【摘要】：目的觀察雷公藤內(nèi)酯醇、沙利度胺對(duì)多發(fā)性骨髓瘤細(xì)胞株及骨髓間充質(zhì)干細(xì)胞的作用,及對(duì)骨髓間充質(zhì)干細(xì)胞細(xì)胞因子的表達(dá)影響,并進(jìn)行機(jī)制的探討,為臨床應(yīng)用提供一定的基礎(chǔ)。方法1.采用MTT比色法(CCK8)檢測(cè)多發(fā)性骨髓瘤細(xì)胞株RPMI8226、ARP-1及骨髓間充質(zhì)干細(xì)胞的增殖活性；2.Annexin V-FITC/PI雙染色,檢測(cè)細(xì)胞凋亡,并采用瑞氏染色法觀察細(xì)胞凋亡形態(tài)學(xué)的變化；流式細(xì)胞術(shù)分析細(xì)胞周期的改變；3.RT-qPCR檢測(cè)經(jīng)雷公藤內(nèi)酯醇、沙利度胺作用于骨髓間充質(zhì)干細(xì)胞后IL-6、IL-1p及SCF mRNA的表達(dá)；4. Western Blot分析經(jīng)雷公藤內(nèi)酯醇、沙利度胺作用于骨髓間充質(zhì)干細(xì)胞后NF-κB/P65蛋白質(zhì)水平表達(dá)；5. ELISA酶聯(lián)免疫吸附實(shí)驗(yàn)測(cè)定經(jīng)雷公藤內(nèi)酯醇、沙利度胺作用于骨髓間充質(zhì)干細(xì)胞后VEGF的表達(dá)。結(jié)果1.MTT比色法(CCK8)檢測(cè)結(jié)果顯示,雷公藤內(nèi)酯醇能顯著抑制RPMI8226、ARP-1的生長(zhǎng),呈濃度依賴,0-8ng/ml的雷公藤內(nèi)酯醇處理RPMI8226、ARP-1細(xì)胞72h后與空白對(duì)照組相比,P0.001, IC50分別為1.187+0.08ng/ml、0.457±0.13ng/ml.2.雷公藤內(nèi)酯醇可誘導(dǎo)RPMI8226及ARP-1細(xì)胞凋亡并伴有典型的細(xì)胞形態(tài)學(xué)改變。雷公藤內(nèi)酯醇阻滯RPMI8226、ARP-1細(xì)胞周期于S期。3.經(jīng)雷公藤內(nèi)酯醇及沙利度胺處理后的骨髓間充質(zhì)干細(xì)胞經(jīng)qPCR檢測(cè),細(xì)胞上清液IL-6,IL-1β,SCF的表達(dá)均下降,與空白對(duì)照組相比,雷公藤內(nèi)酯醇組P0.05,沙利度胺組僅IL-6與空白對(duì)照組相比P0.05。4.經(jīng)VWestern-Blot檢測(cè)經(jīng)雷公藤內(nèi)酯醇處理后的骨髓間充質(zhì)干細(xì)胞其NF-κB/P65表達(dá)下降,P0.05,沙利度胺組無(wú)明顯差異,P0.05。5.ELISA檢測(cè)經(jīng)雷公藤內(nèi)酯醇處理后的骨髓間充質(zhì)干細(xì)胞表達(dá)的VEGF明顯低于沙利度胺組,與空白對(duì)照組相比,雷公藤內(nèi)酯醇組P0.05,有統(tǒng)計(jì)學(xué)意義。沙利度胺組P0.05,無(wú)統(tǒng)計(jì)學(xué)意義。結(jié)論1,TPL可直接抑制MM細(xì)胞RPMI8226、ARP-1及BMMSCs的生長(zhǎng),呈濃度依賴性,對(duì)MM細(xì)胞的增殖抑制作用明顯強(qiáng)于BMMSCs; 2, TPL可誘導(dǎo)MM細(xì)胞RPMI8226、ARP-1及BMMSCs凋亡,呈濃度依賴性,對(duì)MM細(xì)胞的誘導(dǎo)凋亡作用明顯強(qiáng)于BMMSCs;3,TPL可直接殺傷MM細(xì)胞,對(duì)BMMSCs可能不具有直接殺傷作用,但可影響其分化、表達(dá)；減少炎癥介質(zhì)IL-6、IL-1β、SCF、VEGF的表達(dá),改善骨髓造血微環(huán)境,進(jìn)一步抑制MM細(xì)胞增殖。4,TPL可能通過(guò)抑制NF-κB/P65活性誘導(dǎo)細(xì)胞凋亡,抑制腫瘤細(xì)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 鹵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.
【學(xué)位授予單位】：浙江中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R733.3

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