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探討因前驅藥物療法所引發的防禦性免疫反應機制；利用人工分子演化的技術來增進免疫酵素的活性。亦試圖利用分子技術法表現單鏈單株抗體以及活化前驅藥物之酵素。

891601林子云, 2002, Summer Tumor targeting and Prodrug Lab Institute of Biomedical Sciences (IBMS) at the Academia Sinica in Taipei, Taiwan Steve Roffler. 探討因前驅藥物療法所引發的防禦性免疫反應機制；利用人工分子演化的技術來增進免疫酵素的活性。亦試圖利用分子技術法表現單鏈單株抗體以及活化前驅藥物之酵素。

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探討因前驅藥物療法所引發的防禦性免疫反應機制；利用人工分子演化的技術來增進免疫酵素的活性。亦試圖利用分子技術法表現單鏈單株抗體以及活化前驅藥物之酵素。

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  1. 891601林子云, 2002, SummerTumor targeting and Prodrug LabInstitute of Biomedical Sciences (IBMS) at the Academia Sinica in Taipei, TaiwanSteve Roffler • 探討因前驅藥物療法所引發的防禦性免疫反應機制；利用人工分子演化的技術來增進免疫酵素的活性。亦試圖利用分子技術法表現單鏈單株抗體以及活化前驅藥物之酵素。 • 研究將融合蛋白表現在哺乳類細胞表面的基因療法，以應用在特殊的淋巴細胞調控上。同時探討融合蛋白中不同的區域對其運送和滯留於細胞表面的影響。 • 探討細胞腫瘤轉移的機制 ，及調控內皮細胞血管新生的機身。目前著重於探討在癌細胞轉移與血管新生過程中 間的交互作用及其訊息傳遞路徑。

  2. Expression rate of CD13 and L6 on CL1-5 will be induced by growth factors中研院生醫所Steve Roffler 891601林子云 2002, Summer

  3. Purpose and introduction invasion CD13 L6 ? GFs , Hypoxia CL1-5 CL1-5 angiogenesis CD13 ? L6 HUVEC WeStudy how the two surface proteins, CD13 and L6, are induced in different condition of medium. This might be helpful to illustrate roles of the two proteins in invasion of CL1-5.

  4. Fluorescence-Activated Cell Sorter

  5. Fluorescence-Activated Cell Sorter • To define characteristics of unfamiliar cells • To isolate specific cells for growth, cloning or PCR • Performing by the correlation between light scatter patterns and cell properties such as DNA or RNA content, shape, size or texture.

  6. Scattered light and fluorescence signals are generated and the sort logic boards make a decision as to whether the cell is to be sorted or not. • The cytometer waits until that cells has traveled from the intercept to the break-off point and then charges the stream. So as the drop containing the cell of interest leaves the solid fluid stream it will carry a charge, either positive or negative. • The charged drop passes through two high voltage deflection plates and will be attracted towards the plate of opposite polarity.

  7. Dual-parameter plots of combinations of light scatter and fluorescence • Forward light scatter: size • Sideward light scatter: small cellular structures. • The fluorescence:the amount of cellular pigment and its composition.

  8. Methods 0. Protocals I. Test the better condition to harvest CL1-5 Run FACS, 1st abs=4B8, L6, and HB65, 2nd ab=Go  Mo Ig(G+A+M)-FITCExpression level = FL1-intensity of 4B8 or L6 minus that of HB65.

  9. R10+4B8 400 300 R30+4B8 R60+4B8 T10+4B8 T2+4B8 300 V2+4B8 200 V2+HB65 CD13 expression 200 100 100 # Cells 0 0 1 10 100 1000 10000 FL1-H Light trypsin Heavy trypsin recovery 10min recovery 30min recovery 60min vercene

  10. 300 200 R10+L6 R30+L6 R60+L6 T10+L6 150 200 T2+L6 V2+L6 L6 expression V2+HB65 100 100 # Cells 50 0 0 Light trypsin Heavy trypsin recovery 10min recovery 30min recovery 60min vercene 1 10 100 1000 10000 FL1-H

  11. II. The influence of growth factors to CD13 expression on HMEC-1 cell 1. Starve the cells for 24 hr. 2. Culture cells in the medium above for another 36 hr. • 3. Harvest cells by versene**. Run FACS 1st atbs=4B8, L6, and HB65, 2nd atb=Go  Mo Ig(G+A+M) Expression rate = FL1-intensity of 4B8 or L6 minus that of • HB65.

  12. III. the influence of growth factors to CD13 expression on CL1-5 cell 1. Starve the cells for 24 hr. 2. Culture cells in the medium above for another 36 hr. • 3. Harvest cells by versene**. Run FACS 1st atbs=4B8, L6, and HB65, 2nd atb=Go  Mo Ig(G+A+M) Expression rate = FL1-intensity of 4B8 or L6 minus that of • HB65.

  13. HMEC-1 with growth factors 80 60 50 40 40 CD13expressionrate L6expressionrate 30 20 20 10 0 0 TNF- CoCl2 VEGF NONE TNF- VEGF NONE CoCl2 condition condition Result

  14. CL1-5 serum starvation 500 250 400 200 300 150 CD13 expression L6 expression 200 100 100 50 0 0 5%BCS 1%BCS 0%BCS 5%BCS 1%BCS 0%BCS 10%BCS 10%BCS condition condition

  15. CL1-5 with growth factors 300 300 250 200 200 CD13expressionrate 150 # Cells 100 100 50 0 TNF- CoCl2 VEGF NONE 4B8+VEGF 0 4B8+TNF- condition 4B8+None 1 10 100 1000 10000 4B8+CoCl2 FL1-H

  16. 250 250 200 200 L6 expression rate 150 150 # Cells 100 100 50 50 0 0 1 10 100 1000 10000 TNF- CoCl2 VEGF NONE FL1-H condition L6+VEGF L6+TNF- L6+None L6+CoCl2

  17. Conclusion • L6 expressed on HMEC-1 might be induced by some growth factors, including TNF-. • CD13 and L6 expressed on CL1-5 might both be induced by growth factors. • According to the similar trends of CD13 and L6 expression levels on CL1-5, the growth factors might alter the whole CD13-L6 complex expression level. And the invasion of CL1- 5 might be relative to CD13 as well as L6 since they can be induced at the same % of serum.

  18. Future works • Find out the real functions of CD13 and L6 in the process of invasion in CL1-5 by other methods such as genetics. • Test if CD13 and L6 have anything to do with vascularization of HMEC-1 and angiogenesis in tumor formation by other methods.

  19. Immunoenzyme and prodrug treatment of rat malignant ascites effectively controls disseminated tumor growth.

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