過表達Sirt1的骨髓間充質(zhì)干細胞對4T1乳腺癌細胞生長的影響及其機制的探討
發(fā)布時間：2018-11-23 11:23
【摘要】：目的探討過表達Sirt1的骨髓間充質(zhì)干細胞(MSCs-Sirt1)對4T1乳腺癌細胞生長的影響及其潛在的分子機制。方法1、構(gòu)建BALB/c小鼠皮下移植瘤模型,裸鼠成瘤實驗分析MSCs-Sirt1對4T1乳腺癌移植瘤生長的影響;2、Real-time PCR、Western-blot、免疫組化、TUNEL染色、流式細胞術(shù)檢測MSCs-Sirt1對4T1乳腺癌細胞增殖和凋亡的影響;3、酶聯(lián)免疫吸附試驗(ELISA)檢測荷瘤小鼠血清中多種炎癥細胞因子(IL-6、IL-8、IL-10、IFN-γ、TNF-α)的表達水平;4、流式細胞儀檢測荷瘤小鼠腫瘤組織中浸潤的NK細胞數(shù)量,同位素釋放與標記法檢測荷瘤小鼠NK細胞的殺傷活性;5、ELISA檢測荷瘤小鼠血清中與NK細胞聚集相關(guān)的趨化因子CCL3、CCL4和CXCL10的表達水平;Real-time PCR和Western-blot法檢測各組荷瘤小鼠腫瘤中CXCL10的表達情況;6、Transwell實驗檢測CXCL10對NK細胞的趨化作用;7、構(gòu)建CXCL10減少型荷瘤小鼠模型進一步研究CXCL10的作用。結(jié)果1、裸鼠成瘤實驗結(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檢測結(jié)果顯示:與4T1組比較,MSCs組PCNA的表達增加(P0.001),Caspase-3的表達減少(P0.01);相反,MSCs-Sirt1組PCNA的表達減少(P0.01),Caspase-3的表達增加(P0.001);(2)免疫組化、TUNEL染色法檢測結(jié)果顯示:與4T1組相比,MSCs組Ki-67陽性細胞數(shù)明顯增加(P0.001),TUNEL染色陽性細胞數(shù)明顯減少(P0.01),相反,MSCs-Sirt1組TUNEL染色陽性腫瘤細胞數(shù)明顯增多(P0.001),Ki-67陽性腫瘤細胞數(shù)顯著下降(P0.01);(3)流式細胞術(shù)檢測結(jié)果顯示:MSCs-Sirt1組細胞凋亡率明顯大于4T1組(P0.001)。3、ELISA檢測荷瘤小鼠血清中相關(guān)的炎性細胞因子,結(jié)果顯示,與其他組相比,MSCs-Sirt1組小鼠血清中IFN-γ的表達水平顯著增高(P0.001),而其他炎性細胞因子(IL-6、IL-8、IL-10、TNF-α)的表達水平在各組間比較差異無統(tǒng)計學意義(P0.5)。4、流式細胞儀檢測NK細胞數(shù)量,結(jié)果顯示:MSCs-Sirt1組NK細胞數(shù)量明顯比對照組多(P0.001),同位素釋放與標記法檢測NK細胞殺傷活力結(jié)果顯示:MSCs-Sirt1組中NK細胞殺傷活性與其他組相比顯著增強。5、(1)ELISA法檢測荷瘤小鼠血清中與NK細胞聚集相關(guān)的趨化因子表達水平,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統(tǒng)計學意義(P0.5);(2)、Real-time PCR和Western-blot法檢測各組CXCL10的表達水平,結(jié)果顯示:MSCs-Sirt1組CXCL10的m RNA及蛋白水平的表達均較其他組顯著增高(P0.001)。6、Transwell實驗結(jié)果顯示:MSCs-Sirt1組Transwell小室下層NK細胞數(shù)量明顯高于對照組(P0.001),而在MSCs-Sirt1組加入兔抗小鼠CXCL10抗體后,NK細胞的數(shù)量明顯減少(P0.001)。7、CXCL10減少型荷瘤小鼠實驗結(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4T1乳腺癌細胞的生長有明顯的抑制作用。2、MSCs-Sirt1可能通過CXCL10募集NK細胞增強局部炎癥反應(yīng)抑制4T1乳腺癌細胞生長。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9
本文編號：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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9
【參考文獻】
相關(guān)期刊論文 前8條
1 黃磊;鄭建金;;臍帶血間充質(zhì)干細胞與腫瘤關(guān)系的研究進展[J];現(xiàn)代腫瘤醫(yī)學;2017年06期
2 荊娜;張金超;楊巖麗;吳瓊;孫雪冬;張曉燕;趙來偉;潘欣;蔣昊;丁國梁;王丹紅;陳虎;;自然殺傷細胞治療晚期肝癌的近期臨床療效[J];中國腫瘤生物治療雜志;2016年04期
3 劉瑞磊;陳健寧;王佳妮;湯謐;張艷玲;;沉默信息調(diào)節(jié)因子1與乳腺癌臨床病理指標的相關(guān)性及生存分析[J];中華普通外科學文獻(電子版);2016年01期
4 Justin D Glenn;Katharine A Whartenby;;Mesenchymal stem cells: Emerging mechanisms of immunomodulation and therapy[J];World Journal of Stem Cells;2014年05期
5 顧園龍;王東亮;張艷橋;;NK細胞在腫瘤免疫治療中的進展[J];國際免疫學雜志;2012年03期
6 于津浦;孫海燕;李慧;曹水;辛寧;任秀寶;;異基因反應(yīng)性NK細胞在供者淋巴細胞輸注治療單倍體相合造血干細胞移植后肺癌復發(fā)中的作用研究[J];中國實驗血液學雜志;2009年01期
7 焦順昌;張國慶;林星石;李瑾昱;李方;;自體NK、T混合淋巴細胞擴增后回輸對腫瘤免疫的影響[J];軍醫(yī)進修學院學報;2008年05期
8 吳霞,李大金;趨化性細胞因子對NK細胞生物學功能的調(diào)控作用[J];細胞與分子免疫學雜志;2004年04期
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