MTH1在乳腺癌的表达以及抑制MTH1治疗不同亚型乳腺癌的探索性研究

发布时间：2018-03-02 19:28

本文选题：乳腺癌　切入点：MTH1　出处：《北京协和医学院》2015年博士论文　论文类型：学位论文

【摘要】：第一部分MTH1在人乳腺癌组织中的表达目的：分析MTH1蛋白、mRNA在人乳腺癌组织的表达情况以及其表达和不同临床病理特征的关系。方法：从普通标本库中随机选取Luminal型、Her-2过表达型、Basal-like型浸润性乳腺癌标本、非癌乳腺组织标本各10例,行免疫组织化学检测MTHl蛋白的表达；从液氮标本库随机选取Luminal型、Her-2过表达型、Basal-like型浸润性乳腺癌标本以及其相对应的非癌乳腺组织标本各10例行荧光定量PCR检测MTH1 mRNA的表达。结果：乳腺癌组织中MTH1蛋白强阳/中阳表达率为93.3%,非癌乳腺组织MTH1蛋白强阳/中阳表达率为10%,其余为弱阳/阴性表达。乳腺癌组织与非癌乳腺组织中MTH1蛋白的表达强度存在显著差异(P0.001)。MTH1蛋白的表达在乳腺癌不同分子亚型、不同年龄、不同肿瘤大小、不同淋巴结状况等亚组之间无明显组间差异(P0.05)。乳腺癌组织中MTHl mRNA的相对表达水平是非癌乳腺组织中MTHl mRNA表达水平的3.15(±1.67)倍,差异显著(P=0.003)。 MTH1 mRNA的表达在乳腺癌不同分子亚型、不同年龄、不同肿瘤大小、不同淋巴结状况等亚组之间无明显组间差异(P0.05)。结论：在蛋白和mRNA水平,乳腺癌组织中MTH1的表达显著高于非癌乳腺组织。乳腺癌组织中MTH1的高表达不因分子亚型、年龄、肿瘤大小、淋巴结状况等临床病理特征而出现显著改变。第二部分MTH1途径对不同亚型乳腺癌细胞系的体外抑制作用研究目的：研究MTH1途径抑制剂对不同亚型的乳腺癌细胞系体外生长的影响方法：选取不同乳腺癌亚型有代表性的细胞系,Luminal型乳腺癌细胞系MCF-7、 Basal-like型乳腺癌细胞系MDA-MB-231以及Her-2过表达型乳腺癌细胞系MDA-MB-453进行MTH1的Western Blot检测确定其表达是否阳性。采用CCK-8细胞增殖实验测定不同浓度的MTH1抑制剂对各细胞系体外增殖的影响,采用克隆形成实验测定不同浓度的MTH1抑制剂对各细胞系克隆生存能力的影响。结果：不同亚型乳腺癌细胞系的MTH1蛋白均有较强的表达。CCK-8细胞增殖实验显示,随着MTH1抑制剂药物浓度的上升,乳腺癌细胞系的活力呈下降趋势。Luminal型乳腺癌细胞系MCF-7及Basal-l ike型乳腺癌细胞系MDA-MB-231在试验药物浓度范围内均取得了IC50值,分别为11.91umol/l和12.86umol/l:Her-2过表达型细胞系MDA-MB-453在试验药物浓度范围内未取得IC50值。克隆形成实验显示,不同亚型的乳腺癌细胞系的克隆形成均受到了明显的抑制并且取得了IC50值。Luminal型细胞系MCF-7.Basal-like型乳腺癌细胞系MDA-MB-231、Her-2过表达型细胞系MDA-MB-453的IC50值分别为9.78umol/l、6.96umol/l和8.97umol/l。结论：细胞增殖实验及克隆形成实验显示,在体外条件下,对MTH1途径进行干预,能够显著的抑制Lumirlal型乳腺癌细胞系MCF-7、Basal-l ike型乳腺癌细胞系MDA-MB-231以及Her-2过表达型乳腺癌细胞系MDA-MB-453的生长。第三部分MTH1途径对荷人不同亚型乳腺癌裸鼠的抑瘤作用研究目的：研究MTH1途径抑制剂对荷人不同亚型乳腺癌裸鼠体内移植瘤生长的影响方法：选取不同乳腺癌亚型的有代表性的细胞系,Luminal型乳腺癌细胞系MCF-7、Basal-like型乳腺癌细胞系MDA-MB-231以及Her-2过表达型乳腺癌细胞系MDA-MB-453接种于裸鼠建立裸鼠荷人不同乳腺癌亚型移植瘤模型。对荷瘤裸鼠及正常裸鼠进行MTH1抑制剂干预,绘制肿瘤生长曲线及裸鼠体重曲线,并进行血液和生化学检测,测定MTH1抑制剂对不同亚型乳腺癌裸鼠移植瘤生长的影响,以及MTH1抑制剂的安全性。结果：药物抑制实验显示,MTH1抑制剂疗程结束后,不同亚型乳腺癌细胞系移植瘤的最终体积分别为MCF-7(给药组 vs.对照组,60.1 mm3 vs.820.8 mm3, P0.05), MDA-MB-231(给药组 vs.对照组,30.1 mm3 vs.313.5 mm3, P0.05), MDA-MB-453(给药组 vs.对照组,0 mm3 vs.5.2 mm3, P0.05),差异显著。给药组裸鼠的最终体重与给药前初始体重分别为：MCF-7(最终 vs.给药前,16.81g vs.17.44g,P0.05), MDA-MB-231(最终 vs.给药前,17.42g vs.17.68g, P0.05), MDA-MB-453(最终vs. 给药前,17.23g vs.17.69g, P0.05)。单纯给药组裸鼠的最终体重与给药前体重变化为0.73(±0.34)g,无瘤无药组裸鼠相应的体重变化为2.26(±0.29)g,二者差异显著(P0.05)。单纯给药组与无瘤无药组裸鼠主要血常规和肝肾功指标无明显差别(P0.05)。结论：在裸鼠体内,对MTH1途径进行干预,能够显著的抑制Luminal型乳腺癌细胞系MCF-7、Basa1-like型乳腺癌细胞系MDA-MB-231以及Her-2过表达型乳腺癌细胞系MDA-MB-453移植瘤的生长。裸鼠的生长体重和血液学检查提示,短期应用MTH1抑制剂是安全的。
[Abstract]:The first part of the expression of MTH1 in human breast carcinoma: analysis of MTH1 protein, the expression of mRNA in human breast cancer tissues and the relationship between its expression and different clinical pathological features. Methods: the samples were randomly selected from the general library of Luminal, over expression of Her-2, Basal-like type of invasive breast cancer from non cancer breast tissue samples from 10 patients, the expression of MTHl protein detected by immunohistochemistry; specimens were randomly selected from liquid nitrogen Luminal, Her-2 over expression of type of breast cancer specimens and to detect the expression of MTH1 mRNA in breast cancer tissue samples which correspond to 10 patients each fluorescent quantitative PCR Basal-like. Results: invasive breast cancer in Qiangyang / Zhongyang MTH1 protein expression rate was 93.3%, non cancerous breast tissue MTH1 protein positive expression rate was 10% in Zhongyang, the expression was weak positive / negative. MTH1 breast cancer and non cancerous breast tissue There are significant differences in the expression intensity of protein (P0.001) expression of.MTH1 protein in different molecular subtypes of breast cancer, different age, different tumor size, there was no difference between the different lymph node status subgroup (P0.05). The relative expression of MTHl in breast cancer tissue mRNA level is the level of MTHl mRNA expression in non cancer breast tissues 3.15 (+ 1.67) times, significant difference (P=0.003). The expression of MTH1 mRNA in different molecular subtypes of breast cancer, different age, different tumor size, there was no difference between the different lymph node status subgroup (P0.05). Conclusion: the mRNA and protein level, the expression of MTH1 in breast cancer is significantly higher than that in breast cancer tissue. High expression of MTH1 in breast cancer by molecular subtype, age, tumor size, lymph node status and clinicopathological characteristics had significant change. The second part of the MTH1 pathway in different subtypes of breast cancer Objective to study inhibitory effect of cell lines in vitro: breast cancer cells of MTH1 pathway inhibitors on different subtypes: different subtypes of breast cancer cell lines representative, Luminal type of breast cancer cell line MCF-7 Western Blot detection of Basal-like breast cancer cell line MDA-MB-231 and Her-2 over expression of type MDA-MB-453 breast cancer cell line MTH1 to determine whether the expression is positive. The effects of MTH1 inhibitors in different concentration were determined by CCK-8 cell proliferation assay in vitro proliferation of different cell lines, the effect of clone formation assay of MTH1 inhibitors with different concentrations on the viability of the cell clones. Results: different subtypes of breast cancer cell lines MTH1 protein had strong expression of.CCK-8 cell proliferation experiment, with increasing inhibitor concentration MTH1, breast cancer cell vitality Decreased.Luminal breast cancer cell lines MCF-7 and Basal-l type Ike breast cancer cell line MDA-MB-231 in the experimental drug concentration range were obtained in IC50, respectively 11.91umol/l and 12.86umol/l:Her-2 over expression of type MDA-MB-453 cells in the experimental drug concentration range did not obtain IC50 value. Clone formation assay showed that the cloned human breast cancer cell line different subtypes of formation were inhibited obviously and achieved IC50 values of type MCF-7.Basal-like breast cancer cell line MDA-MB-231 and.Luminal cell lines, expression of Her-2 MDA-MB-453 IC50 cell line values were respectively 9.78umol/l, 6.96umol/l and 8.97umol/l. conclusion: the cell proliferation and clone formation assay, in vitro, intervention on the way to MTH1, can significantly inhibit type Lumirlal breast cancer cell line MCF-7, Basal-l type Ike breast cancer cell line MDA-MB-231 and Her Over expression of -2 in breast cancer cell line MDA-MB-453 growth. Objective to study on anti tumor effect of MTH1 pathway on third different subtypes of breast cancer bearing nude mice: Study of MTH1 pathway inhibitors growth in different subtypes of breast cancer xenograft in nude mice bearing human breast cancer: different subtypes of representative the cell line, Luminal type of breast cancer cell line MCF-7 Basal-like breast cancer cell line MDA-MB-231 and Her-2 in nude mice xenografts in different subtypes of breast cancer xenograft model of breast cancer cell line MDA-MB-453 was expression. For MTH1 inhibitor intervention on nude mice and normal mice, the tumor growth curve and the weight of nude mice the curve, and the blood and Biochemistry test, determination of the effects of MTH1 inhibitors on the growth of different subtypes of breast cancer in nude mice, and the safety of MTH1 inhibitors. Results: drug Inhibition experiment showed that after MTH1 inhibitor treatment, the final volume of different subtypes of breast cancer cell line xenografts were MCF-7 (drug group vs. control group, 60.1 mm3 vs.820.8 mm3, P0.05), MDA-MB-231 (drug group vs. control group, 30.1 mm3 vs.313.5 mm3, P0.05), MDA-MB-453 (group of vs. the control group, 0 mm3 vs.5.2 mm3, P0.05), the difference was significant. To the final weight of nude mice and the medicine group before administration of initial weight were: MCF-7 (vs. 16.81g vs.17.44g, before administration, P0.05 (MDA-MB-231), the final vs. before administration, 17.42g vs.17.68g, P0.05 MDA-MB-453 (vs.), the final delivery before 17.23g, vs.17.69g, P0.05). Only to the final weight of nude mice and medicine group before administration of weight change was 0.73 (+ 0.34) g, the changes of body weight of tumor free drug free mice corresponding to 2.26 (+ 0.29) g, two had significant difference (P0.05). The simple drug group and no tumor free the main blood group of nude mice There was no significant difference between the normal and kidney function index (P0.05). Conclusion: in vivo intervention on MTH1 pathway, inhibited the Luminal breast cancer cell line MCF-7, Basa1-like type of breast cancer cell line MDA-MB-231 and Her-2 overexpression breast cancer cell line MDA-MB-453 on the growth of transplanted tumor growth in nude mice body weight and blood. Examination indicated that short-term use of MTH1 inhibitors is safe.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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