过氧化物酶体增殖因子活化受体δ（PPARδ）在大肠癌发生中的作用研究

【作者】 杨烈；

【导师】 周总光；

【作者基本信息】 四川大学 ， 外科学， 2007， 博士

【摘要】 目的：分析人直肠癌组织中PPARδ基因的表达变化及其与直肠癌临床病理特征的关系。方法：选取2004年4-10月四川大学华西医院普外科手术切除的直肠癌标本86例，以正常直肠粘膜为对照，采用real-time RT-PCR对PPARδmRNA作定量检测，并分析其表达与直肠癌分化程度、病理类型及Dukes分期的关系。结果：在86例直肠癌组织中，PPARδ表达上调占55．8％（48/86），其中，45．3％（39/86）上调1．5-5倍，5．8％（5/86）上调10-20倍，4．7％（4/86）大于20倍，PPARδ表达下调占44．2％（38/86），下调1．5-2．0倍。直肠癌组织中PPARδ的总体表达水平比正常粘膜组平均上调1．046倍，但是两组间并无统计学差异（P=0．863）。在不同分化程度、病理类型及Dukes分期的直肠癌组织中，PPARδ的表达无显著性差异（P＞0．05）。结论：与正常直肠组织相比，直肠癌组织中约55．8％的PPARδ表达上调，其总体表达水平无显著变化，PPARδ的表达与直肠癌分化程度、病理类型及Dukes分期无关。目的：探讨PPARδ基因对大肠癌细胞增殖活力及凋亡机能的影响。方法：构建4条表达短发夹结构RNA（shott-hairpin RNA,shRNA）的质粒载体，其中3条为靶向PPARδ基因的候选载体（pS-shRNA-1，-2，-3），1条为不靶向任何编码序列的阴性干扰载体（pS-shRNA-N）。将人大肠癌细胞株HCT-116分为4个组：①．干扰组：分别用ps-shRNA-1，-2，-3转染细胞；②．阴性干扰组：用pS-shRNA-N转染细胞；③．空白质粒组：用空白pSUPER质粒转染细胞：④．对照组：HCT-116细胞和其它组在相同条件下培养，但不转染质粒。用脂质体2000进行转染，48h后，应用real-time RT-PCR分析PPARδ基因表达，筛选出强效表达载体。采用噻唑蓝（MTT）比色实验、流式细胞术及原位缺口末端标记法（TUNEL）观察PPARδ基因沉默对大肠癌细胞增生、细胞周期及凋亡的影响。结果：pS-shRNA-1质粒能显著抑制HCT-116细胞中PPARδ的表达，使PPARδmRNA水平下调73．5％（P=0．012）。MTT试验显示，转染后24-96h，对照组、pS-shRNA-N及空白质粒组的光吸收值在各时点均无显著性差异（P＞0．05），而pS-shRNA-1干扰组在各时点的光吸收值均显著高于前三组细胞（P＜0．05）。转染后48h，干扰组的G₁期细胞分布比对照组显著降低（26．8％VS 38．6％，P=0．037），而对照组、pS-shRNA-N及空白质粒组均无显著性差异（P＞0．05），各组在S期与G₂/M期的细胞分布比例（S期：G₂/M期）无显著差异（P=0．782）。转染后48h，pS-shRNA-1干扰组、pS-shRNA-N组、空白质粒组及对照组的细胞凋亡指数分别为3．76±1．81％、3．98±2．0％、4．0±1．64％、4．70±1．93％，各组间无显著性差异（P=0．726）。结论：PPARδ基因可增加G₁期的大肠癌细胞分布，并抑制细胞增殖，PPARδ对大肠癌细胞的凋亡无影响。更多还原

【Abstract】 PURPOSE: To clarify the expression change of Peroxisome proliferator-activated receptorδ（PPARδ） gene in human colorectal cancer tissues, and determine the correlation of PPARδexpression with clinical and pathological parameters of colorectal cancer. METHODS: Applying Real-time RT-PCR, we quantified PPARδmRNA in 86 tissues from excised primary rectal cancers. In each case, accompanying normal mucosa was collected for comparison.RESULTS: Among the 86 rectal cancer tissues, 55.8% （48/86） cases showed PPARδoverexpression: 45.3% （39/86） tumors gave an expression level 1.5 to 5.0 times, 5.8% （5/86） tumors 10 to 20 times, and 4.7% （4/86） tumors more than 20 times relative to normal mucosa. However, the general level of PPARδmRNA in rectal cancer tissues is not statistically different from normal mucosa. No significant links were found between PPARδexpression and cell differentiations, pathological categories and Dukes stages.CONCLUSIONS: The expression of PPARδgene in rectal cancers is not statistically different from normal mucosa, and it doesn’t correlate with cell differentiation, pathological categories and Dukes stages. PURPOSE: To investigate the effects of peroxisome proliferator-activated receptorδ（PPARδ） on the proliferation and apoptosis of human colorectal cancer （CRC） cells.METHODS: For RNA interfering （RNAi）, HCT-116 cells were transfected with short hairpin RNA （shRNA）-expressing plasmids against PPARδor negative control vectors. The efficacy of RNAi was assessed by real-time reverse transcription polymerase chain reaction （RT-PCR）. The proliferation, cell cycle and apoptosis of HCT-116 cells treated by RNAi, compared with those containing control vectors or untreated, were analyzed respectively using MTT （methyl thiazolyl tetrazolium）, flow cytometry （FCM） and TdT （terminal deoxynucleotidyl transferase）-mediated dUTP nick end-labeling （TUNEL） assay.RESULTS: RNAi targeting PPARδresulted in substantial suppression of PPARδexpression and significantly promoted the proliferation of HCT-116 cells relative to those with control vectors or untreated, obviously decreasing the frequency of G₁-phase cells, but had no effect on cell apoptosis.CONCLUSIONS: PPARδmay inhibit the proliferation of CRC cells and increase the number of G₁-phase cells, without any function in cellapoptosis, which suggests an inhibitive role of PPARδgene in colorectalcarcinogenesis.更多还原