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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。 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。 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 Purpose and introduction invasion L6 CD13 GFs , Hypoxia CL1-5 CL1-5 angiogenesis HUVEC CD13 L6 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 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. Methods 0. Protocals I. Test the better condition to harvest CL1-5 Cell type Condition Versene treated Light trypsin treated CL1-5 Heavy trypsin treated Recovery for short term after trypsin treated Recovery for medium term after trypsin treated Recovery for long term after trypsin treated Run FACS, 1st abs=4B8, L6, and HB65, 2nd ab=Go Mo Ig(G+A+M)-FITC Expression level = FL1-intensity of 4B8 or L6 minus that of HB65. R10+4B8 R30+4B8 R60+4B8 T10+4B8 T2+4B8 V2+4B8 V2+HB65 300 300 200 100 0 200 # Cells CD13 expression 400 100 0 1 10 100 FL1-H 1000 10000 300 R10+L6 R30+L6 R60+L6 T10+L6 T2+L6 V2+L6 V2+HB65 150 200 100 # Cells L6 expression 200 50 100 0 0 1 10 100 FL1-H 1000 10000 II. The influence of growth factors to CD13 expression on HMEC-1 cell Cell type HMEC-1 Condition i Condition ii 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 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. III. the influence of growth factors to CD13 expression on CL1-5 cell Cell type Condition i Condition ii CL1-5 RPMI+10%BCS RPMI+5%BCS RPMI+1%BCS RPMI+0%BCS RPMI+0%BCS RPMI+VEGF RPMI+TNF- 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. Result HMEC-1 with growth factors 80 40 L6 expression rate CD13 expression rate 50 30 20 10 60 40 20 0 0 condition condition 250 500 200 400 L6 expression CD13 expression CL1-5 serum starvation 150 100 50 0 300 200 100 0 condition condition CL1-5 with growth factors 4B8+VEGF 4B8+TNF- 4B8+None 4B8+CoCl2 300 250 200 200 150 # Cells CD13 expression rate 300 100 100 50 0 0 condition 1 10 100 FL1-H 1000 10000 250 200 L6+VEGF L6+TNF- L6+None L6+CoCl2 200 150 150 # Cells L6 expression rate 250 100 100 50 50 0 0 1 condition 10 100 FL1-H 1000 10000 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 CL15 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.