行政院國家科學委員會專題研究計畫 成果報告
NK 細胞受不表現 MHC 且生產 TGF-b 之 CTVT 腫瘤中之細胞素
的影響,並開發治療不表現 MHC 且生產 TGF-b 腫瘤的基因
(3/3)
計畫類別： 個別型計畫 計畫編號： NSC93-2313-B-002-021- 執行期間： 93 年 08 月 01 日至 94 年 07 月 31 日 執行單位： 國立臺灣大學獸醫學系暨研究所 計畫主持人： 朱瑞民 報告類型： 完整報告 報告附件： 出席國際會議研究心得報告及發表論文 處理方式： 本計畫可公開查詢中 華 民 國 94 年 11 月 1 日
NK
MHC
TGF-
CTVT
,
MHC
Abstract
Canine transmissible venereal tumor (CTVT) has low levels of MHC molecules on the cell surface. CTVT spontaneously regresses after a 4-6 months of growth. CTVT cells secrete high concentrations of TGF- during tumor growth. TGF- inhibits NK cell cytotoxicity. However, CTVT undergoes regression when TGF- concentration remains high because tumor infiltrating lymphocytes (TIL) produce large amounts of IL-6. IL-6 is a strong TGF- antagonist and is one of the key substances that restore NK cell cytotoxicity against CTVT cells. Based on these findings, we devised and tested a new immuno-gene therapy strategy that uses both IL-6 and IL-15. IL-6 antagonized the inhibitory effect of TGF- on NK cell cytotoxicity. In a cell culture system, we evaluated the role of NK cells in tumor regression and the ability of IL-6 and IL-15 to restore TGF- -inhibited NK cell cytotoxicity. Only the treatment using both IL-6 and IL-15 effectively relieved the inhibitory effect of TGF- and activated NK cell cytotoxicity. The IL-6 and IL-15 only treatments did not increase, or only slightly increased, NK cell cytotoxicity. Electroporation of IL-6 and IL-15 plasmids into BALB/c mice increased the ratio of NK cells in the spleen and promoted splenocyte NK cell cytotoxicity. The IL-6 and IL-15 only treatments did not significantly change the NK cell ratio or the cytotoxicity of NK cells. However, in the IL-15 treatment, there was a slight elevation of NK cell ratio. In CB17/SCID mice, electroporation with both IL-6 and IL-15 plasmids strongly inhibited the growth and establishment of CTVT. IL-6 or IL-15 alone had no effect. In CB17/SCID mice treated with anti-asialo GM-1 antibody, which blocks NK cell function, combined electroporation with
IL-6 and IL-15 was unable to suppress CTVT growth. Thus, NK cells play a key role in suppressing tumors with low MHC expression and high TGF- secretion. Gene therapy using both IL-6 and IL-15 effectively suppressed the growth and establishment of the tumor.
A.
B.
CellTiter 96 AQueous One Solution TF-1
A.
B.
CellTiter 96 AQueous One Solution TF-1
IL-15 Balb/3T3
HT-2 PC
pcDNA3.1 /V5-his-TOPO Balb/3T3
E/T ratio 25/1 YAC-1
79.05 0.45 12.5/1 58.02 1.44 6.25/1 37.77 1.39 3.125/1 1.5625/1 23.12 0.73 15.32 0.6
ratio 25/1 E T ratio 100/1 50/1 63.01 3.1 77.03 2.64
TGF- IL-2 NK
TGF-YAC-1 Negative control
YAC-1 IL-6
IL-2 IL-15
TGF-TGF- IL-2 LAK
IL-15 NK TGF- NK IL-2
TGF-TGF- IL-6
NK IL-15
IL-6
TGF-IL-15 TGF-
TGF-IL-6 NK IL-15
TGF- NK IL-6
0 200 400 600 800 1000 1200 1400 1600 D0 D3 D5 D8 D12 D15 Day IL -6 c on c. (p g/ m l) Mock IL-6 IL-15 IL-6+IL-15 IL-6 IL-6 8 12 IL-6 + IL-15 3 IL-6 5
8 Day IL -1 5 co nc . ( pg /m l) IL-6+IL-15 IL-15 IL-6 Mock IL-15 IL-15 IL-6 + IL-15 IL-15
3 5
15 IL-6 + IL-15 IL-15
A. CD3+ T
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80
Days after tumor inoculation
% S ur vi va l Anti-asialo GM-1 Vector IL-6 IL-15 IL-6+IL-15
0 100 200 300 400 500 600 700 800 900 1000
+
-Anti-asialo GM-1 T u m o r vo lu m e (m m 3) P<0.05
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