Patrinia scabiosaefolia Inhibits Growth of 5-FU-Resistant Colorectal Carcinoma Cells via Induction of Apoptosis and Suppression of AKT Pathway

Objective: To investigate the effects of ethanol extract of Patrinia scabiosaefolia(EEPS) on chemo-resistance of colorectal cancer cells(CRC) and explore the possible molecular mechanisms. Methods: 5-fluorouracil(5-FU)-resistant human colorectal carcinoma cell line(HCT-8/5-FU) and its parental cells HCT-8 were treated with EEPS(0, 0.25, 0.50, 1 or 2 mg/mL), or 5-FU(0, 100, 200, 400, 800 or 1600 μmol/L). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was performed to evaluate the cell viability. Cell density was observed by phase-contrast microscope, cell counting and colony formation assay were used to determine the cell proliferation of HCT-8/5-FU cells treated with 0, 0.5, 1 or 2 mg/mL EEPS. Cell apoptosis was determined by Hoechst staining. Western-blot was performed to detect the phosphorylation of AKT as well as the protein expression level of B-cell CLL/lymphoma 2(Bcl-2) and Bcl-2-associated X protein(Bax). Results: Compared with HCT-8 cells, MTT assay results indicated that HCT-8/5-FU cells were resistant to 5-FU treatment(P0.05), and sensitive to EEPS treatment(P0.05). Moreover, compared with untreated HCT-8/5-FU cells, 1 and 2 mg/mL of EEPS treatment significantly reduced cell density, cell number, inhibited cell survival(P0.05), and induced apoptosis in HCT-8/5-FU cells. Furthermore, 1 and 2 mg/mL of EEPS significantly decreased the phosphorylation level of p-AKT and Bcl-2 protein expression, and increased the expression of Bax protein(P0.05). Conclusion: EEPS is a promising therapeutic agent that may overcome chemo-resistance in cancer cells, likely through suppression of the AKT pathway and promotion of cancer cell apoptosis.     

Reduction of G0 phase cells of colon cancer caco-2 cells may enhance 5-fluorouracil efficacy

Objective：A major problem in the chemotherapy of colon caner may be due to those cells that are in residence in the G0 phase where they are less vulnerable to conventional therapy. To overcome this phenomenon, we attempted to recruit the reentry of these cells into the cell cycle via a signaling pathway that manipulates tumor growth. Methods: Epidermal growth factor (EGF) was used to stimulate colon cancer caco-2 cells. FACS analysis and proliferating cell nuclear antigen (PCNA) staining were used to estimate the cell cycle transition and cell proliferation activated by EGF, and a MTT assay was used to evaluate the synergistic effect of EGF and chemotherapy. Results: The percentage of caco-2 cells in the G0/G1 phase was significantly reduced by nearly 20% and the percentages in the S and G2/M phases were increased by EGF. The combined use of EGF and 5-fluorouracil (5-FU) enhanced the caco-2 cell chemosensitivity to 5-FU, reaching a maximum of an approximately threefold greater sensitivity than to 5-FU alone as judged by the 50% inhibiting concentration (IC50). Conclusion: Our study demonstrated that stimulation by EGF enhanced the chemosensitivity of caco-2 cells to 5-FU, which may be a novel therapeutic protocol in colon cancer.     

生存素反义核酸抑制肝癌细胞增殖及对5-FU的增敏作用

目的 探讨生存素反义核酸抑制肝癌细胞增殖及对5-FU的增敏作用.方法 WST法,台盼蓝拒染法、克隆形成抑制实验检测聚乙烯亚胺携带生存素反义核酸(PEI-ASODN)对SMMC-7721细胞的增殖抑制作用.建立小鼠肝癌腋下移植瘤和腹水瘤模型,检测PEI-ASODN联合5-FU对肝癌移植瘤小鼠瘤重、瘤体积的改变,对肝癌腹水瘤小鼠平均存活天数的影响.结果 不同浓度的PEI-ASODN作用于SMMC-7721细胞48 h后,0.75 μmol/L的PEI-ASODN分别作用细胞24、48、72和96 h后,均能对细胞产生明显的抑制作用;PEI-ASODN对细胞的克隆形成也有显著的抑制作用,与细胞对照组相比有显著的统计学差异(P<0.01 ).肝癌腋下移植瘤小鼠,各治疗组的瘤重、瘤体积均得到不同程度的抑制.其中以联合用药组(5-FU+PEI-ASODN)最明显,瘤重抑瘤率高达56.91%,瘤体积抑制率高达57.83%;肝癌腹水瘤小鼠模型,联合用药组生命延长率为94.09%,与生理盐水组相比,具有显著性差异(P<0.01).结论 聚乙烯亚胺携带生存素反义核酸(PEI-ASODN)可显著抑制肝癌细胞生长,并可提高荷肝癌小鼠对5-FU的敏感性.

Abstract：

Objective To investigate the inhibitory effect of survivin antisense oligodeoxynuleotides (ASODN) mediated by polyethylenimine (PEI) on the proliferation of hepatocelluar carcinoma SMMC-7721 cells, and assess its detect on the chemosensitivity of the cells to 5-FU. Methods The inhibitory effect of PE1-ASODN on SMMC-7721 cell proliferation was assessed using WST-8 test, trypan blue staining, and cell clone formation test. In mice bearing transplanted hepatocarcinoma and ascites tumor derived from H22 cells, 5-FU combined with PEI-ASODN was administered, and the weight and volume of the subcutaneous tumors were measured to calculate the tumor inhibition rate, and the average survival time of the mice was calculated. Results Incubation of the cells with different concentrations of PEI-ASODN for 48 h significantly inhibited the cell proliferation as compared with the control group, but PEI or ASODN alone produced no significant inhibitory effects. At 24, 48, 72, 96 h of incubation of the SMMC-7721 cells with 0.75 μmol/L PEI-ASODN, the cell proliferation was suppressed significantly, and incubation with PEI-ASODN at 0.25-0.75 μmol/L for 7 days resulted in significantly inhibited cell clone formation. No significant inhibition was detected in ASODN and PEI group. The tumor weight and volume were reduced in all the treated groups. The tumor inhibition rate was 56.91% and volume inhibition rate was 57.83% in 5-FU+PEI-ASODN group, significantly different from those in the normal saline group (P<0.01). In mice bearing ascites tumor, the average survival time was 22.0 days in saline group, and 42.7 days 5-FU+PEI-ASODN group. The life-prolongation rate of 5-FU+PEI-ASODN was 94.09% when compared with the survival time in saline group. A cooperative effect was detected between 5-FU and PEI-ASODN. Conclusion PEI-ASODN complex can significantly inhibit the proliferation of hepatocarcinoma SMMC-7721 cells and enhance the chemosensitivity of the tumor cells to 5-FU.     

Mechanism of apoptotic effects induced by 5-fluorouracil on human liver carc inoma Bel7402 cell line

Objective To evaluate the effect of endogenous nitric oxide (NO) on the ability of 5-fluourouracil (5-FU) to induce apoptosis in the liver carcinoma Bel7402 cell line, and to observe the anti-tumor mechanism and effective adjuvant of 5-FU. Methods Cells were cultured under routine conditions with Dulbecco’s modified Eagle’s medium (DMEM) without L-arginine (L-Arg).We observed the expression of inducible nitric oxide synthase (iNOS) and apoptosis of cells induced by 5-FU with L-Arg added to the medium. The production of nitric oxide was determined by the cell expression of iNOS detected by immunohistochemical staining, and by the concentrations of nitrite and nitrate in the supernatant. Results 5-fluourouracil significantly increased the iNOS expression to 0.1687±0.01968 (P&lt;0.05, vs control group), and the concentration of nitric oxide to 213±30.2 μmol/L (P&lt;0.05, vs control group) The apoptotic cell rate increased significantly to 17.85±0.78%, while the necrotic cell rate decreased to 32.99±0.83% (P&lt;0.05, compared with the 5-FU group). Nω-nitro-L-Arginine methyl ester (L-NAME), the antagonist of L-Arg, can block the apoptotic effects of endogenous nitric oxide. Conclusions 5-FU had a synergistic effects with L-Arg by increasing the production of endogenous nitric oxide. Endogenous nitric oxide plays an important role in the process where 5-FU induces apoptosis in liver carcinoma cells. L-Arg may be a good adjuvant for chemotherapy with 5-FU.     

Effects of Serum Containing Chinese Medicine Sanpi Pingwei (散癖平胃) Formula on Proliferation and Apoptosis of Human SGC-7901 Cells

Objective:To investigate the effects of serum containing Chinese medicine(CM) Sanpi Pingwei(散癖平胃,SPPW) formula on the proliferation and apoptosis of human SGC-7901 cells and the possible mechanism.Methods:Serum containing CM SPPW formula(SPPW serum) was prepared by a serum pharmacology method.Human SGC-7901 cells were incubated with SPPW serum at three different concentrations and with the anticancer drug 5-fluorouracil(5-FU),respectively.Cell proliferation was assessed by MTT assay,and cell apoptosis was detected by flow cytometry assay.Real-time quantitative polymerase chain reaction(RT-PCR) and Western blot assay were employed to confirm the expressions of Bcl-2,Bax and p53 in SGC-7901 cells at mRNA and protein levels,respectively.Results:SPPW serum suppressed the proliferation of SGC-7901 cells in a time- and dose-dependent manner.The colony forming rate of negative control was 48.2%,while those in the three SPPW serum groups and the 5-FU group decreased significantly (P<0.01).The number of colony forming units in the SPPW high dosage group was significantly smaller than that in the 5-FU group(P<0.01).MTT assay showed that SPPW serum restrained the proliferation of SGC-7901 cells,and the inhibition rate increased significantly in a dose-dependent manner.Annexin V/PI Assay suggested that SPPW serum induced the apoptosis of SGC-7901 cells significantly.RT-PCR and western blot assay indicated that SPPW serum upregulated the protein and mRNA expression levels of Bax and p53 in SGC-7901 cells,but downregulated the protein and mRNA expressions of Bcl-2.Conclusions:SPPW formula inhibits the proliferation of SGC-7901 cells in vitro and induces the cell apoptosis.It plays an anticancer role by regulating the expressions of Bax,p53 and Bcl-2 in SGC-7901 cells.     

Early evaluation for treatment efficacy of 5-fluorouracil and hyperthermia on HCT-116 colon cancer cells by fluorine-18-fluorodeoxyglucose uptake

Backgroud  Fluorine-18-fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography imaging can be used to assess the treatment efficacy of chemotherapy and prognosis. The aim of this study was to determine the uptake rate of ¹⁸F-FDG in colon cancer HCT-116 cells, and to evaluate the treatment efficacy of chemotherapy, hyperthermia and thermo-chemotherapy through the uptake inhibition rate of ¹⁸F-FDG.

Methods  The uptake rate of ¹⁸F-FDG in HCT-116 cells was determined at various experimental conditions. The inhibition rate of cell growth, uptake rate of ¹⁸F-FDG and uptake inhibition rate of ¹⁸F-FDG in HCT-116 cells treated with 5-fluorouracil (5-FU) at various concentrations were determined. In HCT-116 cells subjected to chemotherapy (5-FU, 100 μg/ml), hyperthermia (43°C, 40 minutes) and thermo-chemotherapy for 24 hours, the inhibition rate of cell growth and uptake inhibition rate of ¹⁸F-FDG were determined; early apoptosis, the morphology and ultrastructure of HCT-116 cells were examined; and the contents of glucose and lactate dehydrogenase (LDH) in the cell culture medium of HCT-116 cells were determined. One-way analysis of variance (ANOVA) and correlation analyses were conducted by using SPSS 16.0 software.

Results  The uptake rate of ¹⁸F-FDG in HCT-116 cells was (44.25±2.19)%. Under the condition of adding 5-FU at various concentrations for 24 hours, the uptake rate of ¹⁸F-FDG was negatively correlated with 5-FU dosage (r= –0.879, P <0.01); the inhibition rate of cell growth revealed a positive correlation with the uptake inhibition rate of ¹⁸F-FDG (r=0.831, P <0.01). In HCT-116 cells subjected to hyperthermia, chemotherapy, and thermo-chemotherapy for 24 hours, the uptake inhibition rates of ¹⁸F-FDG were (12.94±2.80)%, (28.25±4.59)%, and (21.60±3.68)%, respectively. The early apoptotic rates of HCT-116 cells were (9.80±0.16)%, (19.80±2.40)%, and (15.70±1.80)%, respectively. Moreover, the contents of glucose and LDH in cell culture medium of HCT-116 cells after treatments were higher than those before treatment.

Conclusion  The uptake inhibition rate of ¹⁸F-FDG can be used for early evaluation of hyperthermia and 5-FU treatment efficacy on cancer cells although hyperthermia (43°C, 40 minutes) does not reveal the synergistic effect on 5-FU at the low dosage.

     

丹参注射液联合5-氟脲嘧啶腹腔化疗抗鼠胃肿瘤的实验研究

目的:观察丹参注射液对5-氟脲嘧啶(5-FU)抗肿瘤活性的增强作用及其对5-FU毒性和不良反应的减轻作用,为中药对抗肿瘤药的增效减毒作用提供实验依据。方法:应用Walker-256细胞株建立鼠种植性胃肿瘤模型,将60只Wistar雄性胃肿瘤大鼠随机分为3组:9.0 g/L氯化钠注射液(NS)组、5-FU组、5-FU 丹参组。接种后第7天,NS组每只每天腹腔注入NS,5-FU组每只每天腹腔注入5-FU,5-FU 丹参组每只每天腹腔注入5-FU 丹参,3组均连用5 d。于腹腔化疗结束后第7天每组处死10只大鼠。观察肿瘤体积抑制率、血常规、肝肾功能、细胞凋亡、大鼠生存时间。结果:①5-FU组和5-FU 丹参组鼠胃肿瘤生长明显缓慢,肿瘤体积较NS组明显减小(P(0.05,P(0.01),肿瘤体积抑制率分别为36.57%、59.44%(P(0.05);5-FU组和5-FU 丹参组鼠生存时间明显延长,生存时间延长率分别为23.7%、38.7%(P(0.05)。②与NS组相比,5-FU组和5-FU 丹参组外周血WBC、PLT明显减少,血清ALT、AST、Cr、BUN明显升高,差异均有显著性(P(0.05,P(0.01);与5-FU组相比,5-FU 丹参组WBC、PLT显著升高,而ALT、AST、Cr、BUN显著下降(P(0.05,P(0.01)。③与NS组相比,5-FU组和5-FU 丹参组凋亡面积增大,光密度明显升高(P(0.05,P(0.01);而5-FU 丹参组与5-FU组比较,差异也有显著性(P(0.05,P(0.01)。结论:丹参注射液可增强5-FU诱导肿瘤细胞分化与凋亡,并能减轻5-FU所致的骨髓抑制和肝肾功能损害。     

Effect of Ursolic Acid on Breast Cancer Resistance Protein-mediated Transport of Rosuvastatin In Vivo and Vitro

Objective To evaluate whether ursolic acid can inhibit breast cancer resistance protein (BCRP)-mediated transport of rosuvastatinin vivo andinvitro. Methods Firstly, we explored the pharmacokinetics of 5-fluorouracil (5-FU, a substrate of BCRP) in rats in the presence or absence of ursolic acid. Secondly, we studied the pharmacokinetics of rosuvastatin in rats in the presence or absence of ursolic acid or Ko143 (inhibitor of BCRP). Finially, the concentration-dependent transport of rosuvastatin and the inhibitory effects of ursolic acid and Ko143 were examined in Madin-Darby Canine Kidney (MDCK)Ⅱ-BCRP421CC (wild type) cells and MDCKⅡ-BCRP421AA (mutant type) cells. Results As a result, significant changes in pharmacokinetics parameters of 5-FU were observed in rats following pretreatment with ursolic acid. Both ursolic acid and Ko143 could significantly affect the pharmacokinetics of rosuvastatin. The rosuvastatin transport in the BCRP overexpressing system was increased in a concentration-dependent manner. However, there was no statistical difference in BCRP-mediated transport of rosuvastatin betweent the wild type cells and mutant cells. The same as Ko143, ursolic acid inhibited BCRP-mediated transport of rosuvastatinin vitro. Conclusion Ursolic acid appears to be a potent modulator of BCRP that affects the pharmacokinetic of rosuvastatinin vivo and inhibits the transport of rosuvastatinin vitro.     

头顶一颗珠对肝癌小鼠免疫功能影响的实验研究

目的研究头顶一颗珠(TTM)注射液对肝癌模型小鼠免疫功能的影响。方法建立H22小鼠肝癌模型,将造模成功的肝癌小鼠依据随机数字表法分为模型组、氟尿嘧啶(5-FU)组、5-FU+TTM组和TTM组,每组10只。模型组、5-FU组每日按10 mg/kg腹腔注射生理盐水及5-FU,5-FU+TTM组在注射5-FU的同时每日腹腔按7.2 g/kg注射TTM,TTM组按7.2 g/kg注射TTM。给药5 d后测定各组胸腺、脾脏指数,检测外周血中白细胞、淋巴细胞及CD3、CD4、CD8细胞的百分比,血清中白细胞介素2(IL-2)、肿瘤坏死因子α(TNF-α)水平。结果 5-FU组和TTM组的胸腺指数与模型组比较差异无统计学意义(P>0.05),而5-FU+TTM组显著高于模型组(P<0.05)。与模型组比较,5-FU组外周血白细胞与淋巴细胞数量明显减少(P<0.05),TTM组显著增加(P<0.05);5-FU+TTM组白细胞数与模型组比较差异无统计学意义(P>0.05);与5-FU组比较,TTM组白细胞与淋巴细胞数量显著增加(P<0.05),5-FU+TTM组白细胞数量明显增多(P<0.05),但淋巴细胞数量变化差异无统计学意义(P>0.05)。相对于模型组,TTM组、5-FU+TTM组及5-FU组CD3、CD4百分比显著增加(P<0.05),CD8百分比变化差异无统计学意义(P>0.05);与较5-FU+TTM组比较,TTM组、5-FU组CD3、CD4百分比差异无统计学意义,但CD8百分比显著增加(P<0.05)。相对于模型组,TTM组、5-FU+TTM组及5-FU组IL-2、TNF-α水平显著增加(P<0.05);与TTM组比较,5-FU+TTM组、5-FU组IL-2水平差异无统计学意义(P>0.05),但5-FU组TNF-α水平显著增加(P<0.05),5-FU+TTM组差异则无统计学意义(P>0.05)。结论 TTM可以促进T细胞的成熟与迁移,提高肝癌小鼠IL-2、TNF-α水平,增强机体免疫力,具有抗肿瘤功能。     

三氧化二砷联合5-氟尿嘧啶对人大肠癌SW480细胞的抑制作用

目的:比较5-氟尿嘧啶(5-FU)单独以及联合三氧化二砷(As2O3)对人大肠癌SW480的细胞毒作用,为临床联合用药提供实验依据。方法:以MTT法观察不同浓度的As2O3、5-FU以及两者合用对大肠癌SW480细胞的细胞毒作用,比较药物对细胞增值的抑制效应。结果:与单用5-FU相比,As2O3与5-FU联用能显著抑制人大肠癌SW480细胞的增值作用。结论:As2O3与5-FU联合应用具有显著抑制人大肠癌SW480细胞的作用。     

靶向热休克蛋白-27基因沉默对5-氟尿嘧啶诱导SW480细胞凋亡的影响及机制

目的：通过RNA干扰抑制热休克蛋白-27（HSP27）基因的表达,探讨沉默该基因对5-氟尿嘧啶（5-FU）诱导大肠癌SW480细胞凋亡的影响及机制。方法：将细胞分为正常对照组（Normal组）、阴性siRNA转染组（NC组）、HSP27-siRNA转染组（siRNA组）、单纯5-FU组（5-FU组）、5-FU+阴性siRNA转染组（NC+5-FU组）、5-FU+HSP27-siRNA转染组（siRNA+5-FU组）,通过脂质体LipofectamineTM 2000将HSP27-siRNA导入SW480细胞,CCK-8法检测靶向HSP27的siRNA和5-FU对SW480细胞增殖的抑制作用,流式细胞技术观察转染后细胞在5-FU作用下的早期凋亡情况,Western blot方法检测凋亡相关蛋白Active-casepase-3、Active-caspase-9及细胞色素C（Cytc）的表达。结果：CCK-8法检测结果显示siRNA+5-FU组细胞增殖抑制率明显高于同期siRNA组和5-FU组,表明联合应用抑制率明显升高（P＜0.05）。流式细胞仪检测结果表明siRNA组和5-FU组均可诱导SW480细胞凋亡,两者联合应用细胞早期凋亡率明显升高,与Normal组及NC组比较,差异均有统计学意义（均P＜0.05）;Western blot检测结果显示siRNA+5-FU组Active-casepase-3、Active-caspase-9、Cytc蛋白相对表达水平均高于siRNA组和5-FU组,差异均有统计学意义（均P＜0.05）。结论：靶向HSP27-siRNA能有效抑制大肠癌SW480细胞HSP27蛋白的表达,上调Active-casepase-3、Active-caspase-9、Cytc蛋白的表达,增强5-FU诱导SW480细胞凋亡,逆转肿瘤细胞对5-FU的耐药。     

可视化观察Tiam-1基因沉默对人大肠癌细胞株SW480转移的影响

目的 应用整体可视化成像技术观察Tiam-1基因沉默对人大肠癌细胞株SW480转移特性的影响.方法 将绿色增强荧光蛋白(EGFP)标记的人大肠癌细胞株SW480/EGFP 与SW480/EGFP /Tiam-1-经裸鼠尾静脉注射和结肠原位接种,分别建立可视化转移动物模型,比较两种细胞株成瘤与转移特性的差别.结果 SW480/EGFP 与SW480/EGFP /Tiam-1-细胞均稳定表达EGFP;SW480/EGFP /Tiam-1-的Tiaml基因干扰率达70%;SW480/EGFP /Tiam-1和SW480/EGFP 比较体外增殖能力无显著差异(P＞0.05),但体内增殖能力差异显著(P＜0.05);SW480/EGFP /Tiam-1转移率明显降低.结论 Tiaml基因可能在人大肠癌侵袭转移中发挥重要作用.     

上皮-间质转化在人结肠癌细胞系SW480对西妥昔单抗及氟尿嘧啶耐药中的作用

目的探讨人结肠癌细胞系SW480上皮-间质转化现象与其在化疗药物及靶向药物耐药中的作用.方法西妥昔单抗(cetuximab,C225)、5-氟尿嘧啶(5-Fu)以及二者联合处理人结肠癌细胞系SW480,观察细胞形态,免疫荧光化学方法检测处理前后SW480细胞骨架改变以及E-钙粘素(E-cardherin)、波形蛋白(vinmentin)表达差异,Western blot方法检测实验组与对照组SW480细胞E-cardherin、Vinmentin以及多药耐药蛋白(MRP)、P-糖蛋白(P-gp)表达差异;体外药物敏感试验(cell Counting Kit-8,CCK-8比色法)分别检测实验组与对照组SW480细胞存活能力.结果 SW480细胞经C225、5-Fu以及二者联合处理后细胞形态由上皮细胞向间质细胞转化;细胞免疫荧光显示处理前后SW480细胞骨架改变,微丝蛋白排列极性增强,E-cardherin表达下降,Vinmentin表达升高,同时伴随MRP、P-gp表达增高.SW480细胞经C225与5-Fu联合处理较单独应用2个药物细胞存活率明显降低(P<0.05).结论 SW480细胞经单独使用靶向药物或化疗药物以及两者联合应用可诱导细胞发生EMT,且此EMT对肿瘤细胞耐药有影响.     

雷公藤内脂醇对SW480细胞的生长抑制作用

目的观察雷公藤内酯醇(Tritolide,TL)对体外培养的SW480细胞(人结直肠癌细胞)的诱导分化机制,为人结直肠癌的治疗提供理论依据。方法应用噻唑蓝(MTT)比色法检测不同浓度的TL对SW480细胞的增殖抑制率,流式细胞仪检测SW480细胞周期进程及凋亡率,相差显微镜观察不同浓度的TL对SW480细胞形态学改变,电子显微镜观察细胞超微结构的变化。结果不同浓度的TL对SW480细胞增殖均有抑制作用,具有明显的剂量依赖性,流式细胞仪结果显示,G1期细胞增多,S期细胞减少,G2/M期细胞相对增多,细胞凋亡率升高,相差显微镜下实验组细胞贴壁不佳,细胞数减少,细胞圆缩,电子显微镜下细胞高度空化,线粒体肿胀,可见凋亡小体。结论TL对SW480细胞有明显的增殖抑制作用,阻止SW480细胞G1期向S期的转化进程并诱导SW480细胞凋亡及超微结构改变。     

FHIT基因转染对人结肠癌细胞系SW480增殖和侵袭力的影响

目的 探讨真核表达质粒pRc/CMV2-FHIT对结肠癌细胞株SW480增殖和侵袭活性的影响。方法 将携有FHIT基因的重组真核表达质粒pRc/CMV2-FHIT转化大肠杆菌DH5α并扩增,抽提纯化质粒并进行酶切鉴定,pRc/CMV2-FHIT体外转染SW480细胞（SW480-FHIT）,筛选稳定转染的细胞并扩增培养,以转染了空质粒pRc/CMV2的SW480细胞（SW480-pRc/CMV2）和正常SW480细胞为对照,用RT-PCR检测FHIT的表达情况,并用四甲基偶氮唑蓝比色法（MTT法）和细胞侵袭实验分别检测FHIT基因转染前、后SW480细胞增殖和侵袭活性的变化。结果 pRc/CMV2-FHIT的酶切鉴定证实含有目的基因FHIT;SW480-FHIT细胞的RT-PCR产物经凝胶电泳有明显的阳性条带,而对照组则无;MTT实验结果显示重组质粒转染组SW480细胞的增殖明显受到抑制;Transwell体外侵袭实验显示转染pRc/CMV2-FHIT能明显抑制SW480细胞的体外侵袭力(P<0.01)。结论 应用基因转染技术重表达FHIT基因能有效抑制细胞的生长、增殖及侵袭活性,为以FHIT为靶向的结肠癌基因治疗提供了新的思路和手段。     

光动力对结肠癌细胞周期阻滞作用的相关研究

目的：探讨ALA-PDT后结肠癌细胞关卡相关因子P21、P27、cyclinE、CDK2mRNA的表达情况。方法：应用RT—PCR技术对ALA—PDT后结肠癌细胞株SW4801921、P27、cyclinE、CDK2mRNA的含量进行检测。结果：ALA-PDT后P21mRNA含量较PDT前明显提高（P〈0．01）,而cyclinE、CDK2mRNA的含量下降（P〈0．01）,P27mRNA含量无明显改变。结论：ALA—PDT对肿瘤细胞的增殖抑制作用可能与其诱导P21mRNA的过表达,进而下调cyclinE、CDK2的表达有关。     

靶向抑制存活素基因对大肠癌细胞SW80侵袭和转移的实验研究

目的 构建靶向存活素(survivin)基因的短发夹干扰RNA(shRNA)表达载体,导入人大肠癌细胞SW480中,研究靶向抑制survivin基因对SW480细胞侵袭和转移的影响.方法 根据siRNA设计原则,在survivin序列中选取设计含19个核苷酸(19 nt)的靶序列,间以9个核苷酸的茎环序列,两端分别加上对应的酶切位点,形成shRNA的DNA模板并克隆到shRNA表达载体pRNAT-U6.1/Neo中,获得靶向抑制survivin基因的sihNA表达载体pRNAT-U6.1/Neo-survivin;采用Lipofectamine 2000TM转染试剂将干扰质粒pRNAT-U6.1/Neo-survivin导入到大肠癌细胞SW480中;用Western blotting从蛋白水平检测干扰效果;分别采用肿瘤侵袭粘附实验和明胶酶谱分析法检测pRNAT-U6.1/Neo-survivin对SW480细胞的侵袭和转移潜能的影响.结果 Survivin基因的蛋白表达均得到显著抑制;沉默SW480的survivin基因可以显著抑制SW480细胞的侵袭和转移潜能,而且survivin基因表达被抑制后,SW480细胞分泌基质金属蛋白酶明显减少.结论 Survivin基因可能在SW480的侵袭和转移潜能中起重要作用,沉默SW480的survivin基因可以显著抑制其侵袭、转移和基质金属蛋白酶分泌,因而survivin影响SW480的侵袭和转移可能与调控基质金属蛋白酶分泌密切相关.     

Increased sensitivity of colorectal cancer cell lines with microsatellite instability to 5-fluorouracil in vitro

Objective To study the relationship between sensitivity to 5-FU and the status of a panel of microsatellite loci in three human colon cancer cell lines.Methods Cell viability in several concentrations of 5-FU was assessed by the MTT test. Expression of hMSH2 and hMLH1 in LoVo, SW480 and SW1116 cells were analyzed by immunocytochemical staining. Ten mononucleotide and dinucleotide microsatellite loci were analyzed by the PCR-SSLP-silver staining method. Results By MTT assay, it showed that LoVo cells were more sensitive than SW480 and SW1116 cells (0.8 μmol/L, 2.2 μmol/L and 1.9 μmol/L, respectively,P&lt;0.05). By immunocytochemical staining, hMSH2 was expressed in SW480 and SW1116 cells but not in LoVo cells, while hMLH1 was positive in all three cell lines. The PCR-SSLP-silver staining of 10 microsatellite loci revealed that LoVo cells had a different pattern of electrophoretic bands compared with SW480 and SW1116 cells, manifesting both additions and band-shifts. Conclusion Together with hMSH2 and hMLH1, the status of a panel of microsatellite loci may be used as convenient predictors for drug-optimization or prognosis-assessment in colorectal cancer patients before chemotherapy.     

环氧化酶2基因异常表达与大肠癌转移的关系

目的 研究环氧化酶2在人大肠癌细胞系及大肠癌组织中的异常表达与意义。方法 培养不同转移能力的人大肠癌细胞系SW480和SW620细胞，收集50例大肠癌石蜡组织，50例淋巴结转移性大肠癌石蜡组织：分别用免疫组织化学、Real-time PCR的方法研究环氧化酶2在不同转移能力人大肠癌细胞系及原发人大肠癌组织和大肠淋巴结转移组织中的表达。结果 环氧化酶2蛋白在SW480和SW620细胞株中均阳性表达；环氧化酶2mRNA在SW620比SW480细胞中表达增高，表达量的平均倍比关系为2．268。环氧化酶2蛋白在淋巴结转移癌组的异常表达高于原发大肠癌石蜡组织，相关性具有统计学意义（P〈0．05）。结论 环氧化酶2异常表达可能与大肠癌淋巴结转移相关。