Control 6 60 100 150 300
Ratio of cyclin E mRNA
0.00 0.50 1.00
Figure 50. RT-PCR analysis of cyclin E and beta-actin in human stomach cancer cell line (SC-M1) for treatment with different doses of flavone. (A) Gel electrophoresis. Lane 1：DMSO；Lane 2：6 µM；Lane 3：60
µM；Lane 4：100 µM；Lane 5：150 µM；
Lane 6：300
µM (B) The statistic figure of p21 mRNA levels. Values are mean±
SD n= 2
Cyclin E (375 bp)
β-actin (353 bp)
A.
1 2 3 4 5 6
B.
Concentration of flavone ( µM)
Control 6 60 100 150 300
Ratio of CDK2 mRNA
0.00 0.50 1.00
Figure 51. RT-PCR analysis of CDK2 and beta-actin in human stomach cancer cell line (SC-M1) for treatment with different doses of flavone. (A) Gel electrophoresis. Lane 1：DMSO；Lane 2：6 µM；Lane 3：60
µM；Lane 4：100 µM；Lane 5：150 µM；
Lane 6：300
µM (B) The statistic figure of p21 mRNA levels. Values are mean±
SD n= 2
CDK2 (330 bp)
β-actin (353 bp)
A.
1 2 3 4 5 6
B.
Concentration of flavone (µM)
Control 6 60 100 150 300
Ratio of p21 mRNA
0.00 0.50 1.00
Figure 52. RT-PCR analysis of p21 and beta-actin in human stomach cancer cell line (SC-M1) for treatment with different doses of flavone. (A) Gel electrophoresis. Lane 1：DMSO；Lane 2：6 µM；Lane 3：60
µM；Lane 4：100 µM；Lane 5：150 µM；
Lane 6：300
µM (B) The statistic figure of p21 mRNA levels. Values are mean±
SD n= 2
p21 (380 bp)
β-actin (353 bp)
A.
1 2 3 4 5 6
B.
Concentration of flavone (µM)
Control 6 60 100 150 300
Ratio of p53 mRNA
0.00 0.50 1.00
Figure 53. RT-PCR analysis of p53 and beta-actin in human stomach cancer cell line (SC-M1) for treatment with different doses of flavone. (A) Gel electrophoresis. Lane 1：DMSO；Lane 2：6 µM；Lane 3：60
µM；Lane 4：100 µM；Lane 5：150 µM；
Lane 6：300
µM (B) The statistic figure of p21 mRNA levels. Values are mean±
SD n= 2
p53 (293 bp)
β-actin (353 bp)
第三節 對人類胃癌細胞株基因表現的方面
1. 利用 DD RT-PCR 方法偵測 apigenin、17
α
-estradiol 和 flavone 對 SC-M1 細胞基 因表現的影響利用 mRNA 鑑別展示(DD RT-PCR)找出所有受藥物影響的基因接著將有差 異的 band 取下後進行核酸序列分析。如表六及表七是經 NCBI 基因庫比對之結 果。如 Figure 54 至 Figure 59 及表六結果顯示胃癌細胞受 17
α
-estradiol (E2)影響 的基因共有 32 個，有 12 個基因被誘導表現，有 20 個基因表現被抑制，其中 9 個基因是未知的。受 flavone 影響基因共有 22 個，有 5 個被誘導表現，有 17 個 基因被抑制其中有 3 個是屬於未知的基因。如 Figure 60 至 64 及表七受 apigenin 影響的基因共有 47 個基因，有 25 個被誘導表現，有 22 個基因被抑制其中有 17 個是屬於未知的基因。Figure 54. The gel electrophoresis of the products from DD RT-PCR after exposure to various doses of 17α-estradiol and flavone for treatment 24 h. Lane 1：DMSO, Lane
Figure 55. The gel electrophoresis of the products from DD RT-PCR after exposure to various doses of 17α-estradiol and flavone for treatment 24 h. Lane 1：DMSO, Lane 2：17α-estradiol 6 µM, Lane 3：17α-estradiol 60 µM, Lane 4：flavone 6 µM, Lane 5：
flavone 60 µM.
Figure 56. The gel electrophoresis of the products from DD RT-PCR after exposure to various doses of 17α-estradiol and flavone for treatment 24 h. Lane 1：DMSO, Lane 2：17α-estradiol 6 µM, Lane 3：17α-estradiol 60 µM, Lane 4：flavone 6 µM, Lane 5：
flavone60 µM.
Figure 57. The gel electrophoresis of the products from DD RT-PCR after exposure to various doses of 17α-estradiol and flavone for treatment 24 h. Lane 1：DMSO, Lane 2：17α-estradiol 6 µM, Lane 3：17α-estradiol 60 µM, Lane 4：flavone 6 µM, Lane 5：
flavone 60 µM.
Figure 58. The gel electrophoresis of the products from DD RT-PCR after exposure to various doses of 17α-estradiol and flavone for treatment 24 h. Lane 1：DMSO, Lane 2：17α-estradiol 6 µM, Lane 3：17α-estradiol 60 µM, Lane 4：flavone 6 µM, Lane 5：
Figure 59. The gel electrophoresis of the products from DD RT-PCR after exposure to various doses of 17α-estradiol and flavone for treatment 24 h. Lane 1：DMSO, Lane 2：17α-estradiol 6 µM, Lane 3：17α-estradiol 60 µM, Lane 4：flavone 6 µM, Lane 5：
flavone 60 µM.
表六：胃癌細胞受 17
α
-estradiol (E)和 flavone (F)影響的所有基因PCR No. Sequence result Induced Inhibit
1. A9a, A9b (E) Cytochrome b (Mitochondrion) + 2. A9c (E) Similared Oncostatin M receptor +
3. A10b (F) Chromodomain- helicase-DNA-binding protein
+
4. A10c (E, F) NADH dehydrogenase subunit 2 +
5.A10d (F) Homo sapiens cDNA: FLJ22135 fis, clone HEP20858
+
6.A10e (F) No-homology +
7. A10f (E, F) Cytochrome C oxidase subunit III + 8.A11a (E, F) Similar to unknow HERV-H protein +
9.C10a (F) Hypothetical protein +
10. C10b (E, F) Sodium bicarbonate cotransporter 2b +
11.C10c (F) Hypothetical protein +
12.C11a (E, F) Similar to unknow HERV-H protein +
13.C13a (E) no-homology +
14.C15b (E) no-homology +
15.C15c (F) Cytochrome c oxidase subunit I +
16.G9a (E) Cytochrome b +
17.G9b (E) Similared Oncostatin M protein + 18.G9c (E) Similared Oncostatin M protein + 19.G12a, G12d (E) Homo sapiens, clone IMAGE: 4081483,
mRNA
+
20.G12b (E) Hypothetical protein +
21.G12c (E) (homo with G12e)
Glycerol-3-phosphate dehydrogenase 2 (mitochondrial)
+
22.G12e (E) Glycerol-3-phosphate dehydrogenase 2 +
23.A74a (E) Hypothetical protein +
24.A75a (E) No-homology +
25.A78a, A78b (E, F)
Adenomatosis polyposis coli (APC) +
26.A79a (F) Chromosome 4 +
27.A79b (F) Chromosome 6q16 2-21 +
表六(續)：胃癌細胞受 17
α
-estradiol (E)和 flavone (F)影響的所有基因31.C73a (F) No-homology +
32.C73b (E) Mycoplasma hyorhinis strain BTS7 16S ribosomal RNA
+
33.C73c (F) Chromosome 6q12-14.3 +
34.C74a (E) Proliferation-associated gene (peroxiredxin 1)
+
35.C74b (F) Procollagen- lysine, 2-oxoglutarate 5-dioxygenase
+
36.C75a (E) No-homology +
37.C75b (F) No-homology +
38.C75c (E) Ubiguitin protein ligase E3A +
39.C76a (E) Chromosome 19 +
43.G74a (E) Ribosomal protein L10 (hypothetical protein)
+
44.G75a (F) No-homology +
45.G75b (E) IQ motif containing GTPase activating protein 1
+
46.G75c (E) No-homology +
47.G75e (E) No-homology +
48.G75g (E) Serine (or cysteine) proteinase inhibitor (monocyte/netrophile elastase inhibitor
gene)
+
49.G78b (E, F) Human DNA sequence from clone 191J18 on chromosome 6q16.1-22.33,
complete sequence
+
50.G80a, G80b (E) ATP synthase 6 +
51G80c, G80d (E) Chromosome 13 +
Figure 60. The gel electrophoresis of the products from DD RT-PCR after exposure to various doses of apigenin for treatment 24 h. Lane 1：DMSO, Lane 2：6 µM, Lane
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Figure 61. The gel electrophoresis of the product from DD RT-PCR after exposure to various doses of apigenin for treatment 24 h. Lane 1：DMSO, Lane 2：6 µM, Lane 3：60 µM.
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Figure 62. The gel electrophoresis of the product from DD RT-PCR after exposure to
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Figure 63. The gel electrophoresis of the product from DD RT-PCR after exposure to various doses of apigenin for treatment 24 h. Lane 1：DMSO, Lane 2：6 µM, Lane 3：60 µM.
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Figure 64. The gel electrophoresis of the product from DD RT-PCR after exposure to
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
表七：胃癌細胞受 apigenin 影響的所有基因
PCR No. Sequence result Induced Inhibited
1. A11 (i) No-homology +
2. A12 (i) Human Xq13 3’ end of PAC 92E23 containing the X inactivation transport
+
8. A74 (ii) Hypothetical protein DKFZp 761C169 +
9. A74 (iii) Chromosome 10 complete sequence +
10.A75 (i) GiGl protein or cystein-rich angiogenic inducer 61
+
11.A75 (iii) No-homology +
12.A75 (v) Similar to programmed cell death 6 or similar to probable calcium-binding
protein ALG-2
+
13.A77 (i) Hypothetical protein +
14.A78 (i) Voltage-dependent anion channel 3 +
15.A78 (ii) No-homology +
16.A78 (iii) No-homology +
17.A79 (i) Eukaryotic translation elongation factor 1 β2 → hypothetical protein
+
18.A80 (ii) Chromosome 7 +
19.C9 (i) Chromosome 14 +
20.C9 (ii) No-homology +
21.C11 (iii) TAR DNA binding protein mRNA +
22.C11 (iv) Hypothetical protein +
23.C11 (v) Hypothetical protein +
24.C11 (vi) Chromosome 19 +
25.C12 (ii) Hypothetical protein +
26.C12 (iii) No-homology +
27.C73 (i) Chromosome 6 +
28.C73 (ii) Mycoplasma hyorinis 16S ribosomal mRNA gene
+
表七(續)：胃癌細胞受 apigenin 影響的所有基因
30.C75 (ii) No-homology +
31.C75 (iii) No-homology +
32.C75 (iv) Serine (orcystein) proteinase inhibitor (monocyte/netrophil elastase inhibitor)
+
33.C75 (v) Ubiquitin protein ligase E3A (Homology with C75c)
+
34.C77 (i) Highly similar to Elongation factor 1-α1 +
35.C77 (iv) Immunoglobulin (CD79A) binding protein 1
39.C79 (ii) Chromosome 4 clone C0315N08 +
40.C79 (iii) Hypothetical protein PRO2822 (PRO2822)
+
41.G12 (i) Hypothetical protein PRO2822 (chromosome 10)
+
42.G12 (ii) No-homology +
43.G13 (i) No-homology +
44.G14 (ii) Heterogeneous nuclear ribonucleoprotein H 1
+
45.G74 (i) No-homology +
46.G76 (i) Chromosome 17 +
47.G78 (i) No-homology +
2. 檢測 17
α
-estradiol 對 SC-M1 細胞的細胞膜上 Oncostatin M receptor 的影響 由 DD RT-PCR 結果 Figure 54、56 和 Table 6 發現 17α
-estradiol 可以誘導 Oncostatin M receptor 基因的表現，因此我們利用抗體檢測 17α
-estradiol 是否真的 會增加細胞膜上 Oncosatin M receptor 的表現，而結果與 DD RT-PCR 結果是相符 合。3. 加入 p38 MAPK 抑制劑 SB 203580 和 MAPK/ERK kinase I 抑制劑 PD 98059 檢測對人類胃癌細胞株 SC-M1 的存活率的影響，由 Figure 66 結果發現在有先加 入 10
µ
M SB 203580 作用 3 小時後的細胞存活率增加 6 %，因此推論 17α
-estradiol 是藉由 oncostatin M receptor 下游訊息調控路徑 p38 MAPK 來導致人類胃癌細胞 株 SC-M1 產生細胞凋亡。A. B.
C. D.
Concentration of 17 α-estradiol (µM)
Control 0.6 30 60 100 150
Concentration of estradiol (µM)
Control 6 30 60 90 150
% cells of positive for OSM receptor
0
Fig 65. The distribution of OSM receptor from SC-M1 cells that growing in the present of different doses of 17a-estradioltreatment for 24 hr. The gel electrophoresis of the products from DD RT-PCR (A) and DNA sequence (B) of oncostatin M receptor. (C) The gel electrophoresis and statistic figure of RT-PCR analysis of OSMR and beta-actin for treatment with different doses of 17α-estradiol. . Lane 1：DMSO；Lane 2：6 µM；
Lane 3：60 µM；Lane 4：100 µM；Lane 5：150 µM；Lane 6：300 µM Values are mean±
SD n= 2. (D) The data of oncostatin M receptor that was analyzed by FACS. Data were analyzed by one-way ANOVA. Value= mean± SD, n=3 *P<0.05, **P<0.005,
***P<0.001