BAMBI基因过表达人结肠癌细胞移植斑马鱼致肝转移模型的构建

目的构建BAMBI基因过表达人结肠癌细胞移植斑马鱼肝转移模型,分析斑马鱼肝内转移性结肠癌细胞BAMBI基因表达水平与肝转移发生率的关联性。方法分别构建BAMBI过表达稳转结肠癌细胞株SW620(BAMBI+组)、空载体红色荧光蛋白基因转染结肠癌细胞株SW620(EV组),分别显微注射到受精后第2天转基因斑马鱼Tg(Apo14∶GFP)胚胎卵黄囊(每组各680条),激光共聚焦荧光显微镜观察、拍摄,采用Image J软件测量转移至斑马鱼肝脏内结肠癌细胞红色荧光表达强度,计算肝转移发生率;采用t检验比较BAMBI+组及EV组斑马鱼肝脏内结肠癌细胞红色荧光表达强度及肝转移发生率的差异。结果与EV组相比,激光共聚焦荧光显微观察显示BAMBI+组斑马鱼肝内结肠癌细胞红色荧光表达量显著增加(t=6.247,P=0.003 3);BAMBI+组斑马鱼肝转移发生率与EV组相比显著增加(t=4.276,P=0.012 9)。结论构建了基因修饰人结肠癌细胞移植斑马鱼致肝转移模型,可较好地模拟活体状态的结肠癌肝转移;BAMBI基因可促进异种移植入斑马鱼的结肠癌细胞发生肝转移。     

姜黄素体外抑制人结肠癌癌细胞Caco-2增殖

目的研究姜黄素在体外对人结肠癌细胞Caco-2生长增殖及其抗肿瘤的相关分子机制。方法体外培养人结肠癌细胞Caco-2,四甲基偶氮唑盐(MTT)法检测姜黄素在相同浓度下、不同时间对于结肠癌细胞增殖的影响,并计算半数抑制量(IC50)值;反转录聚合酶链式反应(RT-PCR)法检环氧合酶-2(COX-2)、基质金属蛋白酶-9(MMP-9)的mRNA的表达影响。结果 MTT:相同干预浓度姜黄素抑制人结肠癌细胞增殖,其24 h,48 h,72 h的IC50分别为:205.737±14.929μmol/L、102.023±6.809μmol/L、86.627±9.005μmol/L;RT-PCR:人结肠癌细胞Caco-2正常对照组及姜黄素干预组MMP-9 mRNA灰度值分别为:0.786±0.101、0.310±0.046,P<0.05;COX-2 mRNA灰度值分别为0.830±0.173、0.063±0.035,(P<0.05)。结论姜黄素可能通过抑制COX-2及MMP-9的mRNA的表达来抑制结肠癌细胞Caco-2增殖。     

MMP-3在人大肠癌组织中的表达及意义

目的:检测基质金属蛋白酶-3(MMP-3)在人大肠癌组织中的表达,以探讨其与人结肠癌进程的相关性。方法:应用光镜、电镜对结肠癌组织进行形态学观察,用免疫组化方法检测MMP-3的表达。结果:结肠癌晚期组MMP-3表达水平高于早期组,差异有非常显著性(P<0.01)。电镜观察可见在结肠癌早期,淋巴细胞和树突状细胞浸润较多,癌细胞穿基膜不明显;晚期,淋巴细胞和树突状细胞浸润较少,癌细胞穿基膜明显。结论:MMP-3在大肠癌晚期组织中的表达高于早期。提示MMP-3与结肠癌进程有直接关系。MMP-3的表达程度可作为判定结肠癌侵袭、转移和预后的指标。     

大肠癌肝转移的外科治疗23例分析

对我科近4 a来手术治疗大肠癌肝转移20例分析如下. 　　1临床资料 　　1.1一般资料本组男14例,女9例,年龄38～72(平均55)岁.原发病灶结肠癌10例,直肠癌10例;原发癌与肝转移癌灶同时发现者11例.原发癌根治与肝转移癌灶同时切除者7例,原发癌灶根治切除后,再切除肝转移灶8例,先切除肝转移灶,再切除原发病灶3例. 　　……     

塞来昔布抑制人结肠癌细胞Caco-2细胞增殖及其分子机制

目的研究塞来昔布在体外抑制人结肠癌细胞Caco-2生长增殖及其抗肿瘤的相关分子机制。方法体外培养人结肠癌细胞Caco-2,分组为正常组(无任何干预)及塞来昔布组。四甲基偶氮唑盐(MTT)法检测塞来昔布在相同浓度下,不同时间对于胃癌细胞增殖的影响,并计算半数抑制浓度(IC50)值;反转录聚合酶链式反应(RT-PCR)法检环氧化酶-2(COX-2)、基质金属蛋白酶9(MMP-9)的mRNA的表达影响。结果 MTT结果显示:相同干预浓度塞来昔布抑制人胃癌细胞增殖,其24 h,48 h,72 h的IC50分别为:99.519±10.355μmol/L、71.546±6.446μmol/L、59.622±15.999μmol/L;RT-PCR结果显示:人结肠癌细胞Caco-2正常对照组及塞来昔布干预组COX-2mRNA灰度值分别为:0.808±0.021,0.101±0.002(t=19.037,P=0.000);MMP-9 mRNA灰度值分别为:0.798±0.031,0.190±0.002(t=18.987,P=0.002)。结论塞来昔布可能通过抑制COX-2、MMP-9的mRNA的表达,从而抑制结肠癌细胞增殖。     

12例结肠癌肝转移同步切除护理体会

目的：探讨结肠癌肝转移同步切除术后护理要点。方法：回顾性分析我科近2年来开展的12例结肠癌肝转移同步切除术后病人的治疗效果和护理体会。结果：12例结肠癌肝转移病人行同步切除手术后,1例术后2个月出现肺转移死亡,2例术后5个月在另一肝叶出现肝转移灶而再次行肝转移灶切除,9例术后恢复良好。结论：结肠癌肝转移病人行同步切除,术后护理极其重要,严密的术后监控和护理是保证结肠癌肝转移病人手术成功的关键。     

结肠癌组织中MMP-2、MMP-9、CEACAM-1的表达水平及其临床意义

[目的]探讨结肠癌组织中的基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)、癌胚抗原相关细胞黏附分子1(CEACAM-1)表达水平及其临床意义.[方法]选取在本院接受结肠癌根治术的60例结肠癌患者,获取其癌组织、癌旁组织以及正常结肠组织样本并检测MMP-9、MMP-2和CEACAM-1的表达水平,分析临床...     

结肠癌肝转移中CD34、D2-40的表达及临床病理意义

目的:探讨结肠癌原发灶、肝转移灶及淋巴结转移灶中CD34和D2-40的表达,研究新生淋巴管的生成与结肠癌肝转移的关系。方法:应用免疫组化对52例有结肠癌肝转移患者的原发灶、肝转移灶及淋巴结转移灶中CD34及D2-40的表达进行检测,并对与肝转移相关的因素进行单因素分析。结果:52例结肠癌肝转移患者的原发灶、肝转移灶及淋巴结转移灶中新生淋巴管密度明显高于新生血管密度(F=129.83,P=0.0003);单因素分析显示,患者肿瘤的浸润深度与合并肝转移有关;血管浸润和淋巴管浸润可能是结肠癌肝转移重要的预测预后的因子。结论:在结肠癌肝转移发生发展过程中淋巴管生成可能起主要作用。     

3D腹腔镜肝Ⅶ段病灶前切除术22例疗效分析

目的 探讨3D腹腔镜肝Ⅶ段病灶前切除术的临床疗效及经验.方法 回顾性分析2016年1月-2019年12月22例在金华市中心医院行3D腹腔镜下肝Ⅶ段病灶前切除术患者的临床资料,其中原发性肝癌11例,结肠癌肝转移1例,血管瘤8例,血管平滑肌脂肪瘤1例,肝细胞局灶性结节性增生1例.统计分析所有患者的手术时间、术中出血量、术后...     

茶多酚对人结肠癌细胞株Caco-2凋亡及RhoA蛋白活性的影响

目的探讨茶多酚对人结肠癌细胞株Caco-2凋亡及RhoA蛋白活性的影响。方法将传代后的人结肠癌细胞株Caco-2细胞分为实验组和对照组,实验组加入不同浓度的茶多酚（25、50、100和200μmol／L）,对照组加入等量RPMI-1640培养液,采用四甲基偶氮唑盐（MTr）法测定茶多酚对Caco-2增殖抑制的影响,流式细胞术（FCM）检测细胞凋亡指数,Pull．down法检测人结肠癌细胞株Caco-2细胞内的RhoA蛋白活性。结果不同浓度的茶多酚对人结肠癌细胞株Caco-2有明显的抑制作用,且呈剂量及时间依赖性;Pull-down法检测结果显示,茶多酚可以降低人结肠癌细胞株Caco-2细胞内的RhoA蛋白活性,且随浓度增加其蛋白活性降低。结论茶多酚可促进人结肠癌细胞株Caco-2的凋亡,其可能通过降低RhoA蛋白活件来实现。     

雌激素对结肠癌肝转移细胞增殖与凋亡及MMP-9表达的影响

目的 研究雌二醇（E2）对结肠癌肝转移肿瘤细胞增殖与凋亡及MMP-9表达的影响.方法 实验动物选用裸鼠,随机分为四组（n=6）：雄性组、雌性组、卵巢切除组、卵巢切除并雌激素注射组.培养MCA-38细胞至对数生长期,制成单细胞悬液,采用门静脉注射法构建裸鼠结肠癌肝转移模型.应用免疫组化SP法检测转移灶中PCNA和Caspase-3表达,计算肿瘤细胞增殖率（CPI）和凋亡率（CAI）,Western blot 法检测转移灶中MMP-9表达量.结果 （1）雄性组细胞的CPI最高（0.912±0.023）,其次为卵巢切除组（0.876±0.031）.雄性组与雌性组之间的CPI差异（=7.30,P＜0.05）以及卵巢切除组与卵巢切除注射雌激素组之间的CPI差异（t=2.74,P＜0.05）有统计学意义.（2）雌性组肿瘤细胞的CAI最高（0.064±0.010）,其他各组依次为卵巢切除注射雌激素组（0.049 ±0.007）、雄性组（0.038 ±0.003）和卵巢切除组（0.023±0.011）.雄性组与雌性组之间的CAI差异（t=4.72,P＜0.05）以及卵巢切除组与卵巢切除注射雌激素组之间的CAI差异（t=5.872,P＜0.05）有统计学意义.（3）雄性组肝转移灶MMP-9表达量最高（0.62±0.05）,其他各组依次为卵巢切除组（0.49±0.03）、雌性组（0.36±0.03）和卵巢切除注射雌激素组（0.28±0.04）.雄性组与雌性组之间的MMP-9表达量差异（t=8.462,P＜0.05）、卵巢切除组与卵巢切除注射E2组之间的MMP-9表达量差异（t=7.620,P＜0.05）以及雌性组与卵巢切除组之间的MMP-9表达量差异（t=5.324,P＜0.05）有统计学意义.结论 结直肠癌肝转移灶细胞凋亡与体内雌激素水平增加有关,转移灶细胞增殖与体内雌激素水平减低有关,MMP-9可能参与其调控机制.     

Gab2对结肠癌细胞细胞外黏附作用的影响

;目的探讨Gab2对结肠癌细胞外黏附作用的影响。方法选取SW620和HCT116两种结肠癌细胞系,建立Gab2表达降低和Gab2表达升高的稳定细胞株。将实验NOD/SCID小鼠分为3组,每组10只。(1)对照组;接种scr/MGC803体结肠癌细胞;(2)Gab2降低组;接种Gab2降低的siGab2/MGC803结肠癌细胞;(3)Gab2升高组;接种Gab2升高的Gab2/MGC803结肠癌细胞。检测细胞迁移能力,测量肿瘤的大小和侵袭范围及细胞凋亡情况。结果处理后3组细胞迁移数计算结果显示,Gab2降低组的细胞迁移数显著低于Gab2升高组和对照组(P<0.05)。各组NOD/SCID小鼠均在接种40 d后处死,比较接种前与接种后小鼠体质量,Gab2降低组显著低于Gab2升高组、对照组,对照组低于Gab2升高组。测量瘤体积,Gab2升高组高于Gab2降低组、对照组。Gab2降低组瘤体积抑制率显著高于对照组(P<0.05)。相关分析发现,MMP-2、MMP-9蛋白的表达与Gab2蛋白表达呈正相关。结论 Gab2可有效调控体外细胞迁移数,显著抑制小鼠结肠癌细胞侵袭及转移,通过调节MMP-2、MMP-9蛋白表达,从而抑制结肠癌生长及转移。     

人羊膜上皮细胞移植急性肝损伤小鼠的定量效果分析

背景：目前已有较多关于人羊膜上皮细胞移植入动物体内的存活、迁徙及相关特性的初步研究,但其对移植效果的定量分析尚未见报道。目的：对脾内移植传代的人羊膜上皮细胞小鼠血清肝生化功能及人血白蛋白的定量分析。方法：40只裸小鼠随机分为4组,每组各10只。肝叶切除＋细胞移植2周组、肝叶切除＋细胞移植4周组、肝叶切除＋盐水组,行半肝叶切除,肝叶切除＋细胞移植组自脾下极移植密度为5×106传代的人羊膜上皮细胞约0.2mL,分别于移植后2周和4周采血;肝叶切除＋盐水组自脾下极注射生理盐水0.2mL;单纯细胞移植组：不行肝叶切除,自脾下极移植密度为5×106传代的人羊膜上皮细胞约0.2mL。检测其各组肝脾组织学、形态学的改变及各组血清谷丙转氨酶、谷草转氨酶、人血白蛋白的变化和人血白蛋白表达定量分析。结果与结论：人羊膜上皮细胞移植急性肝损伤小鼠4周后肝脾形态未见明显改变,组织学可检测到特异性细胞,血清谷丙转氨酶、谷草转氨酶、人血白蛋白有明显改善,血清中能检测到人血白蛋白且移植后4周较移植后2周有明显升高。因此,人羊膜上皮细胞移植入肝受损小鼠体内能存活超过4周且仍表达肝细胞样细胞的部分特性及功能,改善小鼠的肝功能,治疗小鼠急性肝损伤。     

Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis.

To investigate the relationship between angiogenesis and hepatic tumorigenesis, we examined the expression of vascular endothelial growth factor (VEGF) in 8 human colon carcinoma cell lines and in 30 human colorectal cancer liver metastases. Abundant message for VEGF was found in all tumors, localized to the malignant cells within each neoplasm. Two receptors for VEGF, KDR and flt1, were also demonstrated in most of the tumors examined. KDR and flt1 mRNA were limited to tumor endothelial cells and were more strongly expressed in the hepatic metastases than in the sinusoidal endothelium of the surrounding liver parenchyma. VEGF monoclonal antibody administration in tumor-bearing athymic mice led to a dose- and time-dependent inhibition of growth of subcutaneous xenografts and to a marked reduction in the number and size of experimental liver metastases. In hepatic metastases of VEGF antibody-treated mice, neither blood vessels nor expression of the mouse KDR homologue flk-1 could be demonstrated. These data indicate that VEGF is a commonly expressed angiogenic factor in human colorectal cancer metastases, that VEGF receptors are up-regulated as a concomitant of hepatic tumorigenesis, and that modulation of VEGF gene expression or activity may represent a potentially effective antineoplastic therapy in colorectal cancer.     

Platelet aggregation-induced by caco-2 cells: regulation by matrix metalloproteinase-2 and adenosine diphosphate

Formation of tumor cell-platelet aggregates facilitates hematogenous metastases. However, molecular mechanisms implicated in tumor cell-induced platelet aggregation (TCIPA) in colon cancer are unclear. To investigate mechanisms of TCIPA induced by colon adenocarcinoma cells in vitro, human Caco-2 cells were used to study their interactions with platelets using aggregometry, zymography, phase-contrast microscopy, and flow cytometry. Caco-2-induced platelet aggregation in a concentration-dependent manner. This aggregation resulted in the release of matrix metalloproteinase (MMP)-2, as measured by zymography. In addition, flow cytometry showed a significant up-regulation of activated GpIIb/IIIa, total GpIIb/IIIa, GpIb, and P-selectin receptors on platelets. Inhibition of MMP-2 by phenantroline and degradation of ADP by APT102, respectively, resulted in inhibition of TCIPA. Furthermore, both phenantroline and APT102 significantly down-regulated the surface abundance of platelet receptors. Caco-2 cells aggregate platelets, at least in part, via releasing MMP-2 and ADP. Modulation of MMP-2 and ADP actions could have therapeutic value in colonic cancer.     

微波消融、TACE与全身化疗治疗结直肠癌肝转移的疗效

[目的]探讨微波消融、介入栓塞化疗(TACE)序贯方案与全身化疗治疗结直肠癌肝转移的临床疗效.[方法]根据不同治疗方案将80例结直肠癌肝转移患者分为观察组(全身化疗+微波消融+TACE序贯治疗)与对照组(全身化疗),比较两组近期疗效,预后及治疗前后细胞免疫功能变化.[结果]观察组近期客观有效率为66.67％(28/42),高于对照组的42.10％(16/38),且差异有显著性(P<0.05);观察组首次治疗后CD3+、CD4+阳性率、CD4+/CD8+比值均显著大于,CD8+显著小于同组治疗前、对照组治疗后(均P<0.05);观察组中位存活时间26.00个月长于对照组的13.60个月(P<0.05).[结论]在全身化疗基础上,行微波消融+TACE治疗能显著提高结直肠癌肝转移患者的有效率,明显改善机体免疫功能,延长患者存活时间.     

Prolonged survival of mice with multiple liver metastases of human colon cancer by intravenous administration of replicable E1B-55K-deleted adenovirus with E1A expressed by CEA promoter.

Liver is the most preferential site for metastasis of colon cancer. We, in the present study, constructed a self-replicable adenovirus in which E1A is driven by a CEA promoter and E1B-55K is deleted from the E1B region (AdCEAp/Rep) and examined its effects on multiple metastases of a human colon cancer cell in a mouse xenograft model. We first showed effective replication of the virus in various CEA-producing human colon cancer cells (M7609, HT-29) and subsequent lysis of the infected cells in vitro. We then demonstrated that a single intratumoral injection of the virus (1 x 10(8) PFU/100 microl) induced a complete regression of subcutaneous tumors (M7609) inoculated into nude mice. Further, we demonstrated that systemic administration of the virus (1 x 10(8) PFU/100 microl) through the tail vein to nude mice, which 1 week prior had been inoculated with tumor cells (colon carcinoma cell line HT-29) via the spleen and showed apparent multiple metastases in the liver, effectively suppressed the metastasis formation. The mean survival time of the treated mice was significantly longer than that of the controls. Thus, the systemic administration of AdCEAp/Rep was considered to be effective on multiple liver metastases of CEA-positive colon cancer in a xenograft model.     

siRNA沉默细胞周期相关激酶基因对鼠结肠癌生长及其细胞凋亡的影响

目的探讨siRNA沉默细胞周期相关激酶(CCRK)基因对鼠结肠癌生长及其凋亡的影响。 方法将50只BALB/C(nu/nu)裸鼠皮下接种人类结肠癌SW480细胞,将其中建立移植瘤裸鼠模型成功的30只裸小鼠按照单纯随机抽样方法随机分为3组,分别接种siRNACCRK转染的人类结肠癌SW480细胞株(实验组)、正常培养的SW480细胞(空白对照组)和以转染携带无关序列片段sh RNA慢病毒的SW480细胞(阴性对照组)。第42天处死裸鼠后取下肿瘤组织,比较各组肿瘤重量、CCRKmRNA表达量、CCRK蛋白表达和细胞凋亡的变化。 结果转染siRNA组肿瘤重量、CCRK mRNA和CCRK蛋白表达[(0.54±0.15)g,1.14±0.24和0.18±0.05]表达显著低于空白对照组[(1.05 ± 0.14)g,2.41±0.42和0.49±0.07]和空siRNA载体组[(1.07 ±0 .12)g,2.39±0.47,0.47±0.09;F＝21.18,23.47,90.03;P<0.01];空白对照组与空siRNA载体组比较差异无统计学意义(q＝0.98,1.07,1.13;P>0.05)。实验组沉默CCRK基因后组织中细胞凋亡率为(21.23±1.12)%,明显高于空白对照组(6.78±0.37)%和阴性对照组(7.25±0.32)%,差异有统计学意义(F＝56.936,P<0.01);但空白对照组和空siRNA载体组之间差异无统计学意义(q＝1.78,P>0.05)。 结论CCRK靶向siRNA表达载体接种结肠癌裸鼠后能显著降低结肠癌瘤体的增长,抑制CCRK基因和蛋白表达;siRNA沉默CCRK基因能促进其凋亡,CCRK基因有望成为结直肠癌治疗的新靶点。     

Mucin production by human colonic carcinoma cells correlates with their metastatic potential in animal models of colon cancer metastasis.

Patients with mucinous colorectal cancers characteristically present with advanced disease, however, the relationship between mucin production by colon cancer cells and their metastatic potential remains unclear. We therefore sought to define the relationship between mucin production by human colon cancer cells and metastatic ability by employing animal models of colon cancer metastasis. LS LiM 6, a colon carcinoma cell line with high liver metastasizing ability during cecal growth in nude mice produced twofold more metabolically labeled intracellular mucin and secreted four- to fivefold more mucin into the culture medium compared to poorly metastatic parental line LS174T. This was accompanied by a similar elevation in poly(A)+ RNA detected by blot hybridization with a human intestinal mucin cDNA probe, and increases in mucin core carbohydrate antigens determined immunohistochemically. Variants of LS174T selected for high (HM 7) or low (LM 12) mucin synthesizing capacity also yielded metastases after cecal growth and colonized the liver after splenic-portal injection in proportion to their ability to produce mucin. Inhibition of mucin glycosylation by the arylglycoside benzyl-alpha-N-acetyl-galactosamine greatly reduced liver colonization after splenic-portal injection of the tumor cells. These data suggest that mucin production by human colon cancer cells correlates with their metastatic potential and affects their ability to colonize the liver in experimental model systems.