炎癥因子IL-1β或TNF-α預(yù)處理的骨髓間充質(zhì)干細(xì)胞可改變骨髓瘤細(xì)胞株H929對(duì)多西環(huán)素毒性作用的敏感性
發(fā)布時(shí)間：2018-07-16 22:31
【摘要】：目的:在多發(fā)性骨髓瘤(Multiple myeloma,MM)疾病中,骨髓微環(huán)境對(duì)MM細(xì)胞的生存、耐藥、演化等起著重要的支持作用。骨髓微環(huán)境中常有各種炎癥相關(guān)因子水平異常,這些炎癥因子除了直接作用于MM細(xì)胞外,其如何通過微環(huán)境中骨髓間充質(zhì)干細(xì)胞(Bone marrow derived mesenchymal stem cells,BMMSCs)來影響MM細(xì)胞,目前尚未完全闡明。本研究在體外研究炎癥因子作用微環(huán)境中BMMSCs后,其對(duì)MM細(xì)胞對(duì)多西環(huán)素(Doxycycline,DOX)殺傷作用敏感性的變化,以及可能涉及的部分機(jī)制。方法:1.用CCK8法檢測不同時(shí)間(1天、2天、3天)不同濃度多西環(huán)素(DOX)處理H929或BMMSCs后的細(xì)胞增殖活性抑制作用,篩選出適宜的多西環(huán)素濃度梯度。然后再用白細(xì)胞介素-1β(IL-1β)或腫瘤壞死因子-1α(TNF-α)預(yù)處理BMMSCs后,利用CCK8法檢測在與BMMSCs共培養(yǎng)條件下,DOX對(duì)骨髓瘤細(xì)胞株H929的增值抑制作用,隨后與對(duì)照組的數(shù)據(jù)進(jìn)行比較,并進(jìn)行統(tǒng)計(jì)學(xué)分析。2.應(yīng)用實(shí)時(shí)熒光定量PCR(RT-PCR)檢測IL-1β或TNF-α處理后,對(duì)BMMSCs的VCAM-1與BAFF基因m RNA的表達(dá)影響。分為1)對(duì)照組a:無處理BMMSCs。2)實(shí)驗(yàn)組b:BMMSCs用IL-1β處理1天(24h)。3)實(shí)驗(yàn)組c:BMMSCs用TNF-α處理1天(24h)。用Real-Time PCR方法檢測各組基因的變化,并進(jìn)行統(tǒng)計(jì)學(xué)分析。3.首先,利用流式細(xì)胞術(shù)(Flow cytometry,FCM)分別檢測在予炎癥因子IL-1β或TNF-α處理后,對(duì)BMMSCs表面的VCAM-1的表達(dá)的影響。然后,先將BMMSCs予IL-1β或TNF-α預(yù)處理后,再用FCM方法檢測H929細(xì)胞與BMMSCs共培養(yǎng)條件下,不同DOX濃度下H929凋亡的變化。隨后與對(duì)照組的數(shù)據(jù)進(jìn)行比較,并進(jìn)行統(tǒng)計(jì)學(xué)分析。4.先將BMMSCs予IL-1β或TNF-α預(yù)處理后,采用Western Blot方法檢測H929細(xì)胞與BMMSCs共培養(yǎng)時(shí),不同濃度多西環(huán)素條件下,H929細(xì)胞p-Erk1/2的表達(dá)的情況,隨后與對(duì)照組的數(shù)據(jù)進(jìn)行比較,并進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果:1.置于顯微鏡下觀察,不論是否有IL-1β或TNF-α處理,BMMSCs的生長方式均是貼壁生長,細(xì)胞形態(tài)大致主要為長梭形狀。不論是否有IL-1β或TNF-α處理,各組的BMMSCs形態(tài)無明顯的差異。與H929細(xì)胞株共培養(yǎng)后,BMMSCs的細(xì)胞形態(tài)和生長特點(diǎn)與單獨(dú)培養(yǎng)時(shí)比較,也無明顯形態(tài)學(xué)改變。2.根據(jù)DOX對(duì)H929細(xì)胞的增殖抑制曲線,篩選出5和10μg/m L的DOX濃度作為下一步的體外實(shí)驗(yàn)檢測濃度。在5和10μg/m L的DOX濃度下,對(duì)BMMSCs的增殖沒有明顯影響(P0.05)。不論是在5或10μg/m L的DOX濃度下,H929與BMMSCs共培養(yǎng)后,與對(duì)照組比較,經(jīng)過IL-1β或TNF-α預(yù)處理的BMMSCs,可以減少DOX對(duì)H929細(xì)胞的體外增殖抑制(均P0.05)。3.不論是經(jīng)炎癥因子IL-1β還是TNF-α處理后,炎癥因子處理組與對(duì)照組比較,RT-PCR檢測提示BMMSCs的VCAM-1的m RNA表達(dá)水平上調(diào)且具有統(tǒng)計(jì)學(xué)意義(P0.05);但是經(jīng)IL-1β或TNF-α處理后對(duì)BMMSCs的BAFF的m RNA表達(dá)沒有明顯變化(P0.05)。4.用IL-1β或TNF-α處理BMMSCs后,FCM檢測提示表達(dá)VCAM-1的細(xì)胞比例較對(duì)照組明顯增多(P0.05)。在IL-1β和TNF-α處理組比較中,發(fā)現(xiàn)TNF-α組比IL-1β組的VCAM-1細(xì)胞陽性率更高(P0.05)。在5μg/ml的DOX濃度下,與無共培養(yǎng)的對(duì)照組相比,H929與BMMSCs共培養(yǎng)條件下,可以降低H929細(xì)胞凋亡率(P0.05);但在10μg/ml的DOX濃度下,共培養(yǎng)組與對(duì)照組間的H929細(xì)胞的凋亡率沒有明顯差異(P0.05)。隨后在BMMSCs經(jīng)IL-1β或TNF-α預(yù)處理的情況下,H929與BMMSCs在5或10μg/ml DOX下共培養(yǎng)2天后,經(jīng)炎癥因子預(yù)處理組中的H929的凋亡率減少(P0.05)。且TNF-α組比IL-1β組的降低H929細(xì)胞凋亡率的作用更顯著(P0.05)。5.首先,DOX處理后可使H929細(xì)胞的p-Erk1/2表達(dá),經(jīng)Western Blot方法檢測后發(fā)現(xiàn)其水平下降,在實(shí)驗(yàn)濃度下不具有劑量依賴性。其次,H929與BMMSCs共培養(yǎng)時(shí),經(jīng)DOX處理后H929的p-Erk1/2水平,較無共培養(yǎng)的對(duì)照組有所上調(diào)(P0.05)。最后,在與經(jīng)IL-1β或TNF-α預(yù)處理的BMMSCs共培養(yǎng)條件下,DOX處理后H929的p-Erk1/2水平,較無炎癥因子處理的對(duì)照也有所上調(diào)(均P0.05)。結(jié)論:我們的體外研究結(jié)果提示,在經(jīng)炎癥因子如IL-1β和TNF-α作用BMMSCs以后,刺激后的BMMSCs與MM細(xì)胞株相互作用中,可以減少DOX引起H929細(xì)胞的增殖抑制和凋亡,增加MM細(xì)胞株對(duì)DOX的耐藥。炎癥因子這一作用可能與其對(duì)BMMSCs的Mek/Erk信號(hào)通路、細(xì)胞表面黏附分子VCAM-1有關(guān)。
[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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.3
本文編號(hào)：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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.3
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