地西他滨增强NK细胞对白血病干细胞的杀伤作用及其机制研究

发布时间：2018-05-24 05:50

  本文选题：白血病干细胞 + NK-92细胞　； 参考：《南方医科大学》2015年硕士论文

【摘要】：背景与目的急性髓系白血病(Acute myeloid leukemia,AML)起源于造血干细胞,病情凶险,病死率高,易复发。其发病率在中国为4.17/10万,死亡率位居肿瘤第七位,在儿童及35岁以下成人居第一位,是严重危害人类身体健康的恶性疾病之一。目前AML的治疗主要为化疗和异基因造血干细胞移植(Allogeneic stem cell transplantation, allo-SCT)。Allo-SCT是治愈AML的重要手段,它可使大部分患者完全治愈,但仍有约23%的患者最终复发死亡。此外,合适骨髓的配型、高昂的医疗费用、移植后排斥等间题均阻碍着患者行allo-SCT的选择,大部分患者最终只能选择化疗。虽然随着化疗方案的不断改进,患者的完全缓解率可达50%～75%,但是仍有15%～25%的患者因药物抵抗不能达到完全缓解,超过60%的患者最终复发死亡。Bonnet等研究发现白血病难治复发的根源是白血病干细胞(Leukemia stem cells, LSCs)。他们首次从AML患者骨髓中分离出CD34+CD38-和CD34+CD38+两组免疫表型的白血病细胞亚群,并将这两组细胞分别从尾静脉移植给非肥胖型糖尿病／重度联合缺陷小鼠伴NK细胞缺陷(NOD/SCID)小鼠体内,发现只有移植CD34+CD38-亚群的小鼠能够克隆白血病,并且白血病细胞的形态和功能状态和病人一致,证实了LSCs的存在。LSCs起源于正常造血干细胞(Hematopoietic stem cells, HSCs),具有HSCs样自我更新复制功能。研究显示95%的LSCs处于GO期,而常规的化疗药物仅能杀死增殖期的白血病细胞,对静止状态下的LSCs缺乏杀伤作用,使LSCs得以逃避杀伤,最终导致白血病复发,LSCs的存在是白血病复发的根源。早期我们的研究也发现将高表达CD34+CD38-表型的KGla细胞移植入NOD/SCID小鼠体内,可成功克隆出白血病动物模型,因此如何彻底杀灭LSCs是治愈AML的关键。自然杀伤细胞(Nature Killer cell, NK细胞)是人体重要的天然免疫细胞,具有广谱抗肿瘤、抗感染、免疫调节等作用。它是人体免疫系统的第一道防线,不需预先致敏,无MHC限制性,可白发地杀伤MHC-1类分子缺陷或低表达的肿瘤细胞,它也是骨髓移植后最先植入的淋巴细胞(在骨髓移植后前三个月,占人体外周血淋巴细胞70%以上),在杀灭白血病干细胞中发挥着至关重要的作用。然而多项研究发现,肿瘤细胞表面NKG2D配体,如MICA/MICB、 ULBP1、ULBP2、ULBP3等表达均明显降低,甚至低于无法检测,增强NKG2D配体的表达可增强NK细胞的杀伤作用,前期我们的研究也发现AML患者白血病细胞表面NKG2D配体MICA/MICB、ULBP1、ULBP2、ULBP3的表达亦降低,如何增强LSCs细胞表面NKG2D配体的表达为治愈AML指明新的方向。地西他滨(decitabine,5-氮杂-2’-脱氧胞苷酸)是一种天然的脱氧胞苷酸的腺苷类似物,可替代肿瘤内的胞嘧啶与DNA甲基化转移酶共价结合,使DNA甲基化转移酶失活,达到去甲基化的作用,从而抑制增殖、促进肿瘤细胞的凋亡。另外一方面,DNA的去甲基化作用还可抑制肿瘤细胞表面杀伤抑制性受体(Killer inhibitory receptor,KIR)的合成,使肿瘤细胞表面杀伤活化性受体(killer cell activatory receptor,KAR)失去KIR的抑制作用,增强NK细胞对肿瘤细胞的杀伤作用,共同发挥抗肿瘤的作用。此外,有研究还发现,地西他滨具有免疫调节作用,可提高肿瘤细胞表面的NKG2D配体MICA/MICB、ULBP等的表达,提高NK细胞对肿瘤细胞的杀伤作用。由此我们设想地西他滨能否通过增强LSCs表面NKG2D配体的表达来增强allo-NK细胞对LSCs的杀伤作用呢?本课题在前期研究的基础上,以CD34+CD38-LSCs为研究对象,探讨地西他滨的免疫调节作用,为治疗AML提供理论基础及实验依据。方法第一章从KGla细胞株分选LSCs采用免疫磁珠分选方法从KGla细胞株分选CD34+CD38-细胞；流式细胞术检测分选细胞的CD34+CD38-LSCs的纯度。第二章地西他滨增强NK细胞系对LSCs的杀伤作用1)淋巴细胞分离液分离出外周血单个核细胞,Human rIL-2、Human rIL-15诱导NK细胞形成；CCK-8实验方法检测不同浓度地西他滨对LSCs的细胞毒性；LDH法检测NK细胞系(NK-92细胞及NK细胞)对K562细胞及干预前后的LSCs在不同效靶比的杀伤作用；流式细胞术检测NK细胞系对K562细胞及干预前后的LSCs在效靶比为10：1时的杀伤作用；2)运用SPSS 20.0软件进行数据分析,数值以均数±标准差(X±s)表示。不同效靶比间NK细胞系的杀伤活性比较采用析因设计方差分析；方差齐时,组间多重比较采用LSD法,方差不齐时,组间多重比较采用Dunnertt T3法；P0.05为差异有统计学意义。第三章地西他滨增强NK细胞杀伤LSCs的机制1)流式细胞术检测K562细胞、干预前后LSCs表面NKG2D配体(MICA/B、 ULBP1、ULBP2、ULBP3)的表达；Western-blot检测地西他滨对LSCs线粒体凋亡途径相关蛋白的影响；2)运用SPSS 20.0软件进行数据分析,数值以均数±标准差(X±s)表示。K562细胞与未干预组LSCs细胞表面NKG2D配体表达、地西他滨干预前后LSCs细胞表面NKG2D配体表达、干预前后凋亡相关蛋白表达均采用独立样本t检验；P0.05为差异有统计学意义。结果第一章从KGla细胞株分选LSCs采用免疫磁珠分选技术从KGla细胞株成功分选出CD34+CD38-型LSCs;流式细胞术检测分选出的LSCs CD34+CD38-抗原的表达高达99.95%。第二章地西他滨增强NK细胞系对LSCs的杀伤作用1)NK细胞形态健康志愿者外周血分离出的PBMC体积较小,细胞圆亮,经Human rIL-2、 Human rIL-15诱导刺激的第二天即开始分裂增殖,细胞生长较快,镜下观察NK细胞体积较小,圆形透亮,呈团簇状生长。2) LSCs对地西他滨的杀伤敏感性CCK-8细胞毒性结果显示,药物浓度在(0-60 uM)的地西他滨干预LSCs 24h后,LSCs的活性依然大于60%,表明地西他滨对LSCs的杀伤作用不敏感,LSCs对地西他滨抵抗。3)NK细胞系的杀伤活性LDH法检测结果显示,NK细胞系对K562细胞杀伤作用敏感。在效靶比为5：1、10：1、20：1时,NK-92细胞株对K562细胞的杀伤率分别为46.00±3.73%、53.58±3.10%、66.51±1.70%,远高于相同效靶比下对LSCs的杀伤率(24.13±1.26%、27.88±2.04%、34.92±4.22%),不同效靶比间两组细胞的被杀伤率比较差异有统计学意义(F=4.327,P=0.038)；NK细胞在效靶比为5：1、10：1、20：1时对K562细胞的杀伤率为44.38±2.81%、64.77±3.66%、73.91±3.54%,对LSCs的杀伤率为22.08±2.07%、28.99±3.13%、36.44±2.40%,两组细胞不同效靶比间的被杀伤率比较差异亦有统计学意义(F=11.588,P＝0.002),以上提示K562细胞对NK细胞系敏感,LSCs对NK细胞系抵抗。而10umol/L地西他滨干预LSCs后,NK细胞系对LSCs的杀伤作用明显增强,在效靶比为5：1、10：1、20：1时,NK-92细胞对干预后的LSCs的杀伤率分别为40.29±1.72%、55.47±1.86%、66.91±2.08%,均高于相同效靶比下对未干预组LSCs的杀伤率,两组细胞的被杀伤率差异有统计学意义(F=13.845, P=0.001); NK细胞对干预后的LSCs的杀伤率为60.52±3.52%、73.93±2.33%、83.08±1.32%,亦高于同一效靶比下NK细胞对未干预组LSCs细胞的杀伤作用,不同效靶比下两组细胞的被杀伤率差异有统计学意义(F=4.276,P=0.04)。流式细胞术检测结果显示,在效靶比为10：1时,NK-92细胞、NK细胞对K562细胞的杀伤率分别为7.33±1.08%、14.65+1.98%,均高于未干预组LSCs的2.43+0.60%、3.33+0.64%,此结果与LDH实验结果一致,二者组间差异比较有统计学意义(t=6.863, P=0.002; t=9.446, P=0.001)。10umol/L地西他滨干预LSCs后,NK-92细胞、NK细胞对干预后的LSCs的杀伤率分别为7.85+0.88%、7.84+0.34%,亦高于各自相对应的未干预组(2.43+0.60%,3.33±0.64%),二者组间比较差异均有统计学意义(t=8.864,P=0.001, t=10.821, P=0.000)。第三章地西他滨增强NK细胞杀伤LSCs的机制1)K562细胞及干预前后LSCs表面NKG2D配体的表达流式细胞术检测结果显示对NK细胞系敏感的K562细胞表面NKG2D配体MICA/B、ULBP1、ULBP2、ULBP3的表达分别为2.45±0.92%、16.85±1.78%、1.94+0.90%、4.73+0.58%,均高于LSCs表面NKG2D配体的表达(0.17±0.07%、0.28±0.10%、1.244±0.12%、0.48±±0.08%),二者组间比较差异有统计学意义(P0.05);10 umol/L地西他滨干预LSCs后,LSCs表面NKG2D配体的表达增加,分别为2.88±±0.10%、1.26±0.35%、2.16±0.34%、3.37+0.82%,、二者组间比较差异有统计学意义(P0.05),表明地西他滨可增强LSCs表面NKG2D配体的表达。2)地西他滨对LSCs线粒体凋亡途径相关蛋白的影响Western-blot检测法结果显示,与未干预组相比,地西他滨可促进LSCs线粒体凋亡途径caspase-9、caspase-3、 PARP的活化,下调Bcl-2抗凋亡蛋白Bcl-2、Bcl-xl表达,而对促凋亡蛋白Bax无影响。结论1)KGla细胞株富含CD34+CD38-LSCs,可通过免疫磁珠分选技术从中成功分选出纯度极高的LSCs用于后续相关研究；2) LSCs对地西他滨抵抗；3) LSCs对NK细胞系(NK-92细胞及NK细胞)抵抗,地西他滨可增强NK细胞系对LSCs的杀伤作用；4)地西他滨可通过上调LSCs表面NKG2D配体的表达增强NK细胞系对LSCs的杀伤作用；5)地西他滨可能通过线粒体凋亡通路上调NKG2D配体的表达。
[Abstract]:Background and objective Acute myeloid leukemia (AML) originated from hematopoietic stem cells. The disease is dangerous, high mortality and easy to relapse. The incidence of the disease is 4.17/10 million in China, the death rate is the seventh in the tumor, the first in children and under 35 years of age, and is one of the malignant diseases which seriously harm the health of human being. Now AML The treatment is mainly chemotherapy and Allogeneic stem cell transplantation (allo-SCT).Allo-SCT is an important means to cure AML, it can make most patients completely cured, but there are still about 23% of patients eventually relapsed. In addition, appropriate bone marrow matching, high medical costs, and rejection after transplantation. All patients can only choose allo-SCT, most patients can only choose chemotherapy. Although with the continuous improvement of chemotherapy, the total remission rate of patients can reach 50% to 75%, but 15% to 25% of patients are still unable to achieve complete remission due to drug resistance, and more than 60% of patients eventually relapse and die.Bonnet and other studies find leukemia difficult The root cause of recurrence is Leukemia stem cells (LSCs). They first isolated the leukemic subsets of CD34+CD38- and CD34+CD38+ two groups from the bone marrow of AML patients, and transplanted these two groups of cells from the tail vein to non obese diabetes / severe combined deficient mice with NK cell defects (NOD/SCID). Mice were found to be able to clone leukaemia only with the transplanted CD34+CD38- subgroup, and the morphological and functional status of the leukemia cells was consistent with the patients. It was confirmed that the existence of LSCs was derived from the normal hematopoietic stem cells (Hematopoietic stem cells, HSCs), with HSCs like self renewal and replication function. The study showed that 95% of LSCs was in GO. On the other hand, conventional chemotherapeutic drugs only kill the proliferating leukemia cells, lack the killing effect of LSCs in the static state, cause LSCs to escape the killing, and eventually lead to the relapse of leukemia. The existence of LSCs is the root of the relapse of leukaemia. Our early study also found that the KGla cells with high expression of CD34+CD38- phenotype were transplanted into NOD/SCID The mouse model can be successfully cloned, so how to kill LSCs is the key to cure AML. The natural killer cells (Nature Killer cell, NK cells) are the important natural immune cells of the human body. It has broad spectrum anti tumor, anti infection, immune regulation and so on. It is the first line of defense of the human immune system and does not need to be induced in advance. Sensitivity, without MHC restriction, can kill MHC-1 molecular defect or low expression of tumor cells in white hair. It is also the first implantation of lymphocytes after bone marrow transplantation (three months after bone marrow transplantation, accounting for more than 70% of human peripheral blood lymphocytes). It plays a vital role in killing leukemic stem cells. The expression of NKG2D ligand on the surface of the tumor cells, such as MICA/MICB, ULBP1, ULBP2, ULBP3, is obviously reduced, even below the undetectable. The expression of enhanced NKG2D ligand can enhance the killing effect of NK cells. Our study also found that the expression of NKG2D ligand MICA/MICB, ULBP1, ULBP2, and ULBP2, and how to enhance the expression of NKG2D ligand on the surface of AML patients' leukemia cells were also reduced. The expression of NKG2D ligand on the surface of s cells indicates a new direction for the cure of AML. Decitabine, 5- aza -2 '- deoxycytidine acid) is a natural adenosine analogs of deoxycytidine acid, which can replace cytosine in tumor with DNA methyltransferase covalent, make DNA methyltransferase inactivated and achieve demethylation. On the other hand, the demethylation of DNA also inhibits the synthesis of Killer inhibitory receptor (KIR) of tumor cell surface killing receptor (KIR), and causes the tumor cell surface killing activation receptor (killer cell activatory receptor, KAR) to lose the KIR inhibitory effect and strengthen the NK cells. In addition, we also found that the NKG2D ligand, MICA/MICB, ULBP and so on, can increase the killing effect of NK cells on the tumor cells. The expression of ligands to enhance the killing effect of allo-NK cells on LSCs? Based on the previous study, this topic is based on CD34+CD38-LSCs as the research object, to explore the immunomodulatory effect of seashine, to provide theoretical basis and experimental basis for the treatment of AML. Method first chapter from the KGla cell line sorting LSCs using the immunomagnetic beads sorting method from KGla Cell lines were selected to separate CD34+CD38- cells; flow cytometry was used to detect the purity of CD34+CD38-LSCs in the selected cells. Second chapter CD34+CD38-LSCs enhanced the killing effect of NK cell line on LSCs. Lymphocyte separation solution separated out peripheral blood mononuclear cells, Human rIL-2, Human rIL-15 induced NK cell formation; CCK-8 experimental method detected different concentrations in the West. The cytotoxicity of itabine on LSCs; LDH assay to detect the killing effect of NK cell line (NK-92 cell and NK cell) on K562 cells and the LSCs at different target targets before and after intervention; flow cytometry was used to detect the killing effect of NK cell lines on K562 cells and LSCs before and after the target ratio of target to 10:1; 2) data analysis using SPSS 20 software The number of NK cell lines of different target ratio (X + s) was compared by factorial analysis of variance. When the variance was homogeneous, LSD method was used for multiple comparison between groups, and when the variance was not homogeneous, the Dunnertt T3 method was used for multiple comparison between groups. The difference was statistically significant in P0.05. The third chapter was the increase of NK cells to kill L. SCs mechanism 1) flow cytometry was used to detect K562 cells, and the expression of NKG2D ligand (MICA/B, ULBP1, ULBP2, ULBP3) on LSCs surface before and after intervention; Western-blot detected the effect of saitabine on the apoptosis pathway related proteins of LSCs mitochondria; 2) using SPSS 20 software to analyze the data, the numerical value was expressed with the mean number + standard deviation (X +) The expression of NKG2D ligand on the surface of LSCs cells in the intervention group, the expression of NKG2D ligand on the surface of LSCs cells before and after intervention, the expression of apoptosis related proteins before and after intervention were all independent sample t test, and the difference of P0.05 was statistically significant. The first chapter was a successful separation of LSCs from KGla cell lines from the KGla cell line selected from the KGla cell line. CD34+CD38- type LSCs was selected and the expression of LSCs CD34+CD38- antigen was detected by flow cytometry. The expression of LSCs CD34+CD38- antigen was as high as 99.95%., 99.95%., NK cell line and LSCs, and NK cell morphology healthy volunteers were small in PBMC volume, and the cells were round, and Human rIL-2, Human induces stimulation for second days. The cell growth was rapid, and the cell growth was faster. The NK cells were smaller, round and bright and cluster like growth.2). The cytotoxicity of LSCs to the killing sensitivity of LSCs to setiabine showed that the activity of LSCs was still greater than 60% after the concentration of the drug concentration (0-60 uM) of LSCs 24h, indicating the killing effect of setiparin on LSCs. The killing activity of LSCs against.3 NK cell line by LDH assay showed that the NK cell line was sensitive to the killing effect of K562 cells. The killing rate of NK-92 cell lines to K562 cells was 46 + 3.73%, 53.58 + 3.10%, 66.51 + 1.70% at the target target ratio of 5:1,10:1,20:1, which was far higher than the killing of LSCs under the same target ratio. The rate of injury was (24.13 + 1.26%, 27.88 + 2.04%, 34.92 + 4.22%). There was a significant difference in the killing rate of two cells between different target targets (F=4.327, P=0.038). The killing rate of NK cells to K562 cells was 44.38 + 2.81%, 64.77 + 3.66%, 73.91 + 3.54% when the target target ratio was 5:1,10:1,20:1, and the killing rate of LSCs was 27.88. 3%, 36.44 + 2.40%, and there were significant differences in the killing rate of different target ratio between the two groups (F=11.588, P = 0.002), which suggested that K562 cells were sensitive to NK cell lines, LSCs was resistant to NK cell lines. While 10umol/L to LSCs, the killing effect of NK cell lines on LSCs was obviously enhanced and the target ratio was 5:1,10:1,20: At 1, the killing rate of NK-92 cells to LSCs was 40.29 + 1.72%, 55.47 + 1.86%, 66.91 + 2.08%, respectively. The killing rate of LSCs was higher than that under the same target ratio. The killing rate of the two groups was statistically significant (F=13.845, P=0.001), and the killing rate of NK cells to LSCs was 60.52 + 3.52%, 73.93 + 2.33%, 83.08, 83.08. + 1.32%, also higher than the killing effect of NK cells on LSCs cells under the same target ratio, the killing rate of two cells under different target ratios was statistically significant (F=4.276, P=0.04). The results of flow cytometry showed that when the target ratio was 10:1, the killing rate of NK cells to K562 cells was 7.33 + 1.08%, 14.6, respectively, 14.6 5+1.98% was higher than 2.43+0.60%, 3.33+0.64%, and the results were in accordance with the results of LDH experiment. The differences in the two groups were statistically significant (t=6.863, P=0.002; t=9.446, P=0.001).10umol/L. After LSCs, NK-92 cells, respectively, were higher than each other. 2.43+0.60%, 3.33 + 0.64%, the difference between the two groups was statistically significant (t=8.864, P=0.001, t=10.821, P=0.000). Third the mechanism of NK cells killing LSCs by NK cells 1) K562 cells and the flow cytometry of LSCs surface NKG2D ligand before and after intervention showed the sensitivity to NK cell line The expressions of NKG2D ligand MICA/B, ULBP1, ULBP2 and ULBP3 were 2.45 + 0.92%, 16.85 + 1.78%, 1.94+0.90%, 4.73+0.58%, respectively higher than the expression of NKG2D ligand on the LSCs surface (0.17 + 0.07%, 0.28 + 0.10%, 1.244 + 0.12%, 0.48 + 0.08%), and there was a significant difference between the two groups (P0.05). The expression of NKG2D ligand on the surface of s increased, respectively, 2.88 + 0.10%, 1.26 + 0.35%, 2.16 + 0.34%, 3.37+0.82%, and there was a significant difference between the two groups (P0.05), indicating that the NKG2D ligand on the LSCs surface could increase the expression of NKG2D ligand on the LSCs surface. Compared with the unpretreated group, it can promote the activation of LSCs mitochondrial apoptosis pathway caspase-9, Caspase-3, PARP, Bcl-2 anti apoptotic protein Bcl-2, Bcl-xl expression, but no effect on apoptotic protein Bax. Conclusion 1) KGla cell line is rich in CD34+CD38-LSCs, and can be successfully selected from the immunobead sorting technique to separate high purity LSCs. For subsequent related studies; 2) LSCs resistance to DHC; 3) LSCs resistance to NK cell lines (NK-92 cells and NK cells), and dhitribin can enhance the killing effect of NK cell lines on LSCs; 4) to increase the expression of NKG2D ligand on the surface of LSCs to enhance the killing effect of NK cell lines on LSCs; 5) The apoptosis pathway up-regulated the expression of NKG2D ligand.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.7

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3 高冲;陈宝安;刘苒;徐昕;丁家华;王骏;程坚;赵刚;余正平;宋慧慧;鲍文;;地西他滨治疗骨髓增生异常综合征一例报告[J];现代医学;2010年02期

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