臺灣博碩士論文加值系統

眼球化學性灼傷, 是因眼球接觸化學物質而導致眼球角上皮以及輪狀部幹細胞傷害。眼球受到急性化學性灼傷, 應先以大量清水沖洗至少30分鐘 ; 是否施以大量清水沖洗,與其臨床治療預後很有關係. 藥物治療: 其主要目的有促進上皮細胞再生, 控制發炎, 預防角膜感染。嚴重的灼傷，當其輪狀部缺血、上皮持續性缺損時，經過藥物治療而沒有上皮化, 就要考慮手術治療. 手術治療有：清創手術, Tenon成型術,羊膜移植術, 輪部幹細胞移植. 本次實驗, 我們用人類脂肪組織培養出之幹細胞來治療兔子化學灼傷. 實驗中, 藉由分析角膜清澈度, 角膜上皮細胞層數, Real-Time PCR, 發現幹細胞對眼球化學性灼傷角膜修復有促進效果. 我們希望藉由動物實驗的經驗, 進而推廣到以體外培養幹細胞, 來治療人體的眼球化學性角膜灼傷.

Chemical burn is the absolute ocular emergency. It is the most severe injuries to the organ of vision with a very high percentage of unfavorable outcomes. It often causes extensive damage and results in permanent visual impairment. Recurrent epithelial erosions, symblepharon, corneal scar, corneal ulceration, severe stromal inflammation, and corneal neovascularization are common clinical complications of alkali burn .We studied the effect of cultured human adipose tissue-derived stem cells on regeneration of rabbit cornea after alkaline chemical burn.

Human adipose tissue–derived stem cells are served as the source of donor material. The frozen stem cells were defreezing and sub-cultured up to 80% with Keratinocyte -SFM (Invitrogen ) and supplements of EGF , BFE( Bovine pituitary extraction), 10 %FBS ( FBS 50 cc / SFM 500 cc),GM solution 100 ul / 500 cc SFM. The cell suspension was cultured in 25 cm2 culture flasks at 37oC and 5% CO2. Culture medium was replaced by 80-90 % every 2 days. The study was performed on 8 rabbits (2-2.5 kg) with alkaline burns of the cornea. NaOH-impregnated disks (impregnated for 5 minutes, size as 7 mm in diameter) were placed into strictly central cornea area for 40 sec. After removal of the disks, the eyes were washed with 20 ml saline for 30 sec. All the procedures were taken under general anesthesia (5% ketamine plus rompune 1 to 2 mg/kg IM) and local analgesia of the cornea (1% Proparacaine Hydrochloride). Immediately after the chemical burn, experimental animals received a single subconjunctival injection of stem cell suspension (1.3×105 cells/ 0.2 ml ml). Controls were injected with 0.2 ml saline. Topical treatment with gentamycin oint (twice per day) was applied for preventing secondary infections. The eyes were eviscerated after sacrifice on days 30. Histological studies were performed on paraffin sections stained with hematoxylin and eosin. Real-time PCR are performed to detect the surface markers of P 63, E-Catherin, Beta-catenin, and Connexin 43.

We evaluate the corneal functions via the following criteria, corneal opacity assessments, Real-time PCR and Histology, to prove the corneal regeneration effects of human adipose tissue - derived stem cells. The average grading of experimental group is graded as 1-2 while the controlled group is graded as 4.This means the stem cells offer more cell renewal processes than the controlled group.
Real-time for beta- catena, connexin43 (Cx43), E-Catherin, and P63 are checked, which represent as cell membrane, cell membrane, non-neural epithelium and corneal stratified epithelium individually. Positive correlation of beta- catenin, connexin43 (Cx43), E-Catherin means good cell renewal for damage repair of corneal epithelium related to chemical burn. P63 shows non-significant change.
Histologically, there are 5-6 cell layers at epithelium for the experimental group and 2-3 cell layers for the controlled group. Except the 8th rabbits with poor section of histology, 6 experimental groups show 5-6 cell layers and 7 control group show 2-3 cell layers over the epithelia .

Transplantation of cultured human adipose tissue derived stem cells on rabbits corneal chemical burn promotes cell renewal and damage repair. Corneal transparency, Real-time PCR, epithelium cell layers are evaluated and proved as positive for wound healing.

Acknowledgments i
Abstract iii
摘要 vi
Outline vii
Outline for Figures ix
Chap 1. Introduction 1
1-1 Background 1
1-2 Purpose 2
Chapter 2. Review of literatures 3
2-1 Chemical burn in occupational diseases 3
2-2 Ocular chemical burn 4
2-2-1 Pathophysiology 6
2-2-2 Corneal haziness grading 7
2-2-3 Management 9
2-3 Treatments for chemical burn 9
2-4 What are stem cells? 11
2-5 Mesenchymal stem cells (Adipose tissue derived stem cells) 12
2-6 Stem cells for clinical applications 13
2-7 Real -time PCR 16
2-7-1 PCR’s three phases 17
2-7-2 Real-Time PCR VS Traditional PCR 18
2-7-3 Limitations of End-Point PCR 20
2-8 Surface markers: 21
2-9 Corneal Anatomy and Epithelium layers 28
2-10 cDNA transforming techniques 31
2-11 Animal Anesthesia and Animal sacrifice 33
Chapter 3. Materials and Methods 36
3-1 Stem cells preparation 36
3-2 Animal study 37
Chapter 4 . Results 39
4-1 Corneal haziness grading: 39
4-2 Real-time PCR: 41
4-3 Epithelial layer numbers (Fig 6, 7, 8) 43
Chapter 5. Discussions 46
Chapter 6. Limitations of the study 49
Chapter 7. Conclusions 50
Chapter 8. References 51

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