雷公藤内酯醇、沙利度胺对多发性骨髓瘤细胞株及骨髓间充质干细胞作用的对比研究
发布时间：2019-01-07 08:53
【摘要】：目的观察雷公藤内酯醇、沙利度胺对多发性骨髓瘤细胞株及骨髓间充质干细胞的作用,及对骨髓间充质干细胞细胞因子的表达影响,并进行机制的探讨,为临床应用提供一定的基础。方法1.采用MTT比色法(CCK8)检测多发性骨髓瘤细胞株RPMI8226、ARP-1及骨髓间充质干细胞的增殖活性；2.Annexin V-FITC/PI双染色,检测细胞凋亡,并采用瑞氏染色法观察细胞凋亡形态学的变化；流式细胞术分析细胞周期的改变；3.RT-qPCR检测经雷公藤内酯醇、沙利度胺作用于骨髓间充质干细胞后IL-6、IL-1p及SCF mRNA的表达；4. Western Blot分析经雷公藤内酯醇、沙利度胺作用于骨髓间充质干细胞后NF-κB/P65蛋白质水平表达；5. ELISA酶联免疫吸附实验测定经雷公藤内酯醇、沙利度胺作用于骨髓间充质干细胞后VEGF的表达。结果1.MTT比色法(CCK8)检测结果显示,雷公藤内酯醇能显著抑制RPMI8226、ARP-1的生长,呈浓度依赖,0-8ng/ml的雷公藤内酯醇处理RPMI8226、ARP-1细胞72h后与空白对照组相比,P0.001, IC50分别为1.187+0.08ng/ml、0.457±0.13ng/ml.2.雷公藤内酯醇可诱导RPMI8226及ARP-1细胞凋亡并伴有典型的细胞形态学改变。雷公藤内酯醇阻滞RPMI8226、ARP-1细胞周期于S期。3.经雷公藤内酯醇及沙利度胺处理后的骨髓间充质干细胞经qPCR检测,细胞上清液IL-6,IL-1β,SCF的表达均下降,与空白对照组相比,雷公藤内酯醇组P0.05,沙利度胺组仅IL-6与空白对照组相比P0.05。4.经VWestern-Blot检测经雷公藤内酯醇处理后的骨髓间充质干细胞其NF-κB/P65表达下降,P0.05,沙利度胺组无明显差异,P0.05。5.ELISA检测经雷公藤内酯醇处理后的骨髓间充质干细胞表达的VEGF明显低于沙利度胺组,与空白对照组相比,雷公藤内酯醇组P0.05,有统计学意义。沙利度胺组P0.05,无统计学意义。结论1,TPL可直接抑制MM细胞RPMI8226、ARP-1及BMMSCs的生长,呈浓度依赖性,对MM细胞的增殖抑制作用明显强于BMMSCs; 2, TPL可诱导MM细胞RPMI8226、ARP-1及BMMSCs凋亡,呈浓度依赖性,对MM细胞的诱导凋亡作用明显强于BMMSCs;3,TPL可直接杀伤MM细胞,对BMMSCs可能不具有直接杀伤作用,但可影响其分化、表达；减少炎症介质IL-6、IL-1β、SCF、VEGF的表达,改善骨髓造血微环境,进一步抑制MM细胞增殖。4,TPL可能通过抑制NF-κB/P65活性诱导细胞凋亡,抑制肿瘤细胞增殖；
[Abstract]:Objective to investigate the effects of triptolide and thalidomide on multiple myeloma cell lines and bone marrow mesenchymal stem cells and the expression of bone marrow mesenchymal stem cell factor. To provide a basis for clinical application. Method 1. The proliferative activity of multiple myeloma cell line RPMI8226,ARP-1 and bone marrow mesenchymal stem cells was detected by MTT colorimetry (CCK8). 2.Annexin V-FITC/PI double staining was used to detect apoptosis, and the morphological changes of apoptosis were observed by the method of Rayleigh staining, and the changes of cell cycle were analyzed by flow cytometry. 3.RT-qPCR was used to detect the expression of IL-6,IL-1p and SCF mRNA in bone marrow mesenchymal stem cells treated with triptolide and thalidomide. 4. Western Blot was used to analyze the expression of NF- 魏 B/P65 protein in bone marrow mesenchymal stem cells treated with triptolide and thalidomide. ELISA enzyme-linked immunosorbent assay (Elisa) was used to determine the expression of VEGF in bone marrow mesenchymal stem cells treated with triptolide and thalidomide. Results 1.MTT colorimetric assay (CCK8) showed that triptolide could significantly inhibit the growth of RPMI8226,ARP-1 in a dose-dependent manner. Compared with the control group, triptolide of 0-8ng/ml treated RPMI8226,ARP-1 cells for 72 hours. P0.001, IC50 was 1.187 0.08ng / ml 0.57 卤0.13ng / ml. 2, respectively. Triptolide can induce apoptosis of RPMI8226 and ARP-1 cells with typical morphological changes. Triptolide blocked the cell cycle of RPMI8226,ARP-1 cells in S phase. After treated with triptolide and thalidomide, the expression of IL-6,IL-1 尾 and SCF in the supernatant of bone marrow mesenchymal stem cells was detected by qPCR. Compared with the control group, the expression of IL-6,IL-1 尾 and SCF in the mesenchymal stem cells of triptolide group was significantly lower than that in the control group (P 0.05). Only IL-6 in thalidomide group was significantly higher than that in blank control group (P0.05.4). The expression of NF- 魏 B/P65 in bone marrow mesenchymal stem cells treated with triptolide was detected by VWestern-Blot, but there was no significant difference in P0.05 and thalidomide groups. The expression of VEGF in bone marrow mesenchymal stem cells treated with triptolide by P0.05.5.ELISA was significantly lower than that in thalidomide group (P 0.05). There was no statistical significance in thalidomide group (P 0.05). Conclusion 1 TPL can directly inhibit the growth of RPMI8226,ARP-1 and BMMSCs in MM cells in a concentration-dependent manner. The inhibitory effect of TPL on the proliferation of MM cells is stronger than that of BMMSCs;. 2. TPL could induce RPMI8226,ARP-1 and BMMSCs apoptosis in MM cells in a dose-dependent manner. The apoptotic effect on MM cells was stronger than that on BMMSCs; cells. 3TPL can kill MM cells directly, but it may not have direct killing effect on BMMSCs, but it can affect its differentiation and expression. The expression of IL-6,IL-1 尾 and SCF,VEGF was reduced, the hematopoietic microenvironment of bone marrow was improved, and the proliferation of MM cells was further inhibited. 4TPL may induce apoptosis and inhibit the proliferation of tumor cells by inhibiting the activity of NF- 魏 B/P65.
【学位授予单位】：浙江中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.3
[Abstract]:Objective to investigate the effects of triptolide and thalidomide on multiple myeloma cell lines and bone marrow mesenchymal stem cells and the expression of bone marrow mesenchymal stem cell factor. To provide a basis for clinical application. Method 1. The proliferative activity of multiple myeloma cell line RPMI8226,ARP-1 and bone marrow mesenchymal stem cells was detected by MTT colorimetry (CCK8). 2.Annexin V-FITC/PI double staining was used to detect apoptosis, and the morphological changes of apoptosis were observed by the method of Rayleigh staining, and the changes of cell cycle were analyzed by flow cytometry. 3.RT-qPCR was used to detect the expression of IL-6,IL-1p and SCF mRNA in bone marrow mesenchymal stem cells treated with triptolide and thalidomide. 4. Western Blot was used to analyze the expression of NF- 魏 B/P65 protein in bone marrow mesenchymal stem cells treated with triptolide and thalidomide. ELISA enzyme-linked immunosorbent assay (Elisa) was used to determine the expression of VEGF in bone marrow mesenchymal stem cells treated with triptolide and thalidomide. Results 1.MTT colorimetric assay (CCK8) showed that triptolide could significantly inhibit the growth of RPMI8226,ARP-1 in a dose-dependent manner. Compared with the control group, triptolide of 0-8ng/ml treated RPMI8226,ARP-1 cells for 72 hours. P0.001, IC50 was 1.187 0.08ng / ml 0.57 卤0.13ng / ml. 2, respectively. Triptolide can induce apoptosis of RPMI8226 and ARP-1 cells with typical morphological changes. Triptolide blocked the cell cycle of RPMI8226,ARP-1 cells in S phase. After treated with triptolide and thalidomide, the expression of IL-6,IL-1 尾 and SCF in the supernatant of bone marrow mesenchymal stem cells was detected by qPCR. Compared with the control group, the expression of IL-6,IL-1 尾 and SCF in the mesenchymal stem cells of triptolide group was significantly lower than that in the control group (P 0.05). Only IL-6 in thalidomide group was significantly higher than that in blank control group (P0.05.4). The expression of NF- 魏 B/P65 in bone marrow mesenchymal stem cells treated with triptolide was detected by VWestern-Blot, but there was no significant difference in P0.05 and thalidomide groups. The expression of VEGF in bone marrow mesenchymal stem cells treated with triptolide by P0.05.5.ELISA was significantly lower than that in thalidomide group (P 0.05). There was no statistical significance in thalidomide group (P 0.05). Conclusion 1 TPL can directly inhibit the growth of RPMI8226,ARP-1 and BMMSCs in MM cells in a concentration-dependent manner. The inhibitory effect of TPL on the proliferation of MM cells is stronger than that of BMMSCs;. 2. TPL could induce RPMI8226,ARP-1 and BMMSCs apoptosis in MM cells in a dose-dependent manner. The apoptotic effect on MM cells was stronger than that on BMMSCs; cells. 3TPL can kill MM cells directly, but it may not have direct killing effect on BMMSCs, but it can affect its differentiation and expression. The expression of IL-6,IL-1 尾 and SCF,VEGF was reduced, the hematopoietic microenvironment of bone marrow was improved, and the proliferation of MM cells was further inhibited. 4TPL may induce apoptosis and inhibit the proliferation of tumor cells by inhibiting the activity of NF- 魏 B/P65.
【学位授予单位】：浙江中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.3
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