Sorcin在小鼠腹水型肝癌高、低淋巴道转移株中的表达及定位

目的：研究抗药蛋白sorcin（又称可溶性抗药相关蛋白）在小鼠腹水型肝癌高、低淋巴道转移潜能不同的细胞株Hca-F/Hca-cP中的定位及表达差异,进一步探讨其在肿瘤淋巴道转移中的作用。方法：以小鼠腹水肝癌高淋巴道转移株Hca-F和小鼠腹水肝癌低淋巴道转移株Hca-P为研究对象,采用实时定量PCR技术检测sorcin在Hca-F/Hca-P细胞中基因水平的表达差异;采用Western免疫印迹技术检测sorcin在Hca-F/Hca-P细胞中蛋白水平的表达差异;采用免疫荧光技术检测sorcin在细胞中的定位情况;采用细胞计数试剂盒-8检测肿瘤细胞的增殖能力;应用Transwell小室检测肿瘤细胞的迁移和侵袭能力。结果：Hca-F细胞的体外增殖、迁移和侵袭能力都明显高于Hca-P细胞;sorcin在Hca-F细胞中的基因水平和蛋白水平表达分别为Hca-P细胞的1.5和1.8倍;sorcin在两种小鼠腹水型肝癌细胞株中的定位主要位于细胞质。结论： sorcin在Hca-F中呈高表达,主要定位于细胞质,可能在其增殖、迁移和侵袭过程中发挥重要作用,为进一步探讨其作用机制和功能奠定了基础。     

细胞内氯离子通道蛋白1在肝癌高、低淋巴道转移细胞株中的定位及表达差异

目的 研究细胞内氯离子通道蛋白1(CLIC1)在小鼠腹水型肝癌高、低淋巴道转移细胞株中的定位及表达差异.方法 以小鼠腹水型肝癌高淋巴道转移细胞株Hca-F和低淋巴道转移细胞株Hca-P为研究对象,采用双向凝胶电泳和质谱鉴定、细胞免疫荧光、细胞免疫化学染色方法和Western blot方法检测CLIC1在高、低淋巴道转移细胞株中的定位及蛋白表达情况.结果 双向凝胶电泳和质谱鉴定CLIC1在Hca-F细胞中高表达,是Hca-P细胞的1.6倍;细胞免疫荧光和免疫化学结果显示CLIC1定位于Hca-F和Hca-P细胞的细胞质和细胞膜,功能形式的细胞膜定位更多存在于Hca-F细胞中,并且在Hca-F细胞中的表达强于Hca-P细胞.Western blot结果显示CLIC1在Hca-F细胞中的表达是Hca-P细胞的1.6倍.结论 CLIC1在Hca-F和Hca-P细胞中定位于细胞质和细胞膜,在Hca-F细胞中高表达,且在Hca-F中更多定位于细胞膜,可能在肝癌淋巴道转移过程中发挥作用.     

小鼠腹水型肝癌高、低淋巴道转移株中Annexin A7蛋白的表达及意义

目的观察Annexin A7(ANXA7)蛋白在小鼠腹水型肝癌高、低淋巴道转移株(Hca-F、Hca-P)中的表达水平,分析其在两种细胞株中的表达及意义。方法采用免疫细胞化学、Western blot和流式细胞仪检测ANXA7在细胞株Hca-F和Hca-P中的表达。结果免疫细胞化学结果显示,ANXA7表达于Hca-F细胞胞核和胞质,以胞核为主,在Hca-P中表达位于胞质;Western blot结果显示,ANXA7在Hca-F、Hca-P中的表达相对值分别为0.09±0.01和0.23±0.02,ANXA7在Hca-F细胞中的表达明显低于Hca-P,差异有统计学意义(P0.01);流式细胞仪结果显示,ANXA7在Hca-F细胞中的相对荧光强度值(57.71±9.85)显著高于Hca-P(17.61±1.00),差异有统计学意义(P0.001)。结论 ANXA7在Hca-F、Hca-P中的差异性表达可能与肿瘤淋巴道转移能力相关。     

CXCR4对小鼠腹水型肝癌细胞淋巴转移潜能的影响

目的探讨趋化因子受体CXCR4表达水平对小鼠腹水型肝癌细胞淋巴转移潜能的影响。方法采用RT-PCR,流式细胞仪检测趋化因子受体CXCR4在小鼠腹水性肝癌细胞株Hca-F和Hca-P细胞的表达。通过归巢实验测定用抗体封闭CXCR4表达前后细胞特异性的向淋巴管迁移能力。结果CXCR4及其mRNA在高转移潜能小鼠肝癌细胞系Hca-F的表达高于低转移潜能小鼠肝癌细胞系Hca-P的表达。CX-CR4中和抗体能够抑制Hca-F细胞在体内向淋巴结转移。结论CXCR4在Hca-F、Hca-P细胞表面表达水平不同,可能是导致它们向淋巴管转移的潜能不同的影响因子之一,CXCR4表达水平与Hca-F、Hca-P细胞的特异性淋巴管转移潜能有关。     

小鼠肝癌淋巴道转移细胞系中NF-κB基因的表达及意义

目的 探讨NF-κB基因表达在小鼠肝癌淋巴道转移细胞系中的表达及意义.方法 采用RQ-PCR方法检测NF-κB基因在肝癌淋巴道低转移潜能的小鼠肝癌细胞系Hca-P和高转移潜能的小鼠肝癌细胞系Hca-F中的表达水平.结果 NF-κB基因在Hca-P和Hca-F细胞系基因表达分别为(1.41±0.48)×10~(-3)、(2.95±0.22)×10~(-3),Hca-P和 Hca-F之间,NF-κB基因表达差异有统计学意义 (P<0.01),其随转移潜能的增高,表达水平增加.结论 NF-κB基因表达水平增高与肝癌淋巴道侵袭转移相关.     

TGF-β1通过EMT促进肝癌细胞株MHCC97L的转移

目的构建TGF-β1真核表达载体,转染低转移MHCC97L肝癌细胞株,检测该肿瘤细胞的迁移、增殖能力,为阐明TGF-β1在肝癌细胞迁移中的作用奠定基础。方法构建真核表达质粒,转染MHCC97L肝癌细胞株,荧光定量PCR和Western blot检测其上皮-间充质转化(EMT)标志因子的表达,细胞划线和MTT增殖检测细胞的迁移和增殖。结果 MHCC97L-TGF-β1细胞比MHCC97L细胞E-cadherin的表达水平下降,而N-cadherin、Fibronectin和Vimentin的表达水平显著升高;MHCC97L-TGF-β1细胞的迁移能力增强,但细胞的增殖能力下降。结论 TGF-β1在低转移的肿瘤细胞株MHCC97L的表达,增强了细胞的迁移能力,为进一步体外和动物实验奠定了基础,也为肝癌转移的临床治疗提供了借鉴。     

泛素C-末端水解酶-L3在小鼠腹水型肝癌高、低淋巴道转移株中的表达

肿瘤转移机制及防治已成为当今肿瘤研究领域的热点.高淋巴道转移力小鼠腹水型肝癌细胞(Hca-F)、低淋巴道转移力小鼠腹水型肝癌细胞(Hca-P)是由大连医科大学病理教研室自行建立的肿瘤转移机制实验模型.它们是一对高度同源的来自同一小鼠肝癌细胞克隆的不同亚克隆,经615小鼠局部皮下注射后,特异地向引流淋巴结转移.     

不同淋巴道转移潜能小鼠肝癌细胞系中Toll样受体2基因的表达

目的 探讨TLR-2基因表达水平与小鼠肝癌淋巴道转移中的关系.方法 采用RQ-PCR方法检测TLR-2基因在肝癌细胞系H22、淋巴道低转移潜能的小鼠肝癌细胞系Hca-P和高转移潜能的小鼠肝癌细胞系Hca-F中的表达水平.结果 TLR-2基因在H22、Hca-P和HcaF细胞系基因表达分别为(1.08±0.25)×10-3、(2.14±0.42)×10-3、(4.31±0.62)×10-3,差异有统计学意义(P<0.01),其随转移潜能的增高,表达水平增加.结论 TLR-2基因表达水平增高提示其在肝癌的淋巴道侵袭转移中起作用,有可能成为预测肝癌预后新的靶点.     

CpGODN对树突状细胞抗肝癌作用的影响

目的:研究CpGODN在BALB/C小鼠骨髓来源树突状细胞(BMDC)抗肝癌免疫中的作用。方法:CpGODN联合肿瘤抗原(TAg)体外刺激BMDC,流式细胞术检测受刺激BMDC表达CD80的阳性率,ELISA检测受刺激BMDC分泌IL-12(p70)的水平,MTT法检测活化BMDC刺激T细胞的增殖能力,联合CpGODN和TAg体内预激小鼠获得CTL,检测CTL对肝癌细胞株Hca-F的特异性杀伤作用。结果:CpGODN联合TAg体外可明显激活BMDC,诱导BMDC膜表面CD80的表达,刺激BMDC分泌高水平IL-12,促进BMDC刺激T淋巴细胞增殖;体内联合应用CpGODN和TAg诱导的CTL对Hca-F具有强大的特异性杀伤作用,其诱导作用明显强于TAg,与细菌脂多糖和金黄色葡萄球菌肠毒素B相近。结论:CpGODN体外能有效促进小鼠BMDC的分化、增殖和成熟,体内与TAg联合应用可显著增强CTL对肿瘤细胞的特异性杀伤作用。     

微粒体谷胱甘肽S转移酶1过度表达促进肝癌发生发展

目的探讨微粒体谷胱甘肽S转移酶1(MGST1)在肝癌中的表达及其在肝癌细胞增殖、迁移和裸鼠成瘤中的作用。方法 Western blot检测人肝癌样品及肝癌细胞系中MGST1的表达;用慢病毒PLL3.7载体系统构建敲低MGST1的MHCC97H和HCCLM3细胞株及慢病毒p CDH载体系统构建过表达MGST1的SK-Hep-1细胞株后,用克隆形成实验检测细胞增殖能力;用Transwell实验检测细胞迁移能力;用皮下移植瘤实验检测MHCC97H细胞裸鼠成瘤能力。结果 71%(17/24)的肝癌组织中MGST1蛋白表达上调。敲低MGST1抑制MHCC97H和HCCLM3细胞的增殖、迁移能力(P0.05);过表达MGST1促进SK-Hep-1细胞增殖、迁移(P0.05);敲低MGST1的MHCC97H细胞裸鼠成瘤时间滞后(P0.01),肿瘤体积减小(P0.001),裸鼠生存期延长(P0.001)。结论 MGST1过度表达促进肝癌发生发展,是治疗肝癌的一个新靶点。     

Adiponectin exerts antiproliferative effect on human placenta via modulation of the JNK/c-Jun pathway

To determine the effects of adiponectin on human placenta during gestational diabetes mellitus (GDM) and on high glucose (HG)-induced BeWo cell proliferation. We examined the expression levels of adiponectin in control and GDM placenta using quantitative real-time PCR, Western blot, and immunohistochemistry (IHC). Cell proliferation and viability were assessed using a colorimetric assay (cell counting kit-8), PCNA immunocytochemical staining, and Western blot analysis of cyclin D1. Transfection of siRNA against c-jun was performed using Lipofectamine 2000. Cell cycle analysis was performed using propidium iodide staining and flow cytometry. Results show a decreased expression of adiponectin and an increased degree of trophoblast cell proliferation in GDM placenta compared to the normal placenta. Similarly, HG can promote BeWo cell proliferation that is associated with adiponectin down-regulation. This proliferation could be depressed by addition of exogenous adiponectin, i.e. adiponectin exerts antiproliferative effects on HG-induced trophoblast cells. Adiponectin suppresses the HG-induced BeWo cell proliferation by inhibiting the activation of JNK/c-jun. In conclusion, adiponectin inhibits HG-induced proliferation of BeWo cells through down-regulation of JNK/c-jun phosphorylation.     

沉默NANOG表达对人肝癌细胞HepG2中cyclin D1表达及细胞增殖的影响

 目的:探讨RNA干扰沉默NANOG表达后对肝癌细胞HepG2中细胞周期素D1(cyclin D1)表达及细胞增殖的影响。方法:将以NANOG基因为靶点的NANOG-siRNA瞬时转染肝癌细胞HepG2,real-time PCR和Western boltting检测NANOG、cyclin D1 mRNA和蛋白的表达,CCK-8和平板克隆形成实验检测细胞增殖能力,流式细胞术检测细胞周期情况。结果:与mock组比较,转染NANOG-siRNA后,肝癌细胞HepG2中NANOG、cyclin D1 mRNA和蛋白水平均下调(P＜0.05),细胞增殖能力下降(P＜0.05),进入G 0/G 1期的细胞比例增多(P＜0.05)。结论:沉默NANOG表达可引起肝癌细胞HepG2中cyclin D1的表达下降,导致细胞增殖能力下降。     

下调MARCH8基因的表达对宫颈癌Siha细胞侵袭、迁移与增殖的影响

目的：研究下调MARCH8基因表达对人宫颈癌Siha细胞侵袭、迁移、增殖以及克隆的影响,并探讨其可能的作用机制。方法：构建特异性针对MARCH8基因的siRNA片段,并将其通过脂质体介导转染至宫颈癌Siha细胞,同时设立转染无义序列的阴性对照组。分别采用Real-time PCR法与蛋白质印迹法检测转染后,细胞中MARCH8在mRNA和蛋白水平上的表达改变。通过Transwell小室实验、划痕实验检测MARCH8表达下调对细胞侵袭、迁移能力的影响,通过CCK-8法、细胞平板克隆实验检测MARCH8表达下调对细胞增殖、克隆形成能力的影响,并采用蛋白质印迹法检测MARCH8基因表达下调对基质金属蛋白酶9（MMP9）,波形蛋白（Vimentin）以及增殖细胞核抗原（PCNA）蛋白表达的影响。最后采用蛋白质印迹法检测MARCH8基因表达下调,对Wnt/β-catenin信号通路中β-catenin以及E钙黏蛋白（E-cadherin）表达的影响。结果：与转入siRNA-NC组相比,转入siRNA-MARCH8组细胞MARCH8在 mRNA和蛋白表达水平上均明显降低,细胞的侵袭、迁移以及增殖和克隆形成能力明显被抑制（P<0.05）,且侵袭标记蛋白MMP9,Vimentin以及增殖标记蛋白PCNA的表达也明显下降。MARCH8基因表达成功下调后,转入siRNA-MARCH8组细胞中β-catenin蛋白的表达水平较阴性对照组明显下降（P<0.05）,而E-cadherin蛋白的表达水平明显升高（P<0.05）。结论：下调MARCH8基因的表达可以抑制宫颈癌Siha细胞的侵袭、迁移、增殖和克隆能力,其机制可能与Wnt/β-catenin通路的抑制有关。     

慢病毒介导的RNA干扰沉默Slug基因对结肠癌HCT116细胞增殖和凋亡的影响

目的:探讨利用RNA干扰(RNA interference,RNAi)技术沉默Slug基因,观察对结肠癌HCT116细胞增殖和周期的影响。方法:构建Slug基因特异性siRNA慢病毒载体,感染结肠癌HCT116细胞,设立空白对照组、阴性对照组及SlugsiRNA三组,应用Real-time PCR和Western blot方法分别从基因和蛋白质水平检测各组干扰质粒对Slug基因的干扰效果,MTT法检测Slug基因在siRNA作用下的细胞增殖率,流式细胞仪检测细胞凋亡周期变化情况。结果:转染Slug siRNA后,结肠癌HCT116细胞中Slug基因mRNA和蛋白表达明显受到抑制(P<0.05);MTT检测,干扰组细胞增殖水平明显低于阴性对照组;流式细胞仪检测细胞G1期细胞百分比(52.3±0.6)高于阴性对照组(45.1±0.3,P<0.05)。结论:Slug siRNA能明显下调靶基因Slug的表达,在体外可抑制结肠癌HCT116细胞的生长并促进其凋亡。     

Response to hepatocarcinoma Hca-F of mice immunized with heat shock protein 70 from elemene combo tumor cell vaccine

To analyze immune response to murine hepatocarcinoma Hca-F of mice immunized with heat shock protein 70 （HSP70） derived from elemene combo tumor cell vaccine （EC-TCV） of Hca-F, HSP70 was isolated from EC-TCV by ADP affinity chromatography. Mice were immunized with HSP70 intraperitoneally three times and spleen calls were sampled. For cells, their proliferation and cytotoxicity against Hca-F were measured with MTT assay and their phenotypes were analyzed with flow cytometry. Spleen cells of immunized mice with HSP70 exhibited more potent cytotoxicity against Hca-F and proliferation than that of normal control mice, but less potent than that of mice immunized with EC-TCV. Among three groups, the percent of T6 T lymphocytes in the mice immunized with HSP70 （35.5%） was the highest compared with 6.25% in normal mice, and 28.4% in the mice immunized with EC-TCV. Immunization of HSP70 derived from EC-TCV could elicit potent immune response to Hca-F. HSP70 is one of elements inducing anti-tumor immune responses against Hca-F.     

Inhibition of ovarian cancer proliferation and invasion by pachymic acid

To determine the effect of pachymic acid (PA) on proliferation, cell cycle, and invasion in human ovarian carcinoma cell lines HO-8910 and explore some possible mechanisms, HO-8910 cells was treated with different concentrations of PA (0.5, 1, 2 μM). CCK-8 assay, propidium iodide staining, was applied to measuring the growth inhibiting rates of HO-8910 cells. Cell cycle was measured by flow cytometry. In addition, the activity of PA against HO-8910 cells invasion was evaluated in transwell assay. Western blot detected the proteins expression of E-cadherin, β-catenin and COX-2 of different groups treated with PA in different concentrations (0.5, 1, 2 μM) for 48 h. Our results showed that PA could effectively inhibit the in vitro growth of HO-8910 cells in dose-dependent manners in 72 h, suppressed migration and invasion of HO-8910 cells in concentration-dependent manners at 24 h, caused the increased accumulation of G1 phase cells, and caused down-regulation of β-catenin and COX-2 and up-regulation of E-cadherin expression level. Taken together, it could conclude that PA might inhibit proliferation and invasion of ovarian carcinoma cell through decreasing β-catenin and COX-2 expression and increasing E-cadherin exprssion.     

靶向PSMB5基因的shRNA慢病毒载体对神经干细胞增殖和分化潜能的影响

目的:优化20S蛋白酶体β5亚单位(proteasome subunit beta type-5,PSMB5)-shRNA慢病毒感染神经干细胞(neural stem cells,NSCs)的方案,观察PSMB5表达下调对NSCs增殖和分化能力的影响,探讨调控NSCs潜能的分子机制。方法:构建携带绿色荧光蛋白(green fluorescent protein,GFP)基因的PSMB5-shRNA慢病毒载体,并设错义序列对照,感染新生小鼠(postnatal day 0,P0)NSCs。倒置荧光显微镜观察GFP阳性率,计算感染率,RT-PCR、免疫印迹和荧光分光光度法检测PSMB5沉默效率和蛋白酶体活性。比较对照组和shRNA组神经球的数量和直径,利用CCK-8实验观察PSMB5基因沉默对NSCs增殖潜能的影响。Tuj1染色观察PSMB5基因沉默对NSCs分化能力的影响。结果:PSMB5-shRNA慢病毒感染NSCs 24 h后可见GFP荧光表达,48 h达峰值,传代后可见GFP稳定表达。其感染复数MOI为40,polybrene 3μg/ml时,感染48 h GFP阳性率可达92.5%±2.3%;PSMB5-shRNA组PSMB5 mRNA和蛋白表达水平分别较对照组降低66.49%±4.81%(P0.001)和33.1%±2.54%(P0.001)。PSMB5-shRNA组蛋白酶体活性较对照组下降43.4%±1.48%(P0.01)。shRNA组NSCs的增殖能力降低,神经球数量为126.5±8.4显著低于对照组163.5±9.5(P0.01),神经球的平均直径为29.9μm±2.6μm显著低于对照组42.9μm±2.3μm(P0.01)。CCK-8结果表明PSMB5-shRNA组细胞吸光度值0.36±0.04,显著低于对照组0.59±0.03(P0.001),PSMB5-shRNA组Tuj1+阳性率为39.13%±8.14%,较对照组显著降低(P0.01)。结论:PSMB5基因沉默可降低NSCs蛋白酶体活性抑制P0期小鼠NSCs增殖分化能力。     

miR-503 inhibits cell proliferation and induces apoptosis in colorectal cancer cells by targeting E2F3

Objective: Colorectal cancer (CRC) is one of the major healthcare problems worldwide. A lot of miRNAs are aberrantly expressed in CRC and involved in its development and progression. The purpose of this study was to investigate the expression and function of miR-503 in CRC. Methods: miR-503 expression was detected in CRC tissues and cell lines by Quantitative real-time PCR. Cell proliferation was assessed by MTT assay. Cell apoptosis and cell cycle distribution were measured by flow cytometry. Moreover, luciferase reporter assay and western blot were performed to determine the potential target of miR-503 in CRC cells. Results: miR-503 was significantly decreased in CRC tissues and cell lines in comparison with controls. Overexpression of miR-503 in CRC cells remarkably inhibited cell proliferation and induced apoptosis. Furthermore, E2F3 was identified as a direct target of miR-503 in CRC cells and down-regulation of E2F3 had a similar effect as miR-503 overexpression on CRC cells. In addition, the expression of E2F3 was negatively correlated with miR-503 level in CRC tissues. Conclusions: miR-503 inhibits cell proliferation and induces apoptosis by directly targeting E2F3 in CRC cells, indicating its potential application in CRC diagnosis and therapy.