過表達(dá)Sirt1的骨髓間充質(zhì)干細(xì)胞對(duì)4T1乳腺癌細(xì)胞生長(zhǎng)的影響及其機(jī)制的探討
發(fā)布時(shí)間：2018-11-23 11:23
【摘要】：目的探討過表達(dá)Sirt1的骨髓間充質(zhì)干細(xì)胞(MSCs-Sirt1)對(duì)4T1乳腺癌細(xì)胞生長(zhǎng)的影響及其潛在的分子機(jī)制。方法1、構(gòu)建BALB/c小鼠皮下移植瘤模型,裸鼠成瘤實(shí)驗(yàn)分析MSCs-Sirt1對(duì)4T1乳腺癌移植瘤生長(zhǎng)的影響;2、Real-time PCR、Western-blot、免疫組化、TUNEL染色、流式細(xì)胞術(shù)檢測(cè)MSCs-Sirt1對(duì)4T1乳腺癌細(xì)胞增殖和凋亡的影響;3、酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)荷瘤小鼠血清中多種炎癥細(xì)胞因子(IL-6、IL-8、IL-10、IFN-γ、TNF-α)的表達(dá)水平;4、流式細(xì)胞儀檢測(cè)荷瘤小鼠腫瘤組織中浸潤(rùn)的NK細(xì)胞數(shù)量,同位素釋放與標(biāo)記法檢測(cè)荷瘤小鼠NK細(xì)胞的殺傷活性;5、ELISA檢測(cè)荷瘤小鼠血清中與NK細(xì)胞聚集相關(guān)的趨化因子CCL3、CCL4和CXCL10的表達(dá)水平;Real-time PCR和Western-blot法檢測(cè)各組荷瘤小鼠腫瘤中CXCL10的表達(dá)情況;6、Transwell實(shí)驗(yàn)檢測(cè)CXCL10對(duì)NK細(xì)胞的趨化作用;7、構(gòu)建CXCL10減少型荷瘤小鼠模型進(jìn)一步研究CXCL10的作用。結(jié)果1、裸鼠成瘤實(shí)驗(yàn)結(jié)果顯示,與4T1組相比,MSCs組移植瘤的體積和重量明顯增加(P0.05);MSCs-Sirt1組移植瘤的體積和重量明顯減少(P0.01)。2、(1)Real-time PCR、Western-blot檢測(cè)結(jié)果顯示:與4T1組比較,MSCs組PCNA的表達(dá)增加(P0.001),Caspase-3的表達(dá)減少(P0.01);相反,MSCs-Sirt1組PCNA的表達(dá)減少(P0.01),Caspase-3的表達(dá)增加(P0.001);(2)免疫組化、TUNEL染色法檢測(cè)結(jié)果顯示:與4T1組相比,MSCs組Ki-67陽(yáng)性細(xì)胞數(shù)明顯增加(P0.001),TUNEL染色陽(yáng)性細(xì)胞數(shù)明顯減少(P0.01),相反,MSCs-Sirt1組TUNEL染色陽(yáng)性腫瘤細(xì)胞數(shù)明顯增多(P0.001),Ki-67陽(yáng)性腫瘤細(xì)胞數(shù)顯著下降(P0.01);(3)流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示:MSCs-Sirt1組細(xì)胞凋亡率明顯大于4T1組(P0.001)。3、ELISA檢測(cè)荷瘤小鼠血清中相關(guān)的炎性細(xì)胞因子,結(jié)果顯示,與其他組相比,MSCs-Sirt1組小鼠血清中IFN-γ的表達(dá)水平顯著增高(P0.001),而其他炎性細(xì)胞因子(IL-6、IL-8、IL-10、TNF-α)的表達(dá)水平在各組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.5)。4、流式細(xì)胞儀檢測(cè)NK細(xì)胞數(shù)量,結(jié)果顯示:MSCs-Sirt1組NK細(xì)胞數(shù)量明顯比對(duì)照組多(P0.001),同位素釋放與標(biāo)記法檢測(cè)NK細(xì)胞殺傷活力結(jié)果顯示:MSCs-Sirt1組中NK細(xì)胞殺傷活性與其他組相比顯著增強(qiáng)。5、(1)ELISA法檢測(cè)荷瘤小鼠血清中與NK細(xì)胞聚集相關(guān)的趨化因子表達(dá)水平,結(jié)果顯示:CXCL10水平在MSCs-Sirt1組相比其他組顯著上調(diào)(P0.01),而CCL3、CCL4在各組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.5);(2)、Real-time PCR和Western-blot法檢測(cè)各組CXCL10的表達(dá)水平,結(jié)果顯示:MSCs-Sirt1組CXCL10的m RNA及蛋白水平的表達(dá)均較其他組顯著增高(P0.001)。6、Transwell實(shí)驗(yàn)結(jié)果顯示:MSCs-Sirt1組Transwell小室下層NK細(xì)胞數(shù)量明顯高于對(duì)照組(P0.001),而在MSCs-Sirt1組加入兔抗小鼠CXCL10抗體后,NK細(xì)胞的數(shù)量明顯減少(P0.001)。7、CXCL10減少型荷瘤小鼠實(shí)驗(yàn)結(jié)果顯示,MSCs-Sirt1+兔抗小鼠CXCL10抗體組較MSCs-Sirt1組腫瘤體積、重量均有所增加(P0.05)。結(jié)論1、MSCs-Sirt1對(duì)4T1乳腺癌細(xì)胞的生長(zhǎng)有明顯的抑制作用。2、MSCs-Sirt1可能通過CXCL10募集NK細(xì)胞增強(qiáng)局部炎癥反應(yīng)抑制4T1乳腺癌細(xì)胞生長(zhǎng)。
[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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9
本文編號(hào)：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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9
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