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。 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。

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

L6 CD13 CL1-5 invasion HUVEC L6 CD13 angiogenesis CL1-5 GFs, Hypoxia Purpose and introduction We Study 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.

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.

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.

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.

Cell type Condition CL1-5 Versene treated Light trypsin treated Heavy trypsin treated Recovery for short term after trypsin treated Recovery for medium term after trypsin treated Recovery for long term after trypsin treated 0. Protocals I. Test the better condition to harvest CL1-5 Methods Run FACS, 1 st abs=4B8, L6, and HB65, 2 nd ab=Go  Mo Ig(G+A+M)-FITC Expression level = FL1-intensity of 4B8 or L6 minus that of HB65.

vercene Light trypsin Heavy trypsin recovery 10min recovery 30min recovery 60min CD13 expression FL1-H # Cells V2+HB65 V2+4B8 T2+4B8 T10+4B8 R60+4B8 R30+4B8 R10+4B8

FL1-H # Cells V2+HB65 V2+L6 T2+L6 T10+L6 R60+L6 R30+L6 R10+L vercene Light trypsin Heavy trypsin recovery 10min recovery 30min recovery 60min L6 expression

Cell typeCondition iCondition ii HMEC-1 EBM-2+15%FBS+10µM L-glutamine +10nM EGFs+1µg/ml HDC EBM-2+0%FBS EBM-2+5%FBS EBM-2+VEGF EBM-2+1%FBS EBM-2+TNF-  EBM-2+0%FBS 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 1 st atbs=4B8, L6, and HB65, 2 nd atb=Go  Mo Ig(G+A+M) Expression rate = FL1-intensity of 4B8 or L6 minus that of HB65.

III. the influence of growth factors to CD13 expression on CL1-5 cell Cell typeCondition iCondition ii CL1-5 RPMI+10%BCSRPMI+0%BCS RPMI+5%BCSRPMI+VEGF RPMI+1%BCS RPMI+TNF-  RPMI+0%BCS 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 1 st atbs=4B8, L6, and HB65, 2 nd atb=Go  Mo Ig(G+A+M) Expression rate = FL1-intensity of 4B8 or L6 minus that of HB65.

NONE VEGF TNF- CoCl2 CD13 expression rate condition NONE VEGF TNF-  CoCl2 L6 expression rate condition  HMEC-1 with growth factors Result

%BCS 5%BCS1%BCS0%BCS condition CD13 expression %BCS 5%BCS1%BCS0%BCS L6 expression condition  CL1-5 serum starvation

 CL1-5 with growth factors NONE VEGF TNF-  CoCl2 CD13 expression rate condition FL1-H # Cells 4B8+CoCl 2 4B8+None 4B8+TNF-  4B8+VEGF

# Cells FL1-H L6+CoCl 2 L6+None L6+TNF-  L6+VEGF NONE VEGF TNF-  CoCl2 L6 expression rate condition

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.

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.

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