臺灣博碩士論文加值系統

近年來幹細胞運用於各式疾病治療與組織再生，又以間葉幹細胞因其容易取得、不易有倫理問題、穩定且安全、可廣泛應用於臨床的可行性而逐漸受到重視，其中骨髓來源間葉幹細胞發現得較早，所以早期的研究以骨髓來源間葉幹細胞為大宗，然而近十年脂肪來源幹細胞由於其取得更為容易，尤其台灣自2017年起實行零安樂死政策，犬隻骨髓取得更加困難，因此我們選擇犬隻脂肪來源間葉幹細胞作為研究題材，有機會於未來廣泛應用。犬乾眼症屬自體免疫引起，因此乾眼症犬一直被視為自體免疫疾病的重要動物模型，過去犬乾眼症透過免疫抑制劑點眼控制然而效果有限，過去三年有數篇研究使用幹細胞於眼周注射治療有不錯的療效，也因此期待間葉幹細胞之免疫調節的效果能為此疾病帶來新的契機，然而臨床上若要進行眼周注射需要鎮靜、甚至全身麻醉，也可能在注射的過程中導致醫源性傷害，因此本研究在探討使用非侵入式的局部點眼給藥方式能否給予臨床犬乾眼症病患有效的治療。首先，脂肪來源間葉幹細胞成功的被分離並確認其具有三系分化與免疫調節之特性，並測試其製成點眼劑之適當保存方式。在幹細胞臨床試驗中中，患有乾眼症犬隻，根據過去是否有用過點眼免疫抑制劑分為兩組，給予連續六週、一週一次的幹細胞點眼液治療，並於第三、六、九週進行眼科檢查，包括淚液分泌試驗、角膜螢光素染色和淚膜破裂時間與淚液滲透壓測試，並透過分泌物、結膜充血、角膜病變程度來評估臨床上的變化。結果淚液量與品質在接受幹細胞點眼治療後皆有顯著的進步，過半數的治療病患能改善淚液量，尤其是過去對免疫抑制劑無反應的組別中，亦有56.5%的病患淚液量有效的增多，而臨床症狀減緩能增進動物眼睛舒適度、改善生活品質。基於以上結果，連續六週給予脂肪來源間葉幹細胞點眼治療，有潛力成為犬乾眼症傳統治療的替代方案，相較於傳統治療一天需兩到三次的長期頻繁點藥，幹細胞治療僅需六次的療程，對於飼主配合度不高、或對免疫抑制劑反應不佳的病患，局部點眼脂肪來源間葉幹細胞有有助於改善犬乾眼症。

In the past two decades, mesenchymal stem cells have been tested in the treatment of various diseases and tissue regeneration. Among these mesenchymal stem cells have gradually gained much attention because of their availability, stability, safety, low-ethical issue and with wide ranges of potential therapeutic applications. Bone marrow-derived mesenchymal stem cells were found and used earlier, so the bulk of early studies used them. However, adipose-derived stem cells are much easier to access and can be acquired in a large quantity. Especially when the euthanasia of stray animals was banned in Taiwan after early 2017, it is even more difficult to obtain bone marrow from dogs. That is why we chose canine adipose-derived mesenchymal stem cells (cAD-MSCs) as the research subjects with potential future applications.
Canine keratoconjunctivitis sicca (KCS) is thought to be an immune-mediated disease, therefore canine KCS patients provide a useful model of autoimmune-mediated diseases. Current therapy of canine KCS mainly uses immunosuppressants, but the effectiveness was limited in some patients. In the past three years, some studies showed the results of the use of mesenchymal stem cells in treating canine KCS via periocular injections. However, the periocular injection procedure requires sedation or general anesthesia, and may lead to iatrogenic or incidental injury during the injection process. The aim of this study was to investigate the efficacy of topical allogenic adipose-derived stem cells in clinical patients of canine KCS.
First, adipose-derived stem cells were isolated and confirmed for their capability of differentiation and immunomodulatory properties. In addition, preparation methods for eye drops of cAD-MSCs was developed and its optimal preservation was tested. Secondly, canine KCS patients were included and divided into two groups based on history of previous therapy for clinical trial. All patients received topical canine adipose-derived mesenchymal stem cells (cAD-MSCs) therapy weekly for 6 consecutive weeks and complete ophthalmic examinations were performed at baseline and 3rd, 6th, 9th week, respectively. A complete ophthalmic examination included Schirmers tear test-1 (STT-1), tear break-up time(TBUT), fluorescein stain, tear osmolarity measurement by i-PEN and assessments the severity of clinical signs such as mucoid discharge, conjunctival hyperemia, and corneal changes.
Based on the results of the clinical trials, the quantity and quality of tears have improved significantly following topical cAD-MSCs treatment. More than half of the patients were found improved in the tear quantity. In particular, 56.5% of the patients that were unresponsive to prior immunosuppressant therapy had an effective increase in tear volume. As relieved from symptoms and uncomfortable, patients’ quality of life was improved following treatment. Based on these results, weekly administration of cAD-MSCs for six consecutive weeks may serve as a good alternative to cyclosporine A or tacrolimus for treatment of canine KCS. Topical cAD-MSCs therapy requires once weekly for six times. It’s much easier for dog owners when compared with traditional treatment, that required frequent administration with two to three times daily. To sum up, topical cAD-MSCs may be beneficial especially in KCS patients with poor owner compliance for frequent daily use of eye drops or those who are unresponsive to immunosuppressant therapy.

誌謝 ii
中文摘要 iii
ABSTRACT iv
CONTENTS vi
LIST OF FIGURES ix
LIST OF TABLES x
Chapter 1 Introduction 1
1.1 Stem cells 1
1.1.1 Mesenchymal stem cells 2
1.1.2 Adipose-derived stem cells 5
1.1.3 Canine adipose-derived stem cells 7
1.2 Canine keratoconjunctivitis sicca (KCS) 10
1.2.1 The pathogenesis of canine KCS 10
1.2.2 The clinical signs and diagnosis in KCS 11
1.2.3 The medical treatment of KCS 13
Chapter 2 Specific aims 18
Chapter 3 Materials and Methods 20
3.1 Canine adipose-derived mesenchymal stem cells 20
3.1.1 Inclusion criteria of donors 20
3.1.2 Isolation of cAD-MSCs 20
3.1.3 Passaging, cryopreservation, and reviving procedure of cAD-MSCs 22
3.1.4 Colony forming unit (CFU) assay 23
3.1.5 Trilineage differentiation of cAD-MSCs 24
3.1.6 Immunophenotyping of cAD-MSCs 26
3.1.7 Immunomodulatory effect of cAD-MSCs 27
3.2 The cAD-MSCs eye drops 29
3.2.1 Preparation of eye drops 29
3.2.2 Cell viability in eye drops 29
3.3 Clinical trial – the efficacy of topical cAD-MSCs 29
3.3.1 Inclusion and exclusion criteria of patients 29
3.3.2 Grouping and protocols 31
3.3.3 Clinical evaluation 31
3.4 Statistical analysis 33
Chapter 4 Results 40
4.1 Isolation of cAD-MSCs 40
4.2 Colony forming unit (CFU) assay 40
4.3 Trilineage Differentiation of cAD-MSCs 40
4.4 Immunophenotyping of cAD-MSCs 41
4.5 Immunomodulatory effect of cAD-MSCs 42
4.6 Cell viability in the eye drops 43
4.7 Study population in the clinical trial 43
4.8 The efficacy of topical cAD-MSCs 45
4.9 Case presentation 47
4.9.1 Group 1: without history of immunosuppressant therapy 47
4.9.2 Group 2: unresponsive to prior immunosuppressant therapy 48
Chapter 5 Discussion 67
5.1 Harvesting of cAD-MSCs 67
5.2 Characterization of cAD-MSCs 68
5.3 Immunomodulatory effect of cAD-MSCs 70
5.4 The cAD-MSCs eye drops 71
5.5 The efficacy of topical cAD-MSCs 72
Chapter 6 Conclusion 76
References 77
Appendix A 84
Appendix B 87

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