頭頸部腫瘤佔全世界人口每年所癌症發生率的 6 ％ ，此疾病過
去好發生於男性，且發生率有逐年增加的趨勢，在統計學上發現頭頸
部腫瘤病人五年存活率少於 50 ％ ，因此，對頭頸部癌的研究更是
迫在眉睫，其中以找尋更多的新穎標的基因作為未來有效的的診斷及
治療方針。在最近的研究發現 podoplanin 表現會使癌細胞增生、發
育異常，且可能與腫瘤的入侵、轉移、嚴重惡性程度相關。 Podoplanin
是一種粘液素類型的醣蛋白，高度表現於人類淋巴內皮細胞，血管內
皮細胞中不表現。本論文主要探討口腔鱗狀上皮細胞癌中 podoplanin
具有的臨床病理意義，及在頭頸部癌細胞中扮演的角色。研究中首先
收集了臨床樣本組織和相關病歷資料，並利用人類頭頸部細胞癌細胞
株 (Fadu、Hep2) 了解 podoplanin 調控的分子機制。
首先由反轉錄聚合酶連鎖反應及西方墨點法發現 podoplanin 在
口腔鳞狀上皮細胞癌組織相對於鄰近的正常組織有較大量表現的情
形；再藉由免疫組織染色觀察 podoplanin 表現在口腔鳞狀上皮細胞
癌組織的位置，結果顯示， podoplanin 在 T3、T4 stage 腫瘤部位的
表現量大於鄰近的正常組織；且相較於T1、T2 stage， podoplanin 較
好發於 T3、 T4 stage 的腫瘤組織。在此 podoplanin 表現於口腔鳞狀上皮細胞癌腫瘤組織及淋巴管；除此之外，經由臨床統計分析發現，
podoplanin 的大量表現會導致口腔鱗狀上皮細胞癌病患五年存活率
降低；在單項分析中也發現病患的存活率與臨床病理性特徵(腫瘤分
期、淋巴結轉移分期、TNM 分期) 及 podoplanin 的表現量是具有相
關性的，但在多項分析中發現病患的存活率只與臨床病理性特徵的腫
瘤分期和淋巴結轉移分期相關。為了更進一步探討 podoplanin 表現
在細胞的位置以及和訊息傳遞的相關性，因此，利用細胞螢光免疫染
色及細胞轉型方式 (transfection) 得知， podoplanin 表現於 Fadu、
Hep2 此兩株細胞核膜的位置，並從中發現 podoplanin 與 PI3K/AKT
之訊息傳遞具有一定的相關性。 Podoplanin 與口腔鱗狀上皮細胞癌
之間存在的關係是值得更深一步去探討研究的，未來 podoplanin 也
可成為新的治療標的也說不定。

Head and neck cancer (HNC) makes up 6 ％ of the cancer patients
in the world every year. This disease usually occurs in males and the
incidence is increasing year by year. According to statistical analysis,
HNC has less than 50 ％ five-year survival rate. Therefore, the research
of HNC seems imminent so that may lead to the development of new
approaches of diagnosis and therapy.
Recent research had shown that expression of podoplanin caused
cellular proliferation, and may be associated with tumor invasion,
metastasis and malignant prognosis. Podoplanin, a mucin-type
transmembrane sialoglycoprotein, is highly expressed in lymphatic
endothelial cells but not expressed in vascular endothelial cells. The
purpose of this study was to determine the clinical and pathological
significance of podoplanin in oral squamous cell carcinoma (OSCC).
Therefore, we collected clinical specimen and associated patient history
of OSCC. Further, we used the human cell lines of HNSCC (Fadu, Hep2)
to investigate the molecular regulation of podoplanin.
Podoplanin expression was analyzed by RT-PCR and Western blot assay
firstly. As shown, podoplanin was found to be overexpressed in tumors compared with normal adjacent tissues. Further, immunohistochemical
analysis revealed the location of podoplanin expression in OSCC tissues.
The results showed that podoplanin had higher expression in T4 stage
tumor section than in normal adjacent tissues of OSCC samples, or in T1
stage. Here, podoplanin was highly expressed in the OSCC tumor cell
and lymphatics of stage T4 OSCC tissue. Furthermore, we found that
overexpression of podoplanin in OSCC patients was associated with
decreased five-years survival rate. In the univariate analysis, several
factors were statistically significantly associated with disease-specific
survival rate, including Tumor stage, Nodal stage, and podoplanin
expression. In the subsequent multivariate analysis, only Tumor stage and
Nodal stage showed a trend toward worse disease-specific survival. To
further investigate the regulatory mechanism of podoplanin and its
position of expression within the cell, immunofluorescence and
transfection were utilized to assay. The results showed that podoplanin
was expressed in the nuclear membrane of the Fadu and Hep2 cell lines,
and the PI3K/AKT signaling pathway was involved. We suggest that the
role of podoplanin in OSCC should be further investigated for potential
future treatment.

目 錄 …...……………………………………………...1
縮寫字辭 ….…………………………………………...…..2
試 劑 表 ……….…………………………………….........3
中 文 摘 要……………………………………………...…...5
英 文 摘 要……………………………………...…………...7
前 言 …..……………………………………………....9
材 料 方 法………………………………………..…..…….20
結 果 …………………………………………....…….35
討 論 ………………………………………………….39
參 考 文 獻…..…………………………………...…………53
附 錄 …..…………………………………...…………63

1. http://www.bioguider.com/ebook/merck/s19/1907.html
2. Forastiere A, Koch W, Trotti A, Sidransky D. Head and neck cancer. N Engl J Med (2001) 345: 1890–1900
3. http://www.mc.ntu.edu.tw/department/ent/ko/ko-3.html
4. Waldron CA, Shafer WG: Leukoplakia revisited: A clinicopathologic study 3256 oral leukoplakias. Cancer (1975) 36: 1386-1392
5. Hardisson D. Molecular pathogenesis of head and neck squamous cell carcinoma. Eur Arch Otorhinolaryngol (2003) 260: 502–508
6. Hackel PO, Zwick E, Prenzel N, Ullrich A. Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Curr Opin Cell Biol (1999) 11: 184–189
7. Grandis JR, Tweardy DJ. Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer. Cancer Res (1993) 53: 3579–3584
8. Miyaguchi M, Olofsson J, Hellquist HB. Expression of epidermal growth factor receptor in laryngeal dysplasia and carcinoma. Acta Otolaryngol (1990) 110: 309–313
9. Maurizi M, Almadori G, Ferrandina G, Distefano M, Romanini ME
, Cadoni G, Benedetti-Panici P, Paludetti G, Scambia G, Mancuso S. Prognostic significance of epidermal growth factor receptor in laryngeal squamous cell carcinoma. Br J Cancer (1996) 74: 1243–1257
10. Werkmeister R, Brandt B, Joos U. Aberrations of erbB-1 and erbB-2 oncogenes in non-dysplastic leukoplakias of the oral cavity. Br J Oral Maxillofac Surg (1999) 37: 477-480
11. Chen YJ, Lin SC, Kao T, Chang CS, Hong PS, Shieh TM, Chang KW.
Genome-wide profiling of oral squamous cell carcinoma. J Pathol (2004) 204: 326-332
12. Sionov RV, Haupt Y. The cellular response to p53: the decision between life and death. Oncogene (1999) 18: 6145–6157
13. Erber R, Conradt C, Homann N, Enders C, Finckh M, Dietz A, Weidauer H, Bosch FX. TP53 DNA contact mutations are selectively associated with allelic loss and have a strong clinical impact in head and neck cancer. Oncogene (1998) 16: 1671–1679
14. Brennan JA, Mao L, Hruban RH, Boyle JO, Eby YJ, Koch WM, Goodman SN, Sidransky D. Molecular assessment of histopathological staining in squamous-cell carcinoma of the head and neck. N Engl J Med (1995) 332: 429–435
15. Shin DM, Lee JS, Lippman SM, Lee JJ, Tu ZN, Choi G, Heyne K, Shin HJ, Ro JY, Goepfert H, Hong WK, Hittelman WN. p53 expressions: predicting recurrence and second primary tumors in head and neck squamous cell carcinoma. J Natl Cancer Inst (1996) 88: 519–529
16. Jin YT, Kayser S, Kemp BL, Ordonez NG, Tucker SL, Clayman GL, Goepfert H, Luna MA, Batsakis JG, El-Naggar AK. The prognostic significance of the biomarkers p21WAF1(CIP1), PR1WAF1(CIP1), p53, and bcl-2 in laryngeal squamous cell carcinoma. Cancer (1998) 82: 2159–2165
17. Caminero MJ, Nunez F, Suarez C, Ablanedo P, Riera JR, Dominguez F. Detection of p53 protein in oropharyngeal carcinoma. Prognostic implications. Arch Otolaryngol Head Neck Surg (1996) 122: 769–772
18.Pruneri G, Pignataro L, Carboni N, Ronchetti D, Cesana BM, Ottaviani A, Neri A, Buffa R. Clinical relevance of p53 and bcl-2 protein over-expression in laryngeal squamous-cell carcinoma. Int J Cancer (1998) 79: 263–268
19. http://www.supermt.com.tw/edu/molbio/lecture_1/5312.pdf
20. Koontongkaew S, Chareonkitkajorn L, Chanvitan A, Leelakriangsak
M,Amornphimoltham P. Alterations of p53, pRb, cyclin D (1) and
cdk4 in human oral and pharyngeal squamous cell carcinomas. Oral
Oncol (2000) 36: 334-339
21. Miyamoto R, Uzawa N, Nagaoka S, Hirata Y, Amagasa T. Prognostic significance of cyclin D1 amplification and overexpression in oral squamous cell carcinomas. Oral Oncol (2003) 39: 610-618
22.Miracca EC, Kowalski LP, Nagai MA. High prevalence of p16 genetic alterations in head and neck tumours. Br J Cancer (1999) 81: 677–683
23. Werkmeister R, Brandt B, Joos U. Aberrations of erbB-1 and erbB-2 oncogenes in non-dysplastic leukoplakias of the oral cavity. Br J Oral Maxillofac Surg (1999) 37: 477-480
24. Chen YJ, Lin SC, Kao T, Chang CS, Hong PS, Shieh TM, Chang KW.
Genome-wide profiling of oral squamous cell carcinoma. J Pathol (2004) 204: 326-332
25. Thomas GT, Lewis MP, Speight PM. Matrix metalloproteinases and
oral cancer. Oral Oncol (1999) 35: 227-233
26.Thiery JP. Epithelial–mesenchymal transitions in tumour progression. Nat Rev Cancer (2002) 2: 442–454
27.Gupta GP, Massague J. Platelets and metastasis revisited: a novel fatty link. J Clin Invest (2004) 114: 1691–1693
28. Folkman J:Clinical applications of research on angiogensis. N Engl J Med (1995) 333: 1757-1763
29. Houck KA, Leung DW, Rolwand AM, Winer J, Ferrara N. Dual
regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem (1992) 267: 26031 -26037
30. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev (1997) 18: 4-25
31. Makinena T. Molecular mechanisms of lymphatic vascular
development. Cell Mol Life Sci (2007) 64: 1915–1929
32.Wetterwald A, Hoffstetter W, Cecchini MG, Lanske B, Wagner C, Fleisch H, Atkinson M. Characterization and cloning of the E11 antigen, a marker expressed by rat osteoblasts and osteocytes. Bone (1996) 18: 125–132
33. Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P, Poczewski H, Kalt R, Schaffner G, Kerjaschki D. Podoplanin, Novel 43-kd Membrane Protein of Glomerular Epithelial Cells, Is Down-Regulated in Puromycin Nephrosis. Am J Pathol. (1997) 151: 1141-1152
34. Kriehuber E, Breiteneder GS, Groeger M, Soleiman A, Schoppmann SF, Stingl G. Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J Exp Med (2000) 194: 797–808
35. Hong YK, Harvey N, Noh YH, Schacht V, Hirakawa S, Detmar M, Oliver G. Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate. Dev Dyn (2002) 225: 351–357
36. Petrova TV, Makinen T, Makela TP, Saarela J, Virtanen I, Ferrell RE, Finegold DN, Kerjaschki D, Yla-Herttuala S, Alitalo K. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J (2002) 21: 4593–4599
37. Hirakawa S, Hong YK, Harvey N, Schacht V, Matsuda K, Libermann T, Detmar M. Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells. Am J Pathol (2003) 162: 575–586
38. Kaneko MK, Kato Y, Kitano T, Osawa M. Conservation of a platelet
activating domain of Aggrus/podoplanin as a platele aggregation- inducing factor. Gene (2006) 378: 52–57
39. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R, Amann G,
Kriehuber E, Diem K, Weninger W, Tschachler E, Alitalo K,
Kerjaschki D. Angiosarcomas express mixed endothelial phenotypes of
blood and lymphatic capillaries: podoplanin as a specific marker for
lymphatic endothelium. Am J Pathol (1999) 154: 385–394
40. Schacht V, Dadras SS, Johnson LA, Jackson DG, Hong YK,
Detmar M. Up-regulation of the lymphatic marker podoplanin, a
mucin-type transmembrane glycoprotein, in human squamous cell
carcinomas and germ cell tumors. Am J Pathol (2005) 166: 913–166921
41. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R, Amann G, Kriehuber E, Diem K, Weninger W, Tschachler E, Alitalo K, Kerjaschki D. Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am J Pathol (1999) 154: 385–394
42. Martin-Villar E, Scholl FG, Gamallo C, Yurrita MM, Munoz-Guerra M, Cruces J, Quintanilla M. Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas. Int J Cancer (2005) 113: 899–910
43. Ramirez MI, Millien G, Hinds A, Cao YX, Seldin DC, Williams MC.
T1a, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolous formation at birth. Dev Biol (2003) 256: 61–72
44. Schacht V, Ramirez MI, Hong YK, Hirakawa S, Feng D, Harvey N, Williams M, Dvorak AM, Dvorak HF, Oliver G, Detmar M. T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema. EMBO J (2003) 22: 3546–3556
45. Kato Y, Fujita N, Kunita A, Sato S, Kaneko M, Osawa M and Tsuruo T. Molecular identification of Aggrus/T1alpha as a platelet aggregation aggregation-inducing factor expressed in colorectal tumors. J Biol Chem (2003) 278: 51599–51605
46. Kaneko MK, Kato Y, Kitano T, Osawa M. Conservation of a platelet activating domain of Aggrus/podoplanin as a platelet aggregation-inducing factor. Gene (2006) 378: 52–57
47. Kawaguchi H, El-Naggar AK, Papadimitrakopoulou V, Ren H, Fan YH, Feng L, Lee JJ, Kim E, Hong WK, Lippman SM, Mao L.
Podoplanin: A Novel Marker for Oral Cancer Risk in Patients With Oral Premalignancy. J Clin Oncol (2008) 28: 354-360
48. Yuan P, Temam S, El-Naggar A, Zhou X, Liu DD, Lee JJ, Mao L. Overexpression of podoplanin in oral cancer and its association with poor clinical outcome. Cancer (2006) 107: 563- 569
49. Schacht V, Dadras SS, Johnson LA, Jackson DG, Hong YK, Detmar M. Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors. Am J Pathol (2005) 166: 913–921
50.Wicki A, Lehembre F, Wick N, Hantusch B, Kerjaschki D, Christofori G. Tumor invasion in the absence of epithelial–mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton. Cancer Cell (2006) 9: 261–272
51. Mishima K, Matsutani M. Podoplanin expression in primary central nervous system germ cell tumors: a useful histological marker for the diagnosis of germinoma . Acta Neuropathol (2006) 111: 563–568
52. Hanahan D. Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature (1985) 315: 115 –122
53. Vairaktaris E, Spyridonidou S, Papakosta V, Vylliotis A, Lazaris A , Perrea D, Yapijakis C, Patsouris E. The hamster model of sequential oral oncogenesis. Oral Oncol (2008) 44: 315– 324
54. Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfi eld MD. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol (2001) 17: 615-675