RNA干扰沉默DcR3基因对人结肠癌裸鼠皮下移植瘤模型作用的初步研究
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摘要
【目的】
体外研究证实,DcR3 siRNA通过特异、高效地沉默结肠癌SW480细胞DcR3 mRNA的表达,从而抑制该结肠癌细胞增殖,诱导其凋亡。本实验利用这一特点,观察DcR3siRNA的作用下结肠癌SW480细胞裸鼠皮下移植瘤内DCR3的表达情况,分析DcR3siRNA对肿瘤的作用,探讨DcR3 siRNA对人结肠癌SW480细胞裸鼠皮下移植瘤生长的影响及可能的作用机制。
【方法】
1、裸鼠背部皮下种植结肠癌SW480细胞,建立结肠癌SW480细胞裸鼠皮下移植瘤模型。2、针对靶标DcR3基因,利用Dharmacon公司软件设计了合成siRNA序列,通过瘤体局部多点注射脂质体与DcR3 siRNA混合物,将DcR3 siRNA转染到裸鼠背部皮下移植瘤内。同时设立阴性对照组和空白对照组。3、观察肿瘤治疗前后体积变化,评价DcR3 siRNA对肿瘤的抑制作用;比较肿瘤治疗前后的细胞形态学改变;免疫组织化学及RT-PCR检测DcR3基因表达;原位细胞凋亡检测肿瘤的凋亡情况。
【结果】
1、成功建立了结肠癌SW480细胞裸鼠皮下移植瘤模型。
2、RT-PCR检测证实,治疗后各组肿瘤组织中DcR3基因均有不同程度的表达,其中治疗组肿瘤组织相对光密度值明显低于阴性对照组和空白对照组相对光密度值(P＜0.05)。
3、免疫组织化学检测显示,治疗组细胞浆内着色明显浅于阴性对照组和空白对照组,治疗组与阴性对照组及空白对照组有显著差异(P＜0.01)。
4、治疗组裸鼠移植瘤体积显著小于阴性对照组和空白对照组(P＜0.01)。
5、电镜结果显示,治疗组肿瘤细胞出现细胞器损伤,数目减少,完整性降低,细胞调亡。
【结论】
1、人结肠癌SW480细胞在裸鼠皮下有良好的成瘤性。
2、DcR3基因可在结肠癌裸鼠皮下移植瘤内产生特异性表达。
3、脂质体介导的DcR3 siRNA对裸鼠皮下结肠癌SW480细胞移植瘤有明显的抑制、杀伤作用,细胞凋亡是DcR3 siRNA所致肿瘤细胞死亡的重要形式。
[Objective]
DcR3 siRNA suppresses colon carcinoma cells proliferation and induce them apoptosis by specific silencing the expression of DcR3 mRNA gene in colon carcinoma SW480 cells. According to this, by abserving the expression of DcR3 in athymic micetransplantation tumor treated subcutaneouly by human colon carcinoma SW480 cells and analyzing the effect of DcR3 siRNA to investigate the effect of DcR3 siRNA to the growth of transplantation tumor and possible mechanism of action.
[Methods]
1. Athymic mice were subcutaneously transplanted with human colon carcinoma SW480 cells to establish transplanted tumor model.
2. To aim directly at target gene—DcR3, siRNA sequence was synthesized by Dharmacon company software , then transfected into subcut transplantation tumor in athymic mice. To establish negative and blank control group.
3. The suppression effect of DcR3 siRNA to tumor was estimated and the change of morphocytology was compared by observing the tumor volume change on pretherapy and posttherapy. The genetic expression of DcR3 was detected by immunohistochemistry and reverse transcriptase polymerase chain reaction, the tumor apoptosis was detected by TUMEL method.
[Results]
1. The implanted tumor model with human colon carcinoma SW480 cells in athymic mice was susussfully established.
2. By the application of reverse transcriptase polymerase chain reaction, it proved that the expression of DcR3 gene were distinct in each group on posttherapy . The relative optical density was obviously lower in treatment group than in negative and blank control group. (P<0.05).
3. The result of immunohistochemistry displayed that the intracytoplasm color was lighter in treatment group than in negative and blank control group by.it exists significant deviation(P<0. 01).
4. The transplantation tumor volume was significantly smaller in treatment group than in contol groups.
5. It was detected that the tumor cells apoptosis number was much more in treatment group than in control groups by TUNEL method (P<0.01) .
[Conclusions]
1. Human colon carcinoma SW480 cells have satisfactory characteristic with tumorigenesis.
2. DcR3 gene can create specific expression in subcut transplantation tumor of athymic mice.
3. DcR3 siRNA Mediated by liposome distinctly inhibits and kills subcut transplantation tumor with human colon carcinoma SW480 cells in athymic mice.
体外研究证实,DcR3 siRNA通过特异、高效地沉默结肠癌SW480细胞DcR3 mRNA的表达,从而抑制该结肠癌细胞增殖,诱导其凋亡。本实验利用这一特点,观察DcR3siRNA的作用下结肠癌SW480细胞裸鼠皮下移植瘤内DCR3的表达情况,分析DcR3siRNA对肿瘤的作用,探讨DcR3 siRNA对人结肠癌SW480细胞裸鼠皮下移植瘤生长的影响及可能的作用机制。
【方法】
1、裸鼠背部皮下种植结肠癌SW480细胞,建立结肠癌SW480细胞裸鼠皮下移植瘤模型。2、针对靶标DcR3基因,利用Dharmacon公司软件设计了合成siRNA序列,通过瘤体局部多点注射脂质体与DcR3 siRNA混合物,将DcR3 siRNA转染到裸鼠背部皮下移植瘤内。同时设立阴性对照组和空白对照组。3、观察肿瘤治疗前后体积变化,评价DcR3 siRNA对肿瘤的抑制作用;比较肿瘤治疗前后的细胞形态学改变;免疫组织化学及RT-PCR检测DcR3基因表达;原位细胞凋亡检测肿瘤的凋亡情况。
【结果】
1、成功建立了结肠癌SW480细胞裸鼠皮下移植瘤模型。
2、RT-PCR检测证实,治疗后各组肿瘤组织中DcR3基因均有不同程度的表达,其中治疗组肿瘤组织相对光密度值明显低于阴性对照组和空白对照组相对光密度值(P＜0.05)。
3、免疫组织化学检测显示,治疗组细胞浆内着色明显浅于阴性对照组和空白对照组,治疗组与阴性对照组及空白对照组有显著差异(P＜0.01)。
4、治疗组裸鼠移植瘤体积显著小于阴性对照组和空白对照组(P＜0.01)。
5、电镜结果显示,治疗组肿瘤细胞出现细胞器损伤,数目减少,完整性降低,细胞调亡。
【结论】
1、人结肠癌SW480细胞在裸鼠皮下有良好的成瘤性。
2、DcR3基因可在结肠癌裸鼠皮下移植瘤内产生特异性表达。
3、脂质体介导的DcR3 siRNA对裸鼠皮下结肠癌SW480细胞移植瘤有明显的抑制、杀伤作用,细胞凋亡是DcR3 siRNA所致肿瘤细胞死亡的重要形式。
[Objective]
DcR3 siRNA suppresses colon carcinoma cells proliferation and induce them apoptosis by specific silencing the expression of DcR3 mRNA gene in colon carcinoma SW480 cells. According to this, by abserving the expression of DcR3 in athymic micetransplantation tumor treated subcutaneouly by human colon carcinoma SW480 cells and analyzing the effect of DcR3 siRNA to investigate the effect of DcR3 siRNA to the growth of transplantation tumor and possible mechanism of action.
[Methods]
1. Athymic mice were subcutaneously transplanted with human colon carcinoma SW480 cells to establish transplanted tumor model.
2. To aim directly at target gene—DcR3, siRNA sequence was synthesized by Dharmacon company software , then transfected into subcut transplantation tumor in athymic mice. To establish negative and blank control group.
3. The suppression effect of DcR3 siRNA to tumor was estimated and the change of morphocytology was compared by observing the tumor volume change on pretherapy and posttherapy. The genetic expression of DcR3 was detected by immunohistochemistry and reverse transcriptase polymerase chain reaction, the tumor apoptosis was detected by TUMEL method.
[Results]
1. The implanted tumor model with human colon carcinoma SW480 cells in athymic mice was susussfully established.
2. By the application of reverse transcriptase polymerase chain reaction, it proved that the expression of DcR3 gene were distinct in each group on posttherapy . The relative optical density was obviously lower in treatment group than in negative and blank control group. (P<0.05).
3. The result of immunohistochemistry displayed that the intracytoplasm color was lighter in treatment group than in negative and blank control group by.it exists significant deviation(P<0. 01).
4. The transplantation tumor volume was significantly smaller in treatment group than in contol groups.
5. It was detected that the tumor cells apoptosis number was much more in treatment group than in control groups by TUNEL method (P<0.01) .
[Conclusions]
1. Human colon carcinoma SW480 cells have satisfactory characteristic with tumorigenesis.
2. DcR3 gene can create specific expression in subcut transplantation tumor of athymic mice.
3. DcR3 siRNA Mediated by liposome distinctly inhibits and kills subcut transplantation tumor with human colon carcinoma SW480 cells in athymic mice.
引文
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