發(fā)布時(shí)間：2018-05-24 05:50

  本文選題：白血病干細(xì)胞 + NK-92細(xì)胞�。� 參考：《南方醫(yī)科大學(xué)》2015年碩士論文

【摘要】：背景與目的急性髓系白血病(Acute myeloid leukemia,AML)起源于造血干細(xì)胞,病情兇險(xiǎn),病死率高,易復(fù)發(fā)。其發(fā)病率在中國(guó)為4.17/10萬(wàn),死亡率位居腫瘤第七位,在兒童及35歲以下成人居第一位,是嚴(yán)重危害人類(lèi)身體健康的惡性疾病之一。目前AML的治療主要為化療和異基因造血干細(xì)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,但仍有約23%的患者最終復(fù)發(fā)死亡。此外,合適骨髓的配型、高昂的醫(yī)療費(fèi)用、移植后排斥等間題均阻礙著患者行allo-SCT的選擇,大部分患者最終只能選擇化療。雖然隨著化療方案的不斷改進(jìn),患者的完全緩解率可達(dá)50%～75%,但是仍有15%～25%的患者因藥物抵抗不能達(dá)到完全緩解,超過(guò)60%的患者最終復(fù)發(fā)死亡。Bonnet等研究發(fā)現(xiàn)白血病難治復(fù)發(fā)的根源是白血病干細(xì)胞(Leukemia stem cells, LSCs)。他們首次從AML患者骨髓中分離出CD34+CD38-和CD34+CD38+兩組免疫表型的白血病細(xì)胞亞群,并將這兩組細(xì)胞分別從尾靜脈移植給非肥胖型糖尿�。囟嚷�(lián)合缺陷小鼠伴NK細(xì)胞缺陷(NOD/SCID)小鼠體內(nèi),發(fā)現(xiàn)只有移植CD34+CD38-亞群的小鼠能夠克隆白血病,并且白血病細(xì)胞的形態(tài)和功能狀態(tài)和病人一致,證實(shí)了LSCs的存在。LSCs起源于正常造血干細(xì)胞(Hematopoietic stem cells, HSCs),具有HSCs樣自我更新復(fù)制功能。研究顯示95%的LSCs處于GO期,而常規(guī)的化療藥物僅能殺死增殖期的白血病細(xì)胞,對(duì)靜止?fàn)顟B(tài)下的LSCs缺乏殺傷作用,使LSCs得以逃避殺傷,最終導(dǎo)致白血病復(fù)發(fā),LSCs的存在是白血病復(fù)發(fā)的根源。早期我們的研究也發(fā)現(xiàn)將高表達(dá)CD34+CD38-表型的KGla細(xì)胞移植入NOD/SCID小鼠體內(nèi),可成功克隆出白血病動(dòng)物模型,因此如何徹底殺滅LSCs是治愈AML的關(guān)鍵。自然殺傷細(xì)胞(Nature Killer cell, NK細(xì)胞)是人體重要的天然免疫細(xì)胞,具有廣譜抗腫瘤、抗感染、免疫調(diào)節(jié)等作用。它是人體免疫系統(tǒng)的第一道防線(xiàn),不需預(yù)先致敏,無(wú)MHC限制性,可白發(fā)地殺傷MHC-1類(lèi)分子缺陷或低表達(dá)的腫瘤細(xì)胞,它也是骨髓移植后最先植入的淋巴細(xì)胞(在骨髓移植后前三個(gè)月,占人體外周血淋巴細(xì)胞70%以上),在殺滅白血病干細(xì)胞中發(fā)揮著至關(guān)重要的作用。然而多項(xiàng)研究發(fā)現(xiàn),腫瘤細(xì)胞表面NKG2D配體,如MICA/MICB、 ULBP1、ULBP2、ULBP3等表達(dá)均明顯降低,甚至低于無(wú)法檢測(cè),增強(qiáng)NKG2D配體的表達(dá)可增強(qiáng)NK細(xì)胞的殺傷作用,前期我們的研究也發(fā)現(xiàn)AML患者白血病細(xì)胞表面NKG2D配體MICA/MICB、ULBP1、ULBP2、ULBP3的表達(dá)亦降低,如何增強(qiáng)LSCs細(xì)胞表面NKG2D配體的表達(dá)為治愈AML指明新的方向。地西他濱(decitabine,5-氮雜-2’-脫氧胞苷酸)是一種天然的脫氧胞苷酸的腺苷類(lèi)似物,可替代腫瘤內(nèi)的胞嘧啶與DNA甲基化轉(zhuǎn)移酶共價(jià)結(jié)合,使DNA甲基化轉(zhuǎn)移酶失活,達(dá)到去甲基化的作用,從而抑制增殖、促進(jìn)腫瘤細(xì)胞的凋亡。另外一方面,DNA的去甲基化作用還可抑制腫瘤細(xì)胞表面殺傷抑制性受體(Killer inhibitory receptor,KIR)的合成,使腫瘤細(xì)胞表面殺傷活化性受體(killer cell activatory receptor,KAR)失去KIR的抑制作用,增強(qiáng)NK細(xì)胞對(duì)腫瘤細(xì)胞的殺傷作用,共同發(fā)揮抗腫瘤的作用。此外,有研究還發(fā)現(xiàn),地西他濱具有免疫調(diào)節(jié)作用,可提高腫瘤細(xì)胞表面的NKG2D配體MICA/MICB、ULBP等的表達(dá),提高NK細(xì)胞對(duì)腫瘤細(xì)胞的殺傷作用。由此我們?cè)O(shè)想地西他濱能否通過(guò)增強(qiáng)LSCs表面NKG2D配體的表達(dá)來(lái)增強(qiáng)allo-NK細(xì)胞對(duì)LSCs的殺傷作用呢?本課題在前期研究的基礎(chǔ)上,以CD34+CD38-LSCs為研究對(duì)象,探討地西他濱的免疫調(diào)節(jié)作用,為治療AML提供理論基礎(chǔ)及實(shí)驗(yàn)依據(jù)。方法第一章從KGla細(xì)胞株分選LSCs采用免疫磁珠分選方法從KGla細(xì)胞株分選CD34+CD38-細(xì)胞；流式細(xì)胞術(shù)檢測(cè)分選細(xì)胞的CD34+CD38-LSCs的純度。第二章地西他濱增強(qiáng)NK細(xì)胞系對(duì)LSCs的殺傷作用1)淋巴細(xì)胞分離液分離出外周血單個(gè)核細(xì)胞,Human rIL-2、Human rIL-15誘導(dǎo)NK細(xì)胞形成；CCK-8實(shí)驗(yàn)方法檢測(cè)不同濃度地西他濱對(duì)LSCs的細(xì)胞毒性；LDH法檢測(cè)NK細(xì)胞系(NK-92細(xì)胞及NK細(xì)胞)對(duì)K562細(xì)胞及干預(yù)前后的LSCs在不同效靶比的殺傷作用；流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞系對(duì)K562細(xì)胞及干預(yù)前后的LSCs在效靶比為10：1時(shí)的殺傷作用；2)運(yùn)用SPSS 20.0軟件進(jìn)行數(shù)據(jù)分析,數(shù)值以均數(shù)±標(biāo)準(zhǔn)差(X±s)表示。不同效靶比間NK細(xì)胞系的殺傷活性比較采用析因設(shè)計(jì)方差分析；方差齊時(shí),組間多重比較采用LSD法,方差不齊時(shí),組間多重比較采用Dunnertt T3法；P0.05為差異有統(tǒng)計(jì)學(xué)意義。第三章地西他濱增強(qiáng)NK細(xì)胞殺傷LSCs的機(jī)制1)流式細(xì)胞術(shù)檢測(cè)K562細(xì)胞、干預(yù)前后LSCs表面NKG2D配體(MICA/B、 ULBP1、ULBP2、ULBP3)的表達(dá)；Western-blot檢測(cè)地西他濱對(duì)LSCs線(xiàn)粒體凋亡途徑相關(guān)蛋白的影響；2)運(yùn)用SPSS 20.0軟件進(jìn)行數(shù)據(jù)分析,數(shù)值以均數(shù)±標(biāo)準(zhǔn)差(X±s)表示。K562細(xì)胞與未干預(yù)組LSCs細(xì)胞表面NKG2D配體表達(dá)、地西他濱干預(yù)前后LSCs細(xì)胞表面NKG2D配體表達(dá)、干預(yù)前后凋亡相關(guān)蛋白表達(dá)均采用獨(dú)立樣本t檢驗(yàn)；P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果第一章從KGla細(xì)胞株分選LSCs采用免疫磁珠分選技術(shù)從KGla細(xì)胞株成功分選出CD34+CD38-型LSCs;流式細(xì)胞術(shù)檢測(cè)分選出的LSCs CD34+CD38-抗原的表達(dá)高達(dá)99.95%。第二章地西他濱增強(qiáng)NK細(xì)胞系對(duì)LSCs的殺傷作用1)NK細(xì)胞形態(tài)健康志愿者外周血分離出的PBMC體積較小,細(xì)胞圓亮,經(jīng)Human rIL-2、 Human rIL-15誘導(dǎo)刺激的第二天即開(kāi)始分裂增殖,細(xì)胞生長(zhǎng)較快,鏡下觀察NK細(xì)胞體積較小,圓形透亮,呈團(tuán)簇狀生長(zhǎng)。2) LSCs對(duì)地西他濱的殺傷敏感性CCK-8細(xì)胞毒性結(jié)果顯示,藥物濃度在(0-60 uM)的地西他濱干預(yù)LSCs 24h后,LSCs的活性依然大于60%,表明地西他濱對(duì)LSCs的殺傷作用不敏感,LSCs對(duì)地西他濱抵抗。3)NK細(xì)胞系的殺傷活性LDH法檢測(cè)結(jié)果顯示,NK細(xì)胞系對(duì)K562細(xì)胞殺傷作用敏感。在效靶比為5：1、10：1、20：1時(shí),NK-92細(xì)胞株對(duì)K562細(xì)胞的殺傷率分別為46.00±3.73%、53.58±3.10%、66.51±1.70%,遠(yuǎn)高于相同效靶比下對(duì)LSCs的殺傷率(24.13±1.26%、27.88±2.04%、34.92±4.22%),不同效靶比間兩組細(xì)胞的被殺傷率比較差異有統(tǒng)計(jì)學(xué)意義(F=4.327,P=0.038)；NK細(xì)胞在效靶比為5：1、10：1、20：1時(shí)對(duì)K562細(xì)胞的殺傷率為44.38±2.81%、64.77±3.66%、73.91±3.54%,對(duì)LSCs的殺傷率為22.08±2.07%、28.99±3.13%、36.44±2.40%,兩組細(xì)胞不同效靶比間的被殺傷率比較差異亦有統(tǒng)計(jì)學(xué)意義(F=11.588,P＝0.002),以上提示K562細(xì)胞對(duì)NK細(xì)胞系敏感,LSCs對(duì)NK細(xì)胞系抵抗。而10umol/L地西他濱干預(yù)LSCs后,NK細(xì)胞系對(duì)LSCs的殺傷作用明顯增強(qiáng),在效靶比為5：1、10：1、20：1時(shí),NK-92細(xì)胞對(duì)干預(yù)后的LSCs的殺傷率分別為40.29±1.72%、55.47±1.86%、66.91±2.08%,均高于相同效靶比下對(duì)未干預(yù)組LSCs的殺傷率,兩組細(xì)胞的被殺傷率差異有統(tǒng)計(jì)學(xué)意義(F=13.845, P=0.001); NK細(xì)胞對(duì)干預(yù)后的LSCs的殺傷率為60.52±3.52%、73.93±2.33%、83.08±1.32%,亦高于同一效靶比下NK細(xì)胞對(duì)未干預(yù)組LSCs細(xì)胞的殺傷作用,不同效靶比下兩組細(xì)胞的被殺傷率差異有統(tǒng)計(jì)學(xué)意義(F=4.276,P=0.04)。流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示,在效靶比為10：1時(shí),NK-92細(xì)胞、NK細(xì)胞對(duì)K562細(xì)胞的殺傷率分別為7.33±1.08%、14.65+1.98%,均高于未干預(yù)組LSCs的2.43+0.60%、3.33+0.64%,此結(jié)果與LDH實(shí)驗(yàn)結(jié)果一致,二者組間差異比較有統(tǒng)計(jì)學(xué)意義(t=6.863, P=0.002; t=9.446, P=0.001)。10umol/L地西他濱干預(yù)LSCs后,NK-92細(xì)胞、NK細(xì)胞對(duì)干預(yù)后的LSCs的殺傷率分別為7.85+0.88%、7.84+0.34%,亦高于各自相對(duì)應(yīng)的未干預(yù)組(2.43+0.60%,3.33±0.64%),二者組間比較差異均有統(tǒng)計(jì)學(xué)意義(t=8.864,P=0.001, t=10.821, P=0.000)。第三章地西他濱增強(qiáng)NK細(xì)胞殺傷LSCs的機(jī)制1)K562細(xì)胞及干預(yù)前后LSCs表面NKG2D配體的表達(dá)流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示對(duì)NK細(xì)胞系敏感的K562細(xì)胞表面NKG2D配體MICA/B、ULBP1、ULBP2、ULBP3的表達(dá)分別為2.45±0.92%、16.85±1.78%、1.94+0.90%、4.73+0.58%,均高于LSCs表面NKG2D配體的表達(dá)(0.17±0.07%、0.28±0.10%、1.244±0.12%、0.48±±0.08%),二者組間比較差異有統(tǒng)計(jì)學(xué)意義(P0.05);10 umol/L地西他濱干預(yù)LSCs后,LSCs表面NKG2D配體的表達(dá)增加,分別為2.88±±0.10%、1.26±0.35%、2.16±0.34%、3.37+0.82%,、二者組間比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),表明地西他濱可增強(qiáng)LSCs表面NKG2D配體的表達(dá)。2)地西他濱對(duì)LSCs線(xiàn)粒體凋亡途徑相關(guān)蛋白的影響Western-blot檢測(cè)法結(jié)果顯示,與未干預(yù)組相比,地西他濱可促進(jìn)LSCs線(xiàn)粒體凋亡途徑caspase-9、caspase-3、 PARP的活化,下調(diào)Bcl-2抗凋亡蛋白Bcl-2、Bcl-xl表達(dá),而對(duì)促凋亡蛋白Bax無(wú)影響。結(jié)論1)KGla細(xì)胞株富含CD34+CD38-LSCs,可通過(guò)免疫磁珠分選技術(shù)從中成功分選出純度極高的LSCs用于后續(xù)相關(guān)研究；2) LSCs對(duì)地西他濱抵抗；3) LSCs對(duì)NK細(xì)胞系(NK-92細(xì)胞及NK細(xì)胞)抵抗,地西他濱可增強(qiáng)NK細(xì)胞系對(duì)LSCs的殺傷作用；4)地西他濱可通過(guò)上調(diào)LSCs表面NKG2D配體的表達(dá)增強(qiáng)NK細(xì)胞系對(duì)LSCs的殺傷作用；5)地西他濱可能通過(guò)線(xiàn)粒體凋亡通路上調(diào)NKG2D配體的表達(dá)。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R733.7

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