【作者】 王欣；

【导师】 王伟；

【作者基本信息】 青岛大学 ， 内科学（血液病）， 2020， 硕士

【摘要】 多发性骨髓瘤（multiple myeloma,MM）是一种起源于B淋巴细胞异常增生,以血液和尿液中产生异常增多的单克隆免疫球蛋白为特征的恶性肿瘤。MM对于大多数的患者来说是一种不可治愈的恶性血液病。MutT homologue-1酶（MutT homolog 1,MTH1）能水解8-oxo-dGTP以及2-OH-dATP防止其参与DNA复制,保护肿瘤细胞不受损伤及衰老。目的:（1）检测MTH1在多发性骨髓瘤患者CD138阴性细胞及CD138阳性细胞中的表达情况。（2）探究MTH1抑制剂TH588对多发性骨髓瘤细胞株U266细胞的形态、增殖、凋亡等细胞生物学特性的影响。（3）初步探究IL-6对MM细胞株MTH1表达水平的影响。方法:（1）收集并分离来自青岛大学附属医院多发性骨髓瘤初治患者的骨髓样本,获得单个核细胞,分选并得到患者CD138阴性细胞及CD138阳性细胞并提取RNA,使用real-time PCR技术分别检测骨髓瘤患者CD138阴性细胞及CD138阳性细胞的Nudix家族中MTH1、NUDT2、NUDT5、NUDT21的表达情况。（2）利用多发性骨髓瘤细胞系U266观察IL-6应用后其对MTH1的表达变化影响。（3）用不同浓度的MTH1抑制剂TH588处理多发性骨髓瘤U266细胞,48小时后在显微镜下观察U266细胞的形态变化,使用DAPI染色观察荧光显微镜下不同浓度TH588作用于多发性骨髓瘤U266细胞48小时后细胞核的形态变化。（4）应用荧光素酶活性检测不同浓度TH588对多发性骨髓瘤细胞系U266细胞增殖的影响。（5）应用流式细胞术检测不同浓度TH588作用于多发性骨髓瘤U266细胞48h后对U266细胞凋亡的影响。结果:（1）多发性骨髓瘤患者CD138阳性细胞MTH1、NUDT2、NUDT5以及NUDT21表达较CD138阴性细胞高,且均有统计学意义（P<0.05）。（2）根据QPCR的结果分析,对照组多发性骨髓瘤细胞系U266的MTH1表达2^-ΔCT值为0.01775 0.0008,25ng/ml组的2^-ΔCT值为0.04370 0.0016;100ng/ml组2^-ΔCT值为0.02596 0.0008。与对照组相比,应用IL-6的U266细胞MTH1表达水平均不同程度提高,且有统计学意义（P<0.01）。（3）荧光素酶报告基因结果显示,TH588作用于多发性骨髓瘤U266细胞系48小时后,1μmol/L组与对照组相比,差异无统计学意义;当药物浓度达到2.5μmol/L,抑制增殖作用明显,且差异有统计学差异（P<0.05）,且药物浓度越高,荧光值越低（r=-0.93）;应用72h后,与对照组相比,1μmol/L的药物浓度即有抑制细胞增殖的作用（P<0.05）,随着药物浓度进一步升高,抑制作用愈明显（r=-0.91）。（4）TH588处理U266细胞48h后,显微镜下观察,细胞出现皱缩、变小、形状不规则等表现,DAPI染色后,荧光显微镜下观察,TH588处理过的细胞呈现细胞核缩小、染色不均、花瓣状、放射状等凋亡特征。（5）流式细胞学分析TH588处理U266细胞后,对照组存活细胞比例为81.93%,1μmol/L组存活细胞比例下降到74.60%;2.5μmol/L组存活细胞比例下降到68.53%;5μmol/L组存活细胞降到67.33%;10μmol/L组存活细胞比例下降到65.80%,经统计学分析,差异具有统计学意义（P<0.05）。结论:（1）MTH1在部分多发性骨髓瘤患者的CD138阳性细胞中高表达。（2）MTH1抑制剂TH588对多发性骨髓瘤U266细胞株有明显的抑制增殖、诱导细胞凋亡的作用。（3）IL-6可以使MTH1在多发性骨髓瘤U266细胞中的表达水平提高。更多还原

【Abstract】 Multiple myeloma（MM）is a malignant tumor that originates from abnormal proliferation of B lymphocytes.It is characterized by abnormally increased monoclonal immunoglobulin production in the blood and urine.MM is still an incurable hematological malignancy for most patients.The MutT homologue-1 enzyme（MutT homolog 1,MTH1）can hydrolyze 8-oxo-dGTP and 2-OH-dATP and prevent them from participating in DNA replication,and protect tumor cells from damage and aging.Objective:（1）To detect the expression of MTH1（Mut T homolog 1,MTH1）in CD138 negative cells and CD138 positive cells in patients with multiple myeloma（multiple,myeloma,MM）.（2）To explore the effect of MTH1 inhibitor TH588 on the morphology,proliferation and apoptosis of multiple myeloma cell line U266 cells.（3）To explore the effect of IL-6 on the expression level of MTH1 of MM cell line initially.Methods:（1）Obtain and separate bone marrow samples from newly diagnosed patients with multiple myeloma,obtain mononuclear cells,sort and obtain patient CD138negative cells and CD138 positive cells and extract RNA.Using real-time PCR technology to detect the expression of Nudix family members MTH1,NUDT2,NUDT5and NUDT21 of CD138 negative cells and CD138 positive cells in myeloma patients.（2）Observe the effect of IL-6 on the change of MTH1 expression after IL-6 application in multiple myeloma cell line U266.（3）Multiple myeloma U266 cells were treated with different concentrations of MTH1 inhibitor TH588.After 48 hours,the morphological changes of U266 cells were observed under the microscope.DAPI staining was used to observe the morphology changes of the nucleus under the different concentrations of TH588 on multiple myeloma U266 cells after 48 hours.（4）Luciferase activity was used to detect the effect of different concentrations of TH588 on the proliferation of multiple myeloma cell line U266 cells.（5）Flow cytometry was used to detect the effect on apoptosis treating with different concentrations of TH588 on multiple myeloma U266cells after 48h.Results:（1）The expression of MTH1,NUDT2,NUDT5 and NUDT21 in CD138-positive cells of patients with multiple myeloma was higher than that of CD138-negative cells,and all had statistical significance（P<0.05）.（2）According to the analysis of QPCR results,2^-ΔCT value of the MTH1 expression of multiple myeloma cell line U266 without IL-6 applied is 0.01775±0.0008,when the concentration of IL-6 is25ng/ml,2^-ΔCTΔCT value is 0.04370±0.0016;when the IL-6 concentration is 100ng/ml,2^-ΔCT value is 0.02596±0.0008.Compared with the control group,the expression level of MTH1 in U266 cells using IL-6 is increased to varying degrees,and it has statistically significance（P<0.01）.（3）Luciferase reporter gene results showed that there was no statistically significant difference compared with the control group after 48 hours of TH588 on multiple myeloma U266 cell line when the drug concentration was 1μmol/L.When the drug concentration reached 2.5μmol/L,the proliferation inhibition effect was obvious,and the difference was statistically significant（P<0.05）.The higher the drug concentration,the lower the fluorescence value（r=-0.93）.After 72 hours of application,compared with the control group,the drug concentration of 1μmol/L had the effect ofinhibiting cell proliferation（P<0.05）.As the drug concentration further increased,the inhibitory effect was more obvious（r=-0.91）.（4）After 48h treatment of U266 cells with TH588,under the microscope,the cells showed shrinkage,irregular shape and so on.After DAPI staining,observed under a fluorescent microscope,the cells treated with TH588 showed shrinking nuclei,uneven staining,petal-like nuclei,radial nuclei and other apoptotic features.（5）According to flow cytometry analysis,after treatment of U266 cells with TH588,the proportion of double negative cells in the control group was 81.93%.When the drug concentration was 1μmol/L,the proportion of double negative cellsdecreased to 74.60%.When the drug concentration is 2.5μmol/L,the double negative ratio drops to 68.53%.When the drug concentration is 5μmol/L,the double negative ratio drops to 67.33%.When the drug concentration was 10μmol/L,the double negativeratio dropped to 65.80%.After statistical analysis,the difference was statistically significant（P<0.05）.Conclusion:（1）MTH1 is highly expressed in CD138 positive cells of some patients with multiple myeloma.（2）MTH1 inhibitor TH588 has a significant effect on inhibiting proliferation and inducing apoptosis in multiple myeloma U266 cell line.（3）IL-6 can increase the expression level of MTH1 in multiple myeloma U266 cells.更多还原