人参皂苷Rg3抗人肝癌细胞株侵袭和转移的实验研究

目的:探讨人参皂苷Rg3抗人肝癌细胞侵袭和转移的作用及其相关机制。方法:选择人类肝癌细胞株SMMC-7721细胞和建株人脐静脉内皮细胞株ECV304作为研究对象,通过细胞抑制实验(MTT法)、细胞粘附试验和免疫组化方法系统地观察人参皂苷Rg3体外对SMMC-7721细胞及ECV304细胞生长的抑制作用、SMMC-7721细胞与纤维粘连蛋白(FN)粘附以及表达nm23、CD44、VEGF基因蛋白的影响。结果:人参皂苷Rg3能够显著地抑制肝癌细胞株SMMC-7721细胞及内皮细胞株ECV304的生长,抑制SMMC-7721细胞与FN粘附及CD44、VEGF的表达,而增强nm23基因的表达。结论:人参皂苷Rg3具有抗人肝癌细胞侵袭和转移的作用,机制可能与其能够抑制肝癌细胞侵袭活力、调节与肝癌细胞侵袭与转移密切相关的基因表达和抗肿瘤血管形成有关。     

培美曲塞抑制肺腺癌细胞侵袭能力的研究

[目的]探讨纤连蛋白（FN）对非小细胞肺癌的黏附和迁移能力的影响及培美曲塞的干预作用。[方法]以肺腺癌细胞系A549为研究对象，观察FN对癌细胞增殖活性的影响及不同浓度培美曲塞的抑制作用。在FN包被的培养板和FN滤膜的Boyden浸润小室等体外侵袭模型中，观察培美曲塞预处理后细胞与FN黏附及迁移能力的变化。设立牛血清白蛋白作为FN的对照组。[结果]FN能明显提高A549细胞的增殖活性，培美曲塞40nmol／L和80nmol／L能有效抑制这种增殖活性的升高，但低浓度20nmol／L培美曲塞的抑制作用却不显著。A549细胞在FN培养板的黏附数量及在小室中的穿膜数量远高于对照组．而各浓度培美曲塞预处理细胞的黏附及迁移能力明显降低。[结论]FN能促进肺腺癌细胞的增殖活性、黏附和迁移能力，培美曲塞能在一定程度上阻断这种效应。     

纤维连接蛋白在不同类型肺癌细胞侵袭过程中的作用及机制

目的 观察和探讨纤维连接蛋白(FN)对不同类型肺癌细胞侵袭能力的影响及机制。方法 在FN包被的培养板和FN滤膜的Boyden浸润小室等体外侵袭模型中，比较小细胞肿癌细胞系054A和肺腺癌细胞系A549粘附及迁移能力的差别，并检测FN对肿瘤细胞增殖活性的影响。分别给予细胞抗α3整合素、抗α5整合素、抗β1整合素单抗预处理后，观察侵袭性的变化。结果 FN增强A549细胞粘附、迁移及增殖活性的作用明显大于054A细胞。这种作用在A549细胞能被抗α3、抗α5和抗β1单抗抑制，而在054A细胞能被抗α3和抗β1单抗抑制。结论 FN对不同类型肺癌细胞侵袭能力的影响不同，可能与细胞膜整合素受体的差异有关。     

细胞生长相关新基因PP3105的克隆及其初步功能研究

目的 新基因PP3105的克隆及其初步功能研究。方法 分离和克隆新基因PP3105的全长序列，在生物信息学分析的基础上，采用克隆形成、亚细胞定位、生长曲线等实验对该基因进行初步功能研究。结果 实验表明PP3105有两个不同的转录本，并表现出一定的组织特异性；染色体定位于4q22-24；蛋白定位于细胞膜和细胞浆中；克隆形成实验显示有明显的体外集落抑制作用；生长曲线实验表明PP3105在SMMC7721细胞中的表达对细胞增殖有抑制作用。结论 新基因PP3105可能是一个新的金属离子尤其是锌的转运蛋白，并且与肝癌细胞的生长、增殖相关。     

脑源性神经营养因子对肝癌细胞株HepG2体外转移能力的影响

背景与目的:肝细胞性肝癌(hepatocellularcarcinoma,HCC)浸润转移是临床治疗失败的最常见原因,浸润转移的发生与癌细胞的生物学行为(迁移、侵袭、增殖)密不可分。近年来,人们已认识到脑源性神经营养因子(brainderivedneurotrophicfactor,BDNF)作为一种功能性蛋白特异地存在于HCC组织中,并与HCC的进展密切相关,但其确切功能不明。本实验初步探讨BDNF对肝癌细胞株HepG2体外迁移能力的影响及其机制。方法:采用MTT法观察BDNF对HepG2细胞增殖的作用,应用肿瘤细胞体外迁移实验和肿瘤细胞体外侵袭实验评价BDNF对HepG2细胞浸润转移能力的影响。采用RT-PCR法和Westernblot法分别从基因水平和蛋白水平检测HepG2细胞中BDNF特异性受体Tr!B的表达情况。结果:外源性的BDNF能有效促进HepG2细胞的增殖,在<100ng/ml的范围内,这一效应呈明显的浓度依赖性;经过50ng/ml和100ng/mlBDNF处理的HepG2细胞,迁移指数明显高于对照组(167vs.100,203vs.100,P<0.05);Transwell侵袭实验中,50ng/ml和100ng/mlBDNF组的每视野侵袭细胞数分别为167±38和215±51,与对照组(113±22)相比差异有显著性(P<0.05);较之正常肝细胞,HepG2细胞高表达Tr"B。结论:BDNF可调节肝癌细胞的生长、迁移与侵袭,是具有促进肝癌浸润转移的细胞因子。     

沉默ANLN基因对胃癌细胞侵袭、迁移、增殖和凋亡的影响

目的 探讨肌动蛋白结合蛋白ANLN在体外对胃癌细胞的增殖、凋亡、迁移和侵袭的影响。方法 RT-PCR和Western blot检测胃癌细胞株、胃黏膜细胞株中ANLN的表达情况和验证siRNA对ANLN基因的敲减情况。划痕实验和Transwell检测沉默ANLN基因表达对胃癌细胞迁移、侵袭能力的影响。CCK-8实验、细胞荧光染色和流式细胞术检测沉默ANLN基因表达对胃癌细胞增殖和凋亡的变化。结果 胃癌细胞株SGC-7901/MGC-803中的ANLN的mRNA和蛋白表达显著高于正常胃黏膜细胞株GES-1（P<0.001）。siRNA干扰后RT-PCR和Western blot结果证明转染成功,转染组的mRNA和蛋白表达量与对照组差异有统计学意义（P<0.05）。下调ANLN能够显著降低胃癌细胞的迁移、侵袭、增殖和凋亡能力。结论 下调ANLN的表达可以显著降低胃癌细胞侵袭、迁移、增殖和凋亡能力。     

FAK反义寡聚脱氧核苷酸对FN诱导Hela细胞MMP-2表达的影响

目的：利用反义核酸技术探讨纤黏连蛋白(fibronectin，FN)诱导Hela细胞中基质金属蛋白酶(matrix nletalloproteinases，MMPs)基因表达的机制。方法：无血清培养Hela细胞。以不同浓度及作用时间的FN诱导Hela细胞中MMPs基因表达，并用反义寡聚脱氧核苷酸(oligodeoxynucleotides，ODN)封闭焦点黏着激酶(focal adhesion kinase.FAK)，用酶谱分析方法检测MMPs的活性。结果：FN浓度为0μg/mL时，Hela细胞无MMP-2基因表达；FN浓度为5μg/mL-10μg/mL、作用时间为12h，MMP-2活性最大；当在培养液反义ODN后，MMP-2活性明显降低。结论：MMP-2基因表达与FN的浓度以及作用时间有关；FN可通过其整合素受体的信号转导通路启动Hela细胞中MMP-2基因表达、降解ECM,从而促进肿瘤的浸润和转移。     

胃腺癌的免疫组化及粘液组化研究

 采用免疫金银染色方法，在70例胃腺癌中显示纤维连接蛋白(FN)及层粘连蛋白(LN)的分布。观察表明:部分胃腺癌细胞呈FN和/或LN阳性反应。高、中分化腺癌细胞浆FN阳性率略低于低分化腺癌及粘液腺癌，细胞表面FN阳性率高于胞浆。癌细胞中FN与LN的分布明显相关。肿瘤间质中存在丰富的FN。LN则主要存在于癌巢及血管基底膜上。癌细胞浆中FN阳性率与粘液染色分型明显相关，胃型胃癌最低，肠型胃癌最高。结合临床资料分析，癌细胞FN、LN阳性率与肿瘤浸润、转移，发生部位及肉眼类型无关。     

乳腺

乳腺原发癌和转移淋巴结肿瘤浸润淋巴细胞体外抗乳腺癌的研究；survivin基因的RNA干涉抑制人乳腺癌SKBr-3细胞体外增殖的作用；细胞因子对乳腺癌细胞钠碘转运体基因表达的调控；乳腺增生性病变中p63蛋白的表达及意义；尿激酶型纤溶蛋白酶激活剂在浸润型乳腺癌中高表达的临床意义；磁共振成像动态增强对乳腺癌血管生成的研究；乳腺癌根治术与改良根治术对患侧肩关节功能的影响。[编者按]     

磷脂酰肌醇蛋白聚糖-3基因对肝癌细胞SK-Hep-1黏附和侵袭的影响

摘 要 目的: 肝细胞癌中高表达磷脂酰肌醇蛋白聚糖3基因(glypican3,GPC3)，而在非肿瘤肝组织、肝细胞腺瘤、胆管癌、肝内胆管细胞癌、胆囊癌等细胞中低表达甚至不表达；本研究利用携带GPC3重组真核表达载体，探讨GPC3基因对SKHep1肝癌细胞增殖、黏附和侵袭能力的影响。方法：将pEGFP-N2-GPC3通过脂质体方法转染人肝癌细胞SKHep1。RT-PCR检测GPC3SKHep1细胞中GPC3 mRNA的表达；MTT法检测SK-Hep-1细胞的增殖并计算黏附率；Transwell小室实验检测SK-Hep-1肝癌细胞的迁移能力和侵袭能力。结果：pEGFPN2GPC3成功转染SKHep1细胞，转染后GPC3-Hep-1细胞明显表达GPC3mRNA。GPC3转染能显著抑制肝癌细胞SK-Hep-1的增殖（P<0.01）；GPC3转染细胞的黏附能力较对照细胞显著下降[（10.21±0.62）% vs (15.51±095)%,P<0.01];GPC3转染细胞的迁徙和侵袭能力较对照细胞明显增强[（131.7±7.44）vs（69.6±5.25），P<0.01;(220±12.8) vs （130±8.2），P< 0.01]。结论：GPC3基因显著抑制肝癌细胞的增殖和黏附能力，但显著增强后者的迁移和侵袭能力。     

miR-4465 通过靶向下调HMGA1的表达抑制肝细胞癌Hep3B细胞的恶性生物学行为

目的：探讨miR-4465 靶向高迁移率族蛋白A1（HMGA1）对肝细胞癌 Hep3B 细胞增殖、迁移和侵袭能力的影响。方法：收集2020 年5 月至2021 年9 月在皖南医学院第一附属医院确诊为肝细胞癌患者的16 对癌组织和癌旁组织样本，采用qPCR 分析miR-4465 在肝细胞癌组织和Hep3B、Huh7细胞中的表达情况，双荧光素酶报告基因实验验证miR-4465 与HMGA1的调控关系。按转染物的不同将 Hep3B 细胞分为 mimics-NC 组 、miR-4465-mimics 组、inhibitor-NC 组、miR-4465 inhibitor组、si-NC 组、si-HMGA1 组；另外分组转染mimics-NC+pcDNA-NC、miR-4465 mimics+pcDNA-NC 和miR-4465 mimics+pcDNA-HMGA1进行回复实验。采用qPCR和WB法检测各组细胞中HMGA1 mRNA和蛋白水平的变化，CCK-8法检测各组细胞增殖能力的变化，划痕实验检测各组细胞迁移能力的变化，Transwell 实验检测各组细胞侵袭能力的变化。结果：miR-4465 在肝细胞癌组织和细胞中的表达水平显著低于癌旁组织和正常肝细胞（P<0.05或P<0.001）。转染48 h后，过表达miR-4465 的Hep3B细胞增殖、迁移和侵袭能力均显著下降（P<0.05、P<0.01或P<0.001）；敲低miR-4465 后细胞的增殖、迁移和侵袭能力均明显升高（P<0.05、 P<0.01或P<0.001）。双荧光素酶报告实验验证了HMGA1-3''UTR 与miR-4465 的靶向结合关系，miR-4465 可以靶向下调HMGA1 mRNA 和蛋白质的表达（均P<0.01）。过表达HMGA1 能部分逆转过表达 miR-4465 对细胞增殖、迁移、侵袭的抑制作用及HMGA1 表达的抑制作用（均P<0.05）。结论：miR-4465 通过靶向下调HMGA1 在肝细胞癌Hep3B 细胞中的表达，从而抑制Hep3B细胞的恶性生物学行为。     

Inhibition of hepatocarcinoma and tumor metastasis to liver by gene therapy with recombinant CBD-HepII polypeptide of fibronectin

Unlike the intact fibronectin (FN) molecule, some proteolytic or recombinant fragments of FN possess inhibitory activities on tumor, providing potential strategies in tumor therapeutics. Using the hydrodynamics-based gene delivery technique, we demonstrated that the treatment by in vivo expression of a recombinant CBD-HepII polypeptide of FN, designated as CH50, strongly inhibited the tumor growth, tumor invasion and angiogenesis. Such inhibitory effects of CH50 on tumor were partly ascribed to its influence on the activities of MMP-9 and alphavbeta3 integrin. The in vivo expressed CH50 decreased both the production and the activity of MMP-9 in tumor tissues. CH50 also down-regulated alphavbeta3 expression in tumor cells and endothelial cells in vitro. The decreased activity of alphavbeta3 integrin was proved by its reduced binding ability to fibrinogen and the down-regulation of cdc2 expression. The gene therapy with CH50 not only prolonged the survival of mice bearing hepatocarcinoma in the liver, but also suppressed the growth and invasive ability of tumor in spleen and its metastasis to liver. Taken together, these findings suggest a prospective utility of CH50 in the gene therapy of liver cancer.     

miR-122 Inhibits Hepatocarcinoma Cell Progression by Targeting LMNB2

In the present study, we investigated the role of miR-122 in hepatocarcinoma progression and explored the mechanism. In hepatocarcinoma tissues and cells, we used qRT-PCR to validate the miR-122 expression level. Next, we used colony formation by crystal violet staining assay to compare cell proliferation ability, and we used scratch test or Transwell assay to compare cell migration or invasion ability. We then conducted bioinformatics or luciferase reporter gene assay to prove the regulation effect of miR-122 on lamin B2 (LMNB2), and the biological function of LMNB2 was analyzed. We used nude mouse tumorigenicity assay to test the inhibition effect of miR-122 ASO therapy against hepatocarcinoma. miR-122 was reduced in hepatocarcinoma tissues compared to the paracarcinoma tissues, which was relatively low or high in hepatocarcinoma cell line SMMC7721 or Hep3B, and overexpressed miR-122 inhibited proliferation, migration, and invasion in hepatocarcinoma cells. Additionally, some reports showed that LMNB2 was regulated by miR-122, which inhibited the expression of LMNB2. Moreover, LMNB2 functioned to promote cell proliferation, migration, and invasion. We could achieve the inhibition of hepatocarcinoma using miR-122 therapy through decreasing LMNB2 expression in vivo. Our data indicated that miR-122 could inhibit hepatocellular carcinoma cell progression by targeting LMNB2 and as a therapeutic target for hepatocarcinoma treatment.     

Notch1基因转染对人肝癌细胞Hep3B生长的影响及作用机制的研究

目的：研究逆转录病毒介导的Notch1（ICN）基因转染对人肝癌细胞生长的影响，并对其作用机制进行了探讨。方法： 采用磷酸钙沉淀法质粒共转染293T细胞，制备出表达组成性活化形式的Notch1（ICN）和/或绿色荧光蛋白（GFP）的逆转录病毒载体MSCVICN/GFP及对照逆转录病毒载体MSCVGFP，并检测病毒滴度。用逆转录病毒感染人肝癌细胞Hep3B，观察病毒的感染效率，MTT比色法测定瞬时表达Notch1（ICN）对Hep3B人肝癌细胞生长的影响，利用流式细胞仪分析细胞周期分布的变化，Western blot方法检测细胞周期调控蛋白的变化。结果：通过质粒共转染可获得具有较高滴度的重组逆转录病毒。MTT比色法显示瞬时表达Notch1（ICN）基因可以抑制人肝癌细胞Hep3B的生长，瞬时表达Notch1（ICN）基因可使Hep3B细胞周期停滞在G0/G1期并上调细胞周期调控蛋白P53的表达水平。结论：Notch1基因可通过影响细胞周期分布和P53蛋白的表达水平而抑制人肝癌细胞生长。     

miR-3612靶向SEMA4C调控肝细胞癌细胞的恶性生物学行为

目的：探讨miR-3612靶向信号素（SEMA）4C对肝细胞癌细胞增殖与侵袭能力的影响。方法:收集2020年5月至2021年9月间在皖南医学院第一附属医院弋矶山医院手术切除的肝细胞肝癌的40对癌组织和相应癌旁组织,常规培养肝细胞癌Hep3B和Huh7细胞,将其分为对照组、miR-3612 mimics-NC组、miR-3612 mimics组、miR-3612 inhibitor-NC组、miR-3612 inhibitor组、si-NC组、si-SEMA4C组、mimics-NC+pcDNA-NC组、miR-3612 mimics+pcDNA-NC组和miR-3612 mimics+pcDNA-SEMA4C组,用转染试剂将相应的核酸和质粒转染各组细胞。qPCR法检测miR-3612和SEMA4C mRNA在肝细胞癌组织和Hep3B和Huh7细胞中的表达,双荧光素酶报告基因实验和免疫共沉淀（RIP）实验验证miR-3612与SEMA4C的结合及调控关系,qPCR法和WB法检测转染后各组Hep3B和Huh7细胞中miR-3612、SEMA4C mRNA和蛋白的表达,CCK-8法、细胞划痕...     

Wnt7a对肝癌细胞凋亡、迁移及侵袭的影响

目的:检测Wnt7a在肝癌中的表达,分析Wnt7a对肝癌细胞活性、凋亡、迁移及侵袭的影响,探讨Wnt7a在肝癌中的作用。方法:分别采用免疫组织化学染色和Western blot检测组织和细胞系中Wnt7a的表达情况;Hep3B细胞经人重组Wnt7a蛋白（rWnt7a）处理后,MTT法检测细胞活性改变,流式细胞仪检测细胞凋亡变化,Transwell分析细胞迁移及侵袭能力变化。结果:相对于癌旁组织,肿瘤组织低表达Wnt7a蛋白;经rWnt7a处理后,Hep3B细胞活性降低、细胞凋亡增多且迁移与侵袭能力下降。结论:Wnt7a蛋白能抑制Hep3B细胞生长、迁移与侵袭能力,可能在肝癌中发挥抑癌作用。     

The melanoma-associated antigen A3 mediates fibronectin-controlled cancer progression and metastasis

Tumor cells frequently exhibit decreased adhesiveness due to failure to deposit stromal fibronectin (FN), permitting more rapid proliferation, migration, invasion, and metastasis. Although up-regulation of FN has been noted in gene profiles of carcinomas compared with normal tissue, reduced FN expression has been described at the peripheral margins of invading tumors. In this study, we investigate the role of FN in cancer behavior. Using human thyroid carcinoma cells with stably down-regulated FN, we performed gene profiling and created an orthotopic mouse model. We stably overexpressed the FN target, MAGE A3, which has also been identified as a target of the breast cancer risk factor fibroblast growth factor receptor 2, and examined the functional effects in vitro and in vivo in a flank model and an orthotopic model of thyroid cancer. Mouse xenografts showed significantly enhanced tumor growth as well as larger and more numerous lung metastases in response to FN silencing. Gene profiling identified the melanoma-associated antigen (MAGE A3) as significantly up-regulated in response to FN silencing. Forced expression of MAGE A3 resulted in p21 down-regulation, accelerated cell cycle progression, increased cell migration rate, and invasion in vitro and in vivo in an orthotopic mouse model where microcomputed tomography confirmed lung metastases that recapitulate the progression of human thyroid cancer. We conclude that MAGE A3 is a functional integrator of diverse signals, including FGFR2 and FN, to modulate cancer progression.     

Synergistic effects of genetically engineered stem cells expressing cytosine deaminase and interferon-β via their tumor tropism to selectively target human hepatocarcinoma cells

Stem cells have received a great deal of attention for their clinical and therapeutic potential for treating human diseases and disorders. Recent studies have shown that it is possible to genetically engineered stem cells (GESTECs) to produce suicide enzymes that convert non-toxic prodrugs to toxic metabolites, selectively migrate toward tumor sites and reduce tumor growth. In this study, we evaluated whether these GESTECs are capable of migrating to hepatocarcinoma cells and examined the potential therapeutic efficacy of gene-directed enzyme prodrug therapy against liver cancer cells in cellular and animal models. A modified transwell migration assay was performed to determine the migratory capacity of GESTECs to Hep3B hepatocarcinoma cells. GESTECs, that is, HB1.F3.CD or HB1.F3.CD.interferon-β (IFN-β) cells, engineered to express a suicide gene, cytosine deaminase (CD), selectively migrated toward liver cancer cells. Treatment of Hep3B, human liver cancer cells, with the prodrug 5-fluorocytosine (5-FC) in the presence of HB1.F3.CD or HB1.F3.CD.IFN-β cells resulted in the inhibition of Hep3B cell growth. In a xenografted mouse model injected with hepatocarcinoma, we investigated the therapeutic effect of these stem cells. For 9 weeks, the xenografted mice were treated with HB1.F3.CD or HB1.F3.CD.IFN-β in the presence of 5-FC. A growth of tumor mass was inhibited about 40-50% in the mice treated with GESTECs and a prodrug. In addition, we further confirmed the cytotoxic effect on tumor cells by histological analysis and migratory effect of therapeutic stem cells. Taken together, GESTECs expressing a fusion gene encoding CD and IFN-β may exert a synergistic antitumor effect on this type of tumor.     

Soluble fibronectin promotes migration of oral squamous-cell carcinoma cells

We have shown that a fibronectin (FN) matrix is required for the organization of tenascin-C (TN-C) matrices by peritumor fibroblasts (PTF) cultured from tissue surrounding oral squamous-cell carcinoma (SCC). In the present study, we detected alternatively spliced FN containing both the EDA and EDB domains decorating the reactive stroma adjacent to the invading tumor nests in oral SCC biopsies. In vitro, PTF cells organized an extensive FN matrix rich in the EDA domain and containing a small amount of EDB. In contrast, normal human fibroblasts deposited a FN matrix which expressed only the EDA domain. PTF-conditioned medium (CM), shown to enhance migration of oral SCC cells on TN-C, was found to enhance their migration on FN and invasion of a reconstituted basement membrane. Addition of antibodies to FN to the PTF-CM inhibited SCC-cell migration on TN-C, and depletion of FN from the PTF-CM abolished its ability to induce migration or invasion by oral SCC cells, suggesting that FN promotes the migration and invasion of oral SCC cells. Western blots of the PTF-CM identified FN containing the EDA but not the EDB domain. When soluble FN was added to the control medium in the lower chamber of the Transwell system, SCC-cell migration increased significantly. These results demonstrate that both the EDA and the EDB domains of FN are expressed in the extracellular matrix of oral SCC in vivo and PTF in vitro and indicate that FN is the probable chemotactic factor in the PTF-conditioned medium. Int. J. Cancer 78:261–267, 1998.© 1998 Wiley-Liss, Inc.