ 9
2.3.1 間葉幹細胞之簡介 ··································· 9
2.3.2 骨髓來源幹細胞之介紹及應用 ························· 10
第三章 材料與方法 ············································· 12
第一節 實驗材料製備 ······································ 12
3.1.1 富含血小板纖維蛋白釋放液的製備 ····················· 11
3.1.2 間葉幹細胞的製備 ··································· 12
3.1.2.1 骨髓來源幹細胞的製備 ·························· 12
3.1.2.2 幹細胞螢光之標記 ······························ 14
第二節 實驗動物 ·········································· 15
3.2.1 實驗動物的來源及飼養管理 ···························· 15
3.2.2 實驗動物分組 ········································ 15
第三節 實驗動物手術相關事項 ······························ 17
3.3.1 實驗動物麻醉方法及過程 ······························ 17
3.3.2 實驗動物手術步驟 ···································· 17
3.3.3 腎臟組織勻漿的製備 ·································· 17
第四節 幹細胞及富含血小板纖維蛋白釋放液注射 ·············· 18
3.4.1 幹細胞數量及富含血小板纖維蛋白釋放液劑量統計 ········ 18
3.4.2 注射實驗材料 ········································ 18
第五節 樣本收集及檢驗 ···································· 19
3.5.1 尿液及血液之收集和檢驗 ····························· 19
第六節 犧牲實驗動物 ······································ 19
第七節 腎臟功能恢復之評估 ································ 20
3.7.1 腎臟組織勻漿細胞實驗之評估 ·························· 20
3.7.2 生化檢驗結果之分析 ·································· 20
3.7.3 組織學之評估 ········································ 20
7.3.3.1 H&E 染色及損傷評估 ·························· 20
7.3.3.2 冷凍切片及共軛焦顯微鏡螢光觀察 ··············· 21
第八節 統計分析 ········································· 22
第四章 實驗結果 ·············································· 23
第一節 骨髓間葉幹細胞製備結果 ···························· 23
4.1.1 初代培養結果 ······································· 23
4.1.2 流式細胞儀篩選純化出間葉幹細胞 ····················· 23
第二節 腎臟組織勻漿細胞實驗結果 ·························· 24
第三節 在腎臟組織中骨髓幹細胞之細胞定位 ·················· 25
第四節 骨髓幹細胞及PRFr共用使用的治療效果 ··············· 25
4.4.1 生化分析結果 ······································· 25
4.4.2 腎臟組織學之評估 ··································· 26
4.4.3 正常大鼠腎臟 ······································ 27
4.4.4 腎臟損傷評分統計分析結果 ··························· 28
第五章 討論 ·················································· 29
第六章 參考文獻 ·············································· 33
附錄 ·························································· 59

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