论文标题:STGC3基因在鼻咽癌细胞系CNE2中抑瘤机制的初步研究
Primary Investigation of the Tumor Suppressor Mechanism of STGC3 Gene on Nasopharyngeal Carcinoma Cell Line CNE2
论文作者
论文导师 贺修胜,论文学位 硕士,论文专业 病理学与病理生理学
论文单位 南华大学,点击次数 516,论文页数 85页File Size3758K
2007-05-01论文网 http://www.lw23.com/lunwen_322217/
STGC3;; CNE2;; nude mouse;; Tet-on regulating expression system;; two dimensional electrophoresis;; MALDI-TOF mass spectrometry;progesterone;; STGC3;; 2-DE;; MALDI-TOF-MS
第一部分Tet调控STGC3基因CNE2细胞系裸鼠成瘤性实验 目的:前期研究显示STGC3高表达,可以抑制CNE2细胞的生长,为进一步研究STGC3基因的抑瘤作用,观测STGC3基因高表达对CNE2裸鼠体内成瘤的影响,并探讨其可能作用机制。 方法:采用Tet/pTRE-STGC3/CNE2细胞系接种于裸鼠皮下,Dox诱导STGC3基因高表达,观测STGC3基因高表达对CNE2裸鼠体内成瘤的影响。运用RT-PCR、免疫组织化学及蛋白质免疫印迹方法,分别从mRNA和蛋白质水平,分析瘤组织中STGC3基因表达;采用HE染色观察瘤组织形态学改变;用流式细胞仪,检测移植瘤组织中细胞的凋亡情况;用免疫组织化学方法,检测移植瘤组织凋亡相关蛋白Bcl-2和Bax的表达。运用蛋白质组学相关技术,如二维电泳、图像分析、质谱技术等方法,分析STGC3对移植瘤组织中蛋白质表达谱的影响,筛选和鉴定与STGC3抑瘤作用相关的差异蛋白质。应用蛋白质免疫印迹验证热休克蛋白70在裸鼠移植瘤组织中的表达情况。 结果:STGC3蛋白质表达主要定位于细胞核内,Dox诱导STGC3基因在Tet/pTRE-STGC3/CNE2细胞系高表达后,Tet/pTRE-STGC3/CNE2细胞系裸鼠体内成瘤受到明显抑制,与对照组比较,移植瘤成瘤时间晚、生长慢、肿块小、细胞凋亡率高;Bcl-2蛋白表达减弱,Bax蛋白表达增强,差异有显著性意义(P<0.01);蛋白组学实验结果显示:STGC3在移植瘤组织中高表达,肽酰辅氨酰顺反式异构酶A,丝切蛋白-1,抑制素和G蛋白表达上调,丝氨酸-苏氨酸蛋白激酶WNK1,丝氨酸-苏氨酸二肽富含性剪切因子1和热休克蛋白70表达下调。这些蛋白质涉及细胞骨架重构、细胞凋亡、细胞增殖、细胞免疫、分子伴侣和信号转导等功能。应用蛋白质免疫印迹验证热休克蛋白70在裸鼠移植瘤组织中的表达情况,结果与蛋白组学实验结果一致,说明蛋白组学结果较准确的反映了裸鼠移植瘤组织中蛋白质的表达情况。 结论: 1 STGC3高表达,使Bcl-2/Bax比值下降,促进CNE2细胞凋亡,抑制CNE2裸鼠移植瘤生长。 2 STGC3高表达,移植瘤组织肽酰辅氨酰顺反式异构酶A,丝切蛋白-1,抑制素和G蛋白等表达上调;丝氨酸-苏氨酸蛋白激酶WNK1,丝氨酸-苏氨酸二肽富含性剪切因子1和热休克蛋白70等蛋白表达下调, CNE2细胞的裸鼠成瘤能力降低。 第二部分孕酮对Tet调控STGC3基因表达CNE2细胞系生长增殖及蛋白质表达谱的影响 目的:STGC3能在体内外抑制CNE2细胞的生长,另外,前期研究发现,种植转染STGC3基因CNE2细胞组的4只裸鼠中,2只雌性鼠的荷瘤肿块明显小于同组的2只雄性鼠,提示孕激素可能具有促进STGC3基因的抑瘤作用。本文旨在进一步探讨孕酮在STGC3基因的抑瘤过程中发挥的作用。 方法:采用四甲基偶氮唑蓝(MTT)检测法,观察孕酮对CNE2细胞系及Tet/pTRE-STGC3/CNE2细胞系生长增殖的影响。运用蛋白质组学相关技术,如二维电泳、图像分析、质谱技术等方法,分析孕酮处理Tet/pTRE-STGC3/CNE2细胞系的蛋白质表达谱改变。 结果: ①MTT法检测结果显示,用10-9mol/L-10-5 mol/L孕酮处理CNE2细胞后,CNE2细胞的生长被抑制,抑制率为0.8%-34.3%,与对照组相比,差异均有显著性意义(P<0.05),而用上述浓度的孕酮处理Dox诱导的Tet/pTRE-STGC3/CNE2细胞后,其抑制率明显提高,为24.1%-50.9%,且呈浓度依赖关系,各处理组与对照组之间差异有显著性意义(P<0.05)。比较不同浓度的孕酮对CNE2和Tet/pTRE-STGC3/CNE2细胞的抑制率,两者差异有显著性意义(P<0.05),说明孕酮能够促进STGC3基因对CNE2细胞的生长抑制作用。 ②抽提10-6 mol/L孕酮处理和PBS处理的Dox诱导的Tet/pTRE-STGC3/CNE2的两种细胞总蛋白,进行双向电泳,获得重复性较好的双向凝胶电泳图谱,经PDQuest软件分析,在孕酮处理和PBS处理的Dox诱导的Tet/pTRE-STGC3/CNE2细胞系中分别检测到785±38、756±41个蛋白质点,平均匹配率为85.4%。初步鉴定了8个差异表达3倍以上的蛋白质点,孕酮处理Dox诱导的Tet/pTRE-STGC3/CNE2细胞系后,磷酸甘油醛异构酶,丝切蛋白-1,核内不均一性核糖蛋白C2,肽酰辅氨酰顺反式异构酶A和假想蛋白表达上调,核内不均一性核糖蛋A2/B1和细胞角蛋白13表达下调,这些蛋白质涉及细胞骨架重构、细胞分化、细胞免疫和细胞代谢等。 结论: 1孕酮能够增强STGC3基因对CNE2细胞系的生长抑制。 2孕酮处理Tet/pTRE-STGC3/CNE2细胞后,磷酸甘油醛异构酶,丝切蛋白-1,核内不均一性核糖蛋白C2,肽酰辅氨酰顺反式异构酶A和假想蛋白的表达上调;核内不均一性核糖蛋白A2/B1和细胞角蛋白13的表达下调。孕酮可能通过影响上述蛋白质表达,增强STGC3基因对CNE2细胞系的生长抑制。
Objective: Previous investigation indicated that overexpression of STGC3 could inhibit the proliferation of the cultured CNE2 cell line. To examine the effect of STGC3 on tumorigenicity of CNE2 cell line and explore its mechanism in nude mice so as to further investigate the tumor suppressor role of STGC3. Methods: The Tet/pTRE-STGC3/CNE2 cell line was planted under the front leg skin of nude mice induced by Dox via intra-peritoneal injection. The mRNA and protein levels of STGC3 in transplanted tumor tissues were detected with RT-PCR, Western blotting and immunohistochemistry. The apoptosis ratio of the tumor cells was analyzed by flow cytometry. Bcl-2 and Bax proteins were examined by immunohistochemistry method. Immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis, Coomassie brilliant blue staining, PDQuest software analysis, peptide mass fingerpringting based on matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) and Mascot database searching were used to separate and identify differentially expressed proteins when STGC3 was highly expressed in the tumor tissue. Heat shock 70 kDa protein was detected in tumor tissues of Tet/pTRE/CNE2 and Tet/pTRE-STGC3/CNE2 in nude mice. Results: The results indicated that STGC3 was mainly expressed in nucleus. A high level of STGC3 expression could inhibit the tumorigenicity of the CNE2 cell line in nude mice. Tumor grew slowly, later and smaller. Cell apoptotic percentage increased, Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated in Tet/pTRE-STGC3/CNE2 cell line induced by Dox (P<0.01). Proteomic experiment indicated that Peptidyl-prolyl cis-trans isomerase A, Cofilin-1, Prohibitin and Guanine nucleotide-binding protein subunit beta 2-like 1 were up-regulated and Serine/threonine-protein kinase WNK1, Splicing factor arginine/serine-rich 1 and Heat shock 70kDa protein 8 isoform 2 variant were down-regulate when STGC3 was highly expressed. The proteins involved in cytoskeleton, cell apoptosis, cell immunity, cell proliferation and signal pathway. Heat shock 70 kDa proteins detected in the tumor tissues of Tet/pTRE /CNE2 and Tet/pTRE-STGC3/CNE2 in nude mice confirmed the proteomic experimental results, which indicated that proteomic results reflected the protein expression condition in tumor tissues fairly well. Conclusion: 1 STGC3 could suppress the proliferation of the Tet/pTRE-STGC3/CNE2 cell line through raising the apoptosis ratio by up-regulated the Bax protein expression and down-regulated the Bcl-2 protein expression. 2 Over-expression of STGC3 might make the tumorigenisity of CNE2 cell line in nude mice decrease via increasing the expression of Peptidyl-prolyl cis-trans isomerase A,Cofilin-1,prohibitin and Guanine nucleotide-binding protein subunit beta 2-like 1 and down-regulating Serine/threonine-protein kinase WNK1,Splicing factor arginine/serine-rich 1 and Heat shock 70kDa protein 8 isoform 2 variant expression. Objective: STGC3 over-expression could inhibit the proliferation of CNE2 cell line in vitro and vivo. Otherwise, our previous study indicated that there were a delay in tumor formation and a dramatic reduction in tumor size when CNE2 cell line overexpressed STGC3 were injected into nude mice and the tumor size in female nude mice was highly smaller than that in male. This paper was designed to investigate the effect of progesterone on Tet/pTRE-STGC3/CNE2 cell line and its related molecular mechanism. Methods: The proliferative capacity of the CNE2 cell line and Tet/pTRE-STGC3/CNE2 cell line in the culture medium with progesterone was evaluated by the means of the microculture tetrazolium assay (MTT). A series of methods, including immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis, Coomassie brilliant blue staining, Imaging Master software analysis, peptide mass fingerpringting based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and Mascot database searching, were used to separate and identify differentially expressed proteins induced by progesterone on Tet/pTRE-STGC3/CNE2 cells. Results: (1) MTT assay showed that the proliferation of CNE2 cell line and Tet/pTRE-STGC3/CNE2 cell line induced by Dox were significantly inhibited by progesterone (10-9mol/L-10-5mol/L) in concentration-dependent manner. The inhibitive ratios were 0.8%-34.3% and 24.1%-50.9% respectively. The inhibitive ratio of progesterone on Tet/pTRE-STGC3/CNE2 cell line was more obvious than that on CNE2 cell line. (2) After 2-DE, there were 785±38, 756±41 spots in Tet/pTRE-STGC3/CNE2 cell line induced by Dox and treated with progesterone and PBS, respectively. 8 differential proteins were primaryly identified by MALDI-TOF-MS. Progesterone could increase the expression of Triosephosphate isomerase,Cofilin-1,Heterogeneous nuclear ribonucleoprotein C2, Peptidyl-prolyl cis-trans isomerase A and Hypothetical protein and down-regulate Heterogeneous nuclear ribonucleoprotein A2/B1 isoform 2 and Keratin 13 expression. The proteins involved in cytoskeleton, cell differentiation, cell immunity and cell metabolism. Conclusion: 1 Progesterone could enhance the tumor suppressor effect of STGC3 on CNE2 cell proliferation. 2 Progesterone could enhance the suppressive function of STGC3 via increasing the expression of Triosephosphate isomerase,Cofilin-1,Heterogeneous nuclear ribonucleoprotein C2, Peptidyl-prolyl cis-trans isomerase A and Hypothetical protein and down-regulating Heterogeneous nuclear ribonucleoprotein A2/B1 isoform 2 and Keratin 13 expression.
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