过表达Sirt1的骨髓间充质干细胞对4T1乳腺癌细胞生长的影响及其机制的探讨
发布时间：2018-11-23 11:23
【摘要】：目的探讨过表达Sirt1的骨髓间充质干细胞(MSCs-Sirt1)对4T1乳腺癌细胞生长的影响及其潜在的分子机制。方法1、构建BALB/c小鼠皮下移植瘤模型,裸鼠成瘤实验分析MSCs-Sirt1对4T1乳腺癌移植瘤生长的影响;2、Real-time PCR、Western-blot、免疫组化、TUNEL染色、流式细胞术检测MSCs-Sirt1对4T1乳腺癌细胞增殖和凋亡的影响;3、酶联免疫吸附试验(ELISA)检测荷瘤小鼠血清中多种炎症细胞因子(IL-6、IL-8、IL-10、IFN-γ、TNF-α)的表达水平;4、流式细胞仪检测荷瘤小鼠肿瘤组织中浸润的NK细胞数量,同位素释放与标记法检测荷瘤小鼠NK细胞的杀伤活性;5、ELISA检测荷瘤小鼠血清中与NK细胞聚集相关的趋化因子CCL3、CCL4和CXCL10的表达水平;Real-time PCR和Western-blot法检测各组荷瘤小鼠肿瘤中CXCL10的表达情况;6、Transwell实验检测CXCL10对NK细胞的趋化作用;7、构建CXCL10减少型荷瘤小鼠模型进一步研究CXCL10的作用。结果1、裸鼠成瘤实验结果显示,与4T1组相比,MSCs组移植瘤的体积和重量明显增加(P0.05);MSCs-Sirt1组移植瘤的体积和重量明显减少(P0.01)。2、(1)Real-time PCR、Western-blot检测结果显示:与4T1组比较,MSCs组PCNA的表达增加(P0.001),Caspase-3的表达减少(P0.01);相反,MSCs-Sirt1组PCNA的表达减少(P0.01),Caspase-3的表达增加(P0.001);(2)免疫组化、TUNEL染色法检测结果显示:与4T1组相比,MSCs组Ki-67阳性细胞数明显增加(P0.001),TUNEL染色阳性细胞数明显减少(P0.01),相反,MSCs-Sirt1组TUNEL染色阳性肿瘤细胞数明显增多(P0.001),Ki-67阳性肿瘤细胞数显著下降(P0.01);(3)流式细胞术检测结果显示:MSCs-Sirt1组细胞凋亡率明显大于4T1组(P0.001)。3、ELISA检测荷瘤小鼠血清中相关的炎性细胞因子,结果显示,与其他组相比,MSCs-Sirt1组小鼠血清中IFN-γ的表达水平显著增高(P0.001),而其他炎性细胞因子(IL-6、IL-8、IL-10、TNF-α)的表达水平在各组间比较差异无统计学意义(P0.5)。4、流式细胞仪检测NK细胞数量,结果显示:MSCs-Sirt1组NK细胞数量明显比对照组多(P0.001),同位素释放与标记法检测NK细胞杀伤活力结果显示:MSCs-Sirt1组中NK细胞杀伤活性与其他组相比显著增强。5、(1)ELISA法检测荷瘤小鼠血清中与NK细胞聚集相关的趋化因子表达水平,结果显示:CXCL10水平在MSCs-Sirt1组相比其他组显著上调(P0.01),而CCL3、CCL4在各组间比较差异无统计学意义(P0.5);(2)、Real-time PCR和Western-blot法检测各组CXCL10的表达水平,结果显示:MSCs-Sirt1组CXCL10的m RNA及蛋白水平的表达均较其他组显著增高(P0.001)。6、Transwell实验结果显示:MSCs-Sirt1组Transwell小室下层NK细胞数量明显高于对照组(P0.001),而在MSCs-Sirt1组加入兔抗小鼠CXCL10抗体后,NK细胞的数量明显减少(P0.001)。7、CXCL10减少型荷瘤小鼠实验结果显示,MSCs-Sirt1+兔抗小鼠CXCL10抗体组较MSCs-Sirt1组肿瘤体积、重量均有所增加(P0.05)。结论1、MSCs-Sirt1对4T1乳腺癌细胞的生长有明显的抑制作用。2、MSCs-Sirt1可能通过CXCL10募集NK细胞增强局部炎症反应抑制4T1乳腺癌细胞生长。
[Abstract]:Objective To study the effect of bone marrow mesenchymal stem cells (MSCs-Sirt1) on the growth of 4T1 breast cancer cells and its potential molecular mechanism. Method 1. The effect of MSCs-Sirt1 on the growth of 4T1 breast cancer xenografts was analyzed by a model of subcutaneous transplantation of BALB/ c mice, and the effect of MSCs-Sirt1 on the proliferation and apoptosis of 4T1 breast cancer cells was detected by real-time PCR, Western-blot, immunohistochemistry, TUNEL staining and flow cytometry. The expression level of various inflammatory cytokines (IL-6, IL-8, IL-10, IFN-1, TNF-1) in the tumor-bearing mice was detected by enzyme-linked immunosorbent assay (ELISA), and the number of NK cells in the tumor tissues of the tumor-bearing mice was detected by flow cytometry. The anti-killing activity of the NK cells in the tumor-bearing mice was detected by the isotope release and labeling method; 5, the expression levels of the chemokines CCL3, CCL4 and CXCL10 associated with the aggregation of NK cells in the serum of the tumor-bearing mice were detected by ELISA; the expression of the CXCL10 in the tumor of each group was detected by the real-time PCR and the Western-blot method; and 6, Transwell's experiment was used to detect the chemotaxis of CXCL10 on NK cells, and to construct the CXCL10-reduced tumor-bearing mice model to further study the role of CXCL10. Results 1. The experimental results of nude mice showed that the volume and weight of the transplanted tumor in the MSCs increased significantly (P0.05). The volume and weight of the transplanted tumor in the MSCs-Sirt1 group were significantly decreased (P0.01). (1) Real-time PCR and Western-blot analysis showed that the expression of PCNA in the MSCs was increased (P 0.001). The expression of Caspase-3 was decreased (P0.01). In contrast, the expression of PCNA in the group of MSCs-Sirt1 was decreased (P0.01), and the expression of Caspase-3 was increased (P0.01). (2) The results of immunohistochemistry and TUNEL staining showed that the number of Ki-67 positive cells in the MSCs increased significantly (P 0.001) compared with that of the 4T1 group. The number of TUNEL staining positive cells decreased significantly (P0.01), but the number of TUNEL-stained positive tumor cells in the MSCs-Sirt1 group increased significantly (P0.01), and the number of Ki-67 positive tumor cells decreased significantly (P0.01). (3) The results of flow cytometry showed that the apoptosis rate of the MSCs in the MSCs-Sirt1 group was significantly higher than that of the 4T1 group (P0.001). The results showed that the expression of IFN-1 in the serum of the mice with MSCs-Sirt1 was significantly higher than that of other groups (P 0.001) and the other inflammatory cytokines (IL-6, IL-8, IL-10, The number of NK cells was detected by flow cytometry. The results showed that the number of NK cells in the MSCs-Sirt1 group was significantly higher than that of the control group (P0.001), and the results of the anti-killing activity of NK cells by the isotope release and labeling method showed that: The anti-killing activity of NK cells in the MSCs-Sirt1 group was significantly enhanced as compared with the other groups. The expression level of CXCL10 in each group was detected by real-time PCR and Western-blot. The results showed that the expression of mRNA and protein of CXCL10 in the group of MSCs-Sirt1 was significantly higher than that in other groups (P 0.001). The number of NK cells in the lower layer of the Transwell chamber of the MSCs-Sirt1 group was significantly higher than that of the control group (P 0.001). After the addition of the anti-mouse CXCL10 antibody in the MSCs-Sirt1 group, the number of NK cells decreased significantly (P0.01). The experimental results of the CXCL10-reduced tumor-bearing mice showed that the tumor volume of the MSCs-Sirt1 + rabbit anti-mouse CXCL10 antibody group was smaller than that of the MSCs-Sirt1 group. The weight was increased (P0.05). Conclusion 1. MSCs-Sirt1 can inhibit the growth of 4T1 breast cancer cells.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
本文编号：2351413
[Abstract]:Objective To study the effect of bone marrow mesenchymal stem cells (MSCs-Sirt1) on the growth of 4T1 breast cancer cells and its potential molecular mechanism. Method 1. The effect of MSCs-Sirt1 on the growth of 4T1 breast cancer xenografts was analyzed by a model of subcutaneous transplantation of BALB/ c mice, and the effect of MSCs-Sirt1 on the proliferation and apoptosis of 4T1 breast cancer cells was detected by real-time PCR, Western-blot, immunohistochemistry, TUNEL staining and flow cytometry. The expression level of various inflammatory cytokines (IL-6, IL-8, IL-10, IFN-1, TNF-1) in the tumor-bearing mice was detected by enzyme-linked immunosorbent assay (ELISA), and the number of NK cells in the tumor tissues of the tumor-bearing mice was detected by flow cytometry. The anti-killing activity of the NK cells in the tumor-bearing mice was detected by the isotope release and labeling method; 5, the expression levels of the chemokines CCL3, CCL4 and CXCL10 associated with the aggregation of NK cells in the serum of the tumor-bearing mice were detected by ELISA; the expression of the CXCL10 in the tumor of each group was detected by the real-time PCR and the Western-blot method; and 6, Transwell's experiment was used to detect the chemotaxis of CXCL10 on NK cells, and to construct the CXCL10-reduced tumor-bearing mice model to further study the role of CXCL10. Results 1. The experimental results of nude mice showed that the volume and weight of the transplanted tumor in the MSCs increased significantly (P0.05). The volume and weight of the transplanted tumor in the MSCs-Sirt1 group were significantly decreased (P0.01). (1) Real-time PCR and Western-blot analysis showed that the expression of PCNA in the MSCs was increased (P 0.001). The expression of Caspase-3 was decreased (P0.01). In contrast, the expression of PCNA in the group of MSCs-Sirt1 was decreased (P0.01), and the expression of Caspase-3 was increased (P0.01). (2) The results of immunohistochemistry and TUNEL staining showed that the number of Ki-67 positive cells in the MSCs increased significantly (P 0.001) compared with that of the 4T1 group. The number of TUNEL staining positive cells decreased significantly (P0.01), but the number of TUNEL-stained positive tumor cells in the MSCs-Sirt1 group increased significantly (P0.01), and the number of Ki-67 positive tumor cells decreased significantly (P0.01). (3) The results of flow cytometry showed that the apoptosis rate of the MSCs in the MSCs-Sirt1 group was significantly higher than that of the 4T1 group (P0.001). The results showed that the expression of IFN-1 in the serum of the mice with MSCs-Sirt1 was significantly higher than that of other groups (P 0.001) and the other inflammatory cytokines (IL-6, IL-8, IL-10, The number of NK cells was detected by flow cytometry. The results showed that the number of NK cells in the MSCs-Sirt1 group was significantly higher than that of the control group (P0.001), and the results of the anti-killing activity of NK cells by the isotope release and labeling method showed that: The anti-killing activity of NK cells in the MSCs-Sirt1 group was significantly enhanced as compared with the other groups. The expression level of CXCL10 in each group was detected by real-time PCR and Western-blot. The results showed that the expression of mRNA and protein of CXCL10 in the group of MSCs-Sirt1 was significantly higher than that in other groups (P 0.001). The number of NK cells in the lower layer of the Transwell chamber of the MSCs-Sirt1 group was significantly higher than that of the control group (P 0.001). After the addition of the anti-mouse CXCL10 antibody in the MSCs-Sirt1 group, the number of NK cells decreased significantly (P0.01). The experimental results of the CXCL10-reduced tumor-bearing mice showed that the tumor volume of the MSCs-Sirt1 + rabbit anti-mouse CXCL10 antibody group was smaller than that of the MSCs-Sirt1 group. The weight was increased (P0.05). Conclusion 1. MSCs-Sirt1 can inhibit the growth of 4T1 breast cancer cells.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
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