【作者】 吴泽霖；

【导师】 陈福雄；

【作者基本信息】 广州医学院 ， 儿科学， 2010， 硕士

【摘要】 骨髓间充质干细胞（bone marrow messenchymal stem cells, MSCs）是骨髓中除了造血干细胞外的另一种成体干细胞。MSCs不仅在体外可培养无限扩增而不丧失其多向分化潜能[1],并且具有支持造血及免疫调节的功能[2],被越来越多地应用于血液肿瘤等恶性疾病的治疗,同时在再生医学及组织工程中也有广泛应用前景。目前应用于临床的或者进行临床试验的骨髓MSCs包括异基因骨髓MSCs及自体骨髓MSCs。然而,研究发现异基因骨髓MSCs归巢困难[3],而自体的MSCs可有效归巢且自体的MSCs安全性高[4],经自体MSCs输注感染传染病的机会少,因此人们也把更多的注意力放在自体的骨髓MSCs身上。对于白血病儿童来说,临床应用的自体MSCs都是在白血病状态下采集,这种异常状态下的骨髓MSCs回输是否安全呢?吴丽萍等人的研究结果表明[5],白血病患儿的骨髓MSCs在生物学特性方面与其他来源的骨髓MSCs无明显差异。但是白血病儿童骨髓MSCs在分子水平及功能学上是否有改变是值得深入研究的。尤其是在功能学上的研究不可忽视,例如:白血病儿童骨髓MSCs是否会有恶性选择及促进白血病复发的作用呢?是否会减弱NK、T细胞等免疫因子的GVL效应及清除MRD的能力而增加白血病的复发率?这些疑问的解决是白血病儿童自体骨髓MSCs安全应用的前提。NK细胞作为机体天然免疫的重要组成部分,是机体抗肿瘤、抗感染的第一道天然防线。随着对NK细胞生物功能及活化机制的了解,NK细胞在造血干细胞/骨髓移植的辅助治疗及肿瘤过继性免疫治疗方面的作用倍受关注。然而,原始来源的NK细胞含量少（外周血约占淋巴细胞的5%-7%）,且目前尚缺乏有效的体外扩增体系,所以NK细胞的广泛临床应用受到了限制。因此,探索NK细胞的体外高效扩增体系具有重要意义。目前研究认为,MSCs细胞具有免疫调节功能;有研究[6-8]发现MSCs细胞能够抑制异体抗原或丝裂原刺激的T淋巴细胞的增殖。但MSCs对T细胞的这种免疫抑制作用机制仍然未明。NK细胞是固有免疫的主要效应细胞,在抗肿瘤抗病毒感染中也同T细胞一样发挥着举足轻重的作用。目前研究认为MSCs对异体抗原或丝裂原刺激的T淋巴细胞起到免疫抑制的作用,那么,MSCs对NK细胞是否也有相似的影响呢?如果不是,MSCs对NK细胞的作用又如何呢?目前这方面的研究相关文献的报道不多,尤其是有关白血病儿童骨髓MSCs更鲜有报道。因此,带着这些疑问,我们在本课题组之前对白血病儿童骨髓MSCs细胞的生物学特性初步研究的基础上,分离纯化扩增脐血NK细胞,并采用NOD/SCID小鼠建立白血病模型,对白血病儿童骨髓MSCs与NK细胞的相互作用及对白血病细胞K562的影响进行体内外的初步研究,为急性白血病儿童骨髓MSCs及脐血NK细胞在白血病免疫治疗中应用的后续研究做前哨性的探索。目的:探讨从人脐血中分选及扩增高纯度NK细胞的优化技术、扩增前后其功能的变化;以及在本课题组前期研究急性白血病儿童骨髓MSCs的生物学特性的基础上,探讨急性白血病儿童骨髓MSCs与脐血NK细胞在体外的可能相互作用。同时采用NOD/SCID小鼠建立荷K562白血病模型,探索急性白血病儿童骨髓MSCs在体内对K562肿瘤细胞生长的影响及对NK细胞抗瘤作用的影响。本研究旨在为儿童白血病综合治疗中应用自体MSCs或者联合应用NK细胞清除MRD的潜在应用价值做基础性的探索。方法:先用miniMACS及阳性免疫磁珠分选方法,从脐血单个核细胞（CB-MNCs）得到纯化的NK细胞,随后通过IL-2、IL-12和IL-15三个细胞因子的不同组合将培养体系分为IL-2组、IL-2+IL-l2组、IL-2+IL-l5组、IL-12+IL-l5组、IL-2+IL-l5+Il-l2组及对照组（不加任何细胞因子）,分别培养15天,每3天半量换液并补充细胞因子;检测分选及扩增前后CD3^-CD56⁺+16⁺NK细胞含量、扩增倍数及扩增前后各组NK细胞对K562杀伤率的变化。参照吴丽萍等人[5]的方法,通过Ficoll-Hypaque梯度密度离心法结合贴壁培养法体外纯化扩增急性白血病儿童骨髓MSCs,并进行形态学、免疫表型及多向分化能力的鉴定;取P₃细胞用于后续研究。随后按照不同的MSCs︰NK比例,采用直接接触共培养及Transwell培养两种体系进行急性白血病儿童骨髓MSCs与脐血NK细胞的相互作用体外研究。出于偶然的发现,其中在MSCs︰NK比例相同的条件下,均设置了对应的IL2预先处理MSCs的实验组。两者相互作用7天后的检测指标有:细胞形态、NK细胞增殖率、对K562细胞的杀伤活性及细胞因子分泌水平,检测方法主要采用CCK-8试剂盒及ELISA。最后,采用NOD/SCID小鼠,先腹腔注射环磷酰胺预处理24小时后,尾静脉输注K562细胞或同时K562细胞与MSCs联合输注建立白血病模型;2周左右白血病小鼠出现症状后给予尾静脉输注NK细胞或者联合输注MSCs与NK细胞;从生存期分析、病理检查等方面研究急性白血病儿童骨髓MSCs在实验动物体内对K562肿瘤细胞生长的影响及对脐血NK细胞抗瘤作用的影响。结果:纯化前,流式细胞术检测CB-MNC中CD3-CD56++16+细胞含量为14.85±9.1,免疫磁珠纯化后,CD3-CD56++16+细胞含量为92.1±1.1;脐血中的NK细胞含量由纯化前的14.85±9.1%提高到92.1±1.1%,每份标本分离得到的NK细胞纯度均>90%。扩增培养期内台盼蓝染色示细胞存活率均为95%以上。培养前5天,各组细胞增殖均较缓慢;最快增殖时间出现在培养第2周,即第7-15天,此阶段各组培养条件下NK细胞均较培养初期显著扩增。培养至第15天IL-2组、IL-2+IL-12组、IL-2+IL-15组、IL-15+IL-l2组、IL-2+IL-15+IL-l2组NK细胞的扩增倍数分别为15.51±1.09,24.01±3.81,51.45±4.36,20.01±3.88及53.34±6.76,均显著高于对照组的1.64±1.0（p﹤0.01）,IL-2+IL-15组、IL-2+IL-15+IL-l2组细胞的扩增倍数显著高于IL-2组、IL-15+IL-l2组及IL-2+IL-12组（p﹤0.01）。其中对照组NK细胞在培养第15天细胞数较前下降,显微镜下观察可见细胞状态差,死亡细胞增多。各细胞因子组NK细胞杀伤活性均较扩增前明显增强（p<0.01）,NK细胞对K562杀伤活性呈IL-2+IL-15组>IL-2+IL-12组>IL-15+IL-l2组>IL-2组,且IL-2+IL-15组与IL-2+IL-15+IL-l2组差异无统计学意义（p>0.05）。。随着效靶比从1︰1、5︰1到10︰1,各组NK细胞对K562细胞的杀伤率均逐步提高（p<0.01）。培养的白血病儿童BM-MSCs在光镜下观察,呈梭形,平行排列漩涡状生长;不表达造血细胞相关抗原CD34、CD45,而CD29、CD105及CD13则阳性表达;在相应的诱导体系下,该MSCs可以向脂肪及骨细胞分化,油红O染色及西素红染色阳性。MSCs与NK细胞共培养的实验结果显示,MSCs抑制NK细胞的增殖,其抑制作用呈剂量依赖性,但IL2预先刺激24小时的MSCs细胞对NK细胞增殖的抑制作用明显减低,当MSCs︰NK比例为1︰100时,这种抑制作用基本消失;在直接相互接触共培养及Transwell培养体系中均观察到这种相同现象,但Transwell培养体系的抑制作用明显减低,这提示MSCs对NK细胞增殖的抑制作用可能需要MSCs与NK细胞的相互直接接触而实现。与IL-2培养体系单独培养的NK细胞相比,与MSCs共培养的NK细胞的杀伤活性受到了抑制,但是在对应的IL-2预先处理的MSCs共培养组,这种抑制作用明显减弱。实验显示,MSCs对NK细胞杀伤活性的抑制作用呈剂量相关。直接相互接触共培养与Transwell培养组的结果无明显差异,这在一定程度上提示MSCs对NK细胞杀伤活性的抑制可能是通过可溶性因子介导的。MSCs可以抑制NK细胞分泌IFN-γ、perforin （ p< 0. 05） ,且抑制作用呈剂量依赖性（ p< 0. 05） ,直接相互接触共培养与Transwell培养的结果无明显区别,但是,IL-2预先处理过的MSCs组NK细胞的IFN-γ、perforin分泌水平相对高于未处理组（ p< 0. 05）;由此可以看出,MSCs对免疫细胞的抑制作用是可调控的。利用K562细胞建立的NOD/SCID小鼠白血病模型中,可以看到,输注MSCs的小鼠在体重变化、生长迟缓、生存期及病理改变方面与对照组小鼠比较无差异;输注NK细胞组与未输注NK细胞的对照组相比,上述指标存在不同,差异具有统计学意义;NK细胞联合MSCs输注与单纯输注NK细胞组小鼠相比,上述指标无明显差异。全文结论:1.利用miniMACS免疫磁珠阳性分选可从人脐血中获得高纯度NK细胞;在细胞因子存在的合适培养体系中可进行有效扩增。联合应用IL-2+IL-15两种细胞因子的培养体系,可有效的扩增NK细胞,增强NK细胞的细胞毒活性及细胞因子分泌水平。2.急性白血病儿童骨髓MSCs对脐血NK细胞增殖具有抑制作用,呈剂量依赖性,且细胞间的相互接触是参与这种抑制作用的主要机制之一。3.急性白血病儿童骨髓MSCs对脐血NK细胞细胞毒活性具有抑制作用,呈剂量依赖性,且这种抑制作用主要通过可溶性因子介导。4.急性白血病儿童骨髓MSCs对脐血NK细胞细胞因子分泌具有抑制作用,呈剂量依赖性,且这种抑制作用主要通过可溶性因子介导。5.急性白血病儿童骨髓MSCs对脐血NK细胞的免疫调节作用是可调控的,可受到IL-2或其它因素的影响而改变。6.采用NOD/SCID小鼠,环磷酰胺2mg/只腹腔注射预处理后尾静脉移植K562肿瘤细胞可成功建立白血病动物模型。7.急性白血病儿童骨髓MSCs与K562肿瘤细胞共移植至NOD/SCID小鼠,其在体内未发现有促进肿瘤细胞生长的现象。8.急性白血病儿童骨髓MSCs与脐血NK细胞联合输注至NOD/SCID荷K562白血病小鼠,MSCs在体内没有对脐血NK细胞的抗肿瘤效应有明显的抑制作用。更多还原

【Abstract】 Mesenchymal stem cells (MSCs) constitute a rare population of adult stem cells that can be isolated from a simple bone marrow except hematopioetic stem cell. MSCs are known for their characteristic of being multipotent stem cells and capable of forming bone, cartilage, and other mesenchymal tissues[1]. Moreover, MSCs are a component of the bone marrow stroma that have been shown to support hemopoiesis by providing suitable cytokines and growth factors. Besides their regeneration capability, MSCs possess immunomodulatory functions[2], being able to suppress immune reactions both in vitro and in vivo in a non-major histocompatibility complex (MHC) restricted manner. So the therapeutic potential of bone marrow-derived MSCs has recently been brought into the spotlight of many fields of research, not only in blood cancer but also in regenerative medicine and tissue engineering. Currently, both allogeneic bone marrow MSCs and autologous bone marrow MSCs are used in clinic or clinical trials. However, research has showed that the homing of allogeneic bone marrow MSCs are uncertain[3], but autologous MSCs can homing effectively[4]. Moreover, the utility of autologous MSCs should be more safe clinically because fewer infection would occur. So much attention has been put into the autologous bone marrow MSCs. For children with leukemia, the clinical application of autologous MSCs might be unsafe because some leukemia cells would mess into the MSCs during the collection. Research by Li-Ping Wu et al showed that the biological characteristics of bone marrow MSCs from children with leukemia have no significant difference compared with that from normal children[5]. However, it is worthy of further study to evaluate whether there are changes at the molecular level and in the functional representation for this kind of MSCs, especially in the functional studies. Keys for the clinically safe application of autologous bone marrow MSCs from leukemia children may lies on the answers to questions such as whether bone marrow MSCs from leukemia children would be a vicious choice or whethere it would weaken the GVL effects and the ability for eradication of MRD of NK cells, T cells or other immune effector cells and then increasing relapse rate ultimately.Natural killer (NK) cells are major effector cells of the innate immunity and are generally thought to play a fundamental role in antiviral and antitumor responses. As the developing comprehension to their biological and the mechanisms of activation, transfusion of NK cells is becoming a important means to clear residual tumor cells after chemotherapy and HSCT, therfore, more and more efforts are been focusing on its effects as adoptive immunotherapy. Because, the level of NK cells from original source is low (5%-7% in lymphocytes of peripheral blood, and higher in umbilical cord blood), and currently few effective expansion systems ex vivo can be used, its clinical application are restricted. Therefore, it is of great significance to explore efficient expansion system for NK cells in vitro.One of the functions of MSCs is its immunomodulatary effect. Data from researches have demonstrated that MSCs can inhibit T-cell responses induced by mitogens or allo-antigens[6-8]. But the mechanisms underlying such immunosuppressive activity are only in part understood. Acting as major effector cells of innateimmunity, NK cells, like T cells are known to have strong cytolytic activity against tumor or virus-infected cells. As mentioned above, MSCs can inhibit T-cell responses induced by mitogens or alloantigens. However, whether MSCs have a similar impact on the NK cells is still unclear. If the answer is negative, then what is the corelationship between the 2 kinds of cells? In fact, little information is available regarding the cellular interactions between NK cells and MSCs, especially about BM-MSCs from leukemia children.Therefore, with these questions, on the basis of our precursory study about the biological characteristics of MSCs from leukemia children, in this research we attempt firstly to optimize the expansion system for the purified NK cells from cord blood, then delineate the effect of MSCs on NK cells in vitro, with respect to their proliferation, cytotoxic potential and cytokine secretion during activation and then analyze the underlying mechanisms, thereby extending our comprehension on the immunomodulatory properties of MSCs. Furthermore, NOD/SCID mouse are used to establish leukemia model, we exploit this model to investigate the interaction between MSCs and NK cells and their impact on the mouse leukemia model. We hope this research can act as an outpost of exploration for follow-up study of application of immunotherapy relating to CB-NK and leukemia childrens autologous MSCs.Objectives: To investigate optimizing expansion of purified NK cells from cord blood, then delineating the effect of MSCs on NK cells in vitro, with respect to their proliferation, cytotoxic potential and cytokine secretion during activation and then analyzing the underlying mechanisms, thereby extending our knowledge on the immunomodulatory properties of MSCs. Furthermore, NOD/SCID mouse are used to establish leukemia model, to exploit this model to study the interaction between MSCs and NK cells and their impact on the model.Methods: NK cells were isolated from cord blood with miniMACS (magnetic cell-selection) and NK Cell Isolation Kit II. The isolated cells were cultured for 15 days in RPMI-1640 supplemented with 10% FBS and different combinations of IL-2 and IL-12, IL-15. Cultures were fed with flesh media and cytokines every 3 days, and were evaluated for cell expansion, cytotoxicity at the end of the culture period. BM-MSCs from AL children were isolated, cultured and purified in vitro with combination of density gradient centrifugation and adhere segregating culture methods and identified by their morphology characterization, differentiation potential into adipocytes and osteoblasts, phenotype analysis with flow cytometry. MSCs with or without Interleukin-2 (IL-2) were cultured for 5 days with NK cells in 48-well plates or with physically separated NK cells in transwell plates. In above culture system different ratio of NK cells were used. Then the proliferation and cytotoxicty of NK cells were measured with CCK8-kit, the level of IFN-γand Perforin in cultures were dermined by enzyme linked immunosorbant assay (ELISA). Finally, K562 leukemia cells with or without MSCs, were injected into the NOD/SCID mice via lateral tail vein within 24h after the injection of cyclophosphamide in 2mg. Two weeks later, the injected mice begin to showed symptoms of bearing tumor, then NK cells with or without MSCs are injected to the suffering mouse via lateral tail vein to determine whether the tumor was eliminated according to the general situation, survival analysis and pathology tests.Results: The results showed that in group IL-2+IL-l5 and IL2+IL-l5+IL-l2, cells were expanded 50.46±4.31 and 52.35±6.72 fold respectively, much higher than others (p<0.01), but no significant difference between themselves (p>0.05). And the purity of CD3~-CD56~++16~+ NK cells was over 94% in all groups except the control. The cytotoxicity of expanded NK cells cultured with cytokines was significantly higher than those that were not expanded at different E︰T ratio (p<0.01), although the cytotoxicity of IL-2+IL-l5+IL-l2 group was slightly higherthan that of IL-2+IL-l5 group, but no significant difference between themselves (p>0.05). BM-MSCs of acute leukemia children could be sucessfully cultured in vitro, grow in colonys. The phenotypes analysis showed that the MSCs were positive for CD105, CD29 and CD13 but negative for CD34 and CD45. It also could be induced into adipocytes and osteocytes in appropriate conditions. In MSCs-NK co-cultures, compared with those in the control group, at a MSCs︰NK cell ratio of 1︰5, the proliferation of NK cells was strongly inhibited, but in the groups that MSCs stimulated 24h with IL-2 before, such effect is weakened unexpectedly, and if the MSCs︰NK cell ratio was lower than 1︰100, the the effect of inhibition was lossed. In transwell culture system, in which MSCs could not contact with NK cells directly, no inhibitive effect on NK cells’proliferation was showed. Also, it is showed that cytotoxicity of NK cells against K562 are suppressed and the level of IFN-γand Perforin secreted by NK cells are reduced. All of these effects seemed to be in a dose-dependent manner. There was little differce between transwell groups and directed contiguous coculture groups. But in the group of MSCs stimulated 24h with IL-2 before the co-culture, the suppressions were not obsvious. It indicated that the inhibitory effect of MSCs on the immune cells may be reversible and regulatable. Furthermore, In vivo part, the mouse inoculated K562 cells only being compared with the mouse co-infusion of MSCs and K562 cells, no obvious changes in body weight, symptom such as sluggish, survival time and pathological biomarks were observed. According to above observations, compared with the mouse in which non-NK cells were infused, the biomarks in mouse infused with NK cells or NK cells together with MSCs showed significant differences statistically. No differences were showed between mouse infused with NK cells and those infused with NK cells together with MSCs.Conclusions: It is concluded that NK cells can be eficiently expanded in culture with IL-2+IL-l5. And there are differences in the functions of NK cells cultured with different cytokines. IL-2 and IL-15 have synergistic effect on strengthening cytotoxicity of NK cells and promoting cell expansion. We demonstrate that at different NK cells to MSCs ratios, MSCs suppress proliferation, cytokine secretion, and cytotoxicity of NK cells against target K562 cell. Some of these effects require cell-to-cell contact, whereas others are mediated by soluble factors. Our research data suggest that different machanisms lie for different MSCs-mediated NK cell suppressions. And MSCs stimulated by IL-2 are susceptible to lysis by activated NK cells. On the other hand, it indicates that the immunoloregulation of MSCs is reversible and ajustable. Finaly, data of our researches demonstrate that MSCs from children with leukemia exert no effect on engraftment of K562 cell, and does not affect the antitumor effect of NK cells from cord blood in NOD/SCID. Overall, these data improve our knowledge about interactions between MSCs and NK cells and consequently of their effect on innate immune responses and their contribution to the regulation of adaptive immunity, graftrejection, and cancer immunotherapy.更多还原