【作者】 杨娟；

【导师】 张红； 陈奎生；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2011， 硕士

【摘要】 结节硬化症(tuberous sclerosis complex,TSC)是一种常染色体显性遗传病,是以多种器官的组织缺陷和错构瘤为特征的系统性疾病,最常表现为脑、’肾、心脏、眼、肺以及皮肤出现的良性错构瘤,是由抑癌基因Tscl (tuberous sclerosis complex 1,Tscl)或Tsc2(tuberous sclerosis complex 2,Tsc2)突变所引起。Tscl与Tsc2作为肿瘤抑制因子,在细胞生长和增殖过程中起关键性的调节作用。生长因子刺激的PI3K/Akt信号通路通过磷酸化马铃薯球蛋白(tuberous sclerosis complex 2,TSC2)调控下游效应因子,影响细胞的生长和增殖。研究发现,TSC2可通过在大脑中大量存在的一种Ras同源物(Ras homologue enriched in brain,Rheb)来发挥对哺乳动物雷帕霉素靶蛋白(mammalian target of rapamyein,mTOR)的功能,从而调节细胞的生长和增殖。TSC2将Rheb从结合GTP的活化状态转换为结合GDP的非活化状态,达到抑制mTOR的活性。磷酸化的TSC2丧失了TSC1/TSC2复合物作为GAP对具有GTP酶活性Rheb的作用,从而导致Rheb-GTP的聚集,Rheb-GTP可有效激活mTOR,mTOR通过磷酸化抑制真核细胞起始因子4E结合蛋白(eukaryotic translation initiation factor 4E-binding protein,4E-BP)的活性,并激活核糖体s6蛋白激酶1(70 kDa ribosomal protein S6 kinasel, S6K1)和核糖体S6蛋白激酶2(70 kDa ribosomal protein S6 kinase2, S6K2)的活性,从而促进细胞的生长,增值和分化,最终导致肿瘤的发生近年来,有关mTOR信号通路及其通路中相关基因之间的相互作用,以及与肿瘤发生之间的关系已经成为研究热点,其中包括AKT/TSC1-TSC2/mTOR通路及其相关基因。研究表明,Tsc2突变导致的蛋白功能失调可引起AKT/TSC1-TSC2/mTOR信号转导异常,导致非TSC疾病相关肿瘤的发生,如：胆囊癌、鼻咽癌、喉癌、直肠癌、肺腺癌、肺小细胞癌、膀胱癌等。但有关Tsc2肿瘤抑制基因在食管癌发生、发展中的作用研究尚未见文献报道。为了探讨Tsc2基因的异常表达在食管癌发生和发展中的作用,本研究采用TSC2反义寡聚核苷酸(antisense oligonucleotide, ASODN)处理食管癌EC9706细胞,采用MTT法检测细胞增殖情况；采用流式细胞技术、TUNEL法检测细胞凋亡率的变化；采用免疫细胞化学技术、Western Blot检测细胞中TSC2及mTOR蛋白的表达；采用原位杂交RT-PCR技术检测细胞中TSC2和mTOR mRNA的表达。材料和方法1.人食管癌EC9706细胞株由中国医学科学院肿瘤研究所分子肿瘤学国家重点实验室惠赠。2. TSC2 ASODN, TSC2正义寡聚核苷酸(sense oligonucleotide, SODN)及无关寡核苷酸(non oligonucleotide, N-ODN)的设计与合成。3.食管癌EC9706细胞的分组培养：采用终浓度分别为5μmol/L、10μmol/L 15μmol/L的TSC2 ASODN转染EC9706细胞24h、48h、72h；同时以终浓度为15μmool/L的TSC2 SOD、N-ODN及不转染组分别作为正义对照组、无关对照组和正常对照组(常规培养液)。4.采用MTT法检测各组细胞生长情况。5.采用流式细胞技术、TUNEL法检测各组细胞周期及凋亡的变化。6.采用免疫细胞化学技术、Western Blot检测各组细胞中TSC2及mTOR蛋白的表达情况。7.采用原位杂交、RT-PCR技术检测细胞中TSC2和mTOR mRNA的表达情况。8.统计学处理：采用SPSS 17.0软件分析,数据采用x±s数据表示,进行完全随机设计资料的单因素方差分析,以P<0.05为差异有统计学意义。结果1 MTT检测细胞各组细胞增殖能力的比较与各对照组相比,各实验组处理因素均对EC9706细胞增殖具有较强的促进作用(P<0.001),各对照组间细胞增殖率无明显差异(P均>0.05)。该作用具有浓度及时间的依赖性,且不同浓度及不同时间段各实验组两两比较,差异均有统计学意义(P均<0.05)。2流式细胞仪检测各组细胞周期的情况与正常对照组(G0/G1：62.11±4.72,S：17.22±1.11)、无关对照组(G0/G1：64.80±1.25,S：17.28±1.33)和正义对照组(G0/G1：66.75±2.78,S：18.65±1.34)相比,各实验组各期细胞数所占的比例有所不同,G0/G1期细胞所占比例减少,S期细胞所占比例增加,差别均有统计学意义(P均<0.05)。各实验组对比,TSC2 ASODN对EC9706细胞周期的影响具有浓度的依赖性,且不同浓度各实验组两两比较,差异均有统计学意义(P均<0.05)3流式细胞仪及TUNEL检测各组细胞凋亡的情况两种方法结果均显示,浓度为5μmol/L组、10μmol/L组、15μmol/L组的各实验组细胞早期凋亡率分别为：4.27±0.28、3.04±0.26、1.96±0.08,晚期凋亡率分别为：4.66±0.30、3.25±0.30、1.99±0.18。AI分别为13.11±0.13、9.31±0.29、4.38±0.43。与正常对照组(早期凋亡细胞：4.91±0.14%,晚期凋亡细胞：10.35±0.47%。AI：16.23±0.45%)、无关对照组(早期凋亡细胞：5.12±0.12%,晚期凋亡细胞：9.68±0.55%。AI：16.63±0.34%)和正义对照组(早期凋亡细胞：5.13±0.23%,晚期凋亡细胞：9.77±0.54%。AI：16.46±0.43%)相比,均明显减少,且差异均具有统计学意义(P均<0.05)。各凋亡率及AI值随TSC2 ASODN转染浓度的增加而减少,各浓度之间两两比较,差异均有统计学意义(P均<0.05)。4免疫细胞化学及Western Blot结果4.1各组细胞中TSC2蛋白的表达与各对照组相比,TSC2 ASODN转染组(F immunocytochemistry=574.30、732.06、2293.70,F Western blot=50.60、330.69、1221.28,P均<0.001)细胞中TSC2蛋白的表达均有所下降,差异均有统计学意义(P均<0.05),且均有浓度依赖性和时间依赖性(F immunocytochemistry=253.54、270.00、269.64,F Western blot=123.47、83.59、13.29,P均<0.001),即随着TSC2 ASODN转染浓度的增加和时间的延长,抑制TSC2蛋白表达的能力增强。4.2各组细胞中mTOR蛋白的表达与各对照组相比,TSC2 ASODN转染组(F immunocytochemistry=1527.97、3543.79 ,1609.99,F Western blot=72.55、114.27、302.39,P均<0.001)细胞中mTOR蛋白的表达均有所增加,差异均有统计学意义(P均<0.05),且均有浓度依赖性和时间依赖性(F immunocytochemistry=465.04、390.40、150.64, F Western blot=87.15、41.89、36.34,P均<0.001),即随着TSC2 ASODN转染浓度的增加和时间的延长,mTOR蛋白表达的能力逐渐增强。5原位杂交及RT-PCR结果5.1各组细胞中TSC2 mRNA的表达与各对照组相比,TSC2 ASODN转染组(F in-situ hybridization=67.81、139.61、392.57,FRT-PCR=1472.41、2733.14、656.62,P均<0.001)细胞中TSC2 mRNA的表达均有所下降,差异均有统计学意义(P均<0.05),且均有浓度依赖性和时间依赖性(F in-situ hybridization=260.23、572.22、1004.35,F RT-PCR=53.56、260.40、333.24,P均<0.001),即随着TSC2 ASODN转染浓度的增加和时间的延长,抑制TSC2 mRNA表达的能力增强。5.2各组细胞中mTOR mRNA的表达与各对照组相比,TSC2 ASODN转染组(F in-situ hybridization=381.78、772.79、1303.82, F RT-PCR= 140.66、153.57、160.65,P均<0.001)细胞中mTOR mRNA的表达均有所增加,差异均有统计学意义(P均<0.05),且均有浓度依赖性和时间依赖性((F in-situ hybridization= 1064.87、1334.50、2793.36,F RT-PCR= 167.59、247.39、541.54,P均<0.001),即随着TSC2 ASODN转染浓度的增加和时间的延长,mTOR mRNA表达的能力逐渐增强。结论1. TSC2 ASODN可抑制细胞的凋亡,使S期细胞增多,G0/G1期细胞减少,促进细胞的增值。2. TSC2 ASODN可特异性抑制食管癌EC9706细胞TSC2蛋白及mRNA的表达,同时上调mTOR蛋白及mRNA的表达。更多还原

【Abstract】 Tuberous sclerosis complex (TSC) is an autosomal dominant hereditary disease which results from the mutations of Tsc1 and Tsc2 gene. TSC is a multi-systemic disorder characterized by a variety of organs micro structure defects and the development of numerous benign tumours (e.g.hamartomas), most commonly affecting the brain, eyes, kidney, skin, heart and lungs. As tumor suppressor factors, Tscl and Tsc2 genes play a critical adjustment effect in cell growth and proliferation process. PI3K/Akt signaling pathways stimulated by growth factor regulates downstream effects factor through phosphorylation tuberin protein, affecting growth and proliferation of cells.Research found that TSC2 can effect on the function of mTOR through Ras homologue enriched in brain (Rheb) which regulates cell growth and proliferation. TSC2 can expedite the conversion of Rheb-GTP into Rheb-GDP suppressing the downstream gene mTOR activity. The phosphorylated TSC2 lost the function to Rheb which has GTP-ase activity, resulting in Rheb-GTP gathering. Rheb-GTP can effectively activate mTOR, mTOR through phosphorylation inhibit the activity of a 4E-BP (eukaryotic translation initiation factor 4E-binding protein) and activate S6K1 (70kDa ribosomal protein S6 kinasel) and S6K2 (70kDa ribosomal protein S6 kinase2) activity, which promote cell growth, value and the differentiation that ultimately lead to tumors.In recent years, the relationship between the mTOR signaling pathways and cancer has become research hot spot, including AKT/TSC1-TSC2/mTOR pathways and related gene. Research shows that Tsc2 mutations resulting in protein dysfunction can cause AKT/TSCl-TSC2/mTOR signal transduction abnormalities, leading to non-TSC associated tumour, such as the gallbladder carcinoma, nasopharyngeal carcinoma, laryngeal cancer, colorectal cancer, lung adeno-carcinoma, lung small cell carcinoma and bladder cancer in sporadic. But the role of Tsc2 in the occurrence and development of esophageal have not been reported in the literature.In order to discuss the role of Tsc2 abnormal expression in the occurrence and development of esophageal, this study adopts TSC2 ASODN (antisense oligonucleotide) processing esophageal cancer EC9706 cells, using the MTT method detection cell growth situation; With flow cytometric technology and TUNEL method detecting apoptosis rate changes; with immunocytochemistry, Western Blot detecting TSC2 and mTOR protein expression of the EC9706 cells; employing in situ hybridization, RT-PCR technology testing cells Tsc2 mRNA and mTOR mRNA expression.Materials and methods1. Human esophageal carcinoma cell lines EC9706 were present from the Chinese Academy of Medical Sciences Cancer Institute.2. TSC2 ASODN, TSC2 SODN and N-ODN were designed and synthesized.3. The ASODN of TSC2 at 5,10, and 15μmol/L was transfected into EC9706 cells by the cationic liposome for 24,48, and 72 hours. Sense oligonucleotide and nonsense oligonucleotide at15μmol/L was transfected as sense and unrelated control, and EC9706 cells not being transfected as normal control.4. The MTT method was used to detect cell growth situation.5. With flow cytometric technology and TUNEL method detecting apoptosis rate changes. 6. Using immunocytochemistry and Western Blot detecting TSC2 and mTOR protein expression of the EC9706 cells.7. Employing in situ hybridization, RT-PCR technology testing cells Tsc2 mRNA and mTOR mRNA expression.8. Statistical analysis:The SPSS 17.0 statistical software was used.Results1 MTT test resultsCompared with the control group, the TSC2 ASODN can strongly promote the EC9706 cell proliferation (all P<0.05), and there were no obvious difference between the control groups (all P>0.05). In TSC2 ASODN transfect-ion group, the effect has concentration and time dependence, there were statistical significances differences between the various concentrations of transfection and between the different time (all P<0.05).2 The cell proliferation was detected by flow cytometryCompared with the normal control group (G0/G1:62.11±4.72%, S:17.22±1.11%)、unrelated control group (G0/G1:64.80±1.25%, S:17.28±1.33%) and sense control group (G0/G1:66.75±2.78%,S:18.65±1.34%), the number of cells in each period was significantly different:the number in GO/G1 phase remarkably decreased, the one in S phase increased, and there were statistical significances (all P<0.05). In TSC2 ASODN transfection group, the effect has concentration and time dependence, there were statistical significances between the three concentrations (all P<0.05).3 The apoptotic was detected by TUNEL and flow cytometryTwo methods both results show that cell apoptosis rates of experimental groups concentration at 5、10、and 15μmol/L respectively are:early apoptosis:4.27±0.28 3.04±0.26 and 1.96±0.08%, late apoptosis:4.66±0.30,3.25±0.3 and 1.99±0.18%, AI:13.11±0.13、9.31±0.29 and 4.38±0.43%. Compared with the normal control group (early apoptosis:4.91±0.14%, late apoptosis:10.35±0.47%, AI:16.23±0.45%), unrelated control group (early apoptosis:5.12±0.12%, late apoptosis:9.68±0.55%, AI: 16.63±0.34%) and sense control group (early apoptosis:5.13±0.23%, late apoptosis: 9.77±0.54%, AI:16.46±0.43%), The apoptosis rates were significantly reduce, and the difference was statistically significant (all P<0.05). The apoptosis rate reduced with the concentration of TSC2 ASODN, there were statistical significances between the different concentrations (all P<0.05).4 The immunocytochemistry and the Western Blotting results4.1 The expressions of TSC2 protein in each group of cellsCompared with the control group, the expressions of TSC2 protein in TSC2 ASODN transfection group was decreased significant, and the differences each other were statistically significant(F immunocytochemistry=574.30、732.06、2293.70, F western blotting=50.6、330.69、1221.28,all P<0.001), and change with concentration-dependent and time-dependent(F immunocytochemistry=253.54、270.00、269.64, F Western blotting=123.47、83.59、13.29, all P<0.001). Namely, with the increase of TSC2 ASODN concentration and the extension of time, restrain TSC2 protein expression ability enhancement.4.2 The expressions of mTOR protein in each group of cellsCompared with the control group, the expressions of mTOR protein in TSC2 ASODN transfection group was increased significant, and the differences each other were statistically significant(F immunocytochemistry=87.15.41.89、36.34, F western blotting=72.55、114.27.302.39 all P<0.001), and change with concentration-dependent and time-dependent(F immunocytochemist,y=72.55,114.27,302.39, F Western blotting=87.15. 41.89.36.34,all P<0.001).Namely, with the increase of TSC2 ASODN concentration and the extension of time, promote mTOR protein expression ability enhancement.5 The in situ hybridization and RT-PCR results5.1 The expressions of TSC2 mRNA in each group of cellsCompared with the control group, the expressions of TSC2 mRNA in TSC2 ASODN transfection group was decreased significant, and the differences each other were statistically significant(F in-situ hybridization=67.81.139.61.392.57, F RT-PCR=1472.41、2733.14.656.62,all P<0.001), and change with concentration-dependent and time-dependent (F in-situ hybridization=260.23.572.22、1004.35, F RT-PCR=53.56.260.40,333.24,all P<0.001.Namely, with the increase of TSC2 ASODN concentration and the extension of time, restrain TSC2 mRNA expression ability enhancement. 5.2 The expressions of mTOR mRNA in each group of cellsCompared with the control group, the expressions of mTOR mRNA in TSC2 ASODN transfection group was increased significant, and the differences each other were statistically significant(F in.situ hybridization=381.78,772.79,1303.82, F RT-PCR=140.66,153.57、160.65,all P<0.001), and change with concentration-dependent and time-dependent (F in-situ hybridization=1064.87,1334.50,2793.36, F RT-PCR= 167.59、247.39、541.54, P<0.001).Namely, with the increase of TSC2 ASODN concentration and the extension of time, promote mTOR mRNA expression ability enhancement.Conclusion1. TSC2 ASODN inhibits cell apoptosis, make S period cells increased, G0/G1 phase cells reduce, strengthen the cell growth.2. TSC2 ASODN can specific inhibit TSC2 expression of esophageal cancer cells EC9706, and raised its mTOR expression.更多还原