Maternal side Fetal side
圖二 Interstitial 及 endovascular trophoblast invasion 的示意圖。A 圖 是六週內的胎盤。B 圖是 在二十週以後的正常胎盤
，可以看到 spiral artery 有 remodeling。C 圖是二十週 以後的 Preeclampsia 的胎 盤，在 placental bed 的 spiral artery 沒有 remodeling。
(參考自 Kaufmann P et al,.
Biol Reprod 2003,69:1-7)
圖三 黏液蛋白與不同醣蛋白質示意圖。MUC2 是 secretory mucin，MUC1、4、
15、16 是 membrane bound mucins。我們可以比較它們的相對分子大小。
圖四 MUC1 訊息傳遞的示意圖。ICAM-1，Pseudomonas aeruginosa 等外來物均 可能是配體，均可能引發如圖中所示之訊息傳遞。 (參考自 TRENDS in Cell Biology 2006, 16 (9); 467-476)
50 nm 細胞膜
MUC 2
MUC16 MUC1 MUC4 integrins MUC15
圖五 MUC1CT 的示意圖。上方顯示是 kinase 接合點，可以造成該點之胺基酸的 磷酸化。下方顯示是與其他蛋白質的互動點。 (參考自 TRENDS in Cell Biology 2006, 16 (9); 467-476)
圖六 人體器官組織的 MUC15 表現。(A) Expression of membrane-bound mucins in the human placenta at term. RT–PCR was used to detect membrane-bound mucins MUC1, 3, 4, 12, 13, 15, 16, 17, and 20, as indicated. (B) Northern blot of MUC15 in human tissues. Multiple human tissue Northern blot (MTN I) was probed with a32P-labeled probe corresponding to full-length MUC15 complementary DNA. Note that MUC15 mRNA was expressed most abundantly in the placenta. (C) Expression of membrane-bound and secreted forms of MUC15 mRNA in human term placentas.
Membrane-bound (m) and secreted(s) forms of MUC15 transcripts were detected by RT–PCR, as indicated by arrows (255 bp and 102 bp). Markers are shown at the left.
圖七 MUC1 mRNA expression的Q-PCR。表現量隨著懷孕週數而增加。A) Quantitative RT-PCR showed that MUC1 mRNA expression in the human placenta was increased during placental development. The real-time RT-PCR signals of MUC1 were normalized to ACTB(=
actin) andanalyzed with MxPro Software; n indicates the placenta number. Mann-Whitney U-test was used for statistical analyses of relative MUC1 mRNA expressions. Results are presented as mean±SD.
** P< 0.01; * P<0.05; 1st, first trimester; 2nd, second trimester; 3rd, third trimester. B) The
representative amplification plots are shown. The arrows indicate the amplification plots of MUC1 for the first (1st, black circle), the second (2nd, blue diamond), and the third trimester (3rd, red square) placenta, as indicated (upper panel). ACTB signals were used as internal controls (lower panel).
圖八 MUC15 mRNA expression 的 Q-PCR。表現量亦隨著懷孕週數而增加。 (A) Real-time RT–PCR revealed that MUC15 mRNA expression in placentas was increased from the first (first Tri) to the third trimester (third Tri). The real-time RT–PCR signals of MUC15 were normalized to b-actin and analyzed with MxPro Software. n indicates the placenta number. Mann–Whitney U-test was used for statistical analyses of relative MUC15 mRNA expression. Results are presented as mean
± SD. *P< 0.05; **P<0.01. (B) Representative amplification plots are shown. The arrows indicate the amplification plots of MUC15 for the first (first Tri), second (second Tri), and third trimester (third Tri).
b-actin signals were used as internal controls.
圖九 MUC1 protein的expression level。表現量隨著懷孕週數而增加。Protein extracts of human placental tissues from the first trimester (1st), second trimester (2nd), and third trimester (3rd) were subjected to SDS-PAGE analysis and immunoblotted with anti-human MUC1 mAbs VU4H5 (A) and M2C5 (B) as well as anti-ACTB mAb. Representative immunoblots were shown. Arrows and the line at the right indicate MUC1 protein bands. ACTB was used as an internal control (lower panel of A and B). C) The MUC1 signals in the representative immunoblots of A and B were quantified and normalized ACTB signals by Image- Quant 5.1 software. *P<0.05; ** P<0.01.
圖十 MUC15 protein的expression level。表現量隨著懷孕週數而增加。(A) Specificity of anti-human MUC15 polyclonal antibodies. Lysates from E. coli (BL21) with (t) or without (2) IPTG induction of MUC15 recombinant protein expression (37 kDa) were western blotted with anti-MUC15 polyclonal antibodies. The arrow indicates MUC15 recombinant proteins. The arrow head indicates the aggregated MUC15. Marker scales (M) are shown at the left. (B) Western blot of MUC15 in human placenta. Protein extracts of human placental tissues from the first trimester and third trimester were subjected to SDS-PAGE analysis under reducing conditions and western blotted with antihuman MUC15 polyclonal and anti-actin monoclonal antibody. A representative immunoblot is shown.
Molecular weight markers are shown at the right. Actin was used as an internal control (lower panel).
(C) The MUC15 signals were quantified and normalized to actin signals by ImageQuant 5.1 software.
*P<0.05, compared with the signal of first trimester (first Tri) placenta.
圖十一 胎盤絨毛的MUC1的Immunohistochemistry。MUC1 expression in the
representative first trimester placentas (A and D: 9 wk), second trimester placentas (B: 23 wk and E: 19 wk), and third trimester placentas (C: 38 wk and F: 39 wk). The placental tissues were immunostained by VU4H5 (A–C) and M2C5 (D–F). Original magnification x 200. The magnification in the inset of D is x 400. The negative control did not show any specific signals (F, inset).
圖十二 胎盤絨毛的MUC15的Immunohistochemistry。Human placentas from the first (A–C), second (D–F), and third trimester (G–I) were stained with anti-MUC15 polyclonal antibodies (A, D, G, H, and I), anti-CK7 (B and E), anti-Ki67 (C) monoclonal antibodies, or without primary antibodies (F), as indicated. Scale bars＝50m. Original magnification: ×200. (A) MUC15 was weakly stained in cytotrophoblasts (CT) and syncytiotrophoblasts (ST). (B) CT were positively stained for CK7. (C) CT were stained for Ki67. (D) MUC15 was present in ST, but not extravillous trophoblasts (ET). (E) Extravillous trophoblasts (ET) in the decidua were positive for CK7 staining. (F) Negative (-) control did not show any signals. (G) In the representative term placenta (38 weeks gestation), MUC15 was highly expressed on the apical membrane of ST. (H) In the decidua, MUC15 was not detected in the luminal epithelium (LE), blood vessels (V), and decidual cells (DC). (I) MUC15 was expressed in the glandular epithelium (GE)
圖十三 蛻膜層的MUC1的Immunohistochemistry。A) Extravillous trophoblasts in the representative decidua (23 wk gestation) were immunostained by anti-KRT7 mAb. B) The serial section of the decidua was stained by anti-MUC1 mAb VU4H5. The arrows indicate MUC1-positive extravillous trophoblasts. The arrowheads indicate MUC1-negative extravillous trophoblasts; only some are pointed out. Magnification × 400.
圖十四 不同週數的蛻膜層的MUC1 Immunohistochemistry。Serial sections of the first (1st), second (2nd), and third (3rd) trimester placenta were immunostained for KRT7(=CK7) (A, C, and E) or VU4H5-reactive MUC1 (B, D, and F). The numbers of MUC1-positive extravillous
trophoblasts were increased during placental development (right panels). Glandular epithelia (GE) were also positive for KRT7 (C, arrow) and MUC1 (D, arrow). Original magnification × 100. The relative regions in the serial sections for KRT7 and MUC1 staining are shown by small rectangles.
圖十五 MUC1 overexpression抑制JAR cells的invasion及可能的mechanism。A) JAR cells were transiently transfected with pHb-Apr1-neo (Mock) or pHb-Apr1-neo/MUC1 (MUC1) for 48 h. The arrow at the right indicates MUC1 protein. B) JAR (1×105) cells transfected with mock or MUC1 plasmids were seeded in each chamber and incubated for 48 h. The representative images of invaded JAR are shown (upper panel).Original magnification ×200. The invaded cell numbers are presented as schematic diagrams (lower panel). The results are presented as mean ±SD. **, P< 0.01. C) The percentage of MUC1-high expressers on the upper side (grey bar) and lower side (black bar) is shown. D) MMP9, but not MMP2, activity was suppressed by MUC1 overexpression.
圖十六 MUC15 overexpression抑制JAR及JEG-3 cells的invasion。JAR and JEG-3 cells were transiently transfected with pcDNA3.1 (Mock), MUC15/pcDNA3.1 (MUC15), MUC15/
pcDNA3.1 t control siRNA (MUC15 t control siRNA) or MUC15/pcDNA3.1 t MUC15 siRNA (MUC15 t MUC15 siRNA) for 48 h. JAR (5×104) or JEG-3 (2×105) cells were seeded in each chamber and incubated for another 48 h. The representative images of invaded JAR (A) and JEG-3 (B) are shown. The numbers of invaded JAR (C) and JEG-3 cells (D) are shown as mean±SD. **P<0.01
圖十七 MUC15 overexpression抑制JAR及JEG-3 cells的invasion。JAR and JEG-3 cells were transiently transfected with Mock, MUC15, MUC15 t control siRNA or MUC15 t MUC15 siRNA. Gelatin zymography showed that MUC15 overexpression did not significantly affect MMP-2 and MMP-9 activity in the conditioned media of JAR (A) and JEG-3 (B) cells. Real-time RT–PCR showed that MUC15 overexpression increased TIMP-1 and TIMP-2 mRNA expression in both JAR (C) and JEG-3 (D) cells. The induction of TIMP-1 and TIMP-2 was significantly blocked by MUC15 siRNA, but not control siRNA. Data are presented as mean± SD. *P<0.05.
圖十八 MUC1mRNA 在 severe preeclampsia 的胎盤的表現量增加。圖示內容請參 考圖七。
圖十九 MUC1 protein 在 severe preeclampsia 的胎盤的 western blot 表現量比 first(1st)、second(2nd)、third(3rd) trimesters 增加。
圖二十 MUC1 (+) trophoblasts 在 mild preeclampsia 的蛻膜層的數量略增加。
100x MUC1 CK7
表一 PCR 的 primers。
圖六、八、十、十二、十六、十七取材自 Shyu MK et.al 之 Human Reproduction.
22(10):2723-32, 2007 Oct.
圖七、九、十一、十三、十四、十五取材自 Shyu MK et.al 之 Biology of Reproduction.
79(2):233-9, 2008 Aug.
第九章 附錄
列出個人在博士班修業期間所發表之相關論文
1. Shyu MK. Lin MC. Liu CH. Fu YR. Shih JC. Lee CN. Chen HY. Huang J. Huang MC. Hsieh FJ. MUC1 expression is increased during human placental development and suppresses trophoblast-like cell invasion in vitro.
Biology of Reproduction. 79(2):233-9, 2008 Aug.
2. Shyu MK. Lin MC. Shih JC. Lee CN. Huang J. Liao CH. Huang IF. Chen HY.
Huang MC. Hsieh FJ. Mucin 15 is expressed in human placenta and suppresses invasion of trophoblast-like cells in vitro.
Human Reproduction. 22(10):2723-32, 2007 Oct.