Transcript Condition i

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.