内、外源性趋化因子CCL5对MCF-7细胞增殖能力影响的比较

目的 观察人乳腺癌MCF-7细胞表达的内源性CC类趋化因子5(CC chemokine ligand 5,CCL5)和细胞周围环境中的外源性CC类趋化因子5对MCF-7细胞增殖能力影响的差异.方法不同浓度的外源性rhCCL5(recombinant human CCL5)处理MCF-7细胞24、48h后,用MTT法检测细胞增殖能力的变化;CCL5特异性siRNA慢病毒载体转染人乳腺癌MCF-7细胞,RT-PCR检测细胞内源性CCL5 mRNA表达水平的变化,同时用MTT法检测转染前后细胞增殖能力的变化.结果 浓度为10、50、100、500ng/ml的外源性rhCCL5分别处理于MCF-7细胞24、48h后,MTT试验检测到rhCCL5处理组细胞增殖水平均高于对照组(P<0.05),外源性rhCCL5表现出促进细胞增殖的效果,并呈浓度依赖的趋势.CCL5-siRNA慢病毒载体感染MCF-7细胞后,RT-PCR检测到细胞内源性CCL5 mRNA的表达被有效抑制,明显少于阴性对照组(P<0.05),但是MTT试验结果显示MCF-7细胞内源性CCL5表达被干扰后,细胞增殖能力与阴性对照组相比差异无统计学意义(P>0.05).结论外源性CCL5可以有效促进人乳腺癌细胞MCF-7的增殖,而细胞内源性CCL5的表达水平对细胞的增殖能力没有明显影响.     

趋化因子2对乳腺癌细胞MCF-7趋化活性及趋化因子5mRNA表达的影响

目的 观察趋化因子2(CCL2)对MCF-7表达趋化因子5(CCL5)mRNA的影响,并观察CCL2对MCF-7趋化活性的影响.方法 使用不同浓度的CCL2作用于MCF-7细胞,通过实时荧光定量聚合酶链反应(RTFQ-PCR)测定不同时间CCL5 mRNA的表达,并使用趋化小室法检测CCL2作用下MCF-7趋化活性的改变.结果 当外源性CCL2浓度为200μg/L时,MCF-7中CCL5 mRNA相对表达量最大(15.22±2.32)(P＜0.01),随着作用时间的延长,CCL5 mRNA表达量增加;MCF-7的趋化活性与CCL2浓度呈正相关,当CCL2浓度为300μg/L时,穿膜细胞数最多(88.00±11.53)(P＜0.01);MCF-7的趋化活性与CCL2作用时间呈正相关,当CCL2作用时间为30 h时,穿膜细胞数最多(81.00±9.54)(P＜0.05).结论 加入外源性CCL2,MCF-7中CCL2 mRNA表达量增加,MCF-7趋化活性增强.

Abstract：

Objective To observe the effects of chemokine 2 (CCL2) on the expression of chemokine 5 ( CCL5) mRNA and chemotactic activity of MCF-7 cells. Methods MCF-7 cells were treated with different concentrations of CCL2, the expression of CCL5 mRNA was detected by using real-time fluorescence quantitative polymerase chain reaction ( RTFQ-PCR), and the chemotactic activity of MCF-7 was measured by using chemotaxis chamber method. Results When the concentration of exogenous CCL2 was 200 μg/L, the MCF-7 cells expressed the highest CCL5 mRNA (15. 22 ± 2. 3, P ＜0. 01). With the prolongation of CCL2 action time, the expression levels of CCL5 mRNA were increased. There was a positive correlation between the chemotactic activity of MCF-7 cells and the concentration of CCL2. When the concentration of exogenous CCL2 was 300 μg/L, the number of penetrating cells was the greatest (88.00 ±11. 53, P ＜0. 01). With the prolongation of CCL2 action time, the chemotactic activity of MCF-7 cells was enhanced. When the action time was 30 h, the number of penetrating cells was the greatest (81.00 ±9. 54, P ＜ 0.05 ). Conclusion Exogenous CCL2 could increase the expression of CCL5 mRNA and the chemotactic activity of MCF-7 cells.     

趋化因子2对乳腺癌细胞MCF-7趋化活性及趋化因子5mRNA表达的影响

Objective To observe the effects of chemokine 2 (CCL2) on the expression of chemokine 5 ( CCL5) mRNA and chemotactic activity of MCF-7 cells. Methods MCF-7 cells were treated with different concentrations of CCL2, the expression of CCL5 mRNA was detected by using real-time fluorescence quantitative polymerase chain reaction ( RTFQ-PCR), and the chemotactic activity of MCF-7 was measured by using chemotaxis chamber method. Results When the concentration of exogenous CCL2 was 200 μg/L, the MCF-7 cells expressed the highest CCL5 mRNA (15. 22 ± 2. 3, P ＜0. 01). With the prolongation of CCL2 action time, the expression levels of CCL5 mRNA were increased. There was a positive correlation between the chemotactic activity of MCF-7 cells and the concentration of CCL2. When the concentration of exogenous CCL2 was 300 μg/L, the number of penetrating cells was the greatest (88.00 ±11. 53, P ＜0. 01). With the prolongation of CCL2 action time, the chemotactic activity of MCF-7 cells was enhanced. When the action time was 30 h, the number of penetrating cells was the greatest (81.00 ±9. 54, P ＜ 0.05 ). Conclusion Exogenous CCL2 could increase the expression of CCL5 mRNA and the chemotactic activity of MCF-7 cells.     

趋化因子2对乳腺癌细胞MCF-7趋化活性及趋化因子5mRNA表达的影响

Objective To observe the effects of chemokine 2 (CCL2) on the expression of chemokine 5 ( CCL5) mRNA and chemotactic activity of MCF-7 cells. Methods MCF-7 cells were treated with different concentrations of CCL2, the expression of CCL5 mRNA was detected by using real-time fluorescence quantitative polymerase chain reaction ( RTFQ-PCR), and the chemotactic activity of MCF-7 was measured by using chemotaxis chamber method. Results When the concentration of exogenous CCL2 was 200 μg/L, the MCF-7 cells expressed the highest CCL5 mRNA (15. 22 ± 2. 3, P ＜0. 01). With the prolongation of CCL2 action time, the expression levels of CCL5 mRNA were increased. There was a positive correlation between the chemotactic activity of MCF-7 cells and the concentration of CCL2. When the concentration of exogenous CCL2 was 300 μg/L, the number of penetrating cells was the greatest (88.00 ±11. 53, P ＜0. 01). With the prolongation of CCL2 action time, the chemotactic activity of MCF-7 cells was enhanced. When the action time was 30 h, the number of penetrating cells was the greatest (81.00 ±9. 54, P ＜ 0.05 ). Conclusion Exogenous CCL2 could increase the expression of CCL5 mRNA and the chemotactic activity of MCF-7 cells.     

Thapsigargin对乳腺癌MCF-7细胞凋亡的诱导作用研究

目的 探讨Thapsigargin对人乳腺癌MCF-7细胞株雌激素受体阳性的凋亡诱导作用。方法 在体外培养条件下，应用Thapsigargin与5-FU处理MCF-7细胞株，采用流式细胞仪检测其细胞凋亡率和周期分布；MTT比色法分析其细胞生长抑制率；透射电镜观察细胞超微结构。结果 Thapsigargin可使5-FU诱导的MCF-7细胞凋亡率和生长抑制率明显增加，并改变细胞周期的分布。电镜下可见MCF-7细胞凋亡小体明显增加。结论 Thapsigargin具有明显加强5-FU对人乳腺癌MCF-7细胞株的凋亡诱导作用，值得进一步研究。     

芹黄素对乳腺癌细胞MCF-7、MDA-MB-435S侵袭和增殖能力的影响

透明质酸酶（Hyalase）是一种基质降解酶，它参与了肿瘤的侵袭转移过程。有研究表明，芹黄素可以抑制Hyalase的活性。我们通过建立乳腺癌细胞体外侵袭模型。观察芹黄索对两种乳腺癌细胞株侵袭和增殖能力的影响。     

脂氧合酶抑制剂对乳腺癌细胞MCF-7增殖和凋亡的影响

目的 探讨脂氧合酶抑制剂去甲二氢愈创木酸（NDGA）对乳腺癌细胞MCF-7增殖和凋亡的影响。方法 噻唑蓝（MTT）法绘制细胞生长曲线及检测细胞存活率；流式细胞仪检测细胞周期和凋亡率；倒置相差显微镜和扫描电镜观察细胞凋亡。结果 MTT法显示NDGA对MCF-7细胞生长增殖有明显的抑制作用（P〈0.05），呈剂量和时间依赖效应；流式细胞仪结果显示NDGA阻滞细胞周期于S期，诱导细胞凋亡；在光镜和扫描电镜下，NDGA诱导细胞发生凋亡形态学变化，导致细胞表面超微结构的改变和凋亡小体的形成。结论 NDGA能够抑制MCF-7细胞的增殖，诱导细胞凋亡，为脂氧合酶抑制剂临床应用于乳腺癌提供了科学依据。     

miR-432-5p靶向UCK2调控乳腺癌细胞MCF-7凋亡的研究

目的:探讨miR-432-5p调控乳腺癌细胞MCF-7凋亡的分子机制.方法:过表达或敲低miR-432-5p后,检测乳腺癌细胞MCF-7的凋亡水平.敲低miR-432-5p后,检测miRDB数据库在线分析的靶标的mRNA水平.通过荧光素酶报告系统检测miRNA是否与潜在靶标直接结合.过表达或敲低靶标后,分析乳腺癌细胞M...     

血清可溶性CD105、CC类趋化因子配体20、CC类趋化因子配体5水平与肺癌手术病人预后的关系

目的 探讨血清可溶性CD105(S-CD105)、CC类趋化因子配体20(CCL20)、CC类趋化因子配体5(CCL5)水平与肺癌手术病人预后的关系。方法 选取2018年3月～2019年3月于本院接受根治术治疗的86例肺癌病人为观察组,术后随访至2022年3月,根据病人是否复发或死亡分为预后良好组(54例)与预后不良组(32例),同期选取43例健康体检者为对照组。对比S-CD105、CCL20、CCL5水平,观察S-CD105、CCL20、CCL5不同水平的复发率、转移率及术后180天后死亡率;采用Kendall’s tau-b法分析SCD105、CCL20、CCL5与肺癌手术病人预后的相关性;采用二元Logistic回归模型分析SCD105、CCL20、CCL5对肺癌手术病人预后影响;采用ROC曲线模式分析S-CD105、CCL20、CCL5预测肺癌手术病人预后的AUC值、敏感度、特异度。结果 观察组的S-CD105为(4.55±0.86)ng/ml、CCL20为(71.97±13.69)ng/ml和CCL5为(49.50±5.17)ng/ml,对照组分别为(3.38±0.52)ng...     

纳洛酮预处理对吗啡抑制人乳腺癌MCF-7细胞生长增殖的影响

目的观察纳洛酮预处理对吗啡抑制人乳腺癌MCF-7细胞生长增殖的影响。方法人乳腺癌MCF-7细胞培养至对数生长期,采用随机数字表法,将其分为四组:空白对照组(C组),10μmol/L吗啡组(M组),10μmol/L纳洛酮组(N组)及10μmol/L吗啡+10μmol/L纳洛酮组(MN组)。M组在培养液中加入吗啡,使吗啡的终浓度为10μmol/L;N组在培养液中加入纳洛酮,使其终浓度为10μmol/L;MN组则先在培养液中加入纳洛酮,使其浓度为10μmol/L,孵育30min后再将吗啡加入培养液中,使其终浓度为10μmol/L;对照组不加入任何药物。采用四唑盐(MTT)法和克隆形成实验观察细胞生长增殖的变化,流式细胞仪检测细胞周期分布和细胞凋亡率。结果C组与N组细胞活力、克隆形成率、细胞周期分布及细胞凋亡率差异无统计学意义;M组及MN组细胞的生长速度明显慢于C组,克隆形成率、细胞停滞在S期的比例明显低于C组,而细胞凋亡率、细胞停滞在G2/M期的比例明显高于C组(P0.05);M组与MN组细胞活力、克隆形成率、细胞周期分布及细胞凋亡率差异无统计学意义。结论在吗啡抑制人乳腺癌MCF-7细胞生长增殖、促进细胞凋亡的过程中,纳洛酮没有发挥拮抗作用,这说明吗啡抑制乳腺癌MCF-7细胞生长增殖的作用与阿片受体途径无关。     

趋化因子受体5促进乳腺癌干细胞的趋化与侵袭

目的 观察趋化因子受体5(CCR5)对乳腺癌干细胞(CD44+CD24-/low)趋化和侵袭作用.方法 应用流式细胞仪分选获得乳腺癌干细胞,逆转录-聚合酶链反应(RT-PCR)和酶联免疫吸附试验(ELISA)检测乳腺癌MCF-72个SP侧群细胞(side population)6例样本CCR5 mRNA和蛋白质,在趋化因子5(CCL5)作用下通过趋化小室法检测2个SP侧群细胞趋化活性和侵袭活性,并与CCR5的封闭进行对照研究.结果 CCL5对MCF-7肿瘤干细胞有明显趋化[乳腺癌干细胞(86.0±14.8)个/HP与CD44+CD24+(72.0±13.5)个/HP,t=7.461,P<0.05]和侵袭作用[乳腺癌干细胞(25.0±8.3)个/HP与CD44+CD24+(16.0±5.4)个/HP,t=6.665,P<0.05].结论 CCB5表达能够促进乳腺癌干细胞趋化和侵袭.     

熊果酸对MCF-7乳腺癌细胞生长的影响

目的 观察熊果酸对MCF-7乳腺癌细胞增殖、凋亡、细胞侵袭及裸鼠移植瘤生长的影响.方法 将1、10、100 μmol/L的熊果酸分别作用于乳腺癌MCF-7细胞12、24、48 h,噻唑蓝(MTT)比色法检测细胞活力变化及生长抑制率,流式细胞术及Transwell小室法检测熊果酸作用48h时MCF-7细胞周期、细胞凋亡率及细胞侵袭力.将乳腺癌MCF-7细胞接种于裸鼠,成瘤后分为生理盐水对照组、熊果酸低剂量组(每日1 mg/kg)、中剂量组(每日5 mg/kg)和高剂量组(每日25 mg/kg),检测5、10、15 d裸鼠移植瘤体积、肿瘤生长抑制率及15 d时肝肾功能、血常规变化.结果 随熊果酸浓度增加及作用时间延长,MCF-7细胞生长及凋亡发生受到显著影响,100 μmol/L熊果酸作用48 h时,细胞生长抑制率为46.0%,生长周期阻滞于G0/G1期,细胞凋亡率为(16.48±2.46)%,细胞侵袭力显著下降.熊果酸组裸鼠移植瘤体积显著小于对照组,15 d时熊果酸高剂量组肿瘤体积为(323.5±33.5)mm3生长抑制率为50.9%.各组肝肾功能和血常规无明显变化.结论 熊果酸可引起体外乳腺癌McF-7细胞增殖抑制、细胞凋亡率增加及细胞侵袭力下降,可显著抑制裸鼠乳腺癌移植瘤生长.     

雄激素对MCF-7乳腺癌细胞株增殖凋亡的影响

目的 观察睾丸酮对MCF-7乳腺癌细胞株增殖、凋亡的影响,并探讨其机制.方法 将1×10~(-5)、1×10~(-7)、1×10~(-9)、1×10~(-11) mol/L的睾丸酮分别作用于乳腺癌MCF-7细胞24、48、72 h,噻唑蓝(MTT)比色法检测细胞生长,流式细胞术检测不同浓度睾丸酮作用48 h时MCF-7细胞的周期分布和凋亡以及该细胞株中细胞周期素D1蛋白和雄激素受体的表达.结果 高浓度睾丸酮抑制MCF-7乳腺癌细胞株的增殖,1×10~(-5) mol/L睾丸酮作用48 h时,细胞生长抑制率为22.21%,细胞凋亡率为(58.60±5.41)%,但可使细胞周期由G_1 期进入S期,并可使细胞周期素D1蛋白表达量增加,雄激素受体蛋白表达量下降.低浓度睾丸酮(1×10~(-9) mol/L)作用后细胞周期素D1蛋白表达量无明显变化,雄激素受体蛋白表达量升高,在短时间内(24 h)可促进MCF-7细胞增殖.结论 高浓度睾丸酮在体外可使MCF-7乳腺癌细胞株细胞周期素D1蛋白表达增加,使细胞周期由G_1进入S期,而同时促使细胞凋亡,表现出抗肿瘤作用.低浓度睾丸酮有短暂的促进MCF-7乳腺癌细胞增殖的作用.     

慢病毒介导CCL5-siRNA对乳腺癌细胞生物学行为的影响

Objective To study the effect of lentivirus-mediated CCL5-RNAi on the biological behaviors of human breast cancer cells. Methods CCL5-specific siRNA gene was synthesized and cloned into the recombinant lentiviral vector, pGCSIL-GFP. Human high-metastatic breast cancer cells, MDA-MB-231, were infected by CCL5-siRNA recombinant lentivirus, which was set as KD group. Cells infected with CCL5-NC was as NC group, and cells cultured was as CON group. The expression of CCL5 mRNA and protein in MDA-MB-231 cells was detected by RT-PCR and western blot, respectively. Cell growth suppression and cell cycle was observed by MTT assay and fluorescence activated cell sorting (FACS). Colony formation and migration ability were determined by colony-rorming assay and Boyden chamber method. Results After infection of CCL5-siRNA recombinant lentivirus, the expression level of CCL5 mRNA and protein in MDA-MB-231 cells as well as the colony formation and migration ability decreased significantly, but cell's proliferation was not affected obviously. Compared with MDA-MB-231 (0.88± 0.15) and MDA-MB-231/CCL5-NC (1.00±0.07) cells, the expression of CCL5 mRNA in MDA-MB-231/ CCL5-siRNA decreased to 0.18±0.03, P<0.01. Compared with MDA-MB-231/CCL5-NC (1.82±0.18) cells, the expression of CCL5 protein in MDA-MB-231/CCL5-siRNA decreased to 0.33±0.13, P <0.01. Colony-forming assay and Boyden chamber method showed that the colony formation and migration ability of MDA-MB-231/CCL5-siRNA decreased markedly (P<0.05). The clone count in KD group was (0.33± 0.10), which was a significant decrease from (0.97±0.09) (NC group) and (1.04±0.07) (CON group), P<0.05. The number of cells that migrated through the chamber membrane of KD group (38± 15) was less than that of NC group (77±11, P <0.05) and CON group (69±9, P <0.05). However, MTT assay and FACS revealed that the proliferation of MDA-MB-231/CCL5-siRNA was not different from MDA-MB-231/CCL5-NC and MDA-MB-231 (P>0.05), the proliferation index (PI) of group KD, NC and CON were (0.48±0.02), (0.44±0.05) and (0.47±0.02) respectively. The difference was not statistically significant by multiple comparison (P>0.05). Conclusion CCL5-specific siRNA can specifically suppress the colony formation and migration of human high-matastatic breast cancer cells.     

慢病毒介导CCL5-siRNA对乳腺癌细胞生物学行为的影响

Objective To study the effect of lentivirus-mediated CCL5-RNAi on the biological behaviors of human breast cancer cells. Methods CCL5-specific siRNA gene was synthesized and cloned into the recombinant lentiviral vector, pGCSIL-GFP. Human high-metastatic breast cancer cells, MDA-MB-231, were infected by CCL5-siRNA recombinant lentivirus, which was set as KD group. Cells infected with CCL5-NC was as NC group, and cells cultured was as CON group. The expression of CCL5 mRNA and protein in MDA-MB-231 cells was detected by RT-PCR and western blot, respectively. Cell growth suppression and cell cycle was observed by MTT assay and fluorescence activated cell sorting (FACS). Colony formation and migration ability were determined by colony-rorming assay and Boyden chamber method. Results After infection of CCL5-siRNA recombinant lentivirus, the expression level of CCL5 mRNA and protein in MDA-MB-231 cells as well as the colony formation and migration ability decreased significantly, but cell's proliferation was not affected obviously. Compared with MDA-MB-231 (0.88± 0.15) and MDA-MB-231/CCL5-NC (1.00±0.07) cells, the expression of CCL5 mRNA in MDA-MB-231/ CCL5-siRNA decreased to 0.18±0.03, P<0.01. Compared with MDA-MB-231/CCL5-NC (1.82±0.18) cells, the expression of CCL5 protein in MDA-MB-231/CCL5-siRNA decreased to 0.33±0.13, P <0.01. Colony-forming assay and Boyden chamber method showed that the colony formation and migration ability of MDA-MB-231/CCL5-siRNA decreased markedly (P<0.05). The clone count in KD group was (0.33± 0.10), which was a significant decrease from (0.97±0.09) (NC group) and (1.04±0.07) (CON group), P<0.05. The number of cells that migrated through the chamber membrane of KD group (38± 15) was less than that of NC group (77±11, P <0.05) and CON group (69±9, P <0.05). However, MTT assay and FACS revealed that the proliferation of MDA-MB-231/CCL5-siRNA was not different from MDA-MB-231/CCL5-NC and MDA-MB-231 (P>0.05), the proliferation index (PI) of group KD, NC and CON were (0.48±0.02), (0.44±0.05) and (0.47±0.02) respectively. The difference was not statistically significant by multiple comparison (P>0.05). Conclusion CCL5-specific siRNA can specifically suppress the colony formation and migration of human high-matastatic breast cancer cells.