臺灣博碩士論文加值系統

反轉錄病毒，目前普遍被應用作為哺乳類動物細胞基因轉殖的工具。與一般轉染（transfection）不同的是，反轉錄病毒所攜帶的基因直接嵌入標的細胞的染色體中表達，而非短暫存在於細胞中。本篇論文主要建立反轉錄病毒感染小鼠初級T細胞（primary T cell）的基因轉殖系統。第一階段，生產高效價（high titer）之反轉錄病毒。我們利用PhoenixE包裝細胞株(package cell line)提供病毒結構gag/pol、env，再以pBMN-EGFP 或pGC-YFP兩個攜帶病毒包裝訊息（y）並會表達螢光蛋白的病毒載體DNA，經轉染法生產病毒。將所生產之病毒感染3T3細胞後，以流式細胞儀測定感染率（即細胞表達螢光蛋白之比率），再依其感染率計算病毒效價。實驗結果顯示，轉染時放入Chloroquine可提高病毒產量，而以離心方式感染3T3則可提高病毒對3T3的感染率。此外，病毒冷凍儲存4個月內效價並未下降。仍可穩定地製造出效價達到1x106 unit/ml的病毒。
第二階段，以初級T細胞為感染對象，分別使用三種T細胞進行實驗。一. 小鼠脾臟細胞經過PMA＆ionomycin活化後，以反轉錄病毒感染，可得到22％的細胞表達黃色螢光蛋白。二. 我們利用過去實驗室所建立小鼠腸道表皮間隙gdT細胞的活化系統，以抗gd型T細胞受器抗體活化小鼠腸道表皮間隙gdT細胞，不同活化時間點給予病毒感染，以流式細胞儀偵測黃色螢光蛋白質，發現在活化的同時（0小時）感染細胞，可以得到最佳的感染率為10％。三. 來自脾臟及淋巴結的CD8細胞，經過抗b型T細胞受器抗體和抗CD28抗體活化，24小時後，以反轉錄病毒感染，得到15％感染率。
未來，將會利用已建立之反轉錄病毒傳導系統，把有興趣的基因放入小鼠腸道表皮間隙gdT細胞和CD8細胞，研究這些基因對於細胞活化及存活的影響。

Retroviral transduction is a technique for introducing genes of interest into mammalian cells. The genes introduced this way can integrate into host chromosome and express stably. Moreover, retrovirus can transduce primary cells that are difficult to receive genes delivered by conventional methods. My project has aimed to establish retroviral transduction of murine primary lymphocytes. The first part of my work was to produce high titer recombinant retrovirus. PhoenixE cell line carrying plasmids encoding retroviral core(gag)/polymerase(pol) or envelope(env) genes was used to produce recombinant virus after transfecting viral vector which contains retroviral packaging signal(y) and the gene of insterest. Two viral vectors, pBMN-EGFP and pGC-YFP, were used to transfect the packaging cell line. I determined the optimal amounts of plasmid DNA and chloroquine for CaPO4 transfection. Then, I tested conditions for spin infection. Finally, I could produce the recombinant retrovirus with a titer around 1x106 unit/ml.
The second part of my work was using the retrovirus to transduce murine lymphocytes. Three types of murine lymphocytes were used. The first cell type was total spleen cells activated by PMA&ionophore. Twenty-two percent of activated spleenocytes were transduced as determined by YFP expression. The second cell type was TCRgd+ intestinal intraepithelial lymphocytes (gdiIEL) activated by anti-TCRgd Ab. Ten percent of gd-iIEL expressing YFP during primary activation were transduced. The third cell type was spleen and lymph node CD8 cells activated with immobilized anti-TCRb Ab&anti-CD28 Ab. After retrovirus transduction, fifteen percent of activated CD8 cells expressed YFP.
The established experimental conditions will allow us to introduce gene of interest into gdiIEL and CD8 cells to study gene function.

中文摘要…………………………………………………………...第i頁
英文摘要………………………………………………………….第iii頁
目次………………………………………………………………….第V頁
表目錄…………………………………………………………….第Vii頁
圖目錄……………………………………………………………第Viii頁
第壹章 緒論
一、小腸表皮間隙gd型T細胞（gd-iIEL）活化致死模式…第1頁
二、介白素十五號受器缺失小鼠CD8細胞之活化模式…….第2頁
三、反轉錄病毒介紹…………………………………………第3頁
四、研究策略…………………………………………………第5頁
第貳章研究材料與方法
一、 細胞與細胞培養…………………………………………....第6頁
二、 藥品、試劑………………………………………………....第7頁
三、 反轉錄病毒載體、質體DNA………………………….......第8頁
四、 小鼠脾臟細胞之分離與活化……………………………....第9頁
五、 小鼠腸道表皮間隙gdT細胞之分離與活化……………....第10頁
六、 小鼠CD8細胞群之分離與活化………………………......第11頁
七、 小鼠腸道表皮間隙gdT細胞增殖分析…………………....第12頁
八、 轉染分析……………………………………………….....第12頁
九、 感染分析……………………………………………….....第14頁
十、 流式細胞儀分析……………………………………….....第15頁
十一、 建構pGC-YFP-Bcl-xL載體………………………….....第16頁
第參章結果
五、生產高效價的反轉錄病毒…………………………….第18頁
六、反轉錄病毒感染小鼠脾臟細胞……………………….第21頁
七、反轉錄病毒感染小鼠腸道表皮間隙gdT細胞………..第22頁
八、反轉錄病毒感染CD8細胞……………………………..第23頁
九、pGC-YFP-Bcl-xL 質體的選殖…………………………第24頁
第肆章討論 ……………………..……………………………第27頁
圖表 ……………………………………………………………….第30頁
參考文獻………..…………………………………………………第58頁
附錄…………………………………………………………………第63頁

Alberts B., Bray D., Lewis J., Raff M., Roberts K.,Waston J. D. 1993. Molecular Biology of The Cell. pp282-286
Allison J. P. and Havran W. L. 1991. The immunobiology of T cells with invariant gd antigen receptors. Annu. Rev. Immunol. 9:679-705 3:
Beagley K. W., Fujihashi K, Black C. A., Lagoo A. S., Yamamoto M., McGhee J. R., Kiyono H. 1993. The Mycobacterium tuberculosis 71-kDa heat-shock protein induces proliferation and cytokine secretion by murine gut intraepithelial lymphocytes. Eur J Immunol. 23(8):2049-52.
Chu C. L., Chen S. S., Wu T. S., Kuo S. C., Liao N. S. 1999. Differential effects of IL-2 and IL-15 on the death and survival of activated TCR gamma delta+ intestinal intraepithelial lymphocytes. J Immunol. 162(4):1896-903.
Cone RD, Mulligan RC. 1984. High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range. Proc Natl Acad Sci U S A. 81(20):6349-53.
Costa G. L., Benson J. M., Seroogy C. M., Achacoso P., Fathman C. G., Nolan G. P. 2000. Targeting rare populations of murine antigen-specific T lymphocytes by retroviral transduction for potential application in gene therapy for autoimmune disease. J Immunol. 164(7):3581-90.
Danos O, Mulligan RC. 1988. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci U S A. 85(17):6460-4.
Dal Canto R. A., Shaw M. K., Nolan G. P., Steinman L., Fathman C. G. 1999. Local delivery of TNF by retrovirus-transduced T lymphocytes exacerbates experimental autoimmune encephalomyelitis. Clin Immunol. 90(1):10-4.
Davey RA, Hamson CA, Healey JJ, Cunningham JM. 1997. In vitro binding of purified murine ecotropic retrovirus envelope surface protein to its receptor, MCAT-1. J Virol. 71(11):8096-102.
DuBridge R. B., Tang P., Hsia H. C., Leong P. M., Miller J. H., Calos M. P. 1987. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol. 7(1):379-87.
Fehniger TA, Caligiuri MA.Interleukin 15: biology and relevance to human disease. 2001. Blood. 97(1):14-32
Ferguson A., Parrott D. M. 1972. The effect of antigen deprivation on thymus-dependent and thymus-independent lymphocytes in the small intestine of the mouse. Clin Exp Immunol. 12(4):477-88.
Fritz J. 1996 set up www.mc.vanderbilt.edu/gcrc/gene/retro.htm
Giri JG, Ahdieh M, Eisenman J, Shanebeck K, Grabstein K, Kumaki S, Namen A, Park LS, Cosman D, Anderson D. 1994. Utilization of the beta and gamma chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13(12):2822-30.
Giri JG, Kumaki S, Ahdieh M, Friend DJ, Loomis A, Shanebeck K, DuBose R, Cosman D, Park LS, Anderson DM. 1995. Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor. EMBO J. 14(15):3654-63.
Goodman T., Lefrancois L. 1988. Expression of the gamma-delta T-cell receptor on intestinal CD8+ intraepithelial lymphocytes. Nature. 333(6176):855-8.
Hayday A.C. 2000. gd cells: a right time and a right place for a conserved third way of protection. Annu Rev Immunol. 18:975-1026.
Kim JW, Closs EI, Albritton LM, Cunningham JM. 1991. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. Nature. 352(6337):725-8.
King D. P., Hyde D. M., Jackson K. A., Novosad D. M., Ellis T. N., Putney L, Stovall M.Y., Van Winkle L. S., Beaman B. L., Ferrick D. A. 1999. Cutting edge: protective response to pulmonary injury requires gamma delta T lymphocytes. J Immunol. 162(9):5033-6
Lefrancois L. 1991. Phenotypic complexity of intraepithelial lymphocytes of the small intestine. J Immunol. 147(6):1746-51.
Lepage A. C, Buzoni-Gatel D, Bout D. T., Kasper L. H. 1998. Gut-derived intraepithelial lymphocytes induce long term immunity against Toxoplasma gondii. J Immunol. 161(9):4902-8.
Mann R, Mulligan RC, Baltimore D. 1983. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 33(1):153-9.
Miller AD. 1990. Retrovirus packaging cells. Hum Gene Ther. 1(1):5-14
Miller AD, Buttimore C. 1986. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 6(8):2895-902.
Miller AD, Law MF, Verma IM. 1985. Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene. Mol Cell Biol. 5(3):431-7.
Miller AD, Miller DG, Garcia JV, Lynch CM. 1993. Use of retroviral vectors for gene transfer and expression. Methods Enzymol. 217:581-99
Morgan JR, LeDoux JM, Snow RG, Tompkins RG, Yarmush ML. 1995. Retrovirus infection: effect of time and target cell number. J Virol. 69(11):6994-7000.
Movassagh M., Boyer O., Burland M. C., Leclercq V., Klatzmann D., Lemoine F. M. 2000. Retrovirus-mediated gene transfer into T cells: 95% transduction efficiency without further in vitro selection. Hum Gene Ther. 11(8):1189-200.
Nilssen D. E., Muller F., Oktedalen O., Froland S. S., Fausa O., Halstensen T. S., Brandtzaeg P. 1996. Intraepithelial gamma/delta T cells in duodenal mucosa are related to the immune state and survival time in AIDS. J Virol. 70(6):3545-50.
Nolan G. P., www.stanford.edu/group/nolan/phoenix_info.html 1:
Pear W. S., Nolan G. P., Scott M.L., Baltimore D. 1993. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 90(18):8392-6.
Sambrook J., Fritsch E. F., Maniatis T. 1989. Molecular Colning ,p.B11-12
Wang H, Kavanaugh MP, North RA, Kabat D. 1991. Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter. Nature. 352(6337):729-31.
Yang S., Delgado R., King S. R., Woffendin C., Barker C. S., Yang Z. Y., Xu L., Nolan G. P., Nabel G. J. 1999. Generation of retroviral vector for clinical studies using transient transfection. Hum Gene Ther. 10(1):123-32.
Zheng L, Dengler TJ, Kluger MS, Madge LA, Schechner JS, Maher SE, Pober JS, Bothwell AL. 2000. Cytoprotection of human umbilical vein endothelial cells against apoptosis and CTL-mediated lysis provided by caspase-resistant Bcl-2 without alterations in growth or activation responses. J Immunol. 164(9):4665-71.