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研究生:陳鞍修
研究生(外文):An-Hsiu Chen
論文名稱:偵測貓腫瘤組織中FIV及FeLV之核酸
論文名稱(外文):Detection of nucleic acid of FIV or FeLV in feline neoplastic tissues
指導教授:張仕杰徐維莉
指導教授(外文):Shih-Chieh ChangWei-Li Hsu
口試委員:廖泰慶
口試日期:2016-06-28
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:44
中文關鍵詞:貓免疫不全病毒貓白血病病毒貓腫瘤
外文關鍵詞:FIVFeLVcat neoplastic tissue
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貓免疫不全病毒(FIV)與貓白血病病毒(FeLV)在貓可誘發一部分的淋巴瘤,這兩種病毒的感染是貓罹患腫瘤的主因之一,相較於正常族群,受FIV及FLV感染的貓罹患淋巴瘤的風險較高。然而目前FIV及FeLV感染對非淋巴樣腫瘤的影響所知仍少。為了解此議題,本研究利用巢式聚合酶連鎖反應(Nested-PCR)偵測腫瘤組織中FIV及FeLV的前病毒序列,其中共收集50個非淋巴樣腫瘤樣本及5個正常組織樣本,均來自國立國立中興大學獸醫教學醫院。Nested-PCR所用的核酸引子針對FIV及 FeLV前病毒及內源性FeLV (enFeLV)序列pol基因中保留度最高的區域設計。為確保實驗品質,在偵測腫瘤樣本之前,本實驗建立一重組質體作為後續nested-PCR偵測FIV及FeLV前病毒序列的陽性對照組。結果顯示,FIV及FeLV的前病毒序列在所收集的腫瘤樣本中並未測得,但可在一些樣本測得enFeLV序列。enFeLV序列的總體陽性率為50% (25/50),而在乳腺癌為51% (15/29),在貓注射部位肉瘤為58% (7/12),在其他腫瘤為33% (3/9),而在正常組織中的陽性率為40% (2/5)。綜上所述,可排除前病毒序列的插入導致本研究所收集的腫瘤,亦發現enFeLV在乳腺癌、貓注射部位肉瘤、其他腫瘤與正常組織間的陽性率沒有顯著差異,因此沒有哪一類的腫瘤被認為與此enFeLV有關。

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections induce the development of a part of lymphomas, which are one of the important causes of neoplasms in cats. Comparing to the normal population, cats infected with FIV and FeLV have higher risk to develop lymphomas. However, little is known about the impact of FIV or FeLV infection to non-lymphoid neoplasms. To address this issue, the nested polymerase chain reaction (nested-PCR) was used to detect FIV/FeLV provial sequences in feline neoplastic tissues that were collected from the Veterinary Medical Teaching Hospital, National Chung Hsing University. They included fifty non-lymphoid neoplastic specimens and five normal tissues. Primers were designed for targeting the most conserved region of pol gene of FIV, FeLV and endogenous FeLV (enFeLV). For quality control, before performing the detection for neoplastic specimens, a recombinant vector has been established to serve as the positive control in the nested-PCR for the detection of proviral sequences of FIV and FeLV. Results showed that no proviral sequences of FIV and FeLV could be detected in both non-lymphoid neoplastic specimens and normal tissues, while enFeLV could be detected in specimens. The mean positive rate of enFeLV was 50% (25/50) in all neoplastic specimens, and 51% (15/29) in mammary simple carcinomas, 58% (7/12) in feline injection site sarcomas, 33% (3/9) in other neoplasms, respectively, and 40% (2/5) in normal tissues. In conclusion, the factor of proviral sequences integration could be excluded in the oncogenesis of the neoplasms in this study. And there was no significant difference in the enFeLV positive rate among mammary carcinomas, feline injection site sarcomas, other neoplasms and normal tissues, in which no neoplastic type was considered related to the existence of this enFeLV.

摘要 i
Abstract ii
Contents iii
List of Tables v
List of Figures vi
Chapter 1 Introduction 1
Chapter 2 Materials and Methods 7
2.1 Feline neoplastic and normal tissue specimens 7
2.2 Preparation of chromosomal DNA from neoplastic and normal tissue specimens 8
2.2.1 Chromosomal DNA extraction of neoplastic and normal tissue specimens 8
2.2.2 Evaluation of chromosomal DNA OD and concentration 8
2.2.3 PCR for GAPDH gene as the quality control 9
2.3 Primers designing 10
2.4 Establishment of an artificial positive control plasmid 12
2.4.1 Vector 12
2.4.2 Preparation of double stranded primer sets for insertion 12
2.4.3 Cloning of the forward and reverse primer sets into the vector 15
2.4.4 PCR of the artificial positive control to insure efficiency 19
2.5 Nested-PCR for detection of proviral sequences of FIV and FeLV 21

2.5.1 Optimization of the annealing temperature of FIV and FeLV
Primers 21
2.5.2 Detection limit of each primer and spiking of canine chromosomal DNA to the positive control 23
2.5.3 Detection of FIV and FeLV proviral sequences in feline neoplastic
and normal tissue specimens 25
Chapter 3 Results 26
3.1 The prevalence of FIV and FeLV proviral sequences in neoplastic and normal tissue specimens 26
3.2 The positive rate of enFeLV sequences in neoplastic and normal tissues specimens 26
Chapter 4 Discussion 33
References 38


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