摘要
妊娠滋养细胞疾病(GTD)包括葡萄胎、侵蚀性葡萄胎、绒毛膜癌等一系列与妊娠相关的疾病谱，目前为止确切的发病机制仍不清。从正常妊娠到葡萄胎，葡萄胎再恶变为侵蚀性葡萄胎，然后继续发展为绒癌的整个过程类似于一个上皮性肿瘤的癌变过程。具有大多数肿瘤无限增殖和转化及多阶段性发展的特征，并不是某一个或某几个基因异常所引起。
基因芯片技术是伴随着人类基因组计划实施而出现的新的基因分析技术。具有大规模和高通量的特点，是当前功能基因组学重要研究手段，它明显优于过去的单一基因研究模式，可以在整体的、基因组的水平对基因表达调控的网络机制，进行系统、全面地分析。
反义核酸技术是一种以应用反义核酸类药物来抑制特定基因表达为目的的基因治疗技术。反义核酸可以作为研究工具，用于对一些特定蛋白和基因的生理功能的研究。为我们特异性降调细胞内基因表达提供一重要方法。
为确定在GTD发病中的重要相关基因，我们首先利用DNA芯片技术，初步确定在葡萄胎和绒毛癌细胞中不同表达的基因；然后，利用传统的RT-PCR、免疫印迹、免疫组化方法，对基因芯片检测结果正确性和可靠性进行分析。最后，利用反义核苷酸技术，对肿瘤生物学行为有重要影响的基因进行绒癌细胞体外反义寡核苷酸干预实验，快速确定新的治疗靶向。
第一部分 葡萄胎和绒毛膜癌发病相关基因筛选
方法：用含4096条基因的表达谱芯片(cDNA基因芯片)，对2例完全性葡萄胎和2例正常绒毛及原代培养的早孕绒毛滋养细胞和绒癌JAR细胞株滋养细胞进行基因差异表达谱分析。
结果：葡萄胎2对临床标本中均有显著差异表达基因89条，占所检基因总数的2.2％。与正常绒毛相比，葡萄胎中24条基因表达显著增高(上调)，65条基因显著降低(下调)。JAR细胞与正常原代培养滋养细胞相比，432条基因下调，380条基因上调。葡萄胎和绒癌细胞中共同下调基因有46条，共同上调的有13条。抑制细胞生长的基因在葡萄胎和绒癌中下调，而与细胞增生、恶性转化、转移等
浙江大学博士生毕业论文
有关的基因上调。
第二部分芯片结果验证及滋养细胞增生机制探讨
方法:利用传统的分子生物学研究手段免疫组织化学、免疫印迹及RT-PCR，对
基因芯片证实葡萄胎和绒毛膜癌细胞中高表达的核昔酸还原酶小亚单位
(RRMZ)、胸腺嚓陡核昔激酶一(TKI)、复制因子C小亚单位2(孙CZ)、CyelihBI
基因，在巧例正常绒毛、20例新鲜葡萄胎组织、98例存档石蜡包埋滋养细胞疾
病组织及JAR、JEG一3绒癌细胞中的表达情况进行较为详尽的检测。PCNA在98
例存档石蜡包埋滋养细胞疾病组织的表达情况进行分析。
结果:JAR、JEG一3细胞、葡萄胎组织RRMZ、TKlm丑NA和蛋白水平显著高于
正常原代滋养细胞或正常绒毛组织，RFCZ在JAR、JEG一3细胞、葡萄胎中蛋白
的表达显著高于正常绒毛和正常原代滋养细胞。Cyclin BI蛋白在JAR、JEG一3
细胞、葡萄胎组织表达增加。
五项指标在石蜡包埋GTD组织中的表达显著高于正常绒毛，妊娠滋养细胞
肿瘤未化疗者TKI、Cyclin Bl、PCNA表达显著高于葡萄胎，葡萄胎恶变者
RRMZ、TKI、PCNA、cyclinBI表达显著高于自然恢复者，化疗影响TKI、RFCZ、
Cyclin Bl表达，期别增加R侧MZ、TKI、RFCZ表达增强，wHO预后评分越高
RRMZ、TKI、盯CZ、Cyelin BI表达越强，pCNA与Cyelin BI、TKI的表达呈
显著正相关。
第三部分RRMZ反义寡核昔酸对人绒毛膜癌细胞株体外生长影响
t了
方法:以脂质体(oligofectamjne)为载体，将人工合成的分别位于RR五江ZmRNA
核昔酸序列626一645(编码区)和1572一1591(3’端非编码区)位点且全程硫代修饰的
两条反义寡核营酸(antisense oligodeoxynucleotide，ASODN)片断导入绒毛膜癌
JAR细胞中，用MTT法检测细胞成活率，免疫印迹和R下PCR方法检测ASODN
对R侧MZ蛋白和mRNA表达的影响。
结果:RRMZ编码区的ASODN(ASODNI)以量效和时效形式显著抑制JAR细胞
增殖，以时效形式降调RRMZ的表达。3’端非编码区ASODN(ASODN2)对细胞
增殖及RRMZ表达无明显的影响，但与ASODNI联合应用，对细胞的生长抑制
作用和对RRMZ表达的影响显著加强。ASODNI作用12h，RRMZ表达水平开始
下降;作用24h，细胞RRMZ表达和生长显著抑制，48h细胞生长和RR办滩2表达
达最低水平，72h均又有所恢复。
结论
胰酶消化法是获得正常高纯度早孕绒毛滋养细胞的快速有效方法。
表达谱基因芯片是一种快速、高效筛选滋养细胞疾病发病相关基因的方法。
R侧MZ、TKI、盯cZ、PCNA、cycljllBI在GTD中存在过度表达。
DNA合成酶TKI、RRMZ异常过度表达可能是导致GTD滋养细胞异常增生
的因素。
细胞周期蛋白Bl及复制因子C的高表达，说明GTD存在细胞周期检查点
2内j
浙江大学博士生毕业论文
制。
RR]MZ反义寡核昔酸通过特异性降调RRMZ蛋白和mRNA的表达，抑制绒
毛膜癌JAR细胞的生长。联合应用针对RRMZ基因不同位点的ASODN是
提高反义药物有效性的重要方法之一。
联合应用基因芯片技术和反义寡核昔酸技术能够有效的筛选滋养细胞疾病
发病相关基因，并快速确定治疗靶向
Gestational trophoblastic diseases (GTD) include the abnormal pregnancy, hydatidiform mole (HM), invasive mole, the overtly malignant tumors, choriocarcinoma and placental site trophoblastic tumor. The molecular pathogenesis of GTD remained largely unknown. Normal pregnancy can develop to hydatiditform mole, which can be transformed further to the malignant invasive mole. Choriocarcinoma is consequently evolved from invasive mole. This whole course is highly similar to the cancinogenesis of epithelial tumor. Therefore the developing course of GTD is of the characters of unlimited proliferation, transformation, and multi-step evolvement like most other tumors. It is not one gene or several genes that abnormally express and cause this transformation course.
The technology of gene chip is a new technique of gene analysis, going with practicing human genome project. Due to its low cost, high sensitivity and high flux, gene chip is one of the important tools for the study of functional genome, which is obviously better than the previous research model of single gene. The network mechanisms of gene expression regulation are thoroughly studied in the level of whole genome using gene chip.
The technique of antisense oligodeoxynucleotides (ASODNs) is one of gene therapies through specifically decreasing the gene expression. ASODNs are used to study the physiological function of the specifically protein and gene as a tool. ASODNs also provide us an important method to down-regulate the gene expression.
To identify relevant genes important in the pathogenesis of GTDs, we firstly use microarray (one of gene chips) to identify genes differentially expressed in HM versus normal villi and choriocarcinoma cells versus normal primary culture trophoblasts. Then for validation of microarray results, the traditional methods of RT-PCR, immunoblot, and immunohistochemistry are used. Finally, for the identification of novel therapeutic targets, we investigate whether ASOND-mediated reduction in the
mRNA level of genes which have the important effect on tumor biological behaviors may resulted the decreased proliferation of choriocarcinoma cells in vitro.
Part one Screening of associated genes of hydatidform mole and choriocarcinoma cells
Methods: The differential expressions of 4096 genes were analyzed in two pairs of the tissues of hydatidiform mole versus normal villi, and in two pairs of normal primary culture trophoblasts versus JAR cell line of chariocarcinoma, using cDNA microarray.
Results: 89 genes were found differently expressed in all hydatidiform moles, counting for 2.2% of total genes. Among them, 24 genes were up-regulated and 65 genes were down-regulated. Compared with normal primary trophoblasts, 433 genes were up-regulated and 380 genes down-regulated in JAR cells. 46 genes were up-regulated in both hydatidiform mole(HM) and choriocarcinoma, while 13 genes were down-regulated. Genes associated with the inhibition of cell proliferative were significantly down-regulated, whereas genes associated with cell proliferation, malignant transformation and tumor metastasis and drug resistance were up-regulated.
Part two Validation of microarray results and mechanism investigation of trophoblastic hyperplasia
Methods: The expressions of the up-regulated genes in microarray analysis, including small subunit ribonucleotide reductase (RRM2), thymidine kinasel (TK1), subunit 2 of replication factor C (RFC2), CyclinBl were examined by immunohistochemistry, immunoblot and RT-PCR in 15 cases of normal villi, 20 cases of fresh tissuses of hydatidiform mole, 98 cases of archived tissuses of GTDs, JAR and JEG-3 cell lines of Choriocarcinoma. Proliferating cell nuclear antigen (PCNA) was detected with immunohistochemistry in 98 cases of archived tissuses of GTDs.
Results: Compared with normal villi and primary culture trophoblasts, the levels of mRNA and proteins of TK1 and RRM2 were significantly increased in hydatidiform moles, JAR and JEG-3 cells. The expression of RFC2 protein was also significantly higher. The expre
引文
1. Yang X, Zhang Z, Jia C, et al. The relationship between expression of c-ras, c-erbB-2, nm23,and p53 gene products and development of trophoblastic tumor and their predictive significance for the malignant transformation of complete hydatidiform mole[J]. Gynecol Oncol, 2002, 85(3): 438-844.
2. Li HW, Tsao SW, Cheung AN. Current understandings of the molecular genetics of gestational trophoblastic diseases[J]. Placenta, 2002, 23(1): 20-31.
3. Tuncer ZS, Vegh GL, Fulop V, et al. Expression of epidermal growth factor receptor-related family products in gestational trophoblastic diseases and normal placenta and its relationship with development ofpostmolar tumor[J]. Gynecol Oncol, 2000, 77(3): 389-393.
4. Fulop V, Mok SC, Genest DR, et al. p53, p21, Rb and mdm2 oncoproteins expression in normal placenta, partial and complete mole, and choriocarcinoma[J]. J Reprod Med, 1998, 43(2): 119-127.
5. Saxena A, Frank D, Panichkul P, et al. The product of the imprinted gene IPL marks human villous cytotrophoblast and is lost in complete hydatidiform mole[J]. Placenta, 2003, 24(8-9): 835-842.
6. Fukunaga M. Immunohistochemical characterization of p57(KIP2) expression in early hydatidiform moles[J]. Hum Pathol, 2002, 33(12): 1188-1192.
7. Kim SJ, Park SE, Lee C, et al. Altered imprinting, promoter usage, and expression of insulin-like growth factor-Ⅱ gene in gestational trophoblastic diseases[J]. Gynecol Oncol,2003, 88(3): 411-418.
8. Fisher PA, Hodges RA. Genomic imprinting in gestational trophoblastic disease—a review[J]. Placenta, 2003, 24(suppl A): S111-S118.
9. Reed JC, Stein C, Subasinghe C et al. Antisense-mediated inhibition of BCL-2 protooncogene expression and leukemic cell growth and survival: Comprisons of phosphodiester and phosphorothioate oligodeoxynucleitides[J]. Cancer Res, 1990, 50(20): 6565-6570.
10. Clarke PA, Poele R, Wooster R, et al. Gene expression microarray analysis in cancer biology,pharmacology, and drug development: progress and potential[J]. Biochem Pharmacol,2001, 62(10): 1311-1336.
11. Williams NS, Gaynor RB, Scoggin S, et al. Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNA interference[J]. Clin Cancer Res, 2003, 9(3): 931-946.
12. Dooley TP, Reddy SP, Wilborn TW, et al. Biomarkers of human cutaneous squamous cell carcinoma from tissues and cell lines identified by DNA microarrays and qRT-PCR[J]. Biochem Biophys Res Commun, 2003, 306(4): 1026-1036.
13． 周怀君，崔金全，石一复．生物芯片技术在肿瘤学研究中的应用[J]．国外医学妇产科分册，2002，29(2)：101-104．
14. Vegh GL, Fulop V, Liu Y, et al. Differential gene expression pattern between normal human trophoblast and choriocarcinoma cell lines: downregulation of heat shock protein-27 in choriocarcinoma in vitro and in vivo[J]. Gynecol Oncol, 1999, 75(3): 391-396.
15. Kato HD, Terao Y, Ogawa M, et al. Growth-associated gene expression profiles by microarray analysis of trophoblast of molar pregnancies and normal villi[J]. Int J Gynecol Pathol, 2002; 21(3): 255-260.
16． 潘光锦，韩金祥．反义核酸类药物的药理学研究进展[J]．医学综述，2002，8(5)302-303．