靶向apollon反义核酸对人结肠癌Lovo细胞的生物学作用
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摘要
目的:
筛选出IAP家族成员中其ASODN对消化系肿瘤细胞HepG_2、Lovo、MGC-803细胞增殖抑制和促凋亡作用较强的成员。将筛选出的成员apollon ASODN作用于结肠癌Lovo细胞,并观察该反义核酸对结肠癌细胞的部分生物学作用(增殖抑制、促凋亡、对化疗药物的敏感性、基因表达的影响),为结肠癌的治疗提供一种新的靶标。
方法:
第一部分探讨脂质体介导的人凋亡抑制蛋白家族(Inhibitor of apoptosis proteins,IAPs)反义寡核苷酸(ASODN)对人消化系肿瘤细胞增殖、凋亡的影响。人工合成survivin、xiap、apollon、livin反义寡核苷酸,经脂质体包裹后作用于肝癌细胞、胃癌细胞、结肠癌细胞48小时后,采用WST-8法观察对细胞增殖的影响。流式细胞术检测细胞凋亡率,并对其中的成员进行筛选。
第二部分靶向apollon反义寡核苷酸对人结肠癌细胞增殖及凋亡的影响。以脂质体为载体把apollon ASODN转染体外培养的结肠癌Lovo细胞。采用WST-8法与克隆形成抑制试验检测不同浓度的apollon ASODN对Lovo细胞增殖抑制率;流式细胞术检测apollon ASODN对Lovo细胞周期和凋亡的影响;Hoechst3325染色观察Lovo细胞凋亡的形态学改变;实时荧光定量RT-PCR检测细胞apollon mRNA的表达水平。
第三部分Apollon ASODN联合化疗药物对人结肠癌细胞增殖的影响以脂质体为载体把apollon ASODN转染入体外培养的结肠癌Lovo细胞,联合临床常用化疗药物(顺铂、表柔比星、5-氟尿嘧啶),采用WST-8法检测细胞增殖抑制作用,计算各组药物的增殖抑制率和半数抑制浓度(IC_(50)),并通过金正均的概率和法,计算联合用药的效果(作用拮抗、作用相加、作用协同)。
结果:
第一部分
IAPs ASODN作用于肝癌细胞(HepG_2)、胃癌细胞(MGC-803)、结肠癌细胞(Lovo)48小时后,与空白对照组(control)和随机寡核苷酸(random oligodeoxynucleotide,RODN)对照组相比,IAPs ASODN能明显抑制肝癌细胞、胃癌细胞、结肠癌细胞的增殖和诱导细胞凋亡(P＜0.05)。其中,在作用浓度为0.2μmol/L时,survivin ASODN抑制HepG_2、Lovo细胞增殖的作用最强,抑制率分别为84.2%、82.6%。apollon ASODN抑制Lovo细胞的增殖的作用最强,抑制率为82.27%。xiap ASODN抑制MGC-803细胞增殖作用最强,抑制率为86.56%,并呈时间-剂量依赖效应。Survivin ASODN诱导Lovo细胞、MGC-803细胞凋亡作用最强,凋亡率为34.95%、27.2%。livin ASODN诱导HepG_2细胞凋亡作用最强,凋亡率为22.5%。
第二部分
1.脂质体介导apollon ASODN转染Lovo细胞48小时后,细胞生长增殖和克隆形成均显著被抑制(P＜0.05),并呈浓度依赖关系。
2.运用AnnexinV-PI双染检测细胞的早期凋亡率显示:apollon ASODN作用于Lovo细胞48 h后,与RODN组,以及control组相比,ASODN组的早期凋亡率明显高于control组(P＜0.05),RODN组与对照组相比对凋亡无明显影响(P＞0.05)。
3.PI单染流式细胞术检测细胞周期百分比显示:与control、RODN组相比,apollonASODN组G0/G1期细胞减少,S期细胞增多,出现明显的S期阻滞。
4.Hoechst33258染色结果显示:apollon ASODN作用于Lovo细胞48 h后,空白对照组细胞形态完整,RODN组与空白对照组相比未见明显差异,而ASODN组可见核固缩、边聚、裂解等细胞凋亡形态学变化。
5.实时荧光定量RT-PCR结果表明:脂质体介导的apollon ASODN转染Lovo细胞能明显下调apollon mRNA的表达,与空白对照组、RODN组相比,有统计学意义(P＜0.05)。
第三部分
0.08μmol/L apollon ASODN与不同浓度的化疗药物(5-FU、EPI、DDP)联合作用于结肠癌细胞48小时后,可以提高结肠癌细胞对这些化疗药物的敏感性,增敏倍数分别为2.5、5.33、4.47。
结论:
1.脂质体介导的IAPs ASODN能够抑制消化系肿瘤细胞增殖、并诱导细胞凋亡。
2.不同浓度的apollon ASODN均能下调apollon基因的mRNA表达水平,抑制Lovo细胞增殖,并诱导Lovo细胞凋亡。
3.低浓度的apollon ASODN复合物联合化疗药物5-FU、DDP、EPI作用于Lovo细胞后,可以提高细胞对这些化疗药物的敏感性。
Objective:
To screen out the antisense oligodeoxynucleotide of inhibitor of apoptosis proteins that has a better suppressing effects on the cell proliferation and promoting apoptosis on gastrointestinal tumor cells(HepG2、Lovo、MGC-803),from the IAP family members ASODN.using the selected member apollon ASODN to act on colon cancer Lovo cells,and then observe particular biochemical reactions(proliferation inhibition,inducing apoptosis,sensitivity to the chemotherapeutic drugs on the treatment of colorectal cancer, and effect on the gene expression),To provide a new target site for the treatment of colorectal cancer.
Methods:
PartⅠ
IAPs ASODN and random oligonucleotides(RODN) were transfected into Gastrointestinal cancer cells(Lovo,HepG_2,MGC-803) treated with some concentration for 48 hours,MTT assay was applied to the determination of cell proliferation,apoptotic index was examined by flow cytometry.
PartⅡ
Apollon ASODN and random oligonucleotides(RODN) were transfected into Lovo cells for 48 hours,proliferation and the clone formation of Lovo cells were detected by CCK-8 and clone formation assay,respectively.The morphology was examined by fluorescence microscope.The percentage of hypodiploid cells of Lovo cells treated with apollon ASODN was determined with propidium iodide(PI) staining by flow cytometry.Early apoptosis was detected with Annexin V-FITC and PI dual parameter by flow cytometry.Furthermore,The expression of apollon gene was analyzed by real time fluorescent quantitativ reverse transcription-polymerase chain reaction.
PartⅢ
Apollon ASODN and random oligonucleotides(RODN) were transfected into Lovo cells, Lovo cells were incubated treated by 5-fluorouracil(5-FU) or Epirubicin(EPI) or Cisplatin (DDP),and their combination of ASODN at different concentrations for 48 hours respectively, then the cell proliferation was detected by WST-8 test,finally their IC_(50) and their drug-sensitivity were calculated statistically.
Results:
PartⅠ
After treated with antisense oligodeoxynucleotide targeting 48 hours,compared with blank control and random oligodeoxynucleotide groups,antisense oligodeoxynucleotide targeting IAPs can significantly suppressed the growth of gastrointestinal cancer cells and induce their apoptosis(P＜0.05),at a concentration of 0.2μmol/L,survivin ASODN has the significantly inhibited effects on the HepG_2,Lovo cells,the inhibitory rates were 84.2%,82.6%.respectively. the xiap ASODN has the significantly inhibited effects on the MGC-803 cell,inhibitory rates were 86.56%.they showed time and dose-dependent fashions.Survivin ASODN inducing the apoptosis of Lovo cell,MGC-803 cell was the strongest than others,the apoptotic rate were 34.95%、27.2%.respectively.livin ASODN inducing the apoptosis of HepG_2 cell was the strongest than others,the apoptotic rate were 22.5%.
PartⅡ
After treated with antisense oligodeoxynucleotide targeting apollon 48 hours,compared with blank control and random oligodeoxynucleotide groups,antisense oligodeoxynucleotide targeting apollon can significantly suppressed the growth of colorectal cancer cells and induce their apoptosis(P＜0.05).It was found that all concentrations of the apollon ASODN used in this study showed to be able to suppress the expression of the apollon gene.antisense oligodeoxynucleotide targeting apollon induce significant increase in s-phase cells,many Lovo cells stained with Hoechst 33258 exhibited apoptotic morphology such as cell shrinkage, nuclear condensation and nuclear fragmentation.
PartⅢ
Compared with single 5-FU,EPI and DDP group,The combition of 5-FU,EPI,DDP with apollon ASODN have enhanced their chemotherapeutic effects on Lovo cells,and the sensitivity enhance to 2.5,4.47,and 5.33 times respectively.
Conclusion:
1.Antisense oligodeoxynucleotide targeting IAPs can inhibit the proliferation and induce the apoptosis of gastrointestinal cancer cells in vitro.
2.The different antisense oligodeoxynucleotide targeting apollon can suppress the express levels of gene,and inhibit the proliferation and induce the apoptosis of colorectal cancer cells in vitro.
3.0.08μmol/L apollon ASODN can enhance the chemotherapeutic drug's sensitivity of Lovo cells to 5-FU,EPI and DDP.
筛选出IAP家族成员中其ASODN对消化系肿瘤细胞HepG_2、Lovo、MGC-803细胞增殖抑制和促凋亡作用较强的成员。将筛选出的成员apollon ASODN作用于结肠癌Lovo细胞,并观察该反义核酸对结肠癌细胞的部分生物学作用(增殖抑制、促凋亡、对化疗药物的敏感性、基因表达的影响),为结肠癌的治疗提供一种新的靶标。
方法:
第一部分探讨脂质体介导的人凋亡抑制蛋白家族(Inhibitor of apoptosis proteins,IAPs)反义寡核苷酸(ASODN)对人消化系肿瘤细胞增殖、凋亡的影响。人工合成survivin、xiap、apollon、livin反义寡核苷酸,经脂质体包裹后作用于肝癌细胞、胃癌细胞、结肠癌细胞48小时后,采用WST-8法观察对细胞增殖的影响。流式细胞术检测细胞凋亡率,并对其中的成员进行筛选。
第二部分靶向apollon反义寡核苷酸对人结肠癌细胞增殖及凋亡的影响。以脂质体为载体把apollon ASODN转染体外培养的结肠癌Lovo细胞。采用WST-8法与克隆形成抑制试验检测不同浓度的apollon ASODN对Lovo细胞增殖抑制率;流式细胞术检测apollon ASODN对Lovo细胞周期和凋亡的影响;Hoechst3325染色观察Lovo细胞凋亡的形态学改变;实时荧光定量RT-PCR检测细胞apollon mRNA的表达水平。
第三部分Apollon ASODN联合化疗药物对人结肠癌细胞增殖的影响以脂质体为载体把apollon ASODN转染入体外培养的结肠癌Lovo细胞,联合临床常用化疗药物(顺铂、表柔比星、5-氟尿嘧啶),采用WST-8法检测细胞增殖抑制作用,计算各组药物的增殖抑制率和半数抑制浓度(IC_(50)),并通过金正均的概率和法,计算联合用药的效果(作用拮抗、作用相加、作用协同)。
结果:
第一部分
IAPs ASODN作用于肝癌细胞(HepG_2)、胃癌细胞(MGC-803)、结肠癌细胞(Lovo)48小时后,与空白对照组(control)和随机寡核苷酸(random oligodeoxynucleotide,RODN)对照组相比,IAPs ASODN能明显抑制肝癌细胞、胃癌细胞、结肠癌细胞的增殖和诱导细胞凋亡(P＜0.05)。其中,在作用浓度为0.2μmol/L时,survivin ASODN抑制HepG_2、Lovo细胞增殖的作用最强,抑制率分别为84.2%、82.6%。apollon ASODN抑制Lovo细胞的增殖的作用最强,抑制率为82.27%。xiap ASODN抑制MGC-803细胞增殖作用最强,抑制率为86.56%,并呈时间-剂量依赖效应。Survivin ASODN诱导Lovo细胞、MGC-803细胞凋亡作用最强,凋亡率为34.95%、27.2%。livin ASODN诱导HepG_2细胞凋亡作用最强,凋亡率为22.5%。
第二部分
1.脂质体介导apollon ASODN转染Lovo细胞48小时后,细胞生长增殖和克隆形成均显著被抑制(P＜0.05),并呈浓度依赖关系。
2.运用AnnexinV-PI双染检测细胞的早期凋亡率显示:apollon ASODN作用于Lovo细胞48 h后,与RODN组,以及control组相比,ASODN组的早期凋亡率明显高于control组(P＜0.05),RODN组与对照组相比对凋亡无明显影响(P＞0.05)。
3.PI单染流式细胞术检测细胞周期百分比显示:与control、RODN组相比,apollonASODN组G0/G1期细胞减少,S期细胞增多,出现明显的S期阻滞。
4.Hoechst33258染色结果显示:apollon ASODN作用于Lovo细胞48 h后,空白对照组细胞形态完整,RODN组与空白对照组相比未见明显差异,而ASODN组可见核固缩、边聚、裂解等细胞凋亡形态学变化。
5.实时荧光定量RT-PCR结果表明:脂质体介导的apollon ASODN转染Lovo细胞能明显下调apollon mRNA的表达,与空白对照组、RODN组相比,有统计学意义(P＜0.05)。
第三部分
0.08μmol/L apollon ASODN与不同浓度的化疗药物(5-FU、EPI、DDP)联合作用于结肠癌细胞48小时后,可以提高结肠癌细胞对这些化疗药物的敏感性,增敏倍数分别为2.5、5.33、4.47。
结论:
1.脂质体介导的IAPs ASODN能够抑制消化系肿瘤细胞增殖、并诱导细胞凋亡。
2.不同浓度的apollon ASODN均能下调apollon基因的mRNA表达水平,抑制Lovo细胞增殖,并诱导Lovo细胞凋亡。
3.低浓度的apollon ASODN复合物联合化疗药物5-FU、DDP、EPI作用于Lovo细胞后,可以提高细胞对这些化疗药物的敏感性。
Objective:
To screen out the antisense oligodeoxynucleotide of inhibitor of apoptosis proteins that has a better suppressing effects on the cell proliferation and promoting apoptosis on gastrointestinal tumor cells(HepG2、Lovo、MGC-803),from the IAP family members ASODN.using the selected member apollon ASODN to act on colon cancer Lovo cells,and then observe particular biochemical reactions(proliferation inhibition,inducing apoptosis,sensitivity to the chemotherapeutic drugs on the treatment of colorectal cancer, and effect on the gene expression),To provide a new target site for the treatment of colorectal cancer.
Methods:
PartⅠ
IAPs ASODN and random oligonucleotides(RODN) were transfected into Gastrointestinal cancer cells(Lovo,HepG_2,MGC-803) treated with some concentration for 48 hours,MTT assay was applied to the determination of cell proliferation,apoptotic index was examined by flow cytometry.
PartⅡ
Apollon ASODN and random oligonucleotides(RODN) were transfected into Lovo cells for 48 hours,proliferation and the clone formation of Lovo cells were detected by CCK-8 and clone formation assay,respectively.The morphology was examined by fluorescence microscope.The percentage of hypodiploid cells of Lovo cells treated with apollon ASODN was determined with propidium iodide(PI) staining by flow cytometry.Early apoptosis was detected with Annexin V-FITC and PI dual parameter by flow cytometry.Furthermore,The expression of apollon gene was analyzed by real time fluorescent quantitativ reverse transcription-polymerase chain reaction.
PartⅢ
Apollon ASODN and random oligonucleotides(RODN) were transfected into Lovo cells, Lovo cells were incubated treated by 5-fluorouracil(5-FU) or Epirubicin(EPI) or Cisplatin (DDP),and their combination of ASODN at different concentrations for 48 hours respectively, then the cell proliferation was detected by WST-8 test,finally their IC_(50) and their drug-sensitivity were calculated statistically.
Results:
PartⅠ
After treated with antisense oligodeoxynucleotide targeting 48 hours,compared with blank control and random oligodeoxynucleotide groups,antisense oligodeoxynucleotide targeting IAPs can significantly suppressed the growth of gastrointestinal cancer cells and induce their apoptosis(P＜0.05),at a concentration of 0.2μmol/L,survivin ASODN has the significantly inhibited effects on the HepG_2,Lovo cells,the inhibitory rates were 84.2%,82.6%.respectively. the xiap ASODN has the significantly inhibited effects on the MGC-803 cell,inhibitory rates were 86.56%.they showed time and dose-dependent fashions.Survivin ASODN inducing the apoptosis of Lovo cell,MGC-803 cell was the strongest than others,the apoptotic rate were 34.95%、27.2%.respectively.livin ASODN inducing the apoptosis of HepG_2 cell was the strongest than others,the apoptotic rate were 22.5%.
PartⅡ
After treated with antisense oligodeoxynucleotide targeting apollon 48 hours,compared with blank control and random oligodeoxynucleotide groups,antisense oligodeoxynucleotide targeting apollon can significantly suppressed the growth of colorectal cancer cells and induce their apoptosis(P＜0.05).It was found that all concentrations of the apollon ASODN used in this study showed to be able to suppress the expression of the apollon gene.antisense oligodeoxynucleotide targeting apollon induce significant increase in s-phase cells,many Lovo cells stained with Hoechst 33258 exhibited apoptotic morphology such as cell shrinkage, nuclear condensation and nuclear fragmentation.
PartⅢ
Compared with single 5-FU,EPI and DDP group,The combition of 5-FU,EPI,DDP with apollon ASODN have enhanced their chemotherapeutic effects on Lovo cells,and the sensitivity enhance to 2.5,4.47,and 5.33 times respectively.
Conclusion:
1.Antisense oligodeoxynucleotide targeting IAPs can inhibit the proliferation and induce the apoptosis of gastrointestinal cancer cells in vitro.
2.The different antisense oligodeoxynucleotide targeting apollon can suppress the express levels of gene,and inhibit the proliferation and induce the apoptosis of colorectal cancer cells in vitro.
3.0.08μmol/L apollon ASODN can enhance the chemotherapeutic drug's sensitivity of Lovo cells to 5-FU,EPI and DDP.
引文
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