【作者】 杨怡；

【导师】 王娅兰；

【作者基本信息】 重庆医科大学 ， 病理学与病理生理学， 2012， 硕士

【摘要】 目的探讨利用RNA干扰技术沉默PARG基因的表达对小鼠结肠癌CT26细胞肝转移的影响。方法采用侵袭试验和细胞-基质黏附实验，观察PARG基因沉默前后CT26细胞侵袭及基质粘附能力的改变；通过脾脏包膜下注射细胞悬液的方式构建BALB/c小鼠肝转移模型，比较各组脾脏移植瘤、肝脏转移瘤结节及荷瘤小鼠生存期变化；采用Western blot检测PARG、PARP、NF-κB、integrin-β1、MMP-9和MMP-2在CT26细胞及脾脏移植瘤组织中的表达，并用明胶酶谱法分析肿瘤细胞培养上清液中MMP-9和MMP-2活性。结果1.侵袭试验和细胞-基质黏附实验结果显示，与对照组相比，PARG基因沉默后CT26细胞穿过微孔膜的数量明显减少（P<0.05），与FN的粘附率明显降低（P<0.05）。2.成功构建小鼠肝转移模型，脾脏包膜接种14天后，各组脾脏均有移植瘤形成，但PARG基因沉默组脾脏移植瘤的体积与对照组相比均有不同程度的缩小（P<0.05），肝脏转移瘤结节分级也明显低于对照组（P<0.05）。Kaplan-Meier生存曲线显示接种PARG基因沉默CT26细胞的荷瘤小鼠平均生存时间显著延长（P<0.05）。3.Western Blot结果显示，PARG基因沉默CT26细胞integrin-β1、MMP-9和MMP-2的蛋白表达量较对照组明显减少（P<0.05）。同时明胶酶谱法也检测到PARG基因沉默CT26细胞培养上清液中MMP-2和MMP-9的活性也明显被抑制（P<0.05）。4.PARG基因沉默组小鼠脾脏移植瘤组织中PARG、PARP、NF-κB、integrin-β1、MMP-9和MMP-2的蛋白表达量明显低于对照组（P<0.05）。结论1. PARG基因沉默能降低CT26细胞的侵袭及黏附能力，抑制小鼠脾脏移植瘤和肝脏转移瘤结节的形成，其可能在肿瘤细胞的侵袭转移过程中起重要调节作用。2. PARG基因沉默降低CT26细胞侵袭、黏附能力及肝脏转移瘤结节的形成，其机制可能与其降低PARP表达，抑制NF-κB的转录活性进而下调integrin-β1、MMP-9和MMP-2等下游靶基因的表达有关。更多还原

【Abstract】 Objective: The study was to investigate the effect of Poly(ADP-ribose)glycohydrolase (PARG) gene silenced via RNA interference on livermetastasis of CT26cell line in mice.Methods: Compared the migration and invasion abilities of PARGgene silenced CT26cells with untransfected CT26cells by cell invasionassay and cell-matrix adhesion assay. Metastasis models of BALB/c micewere established by intrasplenic inoculation of untransfected CT26cells,CT26cell transfected with empty vector and CT26cell transfected withPARG-shRNA respectively. The changes of splenic transplantation tumors,liver metastases and survival time were observed. The expressions of PARG,PARP, NF-κB, integrin-β1, MMP-9and MMP-2in vitro and in vivo weremeasured by Western blot analysis. The effect of PARG on activities ofMMP-9and MMP-2in various groups supernatant were detected byzymography．Results:1. The results of cell invasion assay and cell-matrix adhesionassay showed that PARG silencing lead to significant reduction in the number of CT26cells, that migrated to lower surface of the membrane(p<0.5) and adhered to fibronectin (p<0.5), compared to the two controlgroups.2. Succeeded to construct experimental metastasis model.14daysafter inoculation, the splenic transplantation tumors were observed in threegroups. But the average volume of splenic transplantation tumors andgrading of liver metastasis nodules were significantly less than that of twocontrol groups (p<0.5). The Kaplan-Meier curves showed that PARGsilencing can efficiently prolong the survival time of mice.3. The expressions of integrin-β1, MMP-9and MMP-2in CT26cellslacking PARG were lower than that of two control groups (p<0.5). Similarresults were attained in activities of MMP-9and MMP-2in various groupssupernatant (p<0.5).4.The expressions of PARG, PARP, NF-κB, integrin-β1, MMP-9andMMP-2in splenic transplantation tumors were significantly reduced inPARG-shRNA treated group in comparison with other two control groups(p<0.5).Conclusion:1.PARG silenced via RNA interference can suppress themigration and invasion abilities of CT26cells. In addition, PARG silencedcan inhibit the formation of splenic transplantation tumors and livermetastases. It suggests that PARG may play an important role in tumorinvasion and metastasis. 2.The inhibitory effect of PARG silenced on CT26cell adhesion,invasion and formation of liver metastases may be attributed by reductionof integrin-β1, MMP-9and MMP-2via its downstream regulation ofPARP and NF-κB-dependent genes.更多还原