摘要
影响子宫内膜癌预后的因素之一是肿瘤细胞发生远处转移。淋巴转移是子宫内膜癌最主要的转移途径,淋巴管发生是肿瘤细胞经淋巴系统发生远处转移的关键问题之一。近年来淋巴管内皮细胞特异性标志物的出现使得对淋巴管的结构以及淋巴管新生的研究成为可能。叉头转录因子FOXC2是一种人胚胎蛋白,有证据表明它参与了胚胎心血管、淋巴管、骨骼等中胚层衍生组织的发生。胚胎和肿瘤组织在许多生物学行为上,特别是细胞远处迁移方面表现出极大的相似性,因此推测在肿瘤淋巴管发生过程和肿瘤细胞增殖过程也需要FOXC2参入。子宫内膜随着卵巢功能的周期性变化表现出脱落、再生的循环。子宫内膜再生过程也类似于胚胎发育过程。因此,本研究的目的在于进一步阐明FOXC2在子宫内膜癌的发生及淋巴转移中的作用,为子宫内膜癌淋巴结转移的预测以及肿瘤的抗淋巴管生成的治疗提供理论基础。
本研究调查分析了子宫内膜癌和宫颈癌淋巴结转移模式,发现子宫内膜癌淋巴结转移方式不同于宫颈癌,虽然内膜癌主要向盆腔淋巴结转移,但是仍然存在直接向腹主动脉旁淋巴结转移的病例,并且各病理分级的内膜癌均有淋巴结转移的可能。应用免疫组化的方法,以D2-40为特异性淋巴管内皮标志物,发现肿瘤病灶和病灶周边新生淋巴管密度与淋巴结转移有密切关联性;癌灶内部淋巴管是内膜癌经淋巴转移的主要途径之一。应用免疫组化和实时荧光定量PCR方法观察到FOXC2在正常和癌变子宫内膜中均有表达,FOXC2不仅与子宫内膜癌的发生有关,而且还参与了癌灶内和癌灶周边淋巴管的形成。通过构建pcDNA3.1-FOXC2重组真核表达载体,并转染人子宫内膜癌HEC-1B细胞系,使细胞FOXC2 mRNA表达水平增加,但未发现细胞迁移能力提高,推测FOXC2促进肿瘤细胞侵袭能力可能依赖于体内复杂的网络,而非单一作用的结果。
Endometrial cancer is one of the three most common malignant carcinoma in femal reproductive system. The attack rate of endometrial cancer has become higher in recent 20 years, according to literatuer showing us that the attack rate was 6.5 per 100,000 person, and there were about 20,000 patients all over the world. Up to now, we haven’t build any accurate statistics system to calculate the attack rate in China. But a retrospective study of endometrial cancer clinic cases from 1969~2003 revealed that the new attack rate of endometrial cancer was increasing at logarithmic speed every ten years. And after 21th century, average number of this cases was forty times than thirty years ago. From all these data, we could see that the attack rate of endometrial cancer was increasing at amazing speed.
Clinical and pathological observations suggest that for endometrial carcinoma, transport of tumor cells via lymphatics is a common pathway of initial dissemination, with patterns of spread via afferent lymphatics following routes of natural drainage. Lymphovascular space invasion (LVSI) was an independence risk factor which could predict bad prognosis. We all know that five years survival rate of patients without lymphatic metastasis was obviously higher than the patients presenting lymphatic metastasis. In the past time, we took it for granted that the malignant cells travelled from inherent lymphatic vessel and planted in local lymph node. But nowadays, we knew the truth that the form of new lymphatic vessel around carcinoma cell was the key step of tumor metastasis. The lymphangiogensis which was derivated and regulated by many growth factors and cytokine network directly participate in cancer metastasis.
The recent identification of lymphatic endothelial cell specific markers, such as vascular endothelial growth factor receptor-3 (VEGF-R3), lymphatic endothelial hyaluronan receptor-1 (LYVE-1) and podoplanin , has provided tools to investigate the discrepancies in endometrial lymphatic distribution. Now lymphatic vessel was found in normal endometrium and myometrium across the menstrual cycle with immunohistochemical stain. Lymphatic vessels of the functionalis were small and sparsely distributed whereas the basalis lymphatics were larger and sometimes closely associated with spiral arterioles and there was no difference in LVD during the whole cycle. The lymphangiogensis in tumor was necessary for malignant cell growth, invading and metastasis, and the LVD could affect the invasion of carcinoma cell and survival rate directly. So more and more doctors are studying if controlling the growth of lymphatic vessel is a kind of method to treatment cancer.
The FOXC2 gene is a member of the forkhead, or‘winged-helix’family of transcription factors, which now number over 30 in mammals. Three groups have previously reported mRNA expression studies of Foxc2 during mouse embryogenesis. The gene was shown to be expressed in the developing mesodermal mesenchyme of the head, kidney, and bones, and to play a role in somite formation. By 8.5 d.p.c., it was also expressed in the developing heart, blood vessels and limbs, and is essential for normal development of the aortic arch and axial skeleton. In adults, Foxc2 is expressed in adipose cells , and polymorphic variants have been associated with diabetes and insulin resistance in some studies.Mutations in the FOXC2 transcription factor cause a major form of hereditary lymphedema, the lymphedema–distichiasis syndrome. Foxc2 is expressed in developing lymphatic vessels and other tissues associated with lymphedema–distichiasis syndrome. Since the process of embryogenesis and malignant genesis had some same characters, we presumed that FOXC2 might take part in the genesis of endometrial carcinima. We would explore the expression mode of FOXC2 in normal endometrium during menstration cycle and endometrial cancer tissue, then find the relationship between FOXC2 mode and the density of lymphatic vessel. Our final purpose was to confirm if FOXC2 gene induce and regulate lymphangiogensis in endometrial cancer. Further more to provide theory for the research of control the growth of lymphatic vessel to cure cancer.
PartⅠ: The pattern of lymphatic spread in endometrial carcinomaai
Objective: The purpose is to determine the rate of lymph node metastases in women with endometrial carcinoma and to compare the pattern if lymphatic spread of endometrial carcinoma and cervical carcinoma. Methods: We retrospectively analyzed the lymphatic spread in 104 patients with endometrial carcinoma and 253 patients with cervical carcinoma. All patients underwent a complete pelvic and para-aortic lymphadenectomy from caudal to the median circumflex to the level of renal vessels. Results: The incidence of lymphatic metastases in endometrial carcinoma was higher than that in cervical carcinoma (22.1% vs 16.2 %). The pathologic grade (G1:12.1%;G2:21.4%; G3:34.5%)and depth of myometrial invasion(≤1/2:11.9%>1/2:29%)but not ages and pathologic types was correlated with nodal positivity. Para-aortic and pelvic lymph node involvement alone and both were observed in 4.3%, 34.8% and 60.9% for endometrial carcinoma and 0%, 68.3% and 31.7% for cervical carcinoma. Both of hem exhibited the highest obturator nodes positive rates (73.9% and 70.7%) with suprainguinal nodes positivity being lowest (8.7% and 7.3%). When compaired with cervical carcinoma, endometrial carcinoma showed higher para-aortic and sacral nodes positive rates (65.2%vs31.7% and 21.7%vs17.1%) and lower external iliac nodes positive rates (17.4%vs41.5%). Conclusion: Endometrial carcinoma has a distinct lymphatic spread pattern compared with cervical carcinoma and can directly metastasize to both pelvic lymph nodes and para-arotic lymph nodes with pelvic lymph nodes metastases being dominant. Positive lymph nodes are common in all grades.
PartⅡ: Lymphangiogensis of normal endometrium and endometrial adenocarcinoma
Objective: Information about lymphatics and lymphangiogenesis in the human endometrium is limited. We investigated the distribution of endometrial lymphatic vessels during the normal menstrual cycle and in association with endometrial adenocarcinoma. Methods: Full thickness uterine samples (n=15 proliferative; n=15 secretory) and endometrial adenocarcinoma samples (n=20 stage IA; n=20 stage IIIC) were collected for the study and analysed by immunohistochemistry using D2-40 as the specific marker of LECs. Results: Lymphatic vessels of normal endometrium were almost located in functionalis across the menstrual cycle with lymphatics of the basalis sometimes intimately associated with spiral arterioles. The LVD of the endometrium was significantly reduced when compared with the myometrium across the cycle (endometrium 4.99±0.84mm–2; secretory 10.70±1.16mm–2 P<0.001). Intra-tumoural LVD was significantly decreased in both stage endometrioid adenocarcinoma when compared with normal endometrium and myometrium (stage IA : 3.49±0.94mm2, stage IIIC: 2.43±1.08mm2, normal endometrium: 4.98±0.84 mm2, normal myometrium: 10.71±1.16mm2, P<0.001). Intra-tumoural LVD had a significantly decreased tendency from stage IA to stage IIIC. Peri-tumoural LVD for stage IA and stage IIIC tumours was significantly increased compared with normal endometrial LVD but decreased compared with normal myometrial LVD (stage IA : 7.09±1.22mm2, stage IIIC: 8.63±2.88mm2, normal endometrium: 4.98±0.84mm2 normal myometrium: 10.71±1.16mm2, P<0.005). There was significant difference in peri-tumoural LVD between stage IA and stage IIIC adenocarcinomas (P=0.012) and stage IIIC had increased peri-tumoural LVD. Conclusion: Endometrial lymphatics are located in normal endometrium and myometrium across the menstrual cycle and located in intra and peri of endometrioid adenocarcinoma, and increases in endometrial adenocarcinoma peri-tumoural lymphatics are associated with increases lymphatic metastasis.
PartⅢ: FOXC2 expressed in normal endometrium and endometrioid adenocarcinoma and assosiatied with lymphangiogensis in endometrial adenocarcinoma
Objective: to investigated the expression of FOXC2 in normal endometrium during the normal menstrual cycle and in association with endometrial adenocarcinoma. Methods: the same samples with partⅡwere analysed by immunohistochemistry using FOXC2 mouse mAb, and FOXC2 mRNA level was detected by real time PCR. The intensity and distribution patterns of the specific immunohistochemical staining was evaluated using a semi- quantitative method (IRS score). The IRS for epithelium and stroma were recorded respectively. Results: Out of 15 proliferative endometrium, 11 showed positive immunoactivity in epithelium (73.3%), as for secretory endometrium, the positive rate was 80% (12 out of 15). When compared the IRS of epithelium, there was no significant difference between proliferative and secretory endometium (proliferative IRS=1.53±1.30, secretory IRS=2.1±1.68, p=0.284). Normal endometrium showed positive immunoactivity in stromal cells. The secretory endometrium showed significantly increased FOXC2 expression compared with proliferative endometrium (proliferative IRS=1.93±1.03, secretory IRS=3.93±1.53, p < 0.01). Endometrioid adenocarcinoma showed significantly increased FOXC2 expression compared with normal endometrial epithelium both in epithelium and stroma (in epithelium: normal endometrium IRS=1.83±1.51, endometrioid adenocarcinoma IRS=5.18±2.82, p<0.01, in stroma: normal endometrium IRS=2.93±1.64, endometrioid adenocarcinoma IRS=6.78±2.15, p<0.01). The FOXC2 expression in stroma significantly increased when pelvic and/or para-aotic lymph node was involved in (stage IA IRS=5.70±1.66, stage IIIC IRS=7.85±2.08, P=0.01). FOXC2 was immunolocalized in LECs only in endometrioid adenocarcinoma not in normal endometrium. The RT-PCR showed the same results with immunohistochemistry. Conclusion: FOXC2 might associate with the genesis of endometrial carcinoam and the lymphangiogensis in endometrial adenocarcinoma intra and peri-tumoural lymphatics.
PartⅣ: Construction of eukaryotic expression plasmid pcDNA3.1(-)-FOXC2
Objective: Construct the expression plasmid recombinate with FOXC2 gene for further study of gene function. Methods: FOXC2 gene fragment was obtained with PCR from human genome followed by putting FOXC2 gene fragment into eukaryotic expression vector pcDNA3.1 (-) with double restriction enzyme. We confirmed the plasmid by DNA sequencing. Result: The 1524bp FOXC2 DNA fragment from human genome DNA was obtained and then the recombined eukaryotic expression vector pcDNA3.1(-)-FOXC2 was built succsessfully. It had the same sequence with Genebank by DNA sequencing. Conclusion: we success build the pcDNA3.1 (-)-FOXC2 eukaryotic expression vector for futher study the function of FOXC2 gene.
PartⅤ: Effects of FOXC2 on HEC-1B migration
Objective: In order to invest the effects of FOXC2 on human endometrial carcinoma cell line HEC-1, we constructed an FOXC2-overpressing cell modle. Methods: pcDNA3.1(-) -FOXC2 eukaryotic expression vector was transfected into HEC-1B with LipofectamineTM 2000 and dthe positive cell clone was selected by G418. FOXC2 mRNA level among untransfected cell, transfected with empty plasmid and with recombined plasmid was compared using RT-PCR. The growth curve was used to compare cell proliferation rate and transwell cabin was used to compare the invasion ability among three kinds of cells.Results: on the 6th day after transfetion, control cells were totally dead because of 500μg/ml G418, and 250μg/ml G418 was used to cultivate transfeced cells continuously. Cells became the logarithmic growth phase on 3rd days followed by the proliferation rate decreasing lightly on 5th day and the cell growth was inhibited after 6th day. FOXC2 mRNA level was higher in recombined plasmid transfected cells than in the other two kinds of cells (P < 0.01), and there was no different between the control cells and the empty plasmid transfected cells. Cells were put on the upper tier of transwell basement. After 24h, cells could penetrate matrigel. There was no defference among three kinds of cells when the penetrating cells were counted with viola crystalline (F=0.674, p=0.518). Conclusion: The pcDNA3.1-FOXC2 eukaryotic expression vector could express in HEC-1B cell line, but the invasion ability had not increased after transfeced with pcDNA3.1-FOXC2 eukaryotic expression vector. We presumed that the invasion ability of tumor cells might depend on the complicated network in vivo rather than depend on some single factor.
引文
[1]曹泽毅.中华妇产科学.第2版.北京.人民卫生出版社, 2004:2120-2128.
[2] Parkin DM, Bray F, Ferlay J,Pisani P. Global Cancer Statistics, 2002.CA Cancer J Clin,2005,55(2):74-108.
[3] Rahaman J, Steiner N, Hayes MP, Chuang L, Fishman D, Gretz Iii H. Chemotherapy for Gynecologic Cancers. Mt Sinai J Med,76(6):577-588.
[4] Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ.Cancer statistics, 2008.CA Cancer J Clin. 2008,58(2):71-96.
[5] Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57:43-66.
[6] Jung KW, Won YJ, Park S, Kong HJ, Sung J, Shin HR, Park EC, Lee JS. Cancer statistics in Korea: incidence, mortality and survival in 2005.J Korean Med Sci. 2009 ,24(6):995-1003.
[7]杨丹,韩立敏. 1969-2003年子宫内膜癌发病率及发病因素分析.复旦学报(医学版). 2005.32(4)479-483.
[8]张惜阴.临床妇科肿瘤学.第2版.上海.复旦大学出版社, 2002: 160
[9]高永良,俞华.子宫内膜癌的放射治疗.实用妇产科杂志[J]. 2008, 24(5):266-268
[10]戴钟英.子宫内膜癌的激素治疗.实用妇产科杂志[J]. 2008, 24 (5):268-271
[11]白萍,程敏,李淑敏,章文华,马莹.子宫内膜癌淋巴取样术的临床意义.中国肿瘤临床, 2007,34(12):686-689
[12] Ben-Shachar I, Pavelka J, Cohn DE, et al. Surgical staging for patients presenting with grade 1 endometrial carcinoma [J]. Obstet Gynecol, 2005,105(3):487-493
[13] Watari H, Todo Y, Takeda M, et al. Lymph-vascular space invasion and number of positive para-aortic node groups predict survival in node-positive patients with endometrial cancer[J]. Gynecol Oncol,2005,96(3):651-657.
[14] M.Hirai, M.Hirono, T.Oosaki, et al.Prognostic factors relating to survival in uterine endometrioid carcinoma.Int J Gyn&Obs, 1999;66:155-162.
[15] Creasman WT, Odicino F, Maisonneuve P, et al. Carcinoma of the corpus uteri.FIGO 6th Annual Report on the Results of Treatment in Gynecological Cancer[J]. Int J Gynaecol Obstet, 2006,95(Suppl 1): S105-143.
[16] Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249-257.
[17] Saharinen P, Tammela T, Karkkainen MJ, Alitalo K. Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation. Trends Immunol 2004;25:387-395.
[18] Karpanen T, Egeblad M, Karkkainen MJ, Kubo H, Yla-Herttuala S, Jaattela M, et al. Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res 2001;61:1786-1790.
[19] He, Y., Rajantie, I., Pajusola, K., Jeltsch, M., Holopainen, T., Yla-Herttuala, S., et al. Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels. Cancer Research, 2005,65(8): 4739-4746.
[20] Ji, R. C., Qu, P., & Kato, S. Application of a new 5′-Nase monoclonal antibody specific for lymphatic endothelial cells.Laboratory Investigation, 2003, 83(11): 1681-1683.
[21] Ji, R. C., Miura, M., Qu, P., & Kato, S. Expression of VEGFR-3 and 5′-Nase in regenerating lymphatic vessels of the cutaneous wound healing. Microscopy Research and Technique. 2004, 64(2), 279–286.
[22] Azzali, G. On the transendothelial passage of tumor cell from extravasal matrix into the lumen of absorbing lymphatic vessel. Microvascular Research, 2006, 72(4): 74–85.
[23] Harrell MI, Iritani BM, Ruddell A. Tumor-induced sentinel lymph node lymphangiogenesis and increased lymph flow precede melanoma metastasis. Am J Pathol 2007;170:774e786.
[24] Oliver G, Alitalo K. The lymphatic vasculature: recent progress and paradigms. Annu Rev Cell Dev Biol. 2005;21(11):457-483.
[25] YokoyamaY, Sato S, FutagamiM, et al. prognostic significance of Vascular endothelial growth factor and its receptor in endometrial carcinoma. Gynecol Oncol 2000,77(3):413-418
[26] Gombos Z, Xu X, Chu CS, Zhang PJ, Acs G. Peritumoral lymphatic vessel density and vascular endothelial growth factor C expression in early-stage squamous cell carcinoma of the uterine cervix. Clin Cancer Res 2005;11(23):8364-8371.
[27] Karkkainen MJ, Haiko P, Sainio K, Partanen J, Taipale J, Petrova TV, et al. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat Immunol 2004;5(1):74-80.
[28] Laakkonen P, Waltari M, Holopainen T, Takahashi T, Pytowski B, Steiner P, et al. Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth. Cancer Res 2007;67(2):593-599.
[29] Ka¨rpa¨nen T, Heckman CA, Keskitalo S, Jeltsch M, Ollila H, Neufeld G, et al. Functional interaction of VEGF-C and VEGF-D with neuropilin receptors. FASEB J 2006;20(9):1462-1472.
[30] Yuan L, Moyon D, Pardanaud L, Bre′ant C, Karkkainen MJ, Alitalo K, et al. Abnormal lymphatic vessel development in neuropilin 2 mutant mice. Development 2002;129(20):4797-4806.
[31] Vlahakis NE, Young BA, Atakilit A, Hawkridge AE, Issaka RB,Boudreau N, et al. Integrinα9β1 directly binds to vascular endothelial growth factor (VEGF)-A and contributes to VEGF-A-induced angiogenesis. J Biol Chem 2007;282(20):15187-15196.
[32] Gale NW, Thurston G, Hackett SF, Renard R, Wang Q, McClain J, et al. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev Cell 2002;3(3):411-423.
[33] Tammela T, Saaristo A, Lohela M, Morisada T, Tornberg J, Norrme′n C, et al.Angiopoietin-1 promotes lymphatic sprouting and hyperplasia. Blood 2005; 105(12):4642-4648.
[34] Ma¨kinen T, Adams RH, Bailey J, Lu Q, Ziemiecki A, Alitalo K, et al. PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature. Genes Dev 2005;19(3):397-410.
[35] Foo SS, Turner CJ, Adams S, Compagni A, Aubyn D, Kogata N, et al. Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly. Cell 2006; 124(1):161-173.
[36] Huber, M. A., Kraut, N., & Beug, H.. Molecular requirements for epithelial– mesenchymal transition during tumor progression. Current Opinion in Cell Biology, 2005, 17: 1–11.
[37] Tsukamoto H, Shibata K, Kajiyama H, Terauchi M, Nawa A, Kikkawa F. Irradiation-induced epithelial–mesenchymal transition (EMT) related to invasive potential in endometrial carcinoma cells. Gynecologic Oncology 107 (2007) 500–504
[38] Thiery, Jean Paul. Epithelial-mesenchymal transitions in tumour progression [J]. Curr Opin Cell Biol, 2003,15(6):740-746.
[39] Boyer Brigitte, Vall s Ana Maria, Edme Natacha. Induction and regulation of epithelial mesenchymal transitions [J]. Biochemical Pharmacology, 2000, 60(8):1091-1099.
[40] Thiery, J. P., & Sleeman, J. P. (2006). Complex networks orchestrate epithelial–mesenchymal transitions. Nature Reviews Molecular Cell Biology, 7, 131–142.
[41] Wang W, Goswami S, Sahai E, Wyckoff JB, Segall JE, Condeelis JS. Tumor cells caught in the act of invading: their strategy for enhanced cell motility.Trends Cell Biol 2005; 15: 138–45.
[42] Miura N, Iida K, Kakinuma H, et al. Isolation of the mouse (MFH-1) and human (FKHL-14) mesenchyme fork head–1 gene reveals conservation of their gene and protein structures [J]. Genomics, 1997, 41(3):489-492.
[43] Kaestner KH, Bleckmann SC, Monaghan AP, et al. Clustered arrangement ofwinged helix genes fkh-6and MFH-1:possible implications for mesoderm development [J]. Development, 1996, 122(6):1751-1758.
[44] Kriederman BM, Myloyde TL, Witte MH, Dagenais SL, Witte CL, Rennels M, Bernas MJ, Lynch MT, Erickson RP, Caulder MS, Miura N, Jackson D,Brooks BP, Glover TW. FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome. Hum Mol Genet, 2003,12(10):1179-1185.
[45] Petrova TV, Karpanen T, Norrme′n C, Mellor R, Tamakoshi T, Finegold D, et al. Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat Med 2004;10(9):974-981.
[46] Fang J, Dagenais SL, Erickson RP, et al. Mutations in FOXC2(MFH21),a forkhead fam ily transcrip tion factor, are responsible forthe hereditary lymphedema-distichiasis syndrome [J]. An J Hum Genet, 2000,67(6):1382-1388.
[47] Erickson R P, Dagenais S L, CaulderM S, et al. Clinical heterogeneity in lymphoedema-distichiasis with FOXC2 truncating mutation [J]. J Med Genet, 2001, 38(11):761-766.
[48] Bell R, Brice G, Child A H, et al. Analysis of lymphoedema-distichiasis families for FOXC2 mutations reveals small insertions and deletions throughtout the gene[J]. Hum Genet, 2001,108(6):546-551.
[49] Berry FB, Tamimi Y, Carle MV, et al. The establishment of a predictive mutational model of the forkhead domain through the analyses of FOXC2 missense mutations identified in patients with hereditary lymphedema with distichiasis[J]. Hum Mol Genet, 2005,14(18):2619-2627.
[50] Pajukanta P, Allayee H, Krass KL, et al. Combined analysis of genome scans of Dutch and Finnish families reveals a susceptibility locus for high-density lipoprotein cholesterol on chromosome 16q. Am J Hum Genet, 2003,72:903-917.
[51] Yang X, Enerback S, Smith U. Reduced expression of FOXC2 and brown adipogenic genes in human subjects with insulin resistance. Obes Res, 2003,11:1182-1191.
[52] Ridderstrale M, Carlssom E, Klannemark M, et al. FOXC2 mRNA expression and a 5'untranslated region polymorphism of the gene are associated with insulin resistance. Diabetes, 2002, 51:3554-3560.
[53] Kovacs P, Lehn-Stefan A, Stumvoll M, et al. Genetic variation in the human winged helix/forkhead transcription factor gene FOXC2 in Pima Indians. Diabetes, 2003,52:1292-1295.
[54] Yanagisawa K, Hingstrup Larsen L, Andersen C, et al. The FOXC2 -512C>T variant is associated with hypertriglyceridaemia and increased serum C-peptide in Danish Caucasian glucose-tolerant subjects.Diabetologia, 2003, 46 :1576-1580.
[55] Shields JW. High points in the history of lymphology 1602-2001. Lymphology, 2001,34(2):51-68.
[56] Mani SA, Yang J, Brooks M, Schwaninger G, Zhou A, Miura N, Kutok JL, Hartwell K, Richardson AL, Weinberg RA. Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci USA, 2007,104(24):10069-10074.
[57] Achen MG, Stacker SA.Molecular Control of Lymphatic Metastasis.Ann. N.Y. Acad. Sci, 2008,1131: 225–234.
[58] Van Trappen PO, Pepper MS. Lymphangiogenesis in human gynaecological cancers. Angiogenesis, 2005,8(2):137-145.
[59] Clarijs R, Ruiter DJ, de Waal RM. Lymphangiogenesis in malignant tumors: does it occur? J Pathol, 2001,193(2):143-146.
[60] van Netten JP, Cann SA, Maxwell CA, Finegan RP. Lymphagenesis and cancer metastasis. Br J Cancer, 1998,78(2):277-278.
[61] Pepper MS. Lymphangiogenesis and tumor metastasis: myth or reality? Clin Cancer Res, 2001,7(3):462-468.
[62]唐磊,张家文,杜昂鹰.微淋巴管计数在子宫内膜癌中的意义.中国妇产科临床杂志, 2007,8 (5)346-348,342.
[63] Da MX, Wu XT, Wang J, Guo TK, Zhao ZG, Luo T, Zhang MM, Qian K. Expression of Cyclooxygenase-2 and Vascular Endothelial Growth Factor-C Correlates with Lymphangiogenesis and Lymphatic Invasion in Human Gastric Cancer. Arch Med Res, 2008,39(1):92-99.
[64] Bard JB, Lam MS, Aitken S. Bioinformatics approach for identifying candidate transcriptional regulators of mesenchyme-to-epithelium transitions in mouse embryos. Dev Dyn, 2008,237(10):2748-2754.
[65] W. Creasman, C. Morrow, B. Bundy, H. Homesley, J. Graham and P. Heller, Surgical pathologic spread patterns of endometrial cancer: a Gynecologic Oncology Group study, Cancer 60 (1987), pp. 2035–2041.
[66] Orr JW Jr, Roland PY, Leichter D, Orr PF. Endometrial cancer: is surgical staging necessary? [J]Curr Opm Oncol 2001;13(5):408-12.
[67] ACOG Committee on Practice Bulletins. Management of endometrial cancer. 2005; 65:1–9.
[68] Chan JK, Urban R, Cheung MK, et al. Lymphadenectomy in endometrioid uterine cancer staging. How many lymph nodes are enough? A study of 11,443 Patients[J]. cancer, 2007, 109: 2454-2460.
[69] Benedelti-Panici P, Maneschi F, Cutillo G, et al. Anatomical and pathological study of retroperitoneal nodes in endometrial cancer[J]. Int J Gynecol Cancer, 1998, 8:322-327.
[70] Chan JK, Wu H, Cheung MK, Shin JY, OsannK, Kapp DS. The out comes of 27,063 women with unstaged endometriod cancer[J]. Gynecol Onco, 2007, 106:282-288.
[71] Kinkel K, KajiY, Yu KK, et al. Radiologic staging in patients with endometrial cancer: a meta-analysis[J]. Radiology, 1999, 212:711-718.
[72] A. Mariani, S. Dowdy, W. Cliby, B. Gostout, M. Jones and T. Wilson et al., Prospective assessment if lymphatic dissemination in endometrial cancer: a paradigm shift in surgical staging, Gynecol. Oncol. 109 (2008), pp. 11–18. However, a reliable intraoperativeevaluation is not available at all centers.
[73] Matsumoto K, Yoshikawa H, Yasugi T, Onda T, Nakagawa S, Yamada M, et al. Distinct lymphatic spread of endometrial carcinoma in comparison with cervical and ovarian carcinomas. [J] Cancer Letters 2002;180(1):83-9.
[74] Look KY. Role of lymphadenectomy in management of adenocarcinoma of the endometrium. [J] Eur J Gynaecol Oncol 2004;25(5):545-51.
[75] Kilgore LC, Partridge EE, Alvarez RD, Austin JM, Shingleton HM, Naajin III F, et al. Adenocarcinoma of the endometrium: survival comparisons of patients with and without pelvic lymph node sampling. [J] Gynecol Oncol 1995;56(1):29-33.
[76] Roland PY, Kelly FJ, Kulwicki CY, Blitzer P, Curcio M, Orr JW Jr. The benefits of a gynecologic oncologist: a pattern of care study for endometrial cancer treatment. [J]Gynecol Oncol 2004, 93(1) ; 125–130.
[77] Marziale P, Atlante G, Pozzi M, Diotallevi F, Iacovelli A. 426 cases of stage I endometrial carcinoma: a clinicopathological analysis. [J] Gynecol Oncol ,1989, 32(3);278–281.
[78] Geisle JP, Linnemeier GC, Manahan KJ. Pelvic and para-aortic lymphadenectomy in patients with endometrioid adenocarcinoma of the endometrium. [J] International Journal of Gynecology & Obstetrics, 2007;98(1): 39-43.
[79] McMeekin DS, Lashbrook D, Gold M, Scribner DR, Kamelle S, Tillmanns TD, Mannel R. Nodal distribution and its significance in FIGO stage IIIc endometrial cancer. [J] Gynecol Oncol ,2001, 82 (2);375–379.
[80] Donoghue JF, Lederman FL, Susil BJ, Rogers PA. Lymphangiogenesis of normal endometrium and endometrial adenocarcinoma. Hum Reprod 2007;22(6):1705-1713.
[81] Red-Horse K, Rivera J, Schanz A, Zhou Y, Winn V, Kapidzic M, et al. Cytotrophoblast induction of arterial apoptosis and lymphangiogenesis in an in vivo model of human placentation. J Clin Invest 2006;116(10):2643-2652.
[82] Koukourakis M, Giatromanolaki A, Sivridis E, Simopoulos C, Gatter K, Harris A,et al. LYVE-1 immunohistochemical assessment of lymphangiogenesis in endometrial and lung cancer. J Clin Pathol 2005;58(2):202-206.
[83] Jaeger TM, Weidner N, Chew K, Moore DH, Kerschmann RL, Waldman FM, Carroll PR. Tumor angiogenesis correlates with lymph node metastases in invasive bladder cancer. J Urol. 1995,154(1):69-71.
[84] Beasley NJ, Prevo R, Banerji S, Leek RD, Moore J, van Trappen P, Cox G, Harris AL, Jackson DG. Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer. Cancer Res, 2002,62(5):1315-1320.
[85] Yang S, Cheng H, Cai J, Cai L, Zhang J, Wang Z. PlGF expression in pre-invasive and invasive lesions of uterine cervix is associated with angiogenesis and lymphangiogenesis. APMIS 2009; 117(11): 831–838.
[86] Leu AJ, Berk DA, Lymboussaki A, Alitalo K, Jain RK.Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation. Cancer Res. 2000 ,60(16):4324-4327.
[87] Padera TP, Kadambi A, di Tomaso E, Carreira CM, Brown EB, Boucher Y, Choi NC, Mathisen D, Wain J, Mark EJ, Munn LL, Jain RK.Lymphatic metastasis in the absence of functional intratumor lymphatics.Science. 2002,296(5574):1883-1886.
[88] Cao Y. Opinion: emerging mechanisms of tumor lymphangiogenesis and lymphatic metastasis. Nat Rev Cancer 2005;5(9):735–743.
[89] Stacker SA, Achen MG. From anti-angiogenesis to anti-lymphangiogenesis: emerging trends in cancer therapy. Lymphat Res Biol. 2008;6(3-4):165-172.
[90] Holmqvist A, Gao J, Adell G, Carstensen J, Sun X F. The location of lymphangiogenesis is an independent prognostic factor in rectal cancers with or without preoperative radiotherapy. Ann Oncol, 2009, Nov 4. [Epub ahead of print].
[91] Mints M, Blomgren B, Falconer C, Palmblad J. Expression of the vascular endothelial growth factor (VEGF) family in human endometrial blood vessels. Scand J Clin Lab Invest 2002;62(3):167-176.
[92] Yokoyama Y, Charnock-Jones DS, Licence D, Yanaihara A, Hastings J, Holland C, et al. Vascular endothelial growth factor-D is an independent prognostic factor in epithelial ovarian carcinoma. Br J Cancer 2003;88(2):237-244.
[93] Yang S, Cheng H, Cai J, Cai L, Zhang J, Wang Z. PlGF expression in pre-invasive and invasive lesions of uterine cervix is associated with angiogenesis and lymphangiogenesis. APMIS 2009; 117(11): 831–838.
[94] Mohammed RAA, Green A, El-Shikh S, Paish EC, Ellis IO, Martin SG. Prognostic significance of vascular endothelial cell growth factors -A, -C and -D in breast cancer and their relationship with angio- and lymphangiogenesis. Br J Cancer. 2007; 96(7): 1092–1100.
[95] Cueni LN, Detmar M. The Lymphatic System in Health and Disease. Lymphat Res Biol. 2008;6(3-4):109-122.
[96] Tian LN,Deng WG,Zou JY,Ma L,Ding YL,Song WL,Fu Y. Expression of FOXC2 during lymphangiogenesis in human embryo. Acta Anat Sinica,2008,39(6):886-889.
[97] G?tte M, Wolf M, Staebler A, Buchweitz O, Kelsch R, Schüring AN, Kiesel L.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma. J Pathol. 2008,215(3):317-329.
[98] Kato K, Takao T, Kuboyama A, Tanaka Y, Ohgami T, Yamaguchi S, Adachi S, Yoneda T, Ueoka Y, Kato K, Hayashi S. Endometrial Cancer Side-Population Cells Show Prominent Migration and Have a Potential to Differentiate into the Mesenchymal Cell Lineage. Am J Pathol, 2010,176(1):381-392.
[99] Nina Iversen, Baard Birkenes, Kari Torsdalen et al. Electroporation by nucleofector is the best nonviral transfection technique in human endothelial and smooth muscle cells. Genetic Vaccines and Therapy 2005;3:2
[100]鄂征主编.组织培养和分子细胞技术.北京:北京出版社, 2001.236.237.
[101] Yori JL, Johnson E, Zhou G, Jain MK, Keri RA.Kruppel-like factor 4 (KLF4) inhibits epithelial-to-mesenchymal transition through regulation of E-cadherin gene expression. J Biol Chem. 2010 Mar 31. [Epub ahead of print]
[102] Baum B, Settleman J, Quinlan MP.Transitions between epithelial andmesenchymal states in development and disease.Semin Cell Dev Biol, 2008,19(3):294-308.
[103] Ouyang G, Wang Z, Fang X, Liu J, Yang CJ. Molecular signaling of the epithelial to mesenchymal transition in generating and maintaining cancer stem cells. Cell Mol Life Sci. 2010 Mar 18. [Epub ahead of print]
[104] Creighton CJ, Chang JC, Rosen JM. Epithelial-Mesenchymal Transition (EMT) in Tumor-Initiating Cells and Its Clinical Implications in Breast Cancer. J Mammary Gland Biol Neoplasia ,2010 Mar 31. [Epub ahead of print]
[105] SarrióD, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, Palacios J.Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. Cancer Res, 2008,68(4):989-997.
[106] He H, Davidson AJ, Wu D, Marshall FF, Chung LW, Zhau HE, He D, Wang R.Phorbol ester phorbol-12-myristate-13-acetate induces epithelial to mesenchymal transition in human prostate cancer ARCaP(E) cells. Prostate,2010 Mar 23. [Epub ahead of print]
