【作者】 汪学非；

【导师】 秦新裕； 孙益红； 汤其群；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【摘要】 第一部分人胃癌侧群细胞检测分选与干细胞基因表型分析[目的]检测和分选胃癌细胞株中的SP细胞,并分析其干细胞相关基因表达情况。[方法]选择4株不同分化程度的人胃癌细胞系MKN-28、SGC-7901、BGC-823和AGS,以Hoechst33342/碘化丙啶荧光染料双染,维拉帕米拮抗对照,应用流式细胞仪荧光激活分选法检测SP细胞比例并进行分选；RT-PCR比较分选所得的SP细胞和nonSP细胞中药物转运蛋白ABCG2、干细胞膜蛋白表型CD133、CD24、CD44和干细胞信号通路相关调控基因OCT-4、NOTCH、NANOG、BMI-1的表达差异。[结果]胃癌细胞MKN-28、SGC7901和BGC-823中SP细胞比例分别为0.46%-2.7%、1.89%-2.33%和1.00%-3.55%；胃癌细胞AGS中未能检测出SP细胞。半定量PCR结果显示,SP细胞中ABCG2的表达量明显高于nonSP细胞；实时荧光定量PCR结果进一步显示,侧群细胞中ABCG2约为非侧群细胞中的8倍。干细胞膜蛋白表型CD24和CD44在胃癌SP细胞中的表达强于nonSP细胞；干细胞信号通路相关调控基因OCT-4、NOTCH、NANOG在SP细胞中的表达强于nonSP细胞：CD133、BMI-1基因在SP和nonSP细胞中的表达无差异。[结论]侧群细胞可在胃癌细胞中被检测和分选,但并非所有的胃癌细胞株中均含有侧群细胞。胃癌侧群细胞高度表达药物转运蛋白ABCG2、干细胞膜蛋白表型CD44、CD24和干细胞调控基因OCT-4、NANOG、NOTCH。第二部分人胃癌侧群细胞生物学特性鉴定[目的]系统分析胃癌SP细胞体内外增殖活力、成瘤能力、侵袭力等基本生物学特性。[方法]通过细胞生长曲线(CCK-8法)、平板克隆形成试验比较胃癌SP与nonSP细胞体外增殖活力；应用细胞侵袭transwell试验比较胃癌SP与nonSP细胞侵袭力差异；应用异种移植Nod/SCID小鼠成瘤实验比较胃癌SP与nonSP细胞体内致瘤能力差异；通过胃癌SP细胞体外培养比例回归和体内成瘤细胞分化现象观察鉴定其不对称分裂和多向分化特性。[结果]细胞生长曲线实验显示,仅胃癌MKN-28细胞株SP细胞增殖活力明显强于nonSP细胞,而BGC-823和SGC-7901细胞株来源的SP与nonSP细胞总体生长曲线差别未能无统计学意义(P>0.05)。平板克隆实验结果显示,胃癌细胞株MKN-28、BGC-823和SGC-7901中SP细胞克隆形成率均高于nonSP细胞,其中MKN-28 SP细胞克隆形成能力明显高于nonSP细胞(P<0.05)。胃癌nonSP细胞侵袭力明显强于SP细胞(P<0.05)。异种移植瘤实验结果显示,Nod/SCID小鼠体内胃癌SP与nonSP细胞成瘤能力相仿(P>0.05)。胃癌SP细胞体外培养后,其中SP比例由起始的100%下降至1.0%,nonSP细胞中SP比例由起始的0%增加到0.3%；胃癌SP与nonSP细胞在体内成瘤后,均可转化成不同分化程度的胃癌细胞。[结论]胃癌SP细胞体外增殖活力、体内成瘤和分化能力nonSP细胞相仿,SP细胞富集胃癌干细胞的依据不足。胃癌SP细胞侵袭能力较弱,胃癌细胞侵袭力可能主要与nonSP细胞有关。第三部分人胃癌侧群细胞化疗药物耐受性的相关分析[目的]研究胃癌SP与nonSP细胞对化疗药物的耐受性差异,并对其表达肿瘤耐药相关基因情况进行分析。[方法]通过CCK-8法检测比较胃癌SP与nonSP细胞对于化疗药物5-FU和DDP的耐受性差异；应用肿瘤化疗耐药相关的功能基因芯片,对胃癌SP与nonSP细胞中表达肿瘤耐药相关基因情况进行比较分析[结果]化疗药物5-FU对胃癌细胞株SGC7901来源的SP的半效抑制浓度IC50为0.3346 mg/ml,明显高于nonSP细胞的0.0142mg/ml (P<0.05);化疗药物DDP对胃癌SP的半效抑制浓度IC50为0.0055 mg/ml,亦明显高于nonSP细胞的0.0032mg/ml (P<0.05)。结果显示胃癌SP细胞对化疗药物5-FU和DDP的耐受性明显强于nonSP细胞。功能基因芯片结果显示,在84个常见的肿瘤化疗耐药相关基因中,1个基因在胃癌SP细胞中表达低于nonSP细胞；15个基因在胃癌SP细胞中表达高于nonSP细胞,其中ABCB1、ABCG2、BAX、MYC、GSTP1、AR等6个基因的表达差异显著(P<0.05)。[结论]胃癌SP细胞对化疗药物的耐受性明显强于胃癌nonSP细胞,并高度表达一系列肿瘤耐药相关基因,可能在胃癌多药耐药机制中发挥重要作用。更多还原

【Abstract】 Part I Isolation and stem cell related gene characterization of the side population cells from human gastric cancer cell linesObjective:To isolate and characterize the side population cells from gastric cancer cell lines with a series of stem cell related gene.Methods:Side populations of four human gastric cancer cell lines with different differentiation grades, named MKN-28, SGC-7901, BGC-823, and AGS were analyzed and isolated by fluorescence-activated cell sorting after being stained by fluorochrome Hoechst33342 and PI; expression of drug transporters ABCG2, stem cell related cellular membrane phenotype(CD 133, CD24, CD44) and signaling molecules(OCT-4, NOTCH, NANOG, BMI-1) were detected by reverse transcriptase PCR.Results:Side population were detected in three of the four gastric cancer cell lines, with a proportion of 0.46%-3.55%, without SP cells detected in AGS. The SP cells showed a higher expression of ABCG2, CD24, CD44, OCT-4, NANOG and NOTCH, compared to nonSP cells.Conclusion:Side population could be detected in the cell lines of gastric cancer, with a higher expression of a series of stem cell related gene compared to nonSP cells.Part II Identification of the side population cells from human gastric cancer cell linesObjective:To identify the side population cells from human gastric cell lines.Methods:Sorted SP and nonSP cells from gastric cancer cell lines were analyzed by cell growth assay(CCK-8), clonogenic assay, and matrigel invasion assay in vitro, furthermore by tumorigenicity assay, differentiation study in vivo.Results:Only SP cells from cell line MKN-28 showed a higher proliferation activity and clonogenicity compared to the nonSP cells in vitro(P<0.05). Sorted SP and nonSP cells from cell line SGC-7901 and BGC-823 showed similar proliferation activity, clonogenicity in vitro, tumorigenicity in vivo(P>0.05). Matrigel invasion assay showed that the number of nonSP cells which penetrated artificial basement membrane was more than SP cells (P<0.05). Additionally, culturing sorted SP and non-SP cells showed that the populations are interconvertible in vitro, and both conversed to different degree of differentiation cells in vivo.Conclusion:Sorted SP and nonSP cells from gastric cancer cell lines showed similar proliferation activity, clonogenicity, tumorigenicity and multi-directional differentiation potential, which standed in contrast to the observations that tumor SP cells were enriched in stem cells.PartⅢChemoresistance study of the side population cells from human gastric cancer cell linesObjective:To examine chemotherapeutic drug resistance of the side population cells from human gastric cancer cell lines.Methods:Cell growth assay was performed for sorted SP and nonSP cells from gastric cancer cell line by drug intervention. Gene profile for tumor chemotherapeutic drug resistance was analyzed with a Quantitative Real-Time PCR Arrays.Results:The half maximal inhibitory concentration (IC50) of 5-FU for SP cells from gastric cancer cell line was significant higher than nonSP cells(0.3346 mg/ml vs 0.0142mg/ml, P<0.05). Similarly, the IC50 value of DDP for SP cells from gastric cancer cell line was significant higher than nonSP cells(0.0055 mg/ml vs 0.0032mg/ml, P<0.05). Quantitative Real-Time PCR Arrays displayed a series of 15 higher expression gene and 1 lower expression gene in SP cells rom gastric cancer cell line compared to nonSP cells, substantially including ABCB1, ABCG2, BAX, MYC, GSTP1 and AR (P<0.05)Conclusion:Sorted SP cells from gastric cancer cell line showed higher resistance to chemotherapeutic drugs, with a stronger expression of a series of tumor drug resisitance related genes.更多还原