【作者】 王绍清；

【导师】 唐建武；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 背景:癌的侵袭与转移是癌细胞与宿主细胞、细胞外基质（ extracellular matrix,ECM）之间一系列复杂的、多步骤的、多因素相互作用的动态过程。细胞骨架是横越在真核细胞细胞核和细胞膜内侧面的一种纤维状蛋白基质,由微丝、微管及中间纤维组成;三者高度协调分布,与胞核、质膜、细胞器相连构成了细胞形态骨架和运动协调系统,以保持细胞的形态和行使运动功能,并对信号传递有重要意义。近年的研究发现,细胞骨架与细胞遗传物质的复制、传递、遗传信息的表达,即与细胞的分裂、分化、凋亡、癌变等细胞生理功能改变密切相关。Hca-F细胞和Hca-P细胞是一对来源于同一小鼠肝癌细胞克隆、淋巴道转移潜能显著不同、能够稳定传代的亚克隆细胞系。经615小鼠局部皮下注射后,均特异地向引流淋巴结转移。Hca-F细胞和Hca-P细胞分别为高淋巴结转移和低淋巴道转移细胞株,在615小鼠内淋巴结转移率分别大于70%和小于30%,是研究癌症淋巴道转移机制难得的细胞株模型。本组前期利用基因芯片技术,比较Hca-F细胞和Hca-P细胞之间差异性基因的表达情况,其中gelsolin基因在Hca-F细胞中的表达是Hca-P细胞表达的21.9倍。然而蛋白质是生命活动的体现者,可以说是执行者;基因对性状的控制,是通过控制蛋白质的合成来实现的,因此基因的功能还需要在蛋白水平予以进一步检验和确认。所以我们又通过荧光差异双向凝胶电泳技术,建立了高低淋巴道转移力小鼠肝癌细胞荧光差异蛋白表达图谱,高通量筛选与肿瘤淋巴道转移相关的蛋白质,gelsolin蛋白在Hca-F细胞中的表达是在Hca-P细胞表达的1.7倍,提示其在肝癌的淋巴道转移中发挥着一定的作用。肝癌的淋巴道转移是一个多基因多步骤的过程,研究gelsolin在淋巴道转移过程中的作用和地位,有利于研究肝癌淋巴道转移机制和治疗。目的:1.从蛋白质水平上比较蛋白质组学筛出的差异蛋白gelsolin在淋巴道转移潜能不同小鼠肝癌细胞系中的表达。2.构建pSilencer-gelsolin-shRNA表达载体,稳定转染Hca-F细胞并获得gelsolin的表达水平稳定下调的Hca-F细胞株,同时建立稳定转染pSilencer-无关序列Hca-F细胞株作为对照。3.研究gelsolin在细胞内的表达和下调其在Hca-F细胞株的表达对小鼠肝癌细胞株Hca-F在细胞增殖、细胞迁移和侵袭能力方面的影响,由此确定gelsolin与小鼠肝癌细胞淋巴道转移潜能间的关系。方法:1.采用细胞免疫组化、蛋白免疫印迹方法检测gelsolin蛋白在Hca-F、Hca-P细胞株中表达有无差异,为进一步研究肝癌淋巴道转移的机制奠定基础。2.依据GeneBank中小鼠gelsolin基因序列（序列登录号:NM₂27753）合成三条shRNA的DNA模板的两条单链,分别命名为gelsolin1、gelsolin2、gelsolin3和gelsolin无关序列,将pSilencer ?3.1-H1neo质粒双酶切后分别与三条shRNA的DNA模板和无关序列的退火后产物连接,连接产物分别命名为pSilencer-shRNA-gelsolin-1、pSilencer-shRNA-gelsolin-2、pSilencer-shRNA-gelsolin-3、pSilencer- shRNA-wg。对三条pSilencer-shRNA表达载体经测序鉴定准确后（引物为5’-GTTTTCCCAGTCACGAC-3’）,将三条pSilencer-shRNA表达载体和无关序列对照组分别以脂质体法稳定转染Hca-F细胞,用含400μg/mlG418的完全培养基筛选,获得pSilencer-shRNA-gelsolin表达载体稳定转染的Hca-F细胞和无关序列对照组细胞。提取未处理的Hca-F细胞株、稳转pSilencer无关序列的Hca-F细胞株、稳pSilencer- gelsolin-shRNA三条表达载体的Hca-F细胞株的RNA、蛋白,RT-PCR和Western-Blotting检测shRNA转染后Hca-F细胞gelsolin在mRNA和蛋白质水平的表达。选出下调效果最好的一组细胞株用于后续实验。3.以三组细胞（分别为未处理的Hca-F细胞株、稳染了pSilencer无关序列的Hca-F细胞株、稳转pSilencer-shRNA-gelsolin-1表达载体的Hca-F细胞株）为实验对象。以CCK-8法检测三组细胞的细胞活力和分裂增殖能力;Transwell实验检测三组细胞的体外迁移能力（Transwell小室不做处理）、和侵袭能力（在Transwell上室中加人工基底膜）;分别在615小鼠足垫内侧接种三组细胞,2×106个/0.05ml细胞,28天后观察各组淋巴结肿瘤转移情况。结果:1.Gelsolin在小鼠腹水型肝癌细胞系Hca-F细胞和Hca-P细胞胞浆中表达,Western-blot分析显示gelsolin在Hca-F细胞中高表达,在Hca-P细胞中表达较低（P<0.05）。2.经酶切电泳分析及DNA测序鉴定,成功构建三条重组质粒,并转入Hca-F细胞;与未作处理的Hca-F细胞相比三组重组质粒gelsolin在mRNA水平的抑制率分别为46.8%、48.2%、20%;在蛋白水平对gelsolin的抑制率分别为76.4%、50.4%、37.4%;其中pSilencer-gelsolin-shRNA-1稳定转染的Hca-F细胞株gelsolin下调效果最明显（P<0.01）,选择作为后续功能实验研究的gelsolin稳定下调的细胞株。3.CCK-8检测细胞增殖活性显示gelsolin不影响细胞的增殖;体外迁移实验,通过比较穿过膜的细胞在A570吸光值由Hca-F的（0.2480±0.0447）、对照组的（0.2239±0.0274）减少到干扰组细胞（0.1716±0.0232）（P<0.05）;体外侵袭实验,比较穿过人工基地膜Hca-F细胞（0.2007±0.0129）、对照组（0.2236±0.0446）和干扰组细胞（0.1557±0.0051）,可以发现下调gelsolin蛋白表达后,干扰组细胞穿过人工基底膜的细胞A570 OD值吸光度值较对照降低（P<0.05）,结果显示下调Hca-F细胞中gelsolin的表达后,转移的肿瘤细胞数量减少,癌细胞的运动侵袭能力下降;体内淋巴结转移实验显示,下调gelsolin抑制了Hca-F细胞在小鼠体内淋巴结的转移率。结论:1.Gelsolin在小鼠腹水型肝癌细胞系Hca-F和Hca-P均有表达,且Hca-F中较高,而在Hca-P中表达较低。2.利用pSilencer?3.1-H1neo质粒和shRNA成功构建了三个pSilencer-gelsolin-shRNA表达载体,该载体转染Hca-F细胞细胞后,经含有400μg/mlG418的完全培养基筛选,获得了稳定转染pSilencer-gelsolin-shRNA表达载体的Hca-F细胞株,在成功构建的shRNA稳定转染的Hca-F细胞中,其中pSilencer- gelsolin-shRNA-1对Hca-F细胞中gelsolin抑制效果最明显,作为后续研究的细胞株。3.Gelsolin具有增强肿瘤细胞迁移和侵袭的能力,抑制gelsolin基因和蛋白的表达,具有下调鼠源肝癌细胞转移和侵袭能力的作用,证明在肝癌的淋巴道转移过程中gelsolin发挥着重要的作用。更多还原

【Abstract】 Background: Many biological processes, such as embryonic morpho- genesis, immune surveillance, and tissue repair and regeneration, are required for cell migration which drives progression of many diseases, including cancer invasion and metastasis.Cell migration is a highly integrated multistep process which is initiated by the protrusion of cell membrane. In the process of tumor invasion and metastasis, the relationship among tumor cell,host cell and extracellular matrix (ECM) is complicated. The actin cytoskeleton is crucial for cell motility, and it connects with cell disintegration, cell differentiation, apoptosis and canceration. Gelsolin is best known for its involvement in dynamic changes in the actin cytoskeleton during a variety of forms of cell motility.Hca-P and Hca-F is a pair of syngenetic mouse hepatocarcinoma ascites cell lines, presenting a specific potential of lymphatic metastasis when inoculated subcutaneously in 615 mice. Hca-F with a high metastasis potential (>70%) and Hca-P with a low one (<30%) are rare and available cell model as a control to each other in the research on the molecular mechanisms of lymphatic metastasis.Based on the gene chip experiment, we found that the expression of gelsolin in Hca-F cells was higher than that in Hca-P cells (21.9 fold compared with Hca-P cells). In order to obtain lymphatic metastasis associated proteins, the protein expressed profiles of Hca-F cells and Hca-P cells were compared using fluorescent two-dimensional difference gel electrophoresis (2D DIGE).Gelsolin protein was up-regulated in Hca-F cells (1.7 fold compared with Hca-P cells) . It was suggestted that gelsolin was a lymph node metastasis-associated protein.Objective: 1. To observed the expression of gelsolin in Hca-F cells and Hca-P cells. 2. Down-regulating the gelsolin expression in Hca-F cells by using pSilencer 3.1-shRNA expressing vectors. 3. To investigate the effect of gelsolin on the proliferation and invasion of mouse hepatocarcinoma cell lines.Method:1. Immunohistochemistry and western blot were utilized to show the expression of gelsolin in Hca-F cells and Hca-P cells. 2. Three shRNAs and scrambled shRNA sequences were designed and synthesized according to gelsolin gene sequences .Then three shRNAs and scrambled shRNA sequences were inserted into the pSilencer-3.1 vector to silence gelsolin gene ,and named gelsolin1、gelsolin2、gelsolin3 and gelsolin wg. The four pSilencer 3.1-shRNA expressing vectors were transfected into Hca-F cells respectively using Lipofectamine 2000 according to the manufacturer,s interfering. For RT-PCR analysis of gelsolin mRNA levels, total RNA was isolated from cells using Trizol and cDNA was synthesized using RT-PCR Kit .PCR analysis was performed. Total cell lysates were separated by SDS-PAGE and transferred to PVDF membranes, which were incubated with the indicated antibodies. Bound antibodies were visualized by ECL. And the most effective pSilencer 3.1-shRNA vector was selected based on the results of RT-PCR and Western blotting. In the end, the Hca-F cells were transfected stably with the most effective pSilencer 3.1- gelsolin- shRNA expressing vector. 3. Hca-F cells, control cells and shRNAi cells were used for next experiment. About 1×106 cells in 100μl -RPMI1640 were seeded in duplicate into each well of the 96-well culture plates, and 10μl CCK-8 was added at 24、48、72 hours respectively. After 1 h incubation at 37℃in 5% CO2, the absorbency A450 was measured . The inhibitory effect of RNAi on Hca-F cells migration in vitro was demonstrated using 24-well transwell units with 8μm pore size polycarbonate filter. cells were harvested in 100μl serum-free medium containing 0.1% BSA and added to the upper chamber. The lower chamber contained 10μg/μl FN in 500μl RPMI 1640. Cells were incubated for 24 h at 37℃, 5% CO2 incubator. At the end of incubation the cells on the upper surface of filter were completely removed by wiping with a cotton swab. Then the filters were fixed in methanol and were stained with 0.1% gentian violet. 33% acetate acid was used to wash filters. The absorbency A450 of elution solution was measured. ECM was added in upper filter when metastasis in vitro was performed .In vivo tumor metastasis assay, three group cells were inoculated into footpad of 615 mouse subcutaneously. After three weeks, the mice were killed and their lymph nodes were isolated.Result: 1.The result of immunofluorescence showed that gelsolin protein was located at cytoplasm of hepatic cells; western blot showed that the expression of gelsolin was positive in Hca-F and Hca-P. Its expression in Hca-F cells was higher than that in Hca-P cells﹙P<0.05﹚. 2. The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing.The shRNA expressing recombinants were transfected into Hca-F cells successfully. Gelsolin transcript (gelsolin1,gelsolin2,gelsolin3) was reduced by 46.8%、48.2%、20.2% respectively, and the protein of GSN was reduced by 73.9%、45.1%、31.2% respectively. shRNA-1 was found to have the best effect against gelsolin protein expression(P<0.01).Hca-F cell line with gelsolin stably down regulated using shRNA technique was successfully established, which laid a favorable foundation on the gene function study of gelsolin. 3. There was no influence of gelsolin to the proliferation of hepatic cells. In vitro transwell migration , the absorbency A450 of elution solution from Hca-F cells was(0.2480±0.0447), control cells (0.2239±0.0274) to shRNAi cells (0.1716±0.0232)(P<0.05) ; In vitro invasion assay the absorbency A450 of elution solution from Hca-F cells (0.2007±0.0129), control cells (0.2236±0.0446)to shRNAi cells (0.1557±0.0051)(P<0.05) ;In vivo tumor metastasis assay, lymph node metastasis rates of Hca-F cells and control cells was 90% and 80%; lymph node metastasis rates of shRNAi cells was 50%.Conclusion: 1.The expression of gelsolin was higher in Hca-F cells than that in Hca-P cells. 2. Hca-F cell lines with gelsolin stably down- regulated using shRNA technique was successfully established. 3. The ability of metastasis and invasion of mouse hepatocarcinoma ascites cell lines Hca-F was reduced by gelsolin after its down-regulation and proved that gelsolin played an important role in process of lymphatic metastasis of hepatocarcinoma.更多还原