【作者】 杨洲；

【导师】 王继德； 张亚历；

【作者基本信息】 第一军医大学 ， 内科学， 2006， 硕士

【摘要】 目的：肿瘤分子生物学和分子遗传学的研究表明原癌基因的激活、抑癌基因的失活和细胞凋亡基因的缺失是许多肿瘤的发生机制。结肠癌的发生发展是一个涉及多种基因参与、多步骤的复杂过程，伴有多种原癌基因和抑癌基因表达与活性异常。FHL2(Four and a half LIM protein 2)是仅有LIM结构域的蛋白质，属于LIM蛋白家族成员。LIM结构域存在于很多蛋白质中并具有扮演多种细胞角色，如调节基因表达，维持细胞结构，介导细胞粘附，细胞运动和信号传导。人类四对半LIM结构蛋白质中的家庭成员包括FHL1，FHL2，FHL3，FHL4和ACT。它们在辅助细胞与组织特异性表达并参与不同细胞的病变，包括细胞存活的调节，转录和信号转导等。目前研究最多的是FHL2，FHL2蛋白主要位于细胞核并能在细胞质和细胞核之间穿梭。是一种适应蛋白，能与多种靶蛋白结合并调控后者的活性，FHL2基因与肌细胞成熟与分化的研究较多，但与肿瘤研究较少。有研究提示在乳腺癌和黑色素瘤中表达升高，但是在消化道肿瘤病例还没有研究。本研究探讨了FHL2基因在大肠癌组织中的表达，及它在细胞分化和肿瘤生长中的作用，评价了抑制FHL2基因在控制肿瘤细胞生长中的作用。方法：1、临床标本中FHL2蛋白的表达检测：收集南方医院消化内科病理室2005年6月～2005年12月住院病人肠镜活检的资料完整的病理标本50例，(1)用免疫组织化学方法检测FHL2蛋白在大肠癌组织中的表达：检测高分化腺癌20例、低分化腺癌15例，以及15例正常结肠粘膜组织，分析FHL2表达与相关临床病理因素的关系，所有患者术前均未做放疗、化疗及免疫治疗；(2)Westernblotting检测结肠癌组织和癌旁组织中FHL2蛋白的表达。2、抑制FHL2基因对大肠癌细胞分化的影响：(1)用Western blotting检测不同大肠癌细胞株SW620、SW1116、SW480、DLD1、HCT15、HT29、LoVo和Colo205中FHL2蛋白的基础表达；(2)建立稳定表达空载体LoVo细胞系(LoVo／Vector)和稳定表达FHL2反义基因LoVo细胞系(LoVo／FHL2-AS)并鉴定；(3)用相差显微镜观察LoVo／Vector和LoVo／FHL2-AS1细胞形态学上的表现；(4)经α-微管蛋白抗体和Hochest 22358分别标记细胞浆和细胞核，用免疫荧光显微镜观察LoVo／Vector和LoVo／FHL2-AS1细胞骨架与细胞核的变化；(5)用逆转录一聚合酶链反应(RT-PCR)检测LoVo／Vector、LoVo／FHL2-AS1和LoVo／FHL2-AS2中细胞分化指标癌胚抗原(CEA)和上皮细胞钙粘蛋白(E-Cadherin)的表达；(6)经罗丹明毒肽标记LoVo／Vector和LoVo／FHL2-AS1细胞中的丝状肌动蛋白(F-actin)，用免疫荧光显微镜观察其细胞核和丝状肌动蛋白的形态变化；(7)用逆转录一聚合酶链反应(RT-PCR)检测LoVo／Vector、LoVo／FHL2-AS1和LoVo／FHL2-AS2，观察大肠癌相关癌基因存活素(survivin)，环氧化酶-2(cox-2)，人端粒逆转录酶(hTERT)和c-jun mRNA的表达；(8)经不同浓度的小牛血清(FBS)培养的LoVo／Vector、LoVo／FHL2-AS1和LoVo／FHL2-AS2细胞，用噻唑蓝(MTT)法测定其对血清的增殖反应。结果用SPSS 12.0统计软件进行统计分析，采用秩和检验，并进行两两比较，P＜0.05表明有统计学意义。结果：1、临床标本中FHL2蛋白的表达检测：(1)用免疫组织化学法检测FHL2蛋白的表达，FHL2蛋白主要定位于细胞核，细胞质有少许表达，正常大肠粘膜组织中不表达或低表达，而在大肠癌组织中表达水平较高，FHL2蛋白表达水平与大肠癌分化程度负相关，分化程度越低其表达程度越高；(2) Western blotting发现FHL2在结肠癌组织中高表达，而在结肠癌旁组织中不表达或低表达。2、抑制FHL2基因对大肠癌分化影响的体外细胞实验：(1)肠癌细胞株SW620、SW1116、SW480、DLD1、HCT15、HT29、LoVo、Colo205均表达相当量的FHL2蛋白，而在部分分化的HT29细胞几乎不表达；(2)成功建立了稳定表达空载体LoVo细胞系(LoVo／Vector)和稳定表达FHL2反义基因的LoVo细胞系(LoVo／FHL2-AS)；(3)相差显微镜发现LoVo／Vector细胞形态与肿瘤细胞类同表现为多边形，部分细胞变圆不规则有较短小突起的圆型或方型，而LoVo／FHL2-AS1细胞形态与正常大肠粘膜细胞类似表现为长梭型，(4)经α-微管蛋白抗体和Hochest 22358分别标记细胞浆和细胞核，用免疫荧光显微镜观察发现与LoVo／Vector相比，LoVo／FHL2-AS1细胞变长，胞浆增大，核浆比例下降，LoVo／FHL2-AS1引起的细胞形态变化与全反式维甲酸(ATRA)相仿，提示抑制FHL2可能诱导了癌细胞的分化；(5) RT-PCR发现CEA、E-Cadherin在LoVo／FHL2-AS1中表达较LoVo／Vector明显增强；(6)用罗丹明毒肽标记LoVo／Vetor和LoVo／FHL2-AS1细胞中的丝状肌动蛋白(F-actin)，免疫荧光显微镜发现LoVo／Vector仅有少量的丛集丝状肌动蛋白，在细胞内分布不均匀呈斑块状；LoVo／FHL2-AS1则可见到均匀着色，且有微绒毛肌动蛋白形成；(7) RT-PCR发现与LoVo／Vetor对照组相比，LoVo／FHL2-AS1和LoVo／FHL2-AS2中的存活素(survivin)，环氧化酶-2(cox-2)，人端粒酶逆转录酶(hTERT)和c-jun mRNA的mRNA表达均下调；(8)噻唑蓝(MTT)法测定LoVo／Vector细胞对含有0.4％、2％和10％小牛血清(生长因子)的增殖系数分别为113.8±5％，126.4±6％和162.5±5％，而LoVo／FHL2-AS1和LoVo／FHL2-AS2细胞分别为103.3±3％，110.3±5％，115.2±9％和07.8±6％，113.5±3％，135.5±4％。结论：1、FHL2蛋白主要在大肠癌细胞核表达，而且其表达水平与大肠癌分化程度负相关，分化程度越低其表达程度越高，提示FHL2基因的表达与大肠癌恶性程度成正相关；2、抑制FHL2基因在大肠癌细胞中表达，可以使大肠癌细胞向正常细胞分化；3、抑制FHL2基因的大肠癌细胞增殖减弱，且降低了其它原癌基因的表达，提示FHL2基因可能与大肠癌发生相关，进而提示FHL2基因有希望作为大肠癌治疗的新分子靶点。更多还原

【Abstract】 [Objective]The research of oncomolecularbiology and molecular genetics manifest the mechanism of activation of many proto-oncogene and deactivation of anti-oncogenes. The development of Colorectal cancer involved the changes of a sets of genes in differentiation stees. There are a lot of kinds of proto-oncogene being actived and anti-oncogene being inactivated. Four and a half LIM protein 2 (FHL2) is the only protein with the LIM domain, belongs to the member of LIM protein family. LIM domains are present in many proteins that have diverse cellular roles as regulators of gene expression, cyto-architecture, cell adhesion, cell motility and signal transduction. The human four-and-a-half-LIM-only protein family consists of the members of FHL1, FHL2, FHL3, FHL4 and ACT. They are expressed in a cell- and tissue-specific manner and participate in various cellular processes, including regulation of cell survival, transcription and signal transduction. Here, we review the current knowledge of the best-studied member of this family, FHL2.It localized mainly in nucleus and can shuttle between cytoplasm and nucleus. FHL2 is a proteinum product of oncogene codogenic.FHL2 is particularly intriguing because it can function as either a repressor or activator of target proteins in a cell type-dependent fashion, and interacts with other proteins. FHL2 protein expression was nearly undetectable in normal tissue samples. However, many cancerous tissues expressed higher levels of FHL2 than normal tissues, suggesting this gene might be a novel oncogene, In this study, we investigated the role of FHL2 in growth and differentiation in colon cancer.[Methods]1、The expression of FHL2 protein in clinical specimens: Normal or colon cancer tissues with different stage of differentiation were obtained in the Nanfang Hospital (Guangzhou, China)from June to December 2005: (1) FHL2 expression was detected by immunohistochemistry. (2) FHL2 expression in colon cancer tissues and matched normal tissues as detected by Western blotting.2、In vitro expression: (1) Protein expression of FHL2 in colon cancer cell lines SW620, SW1116, SW480, DLD1, HCT15, HT29, LoVo and Colo205 was detected by Western blotting. (2) FHL2 expression in stable transfectants of LoVo cells expressing vector or antisense FHL2 was detected by Western blotting. (3) Morphology of stable transfectants of LoVo/Vector and LoVo/FHL2-AS1 was observed under phase-contrast microscope. (4) Stable transfectants of LoVo cells was stained withα-tubulin to show the cytoplasm and Hochest 22358 to show the nuclei, the visualized under by fluorescent microscopy. (5) Expression of CEA and E-cadherin in LoVo/Vector and LoVo/FHL2-AS stable transfectants was detected by RT-PCR. (6) Stable transfectants of LoVo/Vector and LoVo/FHL2-AS1 stained with rhodamine-phallotoxin with F-actin filaments being visualized were under fluorescent microscopy. (7) The expression of cox-2, survivin, c-jun and hTERT in stable transfectants of LoVo cells was detected by RT-PCR. (8) LoVo/Vector and LoVo/FHL2-AS stable transfectants were seeded into a 96 well plate in triplicate at a density of 10,000 cells/well for 12 h. The medium was replaced with RPMI 1640 containing 0.1%, 0.4%, 2% or 10% FBS for additional 48 h, and then cell growth was assessed by MTT assay. The data obtained were statistically analyzed by SPSS 12.0. The results with different treatments were compared using a two-tailed Student’s t test and considered significant if pvalue was less than 0.05.[Results]1、Detection of FHL2 protein in clinical specimen: (1) FHL2 protein was mainly localized in nucleus. FHL2 protein was detectable only in few normal tissues. However, all colon cancer tissues expressed higher levels of FHL2 normal tissues and the expression pattern is negatively correlated with cell differentiation. The lower differentiation cancer tissues expression higher levels of FHL2; (2) Western blotting assay showed that colon cancer tissues expressed higher level of FHL2 than matched normal tissues.2、Suppression of FHL2 expression induced differentiation in colon cancer cells: (1) Protein expression of FHL2 in colon cancer cell lines SW620, SW1116, SW480, DLD1, HCT15, LoVo and Colo205 was relatively high. However, it is nearly undetectable in semi-differentiated HT29 cells. (2) We established stable LoVo transfectant expression vector control or FHL2-AS gene. Western blotting assay showed that LoVo/FHL2-AS expressed lower levels of FHL2 protein compared with LoVo/Vector. (3) The stable transfectants of LoVo/Vector displayed a round or flat morphology with a short cytoplasmic process. However, LoVo/FHL2-AS transfectant exhibited lengthened or shuttle-shape morphology. Long or dendritic-like cytoplasmic processes were visible under phase-contrast microscope. (4) To distinguish cytoplasm and nucleus, we stained the stable transfectants withα-tubulin antibody by immunofluorescence to display the cytoplasm and Hochest22358 to display the nuclei. Similar morphological changes with that observed under phase-contrast microscope were found. Furthermore, LoVo/FHL2-AS stable transfectants exhibited a decreased nuclear:cytoplasmic ratio by mainly increasing the cytoplasm plot and slightly decreasing the size of nuclei. (5) The pretreatment with ATRA induced similar morphological changes to that of LoVo/FHL2-AS transfectant. (6) RT-PCR showed that suppression of FHL2 increased expression of the differentiation markers of colon cancer, CEA and E-cadherin. (7) F-actin staining showed weak and aggregated F-actin filaments exhibited in LoVo/Vector transfectants. Suppression of FHL2 displayed uniform and highly structured array of thick F-actin filaments at cell-adherent regions and throughout the cytoplasm. Importantly, typical microvilli-like actin filaments that protruded out of the cytoplasm plot were observed only in LoVo/FHL2-AS transfectants. (8) The mRNA expression of survivin, cox-2, hTERT, and c-jun were downregulated in stable transfectants of FHL2 antisense. (9) The cell proliferation by MTT assay of LoVo/Vector were 113.8±5%, 126.4±6% and 162.5±5% when cultured in the presence of 0.4%, 2% and 10% FBS, respectively, while those of LoVo/FHL2-AS1 and LoVo/FHL2-AS2 were 103.3±3%, 110.3±5% and 115.2±9%, and 107.8±6%, 113.5±3% and 135.5±4%.[Conclusions]1、Protein expression of FHL2 localized mainly in nucleus, FHL2 expression levels was negatively correlated with the differentiation degree of colon cancer.2、Suppression of FHL2 gene expression in the colon cancer cell. induced morphological changes and the decreased nuclear:cytoplasmic ratio, suggesting that FHL2 might involved in the de-differentiation of colon cancer cells.3、Antisense FHL2 inhibited serum-dependent cancer cell growth and suppressed expression of other oncogenes. It suggested that FHL2 might play important role in carcinogenesis of colon cancer.更多还原