炎症因子IL-1β或TNF-α预处理的骨髓间充质干细胞可改变骨髓瘤细胞株H929对多西环素毒性作用的敏感性
发布时间：2018-07-16 22:31
【摘要】：目的:在多发性骨髓瘤(Multiple myeloma,MM)疾病中,骨髓微环境对MM细胞的生存、耐药、演化等起着重要的支持作用。骨髓微环境中常有各种炎症相关因子水平异常,这些炎症因子除了直接作用于MM细胞外,其如何通过微环境中骨髓间充质干细胞(Bone marrow derived mesenchymal stem cells,BMMSCs)来影响MM细胞,目前尚未完全阐明。本研究在体外研究炎症因子作用微环境中BMMSCs后,其对MM细胞对多西环素(Doxycycline,DOX)杀伤作用敏感性的变化,以及可能涉及的部分机制。方法:1.用CCK8法检测不同时间(1天、2天、3天)不同浓度多西环素(DOX)处理H929或BMMSCs后的细胞增殖活性抑制作用,筛选出适宜的多西环素浓度梯度。然后再用白细胞介素-1β(IL-1β)或肿瘤坏死因子-1α(TNF-α)预处理BMMSCs后,利用CCK8法检测在与BMMSCs共培养条件下,DOX对骨髓瘤细胞株H929的增值抑制作用,随后与对照组的数据进行比较,并进行统计学分析。2.应用实时荧光定量PCR(RT-PCR)检测IL-1β或TNF-α处理后,对BMMSCs的VCAM-1与BAFF基因m RNA的表达影响。分为1)对照组a:无处理BMMSCs。2)实验组b:BMMSCs用IL-1β处理1天(24h)。3)实验组c:BMMSCs用TNF-α处理1天(24h)。用Real-Time PCR方法检测各组基因的变化,并进行统计学分析。3.首先,利用流式细胞术(Flow cytometry,FCM)分别检测在予炎症因子IL-1β或TNF-α处理后,对BMMSCs表面的VCAM-1的表达的影响。然后,先将BMMSCs予IL-1β或TNF-α预处理后,再用FCM方法检测H929细胞与BMMSCs共培养条件下,不同DOX浓度下H929凋亡的变化。随后与对照组的数据进行比较,并进行统计学分析。4.先将BMMSCs予IL-1β或TNF-α预处理后,采用Western Blot方法检测H929细胞与BMMSCs共培养时,不同浓度多西环素条件下,H929细胞p-Erk1/2的表达的情况,随后与对照组的数据进行比较,并进行统计学分析。结果:1.置于显微镜下观察,不论是否有IL-1β或TNF-α处理,BMMSCs的生长方式均是贴壁生长,细胞形态大致主要为长梭形状。不论是否有IL-1β或TNF-α处理,各组的BMMSCs形态无明显的差异。与H929细胞株共培养后,BMMSCs的细胞形态和生长特点与单独培养时比较,也无明显形态学改变。2.根据DOX对H929细胞的增殖抑制曲线,筛选出5和10μg/m L的DOX浓度作为下一步的体外实验检测浓度。在5和10μg/m L的DOX浓度下,对BMMSCs的增殖没有明显影响(P0.05)。不论是在5或10μg/m L的DOX浓度下,H929与BMMSCs共培养后,与对照组比较,经过IL-1β或TNF-α预处理的BMMSCs,可以减少DOX对H929细胞的体外增殖抑制(均P0.05)。3.不论是经炎症因子IL-1β还是TNF-α处理后,炎症因子处理组与对照组比较,RT-PCR检测提示BMMSCs的VCAM-1的m RNA表达水平上调且具有统计学意义(P0.05);但是经IL-1β或TNF-α处理后对BMMSCs的BAFF的m RNA表达没有明显变化(P0.05)。4.用IL-1β或TNF-α处理BMMSCs后,FCM检测提示表达VCAM-1的细胞比例较对照组明显增多(P0.05)。在IL-1β和TNF-α处理组比较中,发现TNF-α组比IL-1β组的VCAM-1细胞阳性率更高(P0.05)。在5μg/ml的DOX浓度下,与无共培养的对照组相比,H929与BMMSCs共培养条件下,可以降低H929细胞凋亡率(P0.05);但在10μg/ml的DOX浓度下,共培养组与对照组间的H929细胞的凋亡率没有明显差异(P0.05)。随后在BMMSCs经IL-1β或TNF-α预处理的情况下,H929与BMMSCs在5或10μg/ml DOX下共培养2天后,经炎症因子预处理组中的H929的凋亡率减少(P0.05)。且TNF-α组比IL-1β组的降低H929细胞凋亡率的作用更显著(P0.05)。5.首先,DOX处理后可使H929细胞的p-Erk1/2表达,经Western Blot方法检测后发现其水平下降,在实验浓度下不具有剂量依赖性。其次,H929与BMMSCs共培养时,经DOX处理后H929的p-Erk1/2水平,较无共培养的对照组有所上调(P0.05)。最后,在与经IL-1β或TNF-α预处理的BMMSCs共培养条件下,DOX处理后H929的p-Erk1/2水平,较无炎症因子处理的对照也有所上调(均P0.05)。结论:我们的体外研究结果提示,在经炎症因子如IL-1β和TNF-α作用BMMSCs以后,刺激后的BMMSCs与MM细胞株相互作用中,可以减少DOX引起H929细胞的增殖抑制和凋亡,增加MM细胞株对DOX的耐药。炎症因子这一作用可能与其对BMMSCs的Mek/Erk信号通路、细胞表面黏附分子VCAM-1有关。
[Abstract]:Objective: in the Multiple myeloma (MM) disease, bone marrow microenvironment plays an important role in the survival, resistance and evolution of MM cells. There are often various levels of inflammation related factors in the bone marrow microenvironment. These inflammatory factors are not only directly acting on the MM cells but also through the microenvironment in the bone marrow mesenchymal stem cells. Bone marrow derived mesenchymal stem cells, BMMSCs, which affects MM cells, has not yet been fully elucidated. In this study, the changes in the sensitivity of MM cells to the cytotoxicity of MM cells to doxycycline (Doxycycline, DOX), and some possible mechanisms involved in the study of inflammatory factors in microenvironment in vitro, were studied in this study. Methods: 1. At different times (1 days, 2 days, 3 days), the inhibitory effects of different concentrations of doxycycline (DOX) on the cell proliferation activity after H929 or BMMSCs were inhibited, and a suitable concentration gradient was screened. Then BMMSCs was pretreated with interleukin -1 beta (IL-1 beta) or tumor necrosis factor -1 alpha (TNF- a), and the CCK8 method was used to detect the DO under co culture with BMMSCs. The inhibitory effect of X on the proliferation of myeloma cell line H929, then compared with the control group, and statistically analyzing the effect of.2. on the expression of IL-1 beta or TNF- alpha by real-time fluorescent quantitative PCR (RT-PCR) detection of BMMSCs's VCAM-1 and BAFF gene m RNA. After 1 days (24h).3) the experimental group c:BMMSCs was treated with TNF- alpha for 1 days (24h). The changes of each gene were detected by Real-Time PCR method, and the statistical analysis of.3. first was carried out by flow cytometry (Flow cytometry, FCM). After preconditioning with IL-1 beta or TNF- alpha, FCM method was used to detect the changes of H929 apoptosis under the co culture condition of H929 cells and BMMSCs, and then compared with the data of the control group, and then the statistical analysis was carried out and the BMMSCs was pretreated with IL-1 beta or TNF- alpha first, and the Western cells were used to detect the co culture of H929. The expression of p-Erk1/2 in H929 cells under the condition of different concentrations of doxycycline was compared with the data of the control group, and the results were statistically analyzed. Results: 1. under the microscope, no matter whether IL-1 beta or TNF- alpha were treated, the growth mode of BMMSCs was adhered to the wall, and the morphology of the cells was mainly the shape of the long spindle. There was no significant difference in the BMMSCs morphology between IL-1 beta or TNF- alpha. After co culture with H929 cell lines, the cell morphology and growth characteristics of BMMSCs were compared with the individual culture, and there was no obvious morphological change of.2. based on the proliferation inhibition curve of H929 cells based on DOX, and the DOX concentration of 5 and 10 mu g/m L was screened as the next test in vitro. Under the concentration of DOX in 5 and 10 g/m L, the proliferation of BMMSCs was not significantly affected (P0.05). No matter at the DOX concentration of 5 or 10 mu L, H929 and BMMSCs were co cultured. After the treatment of L-1 beta or TNF- alpha, the inflammatory factor treatment group was compared with the control group. RT-PCR detection suggested that the m RNA expression level of VCAM-1 in BMMSCs was up and statistically significant (P0.05), but the expression of BMMSCs BAFF was not significantly changed after the treatment of IL-1 beta or TNF- alpha. The proportion of cells expressing VCAM-1 was significantly higher than that in the control group (P0.05). In the comparison of IL-1 beta and TNF- alpha treatment groups, the positive rate of VCAM-1 cells in TNF- a group was higher than that of IL-1 beta group (P0.05). Under 5 u g/ml DOX concentration, the apoptosis rate could be reduced under the co culture condition of H929 and BMMSCs, but at 1, the rate of apoptosis could be reduced. There was no significant difference in the apoptosis rate of H929 cells between the co culture group and the control group at 0 g/ml DOX concentration (P0.05). Subsequently, the apoptotic rate of H929 and BMMSCs under the condition of IL-1 beta or TNF- alpha was 2 days after the co culture of H929 and BMMSCs under the preconditioning of the inflammatory factor preconditioning group. The effect of reducing the apoptosis rate of H929 cells was more significant (P0.05).5. first. After DOX treatment, the p-Erk1/2 expression of H929 cells could be expressed. The level of H929 cells decreased after the Western Blot method. The concentration of H929 and BMMSCs was not dose-dependent. Secondly, when H929 and BMMSCs were co cultured, the level of H929 was compared with the control group without co culture. P0.05. Finally, the p-Erk1/2 level of H929 after DOX treatment with the BMMSCs co culture with IL-1 beta or TNF- alpha was also up up (all P0.05). Conclusion: our in vitro study results suggest that the stimuli after the stimulation of the inflammatory factors such as IL-1 beta and TNF- alpha are BMMSCs. The interaction of M cell lines can reduce the proliferation inhibition and apoptosis of H929 cells by DOX and increase the resistance of MM cell lines to DOX. The role of inflammatory factors may be related to the Mek/Erk signaling pathway of BMMSCs and the adhesion molecule VCAM-1 of the cell surface.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3
本文编号：2127858
[Abstract]:Objective: in the Multiple myeloma (MM) disease, bone marrow microenvironment plays an important role in the survival, resistance and evolution of MM cells. There are often various levels of inflammation related factors in the bone marrow microenvironment. These inflammatory factors are not only directly acting on the MM cells but also through the microenvironment in the bone marrow mesenchymal stem cells. Bone marrow derived mesenchymal stem cells, BMMSCs, which affects MM cells, has not yet been fully elucidated. In this study, the changes in the sensitivity of MM cells to the cytotoxicity of MM cells to doxycycline (Doxycycline, DOX), and some possible mechanisms involved in the study of inflammatory factors in microenvironment in vitro, were studied in this study. Methods: 1. At different times (1 days, 2 days, 3 days), the inhibitory effects of different concentrations of doxycycline (DOX) on the cell proliferation activity after H929 or BMMSCs were inhibited, and a suitable concentration gradient was screened. Then BMMSCs was pretreated with interleukin -1 beta (IL-1 beta) or tumor necrosis factor -1 alpha (TNF- a), and the CCK8 method was used to detect the DO under co culture with BMMSCs. The inhibitory effect of X on the proliferation of myeloma cell line H929, then compared with the control group, and statistically analyzing the effect of.2. on the expression of IL-1 beta or TNF- alpha by real-time fluorescent quantitative PCR (RT-PCR) detection of BMMSCs's VCAM-1 and BAFF gene m RNA. After 1 days (24h).3) the experimental group c:BMMSCs was treated with TNF- alpha for 1 days (24h). The changes of each gene were detected by Real-Time PCR method, and the statistical analysis of.3. first was carried out by flow cytometry (Flow cytometry, FCM). After preconditioning with IL-1 beta or TNF- alpha, FCM method was used to detect the changes of H929 apoptosis under the co culture condition of H929 cells and BMMSCs, and then compared with the data of the control group, and then the statistical analysis was carried out and the BMMSCs was pretreated with IL-1 beta or TNF- alpha first, and the Western cells were used to detect the co culture of H929. The expression of p-Erk1/2 in H929 cells under the condition of different concentrations of doxycycline was compared with the data of the control group, and the results were statistically analyzed. Results: 1. under the microscope, no matter whether IL-1 beta or TNF- alpha were treated, the growth mode of BMMSCs was adhered to the wall, and the morphology of the cells was mainly the shape of the long spindle. There was no significant difference in the BMMSCs morphology between IL-1 beta or TNF- alpha. After co culture with H929 cell lines, the cell morphology and growth characteristics of BMMSCs were compared with the individual culture, and there was no obvious morphological change of.2. based on the proliferation inhibition curve of H929 cells based on DOX, and the DOX concentration of 5 and 10 mu g/m L was screened as the next test in vitro. Under the concentration of DOX in 5 and 10 g/m L, the proliferation of BMMSCs was not significantly affected (P0.05). No matter at the DOX concentration of 5 or 10 mu L, H929 and BMMSCs were co cultured. After the treatment of L-1 beta or TNF- alpha, the inflammatory factor treatment group was compared with the control group. RT-PCR detection suggested that the m RNA expression level of VCAM-1 in BMMSCs was up and statistically significant (P0.05), but the expression of BMMSCs BAFF was not significantly changed after the treatment of IL-1 beta or TNF- alpha. The proportion of cells expressing VCAM-1 was significantly higher than that in the control group (P0.05). In the comparison of IL-1 beta and TNF- alpha treatment groups, the positive rate of VCAM-1 cells in TNF- a group was higher than that of IL-1 beta group (P0.05). Under 5 u g/ml DOX concentration, the apoptosis rate could be reduced under the co culture condition of H929 and BMMSCs, but at 1, the rate of apoptosis could be reduced. There was no significant difference in the apoptosis rate of H929 cells between the co culture group and the control group at 0 g/ml DOX concentration (P0.05). Subsequently, the apoptotic rate of H929 and BMMSCs under the condition of IL-1 beta or TNF- alpha was 2 days after the co culture of H929 and BMMSCs under the preconditioning of the inflammatory factor preconditioning group. The effect of reducing the apoptosis rate of H929 cells was more significant (P0.05).5. first. After DOX treatment, the p-Erk1/2 expression of H929 cells could be expressed. The level of H929 cells decreased after the Western Blot method. The concentration of H929 and BMMSCs was not dose-dependent. Secondly, when H929 and BMMSCs were co cultured, the level of H929 was compared with the control group without co culture. P0.05. Finally, the p-Erk1/2 level of H929 after DOX treatment with the BMMSCs co culture with IL-1 beta or TNF- alpha was also up up (all P0.05). Conclusion: our in vitro study results suggest that the stimuli after the stimulation of the inflammatory factors such as IL-1 beta and TNF- alpha are BMMSCs. The interaction of M cell lines can reduce the proliferation inhibition and apoptosis of H929 cells by DOX and increase the resistance of MM cell lines to DOX. The role of inflammatory factors may be related to the Mek/Erk signaling pathway of BMMSCs and the adhesion molecule VCAM-1 of the cell surface.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3
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