【作者】 李敏；

【导师】 王锐；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 目的：许多抗肿瘤化疗药物虽然结构不同、作用机制不同、作用靶点一不同，但都可能诱导肿瘤细胞的凋亡。本研究通过观察放线菌素D（ActD）对体外培养的胃腺癌BGC823细胞生长增殖、凋亡的影响及表达Survivin、HSP70和Cox-2的变化，探讨ActD与胃腺癌细胞凋亡的关系和ActD诱导细胞凋亡的分子机制。用基因芯片研究放线菌素D诱导胃癌细胞凋亡的相关差异表达的基因。同时检测了胃癌组织中Survivin、Cox-2和HSP-70的表达，探讨三者与胃癌发生发展的可能关系。方法：应用MTT观察了ActD及其类似物对胃癌BGC-823细胞生长增殖的影响，应用基因芯片、流式细胞仪、激光共聚焦显微镜和电子显微镜等技术检测了ActD对胃癌BGC-823细胞的诱导凋亡作用及差异表达的基因；RT-PCR观察了用药前后胃癌细胞表达Survivin、Cox-2、HSP70等几种凋亡相关基因的变化；构建胃癌组织芯片，应用免疫组织化学方法检测Survivin、Cox-2和HSP-70在胃癌组织中的表达并对三者相关性及临床意义进行初步探讨。结果：1．浓度大于5.0×10^-9mol/l时，ActD对胃腺癌细胞的生存有显著的影响，并呈剂量、时间依赖性，随着药物浓度的增高和作用时间的延长，ActD对体外培养的BGC823细胞的生长抑制作用亦增强，且差异有统计学意义（P＜0.05）。ActD类似物中[D-Phe²，D-Me-Phe⁵]₂ AMD，[D-Phe²，D-Me-Val⁵]₂，与母体对BGC823细胞的生长抑制作用相似，但是两种类似物和母体相比较，对胃腺癌BGC823细胞生长的抑制率在各作用时间内、各浓度组中均略低于母体。2．浓度为1.0×10^-7，5.0×10^-8mol/l时，ActD主要以细胞毒作用为主，流式细胞术检测发现其主要表现为坏死峰；浓度为1.0×10^-8，5.0×10^-9，1.0×10^-9mol/l时，ActD作用72h后可诱导BGC823细胞发生凋亡，在G1期前出现明显的凋亡峰，尤以5.0×10^-9mol/l最为显著，凋亡率分别为8.0％、28.0％、7.8％；当作用时间小于72h时，仅有5.0×10^-9mol/l组出现明显的凋亡峰，凋亡率为6.0％；ActD处理BGC823细胞大于24h（48h、72h）后，G1期含量则较对照组均有不同程度的升高，而G2、S期细胞含量均有不同程度的降低。3．经ActD(1.0×10^-8，5.0×10^-9mol/l)处理48h、72h后，胃腺癌细胞发生凋亡，形成凋亡小体。4．1.0×10^-9mol/l、5.0×10^-9mol/l两实验组Survivin mRNA相对表达水平较对照组均降低，而且浓度较高组survivin mRNA相对表达水平更低。5.0×10-9mol/l浓度实验组Cox-2和HSP70mRNA相对表达水平均明显低于浓度1.0×10-9mol/l实验组。5．ActD处理胃癌BGC-823细胞后，基因芯片筛选出差异表达的基因19条，其中16条表达上调，3条表达下调。主要是与DNA修复、免疫、凋亡、细胞增殖、信号转导等多个相关基因的差异表达。6．Survivin、Cox-2和HSP70在胃癌中的阳性表达率分别为58.6％（58/99）、60.6％（60/99）和61.6％（61/99），三者阳性表达与胃癌浸润深度相关（P＜0.05），Cox-2和HSP70阳性表达与淋巴结转移有关（P＜0.05）；Survivin和HSP70在胃癌组织中共表达率为37.4％（37/99），Survivin和Cox-2在胃癌组织中共表达率为35.4％（35/99），Cox-2和HSP70在胃癌组织中共表达率为41.4％（41/99），三项指标在胃癌中的表达无相关性（P＞0.05）；Kaplan-Meier生存曲线显示三项指标阳性表达与胃癌患者的生存率之间差异无统计学意义（P＞0.05）；单因素Cox回归模型分析结果显示，胃癌浸润深度、患者性别、淋巴结转移及Cox-2蛋白表达等四因素与患者预后有关，具有统计学意义（P＜0.05）；多因素Cox回归模型分析结果仅见胃癌浸润深度、胃癌患者性别及淋巴结转移等三因素具有独立的预后意义（P＜0.05）；胃癌多因素Cox回归模型风险函数：结论：1．高浓度组ActD主要以细胞毒作用对胃腺癌细胞造成直接杀伤致死作用，对胃癌细胞的生长有显著的抑制作用，且具有时间和浓度依赖性；低浓度组ActD抑制胃腺癌细胞的生长、增殖，主要通过诱导胃癌细胞发生凋亡来实现，低浓度组ActD对胃癌细胞生长、增殖的影响与细胞周期有一定关系；低浓度ActD可以使胃癌细胞Survivin、Cox-2和HSP70表达下调，提示Survivin、Cox-2和HSP70表达下调可能共同参与ActD诱导的胃癌细胞凋亡。2．ActD类似物中[D-Phe²，D-Me-Phe⁵]₂ AMD，[D-Phe²，D-Me-Val⁵]₂ AMD，对BGC823细胞的生长抑制作用与母体相似，但抑制率低于母体，提示还需对ActD的结构改造作进一步研究。3．ActD可以影响人胃癌BGC823细胞株凋亡等多个相关基因的差异表达。4．Survivin、Cox-2和HSP70在胃癌中均有过度表达。Survivin过度表达可能主要参与胃癌发生、发展的早期过程；Cox-2和HSP70与胃癌的浸润、转移密切相关；胃癌浸润深度、患者性别、淋巴结转移及Cox-2蛋白表达等因素与患者预后有关，而胃癌浸润深度、患者性别及淋巴结转移具有独立的预后意义。更多还原

【Abstract】 Objective In order to investigate the effects of Act D on the growth and proliferation of gastric adenocarcinoma cells and apoptosis of gastric adenocarcinoma cells induced by ActD. To study the effect of ActD on Survivin,Cox-2 and HSP70 mRNA expression of gastric adenocarcinoma cells （BGC823）, in vitro. To investigate the prevalence and their clinical significance of Survivin,Cox-2 and HSP70 in gastric carcinoma and screen the prognostic factors of gastric carcinoma.Methods MTT assay was used to observe the viability and growth inhibition of human gastric adenocarcinoma cells （BGC823） after different time treatment with various concentrations of ActD and ActD analogs. The percentage of apoptotic cells and the cell-cycle distribution of BGC823 cells treated with ActD were measured by flow cytometry （FCM）. Using the transmission electron microscopy （TEM）, the change of ultrastructural organization of BGC823 cells was observed. Expression of Survivin,Cox-2 and HSP70 mRNA was detected using RT-PCR in gastric cancer cells （BGC823）.To identify and analysis the differentially expressed genes profile on gastric cancer cells （BGC823） by cDNA microarray.The gastric cancer tissue microarrays （TMA） containing 99 gastric cancer specimens from different clinical stages were used to determine Survivin,Cox-2 and HSP70 expression by immunohistochemistry.Results 1.When the concentrations of ActD were higher than 5.0×10^-9mol/l, the cell growth of BGC823 was suppressed statistically in dose- and time- dependent manner （P<0.05）. ActD analogs [D-Phe², D-Me-Phe⁵]₂ AMD and [D-Phe², D-Me-Val⁵] AMD₂ were similar with ActD suppressed the cell growth of BGC823.The inhibition ratio of ActD analogs were lower than that ActD.2.After 48h and 72h treatment with the higher concentrations of ActD (1.0×10^-7, 5.0×10^-8mol/l), the cell growth of BGC823 was suppressed remarkably and necrotic peak was observed in all specimens.The percentage of apoptotic cells increased to 8.0%, 28.0% and 7.8% after 72h treatment with 1.0×10^-8, 5.0×10^-9 and 1.0×10^-9 mol/l of ActD, respectively, and increased to 6.0% after 48h treatment with 5.0×10^-9mol/l of ActD, while the percentage of apoptotic cells was unchanged after less than 72h incubation with 1.0×10^-8 and 1.0×10^-9mol/l of ActD.After 24h treatment with 1.0×10^-8, 5.0×10^-9 and 1.0×10^-9mol/l of ActD, the G1 phase content of BGC823 cells decreased and the G2 phase content of BGC823 cells increased as compared with control group. However, the G1 phase content of BGC823 cells increased and the G2, S phase content of BGC823 cells decreased as compared with control group after 48h and 72h treatment.3.After 48h and 72h incubation with the higher concentrations of ActD (1.0×10^-7, 5.0×10^-8mol/l,1.0×l0^-9mol/l), nuclear condensation, nuclear fragmentation and apoptotic bodies were observed.4. After 72h treatment with 1.0×10^-9 and 5.0×10^-9mol/l of ActD, the relative expression level of survivin mRNA was down in gastric cancer cells.The relative expression level of Cox-2 and HSP70 mRNA treatmented with 5.0×10^-9mol/l was lower obviously it treatmented with 1.0×10^-9 mol/l.5. After the gastric cancer cells was treatment with of ActD, 16 genes were up-regulated and 3 genes were down-regulated.The profile of these differentially expressed mainly focus on DNA repair genes,immunologic genes,apoptosis genes,cell proliferation genes,signal transduction genes,etc.6.The expression rates of Survivin,Cox-2 and HSP70 were 58.6% （58/99）,60.6% （60/99） and 61.6%（61/99）, respectively. There were a significant correlation between Survivin,Cox-2 and HSP70 expression and the depth of invasion（P<0.05）.Cox-2 and HSP70 were found to be statistically associated with lymph node metastasis of gastric cancers （P<0.05）. Coexpression rates of survivin and Cox-2 and HSP70 were observed in 37.4%,35/99,41/99,respectively. But no statistically significant correlation was found （P>0.05）. Kaplan-Meier survival plots for 89 patients showed no significant relationship（P>0.05） between Survivin,Cox-2 and HSP70 expression and the 5-year survival rates of patients with gastric carcinoma.In the initial univariate analysis of 89 gastric cancer cases followed up, the depth of invasion, sex, lymph node metastasis,Cox-2 expression were all significant（P<0.05）. Multiple Cox regression analyses demonstrated that the depth of invasion,sex and lymph node metastasis were independent prognosticators（P<0.05）. According to regression coefficients of the depth of invasion,sex and lymph node metastasis in the final Cox regression analysis, a risk function of postoperative patients with gastric cancers was established as follows:h（t）=[h₀（t）]e^{0.411X₁+0.635 X₃+0.630X₆} Conclusions 1. The higher concentrations of ActD cause cell death directly by cytotoxic role and suppress the cell growth of BGC823 notablely in dose- and time- dependent manner. After longer time treatment with the lower concentrations of ActD, the growth and proliferation of gastric adenocarcinoma cells are suppressed significantly due to apoptosis of cells.The lower concentrations of ActD influence the growth of gastric cancer cells by blocking cell cycle. BGC823 cells can be arrested in G2 phase after treatment with the lower concentrations of ActD for shorter time. For longer time, ActD mainly affects G1 phase and arrests cells in G1 phase, i.e, transition from G1 to S phase of gastric cancer cells can be blocked by ActD.The lower concentrations of ActD can cause down-regulated expression of Survivin,Cox-2 and HSP70 mRNA in gastric cancer cells. According to the results of flow cytometry （FCM）, transmission electron microscopy （TEM）, laser confocal scanning microscopy （LCSM） and RT-PCR, down-regulation of Survivin,Cox-2 and HSP70 mRNA possibly participates in apoptosis of gastric cancer cells induced by ActD.2. The inhibition ratio of ActD analogs were lower than that ActD.It is necessary that the reforming frame of ActD are studied further.3. ActD showed significant effecton the differential expression of cells apoptosis genes and other related genes in human gastric carcinoma cells （BGC823）.4.Overexpression of Survivin,Cox-2 and HSP70 are observed in gastric cancers. The upregulated expression of Survivin in gastric carcinoma plays positive role in early stage of human gastric carcinogenesis. Cox-2 and HSP70 may contribute to the growth, the invasion and the lymph node metastasis in gastric carcinoma and display the poor clinical prognosis of patients with gastric carcinoma as an independent prognosticator. The depth of invasion, sex, lymph node metastasis and C0x-2 expression are significant prognostic factors. However, only the depth of invasion, sex and lymph node metastasis can indicate prognosis of patients with gastric carcinoma independently.更多还原