【作者】 陈坚；

【导师】 林庚金；

【作者基本信息】 复旦大学 ， 内科学， 2003， 博士

【摘要】 第一部分不同分化胃癌细胞株内超氧化物歧化酶的表达及内源性活性氧水平目的 检测正常胃粘膜及MKN-28、SGC7901、BGC823、HGC-27四种不同分化的胃癌细胞株内锰型超氧化物歧化酶（MnSOD）mRNA的表达，评价MnSOD转录水平与肿瘤细胞分化程度、胞内活性氧(ROS)水平及增殖活性的相关性。方法 利用酶底物催化方法测定四种胃癌细胞株的碱性磷酸酶（ALP）及乳酸脱氢酶（LDH）活力。RT－PCR方法检测正常胃粘膜组织及MKN-28、SGC7901、BGC823、HGC-27四株高、中、低、未分化胃癌细胞株内的MnSOD mRNA的表达。利用2’7’-二氯氢化荧光素二酯（DCFH－DA）荧光染色方法检测不同分化胃癌细胞株的胞内ROS水平。四唑蓝（MTT）比色法测定不同分化胃癌细胞株的增殖活性并绘制生长曲线。结果 MKN-28、SGC7901、BGC823、HGC-27四株细胞ALP及LDH的酶活性有显著差异。不同分化胃癌细胞内的MnSOD表达普遍低于正常胃粘膜，且随分化程度减低而逐步下降；其胞内ROS水平随MnSOD表达的下降而逐步上升，同时肿瘤增殖活性上升。第二部分MnSOD基因转染对SGC7901胃癌细胞增殖的影响目的 观察改变胞内MnSOD基因表达水平对SGC7901胃癌细胞增殖的影响。方法 采用电穿孔方法将反义和正义MnSOD cDNA真核表达载体pHβA-SOD (-)/pHβA-SOD (+)转染SGC7901胃癌细胞，400mg/LG418筛选稳定表达克隆并用RT-PCR及Western杂交法鉴定后扩大培养。用pHβA空质粒转染作为对照。放射免疫法检测正义、反义及空载SGC7901胃癌细胞株内的铜锌超氧化物歧化酶（CuZnSOD，SOD1）蛋白含量。分别用直接细胞计数法、四唑蓝（MTT）比色法、平皿克隆形成率试验及裸鼠移植瘤模型测定三组细胞在体外、体内的增殖活性。结果 与空载组（Vector－7901）相比，转染正义质粒的MnSOD-7901胃癌细胞呈现抑增殖效应：(1) 生长曲线示增殖速度减慢； (2)在平皿上的集落形成能力下降；(3)在裸鼠体内的成瘤性明显受抑制。转染反义质粒的MnSOD-AS7901胃癌细胞则出现促增殖效应：(1) 生长曲线示增殖速度加快； (2)在平皿上的集落形成率上升；(3)裸鼠移植瘤生长加速。第三部分MnSOD调控SGC7901胃癌细胞增殖的分子机制目的 进一步研究MnSOD高、低表达调控SGC7901胃癌细胞增殖的分子生物学机<WP=4>制。方法 采用DCF－DA荧光染色法检测MnSOD－7901、MnSOD－AS7901及Vector－7901三组细胞的胞内ROS水平；透射电镜观察细胞超微结构改变；流式细胞仪（FCM）检测细胞周期；免疫组化法检测P53、Bcl－2、Ki67、C-erbB2 的蛋白表达。RT－PCR检测三组细胞的P53mRNA表达。结果 与Vector－7901相对照，MnSOD－7901细胞胞内ROS水平下降， G0/G1期细胞比例增高，S期细胞减少， Ki67表达明显抑制， Bcl-2及C-erbB2表达轻度下调。MnSOD－AS7901细胞胞内ROS水平上升， G0/G1期细胞减少，S期细胞增多， Ki67表达明显升高， Bcl-2及C-erbB2表达轻度上升。此外，MnSOD－7901 细胞及MnSOD－AS7901细胞的电镜下超微结构发生改变。第四部分丹参酮ⅡA诱导SGC7901胃癌细胞凋亡的研究目的 观察抗氧化剂丹参酮ⅡA（TanⅡA）在体外对SGC7901胃癌细胞的作用，并对其分子机制作初步研究。。方法 分别采用1.0mg/L、2.0 mg/L、4.0 mg/L、6.0 mg/L、8.0 mg/L、10.0mg/L的不同浓度梯度的TanⅡA干预SGC7901胃癌细胞24、48、72小时后，用MTT比色法绘制肿瘤细胞生长曲线。采用1.0mg/L、2.0 mg/L、4.0 mg/L、6.0 mg/L、8.0 mg/L、10.0mg/L的不同浓度梯度的TanⅡA干预SGC7901细胞24小时，或同样采用10.0mg/L的TanⅡA干预0、12、24、36、48小时，应用碘化丙叮（PI）及Annexin V双重染色法，通过流式细胞仪观察不同浓度梯度及不同时间梯度干预下的SGC7901细胞凋亡率及坏死率。用透射电镜直接观察细胞凋亡形态并拍摄照片记录。用RT－PCR检测1.0mg/L、2.0 mg/L、4.0 mg/L、6.0 mg/L、8.0 mg/L、10.0mg/L的TanⅡA干预24小时后SGC7901细胞P53的mRNA表达。结果 抗氧化剂TanⅡA在体外具有良好的抗肿瘤效应，呈现时间依赖性及浓度依赖性地诱导SGC7901细胞坏死及凋亡。随着TanⅡA干预浓度的逐步上升，胞内 P53mRNA表达亦逐步上升。结 论(1) 胃癌细胞内 MnSOD的表达低于正常，MnSOD的表达水平与胃癌分化程度、胞内ROS水平及肿瘤增殖活性密切相关。(2) 通过基因转染提高胞内MnSOD 的表达可抑制胃癌细胞的增殖，MnSOD可能是胃癌基因治疗的一个新靶点。(3) MnSOD抑制胃癌细胞增殖的机制与下调胞内ROS水平，G0/G1期细胞周期阻滞，上调P53表达，下调Ki67、 Bcl-2及C-erbB2表达有关。抗氧化剂丹参酮ⅡA在一定浓度范围内呈时间依赖性及剂量依赖性地诱导胃<WP=5>(4) 癌细胞坏死及凋亡，机制与P53表达上调有关。更多还原

【Abstract】 Effects of MnSOD Transfection and TanⅡA on the growth of gastric cancer cell lines and the molecular mechanisms Part 1. MnSOD expression is relevant with endogenous ROS differentiation degree and proliferation in gastric cancer cell linesObjective Try to measure maganese superoxide dismutase(MnSOD) expression in normal gastric mucosa and MKN-28 SGC7901 BGC823 HGC-27 gastric cancer cell lines. Furthermore，try to elucidate the relationships among MnSOD expression and endogenous reactive oxygen species(ROS)， tumor differentiation degree and proliferate activities in these cell lines. Methods Enzyme activities of alkaline phosphatase (ALP) lactate dehydrogenase (LDH), which represent the differentiation degree of these cell lines, were measured by enzymatic catalysis assay. RT-PCR and DCFH-DA fluorescence assay were taken to identify the MnSOD transcription and ROS contents respectively. In addition, the proliferate activities were measured by MTT assay and direct cells count. Results Enzymatic activities of ALP and LDH are significantly different in SGC7901、BGC823、HGC-27 cell lines. Meanwhile，it indicates much less MnSOD expressions in these four gastric cancer cell lines compared with that in the normal gastric mucosa. With the decrease of MnSOD transcriptions, the endogenous ROS level rise synchronously as well as the proliferation activities in MKN-28 SGC7901 BGC823 HGC-27 gastric cancer cell lines. Part 2. Effects on the proliferation of SGC7901 via MnSOD transfectionObjective To observe the effects on proliferation of SGC7901 mediated by MnSOD gene transfection.Methods Transferring sense and antisense MnSOD cDNA carrier pHβA-SOD (-)/pHβA-SOD (+) or mock vector into SGC7901 gastric cancer cell by electroporation. Screening stable expression sublines by 400ug/ml G418<WP=7>solution and further confirmed by RT-PCR and western blot. Then the subtypes of SGC7901 were marked as MnSOD-7901 MnSOD-AS7901 and vector-7901 respectively. The Copper-Zinc superoxide dismutase (CuZnSOD) contents in the transfectants were also measured by radioimmunoassay. Evaluate the proliferation activities in these three subtypes by direct cells count, MTT assay, plate clone formation test and nude mice transplanting tumor model respectively. Results Compared with the vector-7901, MnSOD-7901 reveals such characteristics as followed: (a) Partial inhibition of proliferation activities reflected by growth curves；(b) Decreasing clone formation on plate; (d) Remarkable growth retardation of transplanting tumor in nude mice. On the contrary, the MnSOD-AS7901 subline reveals the promotion of tumor proliferation. Part 3. The molecular mechanisms involved in the proliferation modulated by MnSOD transfectionObjective Try to disclose the molecular mechanisms associated with MnSOD transfection, which are involved in modulating the proliferation activities in SGC7901 gastric cancer cell line.Methods DCFH-DA fluorescence assay were used to measure the ROS contents in MnSOD－7901 MnSOD－AS7901 and Vector－7901 sublines. Ultra structures of the transfectants were studied by transmission electron microscopy. Cell cycles were examined by flow cytometry. Immunohistochemistry assay were taken to identify P53 Bcl－2 Ki67 and C-erbB2 protein expression in the slices of nude mice transplanted tumor.P53 mRNA expression was further evaluated by RT-PCR.Results Compared with the vector-7901, MnSOD-7901 subline reveals such genomic or molecular changes: (a) Decreasing endogenous ROS level; (b) G1 phase arrest in cell cycle;(c) Down-regulated expression of Bcl－2 Ki67 and C-erbB2.(d) P53 mRNA expression was up-regulated. Meanwhile, the MnSOD-AS7901 subline show the opposite changes.<WP=8>Part4. Apoptosis induced by TashinoneⅡA in SGC7901 gastric cancer cell Objective To assess the anticancer effects of antioxidant TashinoneⅡA (TanⅡA) on SGC7901 gastric cancer cell in vitro.Methods SGC7901 gastric cancer cells were inoculated into the 96-well-plates. They were trea更多还原