p73α和14-3-3σ抑制乳腺肿瘤生长的体内实验研究
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摘要
目的:乳腺癌是严重威胁妇女健康的一种恶性肿瘤。据统计,全球每年约有120万~140万妇女患乳腺癌,约有50万患者死于该病。研究表明,乳腺癌的发生、发展是多步骤、多基因作用的结果。随着乳腺癌发病率的不断增加,对其易感基因、相关基因及其产物的研究已成为肿瘤学关注的热点问题。研究发现,作为细胞周期负调控因子,14-3-3σ在人类乳腺癌细胞中的表达下降可能是乳腺癌发生的一个重要原因。研究已经证实,p53是14-3-3σ活化的主要调控因子。p73α是p73基因的剪切变异体,之前的体外实验已经证实,当p53基因缺失或突变时,p73α可以替代p53调节下游基因14-3-3σ,提高其抗肿瘤作用。但是至今,关于p73α和14-3-3σ抑制乳腺癌生长的体内实验研究尚未深入开展。本研究应用细胞外源基因转染和免疫缺陷鼠乳腺癌移植模型方法,进一步探讨了活体内p73α和14-3-3σ对乳腺癌生长的抑制作用,确认14-3-3σ的作用机制以及p73α基因对14-3-3σ的调节作用,为乳腺癌的早期发现和靶向治疗提供新的分子生物学指标和靶点。
方法:
1细胞培养和基因转染:将p53缺失型乳腺癌细胞系MDA-MB-231置于含10 %胎牛血清的DMEM培养液中培养,当细胞生长至约70 %融合时,用脂质体介导法分别转染pcDNA3-p53,pcDNA3-73α和pcDNA3-14-3-3σ质粒,按实验分组:对照组、p53组、p73α组、14-3-3σ组、p53+14-3-3σ组和p73α+14-3-3σ组,分别导入p53突变型人类乳腺癌细胞系MDA-MB-231中,以pcDNA3空质粒组为对照组。
2乳腺癌裸鼠移植模型:选取BALB/C-null 3周龄雌性裸鼠12只,在无特定病原体( specific pathogen free, SPF )环境下饲养。分组同前,每组2只,其中标记鼠为a鼠,未标记鼠为b鼠。饲料喂养、2天,待裸鼠状态稳定后,按照分组,于a、b两裸鼠腋下分别接种MDA-MB-231, 2μg p53, 2μg p73α, 2μg 14-3-3σ, 2μg p53+2μg 14-3-3σ和2μg p73α+2μg 14-3-3σ(相当于4×106个细胞)。接种后48 h,b鼠尾静脉内按15 mg/Kg缓慢静注表阿霉素( adriamycin,ADR )[2],观察8周后脱颈椎处死裸鼠,计数腋下肿块数量、形态及大小,解剖乳腺组织取材,标本常规固定,石蜡包埋,4μm切片,HE染色,光镜观察。
结果:
1 MDA-MB-231乳腺癌细胞生长良好。
2细胞外源性基因转染成功。将转染后的细胞提取总RNA进行RT-PCR,检测p53、p73α、14-3-3σ基因在转录水平上的表达。结果表明,( 1 ) p53和p73α均可在转录水平上诱导14-3-3σ的表达,且14-3-3σ可反作用于p53,两者相互诱导;而14-3-3σ对p73α则无此作用。( 2 ) p53可能参与了p73α对14-3-3σ的调节。
3 12只裸鼠在实验全过程中无死亡。在裸鼠接种肿瘤细胞后第30天,发现对照组a鼠(未静注ADR者)皮下接种处可触及5 mm×5 mm肿物,质硬,表面不平,活动差;第40天发现14-3-3σ组a鼠皮下接种处可触及5 mm×3 mm肿物,质硬,活动可。至55天脱颈椎处死裸鼠,对照组a鼠肿瘤长至12 mm×7.2 mm,14-3-3σ组a鼠肿瘤长至7.2 mm×6.2 mm,所有组b鼠和其他组a鼠均未发现肿瘤生长。对裸鼠肿瘤进行形态学观察,肉眼观察:包膜多不完整,解剖肿瘤时有砂粒感,切面为黄白色。镜下观察:癌细胞呈实性团索,无腺腔样结构,细胞体积较小,呈条索状,周围有纤维化的癌性间质围绕,细胞异型性多见,常可见核分裂相,诊断为乳腺癌。
结论:
1在活体内,细胞周期负调控因子14-3-3σ存在活化和非活化两种状态,活化状态下的14-3-3σ可以发挥其抑制肿瘤生长的作用,而非活化状态的14-3-3σ无抑制肿瘤生长的作用。
2 p53基因和p73α基因都是14-3-3σ主要的上游调控基因,当细胞DNA受到损伤时,他们不但可上调14-3-3σ的表达,而且可使其由非活化状态转为活化状态,发挥细胞周期负调控因子的作用,从而抑制肿瘤生长。
3作为抑癌基因,p53基因和p73α基因可以多途径抑制裸鼠乳腺肿瘤细胞的生长,具体机制有待于进一步研究。
Objective: Breast cancer is one of the malignant tumors which threatened the health of women. According to statistics, there are about 1200,000~1400,000 new breast cancer cases every year all over the world, and 500,000 of them died from it. Studies showed that, carcinogenesis and development of breast cancer had multiple steps and was the results of the function of multiple genes. With the increasing of the incidence of breast cancer, it becomes one hot issue in the oncology field to study the susceptibility gene and correlative gene and its product. As a negative regulation factor of cell cycle, the expression of 14-3-3σin human breast carcinoma cell was decreased, and it may be an important reason for the carcinogenesis. It had verified that p53 was the most important regulating factor of 14-3-3σ. p73αwas a shearing variant of p73. In vitro, it had verified that the expression of p73αgene increased to replace p53 to up-regulated the downstream factor 14-3-3σto exert the function of restraining the cell proliferation, when p53 gene deletion or mutation. However, the internal empirical studies about the inhibition of tumor growth of p73αand 14-3-3σhave not been carried out. In this study, we used cell exogenous gene transfection and breast cancer transplanted pattern of mouse of immunodeficiency to investigate the function of p73αand 14-3-3σto restrain the growth of breast cancer cells in vivo, and further to confirm the functional mechanism of 14-3-3σ, and the regulative function of p73αto 14-3-3σ. We anticipated to provide a new molecular biological index and target to the early discovery and targeted therapy of breast cancer.
Methods:
1 Cell culture and gene transfection: p53-mutate cell line MDA-MB-231 cultured by DMEM culture fluid, which contains 10% fetal bovine serum. When MDA-MB-231 mixed together about 70%, we imported pcDNA3-p53 plasmid, pcDNA3-p73αplasmid and pcDNA3-14-3-3σplasmid into the p53-mutate cell line MDA-MB-231 with lipofectamine-mediated. Free pcDNA3 plasmid was used as control.
2 Breast cancer transplanted model in nude mice: We selected 3-week-old female nude mice 12, and bred in the environment of SPF. With the former group, each 2. In each group,‘a’mouse was marked;‘b’mouse was unmarked. After breeding for two days, we inoculated MDA-MB-231 2μg, p53 2μg, p73α2μg, 14-3-3σ2μg, p53+14-3-3σ2μg, p73α+14-3-3σ2μg(about 4×106 cells)into athymic mouse’s mammary fat pad. 48 h after inoculation, we imported the interference of ADM in therapeutic dose into b mice. Tumor volumes were measured and recorded three times a week. At the end of 8 weeks, the animals were sacrificed and mammary gland samples were obtained, fixed, paraffin imbedded, chipped 4μm, HE dyeing, observed by light microscope.
Results:
1 p53-mutate cell line MDA-MB-231 cells grew well.
2 Cell exogenous gene transfected succeed. We extracted the total RNA to detect the expression of p53, p73αand 14-3-3σat the level of transcription by RT- PCR. The result showed that, (1) p53 and p73αcan induce the expression of 14-3-3σat the transcriptional level, and 14-3-3σcan be counterproductive in p53. (2) p53 may be involved in the adjustment of p73αto 14-3-3σ.
3 Tumors which were confirmed as breast cancer by pathology grew in the 14-3-3σtransfected group and the control group, 4 or 6 weeks postinoculation. No tumor grew in the p53 or p73αtransfected group, co-transfected p53/14-3-3σgroup and co-transfected p73α/14-3-3σgroup. No tumor grew in the group whose athymic mouse was injected by ADM through vena caudalis.
Conclusions:
1 The negative regulation factor of cell cycle 14-3-3σhas both activity and inactivity states. The activity state can inhibit the growth of tumor, while the inactivity state can not.
2 p53 and p73αare both major regulation genes of 14-3-3σupstream. They can up-regulate the expression of 14-3-3σand inhibit the growth of tumor by activating it when the cellular DNA was injuried.
3 As anti-oncogene, both p53 and p73αcan inhibit the growth of breast tumor cells of athymic mouse, and the concrete mechanism is to be studied.
方法:
1细胞培养和基因转染:将p53缺失型乳腺癌细胞系MDA-MB-231置于含10 %胎牛血清的DMEM培养液中培养,当细胞生长至约70 %融合时,用脂质体介导法分别转染pcDNA3-p53,pcDNA3-73α和pcDNA3-14-3-3σ质粒,按实验分组:对照组、p53组、p73α组、14-3-3σ组、p53+14-3-3σ组和p73α+14-3-3σ组,分别导入p53突变型人类乳腺癌细胞系MDA-MB-231中,以pcDNA3空质粒组为对照组。
2乳腺癌裸鼠移植模型:选取BALB/C-null 3周龄雌性裸鼠12只,在无特定病原体( specific pathogen free, SPF )环境下饲养。分组同前,每组2只,其中标记鼠为a鼠,未标记鼠为b鼠。饲料喂养、2天,待裸鼠状态稳定后,按照分组,于a、b两裸鼠腋下分别接种MDA-MB-231, 2μg p53, 2μg p73α, 2μg 14-3-3σ, 2μg p53+2μg 14-3-3σ和2μg p73α+2μg 14-3-3σ(相当于4×106个细胞)。接种后48 h,b鼠尾静脉内按15 mg/Kg缓慢静注表阿霉素( adriamycin,ADR )[2],观察8周后脱颈椎处死裸鼠,计数腋下肿块数量、形态及大小,解剖乳腺组织取材,标本常规固定,石蜡包埋,4μm切片,HE染色,光镜观察。
结果:
1 MDA-MB-231乳腺癌细胞生长良好。
2细胞外源性基因转染成功。将转染后的细胞提取总RNA进行RT-PCR,检测p53、p73α、14-3-3σ基因在转录水平上的表达。结果表明,( 1 ) p53和p73α均可在转录水平上诱导14-3-3σ的表达,且14-3-3σ可反作用于p53,两者相互诱导;而14-3-3σ对p73α则无此作用。( 2 ) p53可能参与了p73α对14-3-3σ的调节。
3 12只裸鼠在实验全过程中无死亡。在裸鼠接种肿瘤细胞后第30天,发现对照组a鼠(未静注ADR者)皮下接种处可触及5 mm×5 mm肿物,质硬,表面不平,活动差;第40天发现14-3-3σ组a鼠皮下接种处可触及5 mm×3 mm肿物,质硬,活动可。至55天脱颈椎处死裸鼠,对照组a鼠肿瘤长至12 mm×7.2 mm,14-3-3σ组a鼠肿瘤长至7.2 mm×6.2 mm,所有组b鼠和其他组a鼠均未发现肿瘤生长。对裸鼠肿瘤进行形态学观察,肉眼观察:包膜多不完整,解剖肿瘤时有砂粒感,切面为黄白色。镜下观察:癌细胞呈实性团索,无腺腔样结构,细胞体积较小,呈条索状,周围有纤维化的癌性间质围绕,细胞异型性多见,常可见核分裂相,诊断为乳腺癌。
结论:
1在活体内,细胞周期负调控因子14-3-3σ存在活化和非活化两种状态,活化状态下的14-3-3σ可以发挥其抑制肿瘤生长的作用,而非活化状态的14-3-3σ无抑制肿瘤生长的作用。
2 p53基因和p73α基因都是14-3-3σ主要的上游调控基因,当细胞DNA受到损伤时,他们不但可上调14-3-3σ的表达,而且可使其由非活化状态转为活化状态,发挥细胞周期负调控因子的作用,从而抑制肿瘤生长。
3作为抑癌基因,p53基因和p73α基因可以多途径抑制裸鼠乳腺肿瘤细胞的生长,具体机制有待于进一步研究。
Objective: Breast cancer is one of the malignant tumors which threatened the health of women. According to statistics, there are about 1200,000~1400,000 new breast cancer cases every year all over the world, and 500,000 of them died from it. Studies showed that, carcinogenesis and development of breast cancer had multiple steps and was the results of the function of multiple genes. With the increasing of the incidence of breast cancer, it becomes one hot issue in the oncology field to study the susceptibility gene and correlative gene and its product. As a negative regulation factor of cell cycle, the expression of 14-3-3σin human breast carcinoma cell was decreased, and it may be an important reason for the carcinogenesis. It had verified that p53 was the most important regulating factor of 14-3-3σ. p73αwas a shearing variant of p73. In vitro, it had verified that the expression of p73αgene increased to replace p53 to up-regulated the downstream factor 14-3-3σto exert the function of restraining the cell proliferation, when p53 gene deletion or mutation. However, the internal empirical studies about the inhibition of tumor growth of p73αand 14-3-3σhave not been carried out. In this study, we used cell exogenous gene transfection and breast cancer transplanted pattern of mouse of immunodeficiency to investigate the function of p73αand 14-3-3σto restrain the growth of breast cancer cells in vivo, and further to confirm the functional mechanism of 14-3-3σ, and the regulative function of p73αto 14-3-3σ. We anticipated to provide a new molecular biological index and target to the early discovery and targeted therapy of breast cancer.
Methods:
1 Cell culture and gene transfection: p53-mutate cell line MDA-MB-231 cultured by DMEM culture fluid, which contains 10% fetal bovine serum. When MDA-MB-231 mixed together about 70%, we imported pcDNA3-p53 plasmid, pcDNA3-p73αplasmid and pcDNA3-14-3-3σplasmid into the p53-mutate cell line MDA-MB-231 with lipofectamine-mediated. Free pcDNA3 plasmid was used as control.
2 Breast cancer transplanted model in nude mice: We selected 3-week-old female nude mice 12, and bred in the environment of SPF. With the former group, each 2. In each group,‘a’mouse was marked;‘b’mouse was unmarked. After breeding for two days, we inoculated MDA-MB-231 2μg, p53 2μg, p73α2μg, 14-3-3σ2μg, p53+14-3-3σ2μg, p73α+14-3-3σ2μg(about 4×106 cells)into athymic mouse’s mammary fat pad. 48 h after inoculation, we imported the interference of ADM in therapeutic dose into b mice. Tumor volumes were measured and recorded three times a week. At the end of 8 weeks, the animals were sacrificed and mammary gland samples were obtained, fixed, paraffin imbedded, chipped 4μm, HE dyeing, observed by light microscope.
Results:
1 p53-mutate cell line MDA-MB-231 cells grew well.
2 Cell exogenous gene transfected succeed. We extracted the total RNA to detect the expression of p53, p73αand 14-3-3σat the level of transcription by RT- PCR. The result showed that, (1) p53 and p73αcan induce the expression of 14-3-3σat the transcriptional level, and 14-3-3σcan be counterproductive in p53. (2) p53 may be involved in the adjustment of p73αto 14-3-3σ.
3 Tumors which were confirmed as breast cancer by pathology grew in the 14-3-3σtransfected group and the control group, 4 or 6 weeks postinoculation. No tumor grew in the p53 or p73αtransfected group, co-transfected p53/14-3-3σgroup and co-transfected p73α/14-3-3σgroup. No tumor grew in the group whose athymic mouse was injected by ADM through vena caudalis.
Conclusions:
1 The negative regulation factor of cell cycle 14-3-3σhas both activity and inactivity states. The activity state can inhibit the growth of tumor, while the inactivity state can not.
2 p53 and p73αare both major regulation genes of 14-3-3σupstream. They can up-regulate the expression of 14-3-3σand inhibit the growth of tumor by activating it when the cellular DNA was injuried.
3 As anti-oncogene, both p53 and p73αcan inhibit the growth of breast tumor cells of athymic mouse, and the concrete mechanism is to be studied.
引文
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