人脐带间充质干细胞对不同类型肿瘤细胞恶性表型的调控作用研究
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摘要
研究背景和意义
成体干细胞是存在于胎儿和成人不同组织内的多潜能干细胞,其类型众多、获取相对容易,所受伦理争议较少,为肿瘤等人类重大疾病的治疗提供了崭新的手段。间充质干细胞(mesenchymal stem cells, MSCs)是成体干细胞家族的典型代表和重要成员,这一类成体干细胞主要存在于全身结缔组织或器官间质,能够进一步向间质系细胞分化,可用于不同类型组织器官的修复。因此,人们最初对MSCs的关注主要集中在其多向分化能力上。
随着干细胞研究和应用的不断深入,MSCs在肿瘤治疗中的潜在价值也逐渐为人们所认识。研究发现,MSCs不仅可直接抑制某些肿瘤的体内外生长,而且易于接受外源基因修饰,并具有靶向迁移到肿瘤组织、损伤组织、以及慢性炎性反应部位的特性,同时也是一类兼具造血支持作用和免疫抑制功能的低免疫原性细胞,既可被用作实体瘤基因治疗的载体细胞,又能以"MSCs/造血干细胞共移植”的方式实现对血液系统恶性肿瘤的有效治疗。正因为如此,间充质干细胞在肿瘤治疗中的作用才得到了越来越多研究者的认同,并已被用于多种肿瘤的基础和临床治疗研究,有着广阔的应用前景。但值得注意的是,MSCs在输入体内后,除发挥其应有的治疗作用外,还面临与宿主体内的肿瘤细胞发生相互作用的复杂处境。因此,MSCs在肿瘤发生发展中的作用以及二者关系的研究已成为细胞治疗领域的重要课题。然而,综合现有的报道来看,MSCs对特定类型肿瘤细胞恶性生物学行为的影响目前尚不明确,相关问题亟待进一步研究。
人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells, hUC-MSCs)是间充质干细胞家族的新生代表,具有组织来源原始、生物性能稳定、增殖分化能力强、采集方式对供者无侵害等优点,是实体瘤和白血病等疾病细胞治疗的理想种子细胞。已有研究将hUC-MSCs作为负载外源基因的载体细胞用于实体瘤动物模型的基因治疗,临床上也开始使用hUC-MSCs来治疗高危难治性血液病患者的急性移植物抗宿主病(acute graft-versus-host disease, aGVHD),并取得了良好的效果。尽管如此,hUC-MSCs在肿瘤治疗中的应用研究目前仍处于探索阶段,人们对于这一类间充质干细胞各方面的认识还相对有限,其本身对肿瘤细胞的直接作用和影响尚待研究。
在上述背景下,本论文首先对人脐带组织中的间充质样细胞进行了原代分离和培养,对其做包括细胞表面标志、增殖分化以及免疫调节能力等在内的MSC性质鉴定,在成功制各hUC-MSCs的基础上,又进一步研究了hUC-MSCs对肺癌、肝癌、白血病等不同类型肿瘤细胞恶性表型的调控作用,探讨了与hUC-MSCs对特定肿瘤细胞作用效应相关的机制和解决方案,以期进一步拓宽人们对人脐带间充质干细胞生物学性质的认识,并为这一类间充质干细胞的临床开发利用提供借鉴,具有理论和实际意义。
研究内容及方法
1.通过组织块贴壁法原代分离人脐带组织细胞,进行间充质干细胞的基本性质鉴定,并考察其是否具有免疫调节功能。具体内容包括：在倒置显微镜下观察所获细胞形态,MTT比色法测定细胞增殖情况,流式细胞术检测细胞免疫表型,对所获细胞向成脂、成骨以及成软骨细胞方向的分化能力进行评价,ELISA法测定hUC-MSCs对健康成年人外周血单个核细胞(peripheral blood mononuclear cells, PBMC)受OKT3刺激后γ-干扰素(interferon-γ, IFN-γ)分泌能力的影响,多色荧光标记流式细胞术检测PBMC中的特定T淋巴细胞亚群在hUC-MSCs作用下的变化。
2.收集人脐带间充质干细胞(hUC-MSCs)培养上清,按不同比例与肿瘤细胞培养基RPMI1640混合,制成hUC-MSCs条件培养基,分别作用于人肺癌细胞株A549和人肝癌细胞株BEL7402。通过流式细胞术测定PI掺入情况,判断hUC-MSCs对以上实体瘤细胞周期分布的影响,同时通过Transwell小室实验测定hUC-MSCs对这两种实体瘤细胞体外迁移能力的影响,并采用AnnexinV/PI双染流式细胞术检测肿瘤细胞在hUC-MSCs作用下的存活情况,利用实时荧光定量PCR检测肿瘤休眠相关基因的转录,通过Western blotting测定EphA5、Bcl-2、Caspase-7、β-Catenin和c-Myc等分子蛋白表达的变化。
3.用亲脂性荧光染料DiD标记hUC-MSCs,同时以另一种亲脂性荧光染料DiO标记实体瘤细胞A549和BEL7402,然后分别进行这两种肿瘤细胞与hUC-MSCs的直接接触培养,光镜和共聚焦显微镜观察共培养细胞的形态和位相关系,流式细胞术确认hUC-MSCs与肿瘤细胞的自发融合。
4.分别通过RT-PCR、免疫荧光以及流式细胞术检测Notch信号通路分子在hUC-MSCs和Jurkat白血病细胞的表达；将人白血病细胞株Jurkat接种于已贴壁的单层hUC-MSCs之上,进行体外共培养；通过抗体中和实验以及外源重组蛋白激活实验,结合CD45-APC/Annexin V-FITC/PI三色荧光标记流式细胞术,判断Jagged1分子在hUC-MSCs介导Jurkat白血病细胞耐药中的作用。
5.构建携带人吲哚胺-2,3-双加氧酶(indoleamine2,3-dioxygenase, IDO)基因cDNA的重组腺病毒穿梭质粒,与骨架质粒共转染HEK293细胞,包装出重组腺病毒并感染hUC-MSCs,获得IDO基因修饰型hUC-MSCs,通过CD45-APC/Annexin V-FITC/PI三色荧光标记流式细胞术,分别检测Jurkat或HL-60白血病细胞与IDO修饰或未修饰的hUC-MSCs共培养后的自发凋亡情况,并利用CFSE稀释法测定各组hUC-MSCs对上述两种白血病细胞分裂增殖能力的影响,综合评价IDO基因修饰策略在纠正hUC-MSCs保护白血病细胞这一不利性质中的作用。
结果
1.利用组织块法可在2-3周内从人脐带组织中分离出贴壁细胞,该方法的成功率高、分离效果稳定。所获细胞易于体外扩增,经多次传代后仍保持稳定的MSCs形态特征,均一地表达基质细胞相关的表面标记CD90、CD73、CD105、 CD44、CD29,不表达造血干细胞的表面标记CD34、人类白细胞共同抗原CD45、单核细胞表面标记CD14以及MHC-Ⅱ类分子HLA-DR,并具有向成脂、成骨和成软骨细胞方向分化的能力,符合间充质干细胞的国际通用鉴定标准。
2. hUC-MSCs具有免疫调节特性,可抑制OKT3刺激下PBMC的IFN-γ分泌,促进了PBMC中调节性T淋巴细胞(regulatory T cells, Treg)的生成,并使CD4+和CD8+T淋巴细胞的比值出现下调。
3. hUC-MSCs条件培养基对肺癌细胞A549和肝癌细胞BEL7402的细胞周期均产生了阻滞作用,A549细胞被阻滞于Go/G1期,BEL7402细胞被阻滞于S期。与此同时,hUC-MSCs条件培养基还抑制了A549和BEL7402实体瘤细胞的体外迁移,并诱导了这两种肿瘤细胞的凋亡。以上结果表明,hUC-MSCs能够以非直接接触的方式阻抑A549和BEL7402肿瘤细胞的恶性生物学行为。
4.在抑制A549和BEL7402实体瘤细胞恶性表型的同时,hUC-MSCs亦促进了肿瘤休眠基因EphA5在这两种实体瘤细胞中的转录。蛋白水平的检测显示,A549和BEL7402细胞均存在Wnt/β-catenin信号通路的过度活化,该通路的关键分子(3-catenin及其下游的原癌基因c-Myc在这两种实体瘤细胞中有高水平的表达,而在hUC-MSCs条件培养基作用下,A549和BEL7402细胞内的β-Catenin和c-Myc、以及抗凋亡蛋白Bcl-2和非活化型凋亡相关蛋白酶Caspase-7的表达均出现了显著下降,说明hUC-MSCs至少可通过抑制上述信号通路分子的表达而实现其抗A549肺癌细胞和BEL7402肝癌细胞的体外作用,同时也提示在hUC-MSCs的培养上清中可能存在Wnt信号通路的抑制因子。
5. hUC-MSCs在与实体瘤细胞A549或BEL7402直接接触培养时,紧密包围于肿瘤克隆团四周和侵润至克隆团内部,使肿瘤克隆团内部结构松动进而收缩脱落,并出现肿瘤细胞与间充质干细胞的相互融合,这种自发的细胞融合可能也是hUC-MSCs抑制A549和BEL7402实体瘤细胞的作用机制之一。
6. hUC-MSCs同样可抑制白血病细胞株Jurkat和HL-60的增殖,但这种增殖抑制并未引起白血病细胞的凋亡。相反,hUC-MSCs促进了与之共培养的Jurkat和HL-60白血病细胞的体外存活,还降低了Jurkat细胞对地塞米松的敏感性,有诱导白血病细胞耐药的作用。
7. hUC-MSCs表达Notch信号通路的配体分子Jagged1和Delta-like4,有进一步活化邻近细胞内Notch信号的能力。人白血病细胞株Jurkat组成性地表达Jagged1及其Notch1受体、以及Notch通路下游靶分子Hes-1,具有Notch通路自激活或相互激活的能力。与hUC-MSCs共培养后,Jurkat细胞的"Notch1/Jagged1'作用对、以及CD28分子的表达均出现上调。在共培养体系中加入Jagged1中和抗体能够减轻hUC-MSCs对药物所致Jurkat白血病细胞凋亡的抑制作用。对于单独培养的Jurkat细胞,Jagged1中和抗体和重组Jagged1蛋白则分别引起了地塞米松作用下Jurkat细胞凋亡率的增加和减少。
8.腺病毒表达系统成功介导了IDO基因和GFP报告基因在hUC-MSCs胞内的表达。经IDO基因修饰的hUC-MSCs失去了保护Jurkat或HL-60白血病细胞的能力,却显示出更强的抑制白血病细胞增殖的能力。
结论
1.采用组织块法能够稳定地从人脐带组织中分离间充质样细胞,所获得的细胞满足间充质干细胞鉴定的基本条件,并具有独特的免疫调节功能,可用于进一步的实验研究。
2.人脐带间充质干细胞(hUC-MSCs)对实体瘤细胞株A549(肺癌)和BEL7402(肝癌)的恶性生物学行为产生了全面的抑制,有诱导实体瘤细胞休眠的作用,有望作为新型的载体细胞用于肺癌和肝癌的生物治疗。
3.Wnt经典信号通路活化水平的下调、以及hUC-MSCs与实体瘤间的自发融合,是hUC-MSCs抑制A549和BEL7402实体瘤细胞的重要机制。
4. hUC-MSCs抑制了白血病细胞株Jurkat和HL-60的增殖,但却不会使这两种白血病细胞发生凋亡,反而会发挥抗白血病凋亡的作用。因此在将hUC-MSCs用于白血病的造血干细胞移植治疗时,应充分考虑并尽量避免hUC-MSCs保护白血病细胞的这一不利属性对白血病治疗的可能影响。
5. hUC-MSCs诱导了急性T淋巴细胞白血病细胞株Jurkat的体外耐药,Notch信号通路的配体之一Jagged1参与了hUC-MSCs对Jurkat细胞耐药的诱导,并与Jurkat细胞自身活性的维持有关,是治疗急性T淋巴细胞白血病的潜在分子靶点。
6.外源IDO基因修饰逆转了hUC-MSCs对白血病细胞Jurkat和HL-60的保护效应,是提高间充质干细胞的临床应用安全性的有效途径。
综上,本文综合研究了人脐带间充质干细胞对肺癌、肝癌实体瘤细胞以及白血病非实体瘤细胞体外生物学行为的直接影响,确认了hUC-MSCs对上述不同类型肿瘤细胞恶性表型的差异性调控作用,为hUC-MSCs在肿瘤治疗中的开发利用提供了有价值的实验参考,同时也建立了一套较为完善的评价体系,为hUC-MSCs与其它类型肿瘤细胞相互作用的体内外研究奠定了基础。
Background/Aims
Adult stem cells are undifferentiated cells that can be easily isolated from infants, children, and adults. They have the ability to divide indefinitely and produce different kinds of cells that maintain the body's tissues and organs. Because the production of adult stem cells does not require the destruction of an embryo, the use of adult stem cells in research and therapy is not ethically controversial, which paves a new way for the treatment of major human diseases such as cancer. As one of the representatives of adult stem cells and ideal therapeutic agents for the cell-based therapies, mesenchymal stem cells (MSCs) have been applied in basic research and clinical treatment of various malignant diseases. These cells are of stromal origin and may differentiate into a variety of mesenchymal lineage cells, which makes them applicable for damaged tissue repair or regenerative medicine. Besides, MSCs are capable of suppressing the growth of certain tumors, migrating into tumors and chronic inflammatory sites, amenable to genetic modification, and possess a profound immunosuppressive activity as well as hematopoiesis-supportive function. Such versatile properties have prompted their applications in the treatment of solid tumors and leukemia as gene delivery vehicle and immunosuppressant, respectively. Correspondingly, there have also emerged a great deal of interest in the direct interaction between MSCs and cancer cells over the past decade. However, the existing data on the functions of MSCs in cancer development remain controversial and further evaluation is greatly needed.
It has been reported that MSCs can be isolated from a great variety of tissues, including bone marrow, umbilical cord, placenta, adipose tissue, etc. Compared with bone marrow-derived MSCs (BM-MSCs), human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have several advantages such as better expandability, painless collection procedures, and lower risk of viral contamination. These advantages enable them to be excellent sources for the hematopoietic and non-hematopoietic cancer treatments. In spite of this, little is known about the actual effects or the direct impact of hUC-MSCs on specific cancer cells.
In the present study, hUC-MSCs were prepared and evaluated for their fundamental MSC characteristics. Specifically, the immunomodulatory properties of hUC-MSCs were also verified. Thereafter, they were used to treat cancer cells of hematopoietic and non-hematopoietic origins, followed by the investigation of their influence on the malignant phenotype of these cancer cells as well as the underlying mechanisms. Overall, the findings in this study enrich the knowledge about umbilical cord-derived MSCs and provide some useful clues for the safe development of MSCs as therapeutic tools.
Methods
Human UC-MSC cultures were established from umbilical cords of healthy donors by plastic adherence method. The resulted cells were further expanded and assessed for their morphology, proliferating ability by MTT assay, immunophenotype by flow cytometry, and triple differentiation potentials. The immunomodulatory abilities of hUC-MSCs were determined by evaluating their impacts on lymphocyte activation and T cell subpopulations.
For the observation of the effects of hUC-MSCs on solid tumor cells, human lung cancer cell line A549and human hepatocarcinoma cell line BEL7402were, respectively, cultured in the mixture of tumor cell culture medium and hUC-MSC supernatant at different proportions. As controls, tumor cells were grew in the mixture of tumor cell culture medium and hUC-MSC growth medium not containing FBS. After cultured for72h, tumor cells per well were collected and detected by flow cytometry for cell cycle distribution and cell apoptosis. Tumor migration ability was assessed by using the Transwell chambers. The transcription levels of tumor dormancy-associated genes including EphA5, AMOT, OPN and CD73in either tumor cells were evaluated by RT-qPCR, and the expression of EphA5, Bcl-2, Caspase-7, β-Catenin and c-Myc at protein level were detected by Western blotting analysis. On the other hand, the co-culture experiments were performed by direct contact of DiD-labeled hUC-MSCs and DiO-labeled A549or BEL7402tumor cells, followed by confocal observation-and flow cytometry assay.
For the observation of the effect of hUC-MSCs on leukemia cells, human T cell leukemia cell line Jurkat or human promyelocytic leukemia cell line HL-60were cultured in direct contact with hUC-MSCs. In some experiments, hUC-MSCs were equipped with human IDO gene via the aid of adenovirus expression system before they were used as stromal cells for Jurkat and HL-60leukemia cells. Spontaneous or drug-induced apoptosis in leukemia cells was detected by flow cytometry after cells were triply stained with CD45-APC/AnnexinV-FITC/PI. Leukemia cell proliferation was examined by CFSE dilution assay. Specifically, expression of Notch pathway-related molecules in hUC-MSCs and Jurkat cell line was confirmed by RT-PCR, immunofluorescence staining and flow cytometry, respectively. Blocking of Jagged1with neutralizing antibody and extra stimulation of Notch signaling by recombinant Jagged1were performed to identify the role of Jagged1in the hUC-MSC-induced drug resistance of Jurkat leukemia cell line.
Results
Fibroblast-like cells were successfully isolated from human umbilical cord tissues using the direct plastic adherence method. The cells formed whirlpool-like arrays when a confluent monolayer had developed. The flow cytometry analysis demonstrated that the hUC-MSCs showed good homogeneity and expressed MSC markers CD73, CD90, CD105, CD44and CD29, but were negative for CD34, CD45, HLA-DR and CD14. The same cells showed multilineage differentiation potential, as assessed by culturing in adipogenic, osteogenic or chondrogenic medium. They were able to suppress the production of IFN-y by OKT3-activated PBMC, promote the differentiation CD4+CD25+CD127low Treg cells and down-regulate the ratio of CD4+T cells versus CD8+T cells.
The hUC-MSCs inhibited in vitro migration of A549and BEL7402tumor cells, arrested these tumor cells in the Go/G1or S phase of the cell cycle, and induced their apoptosis independently of direct cell contact. RT-qPCR analysis indicated that the hUC-MSCs elevated the transcription of EphA5gene in these tumor cells. Western blotting data showed that the expressions of Bcl-2, pro-form Caspase-7, β-Catenin and c-Myc were all downregulated in the tumor cells treated with the hUC-MSC conditioned medium. By direct contact with tumor cells, the hUC-MSCs surrounded and then invaded into tumor cell clones, resulting in the loosening of the tumor clones. Of note, cell fusion between hUC-MSCs and tumor cells occurred spontaneously during co-culture.
By contrast, hUC-MSCs were able to maintain the viability of the Jurkat or HL-60leukemia cells by preventing them from apoptosis. Compared with control cells cultured alone, Jurkat cells that were cultured on the monolayer of hUC-MSCs showed an improved cell morphology and underwent far less apoptosis induced by dexamethasone. It was also observed that forced IDO expression in hUC-MSCs abolished the anti-apoptotic effect of hUC-MSCs on these leukemia cells and enhanced their leukemia-growth-inhibitory effect. Both hUC-MSCs and Jurkat cells expressed Jagged1. Furthermore, co-culture with hUC-MSCs led to a significant upregulation of Jagged1, Notch1and CD28in Jurkat cells, indicating that the Notch1/Jagged1pair may play a role in the autonomous or reciprocal activation of Notch signaling in these leukemia cells. Blocking Jagged1using the neutralizing antibody restored drug-induced apoptosis in the Jurkat cells that were co-cultured with hUC-MSCs, and also increased the drug sensitivity of the Jurkat cells that were cultured alone, while direct incubation with exogenously recombinant Jagged1 produced the same protective effects in Jurkat cells as those induced by hUC-MSCs.
Conclusions
We established a tissue adherence method-based protocol to isolate MSCs from human umbilical cords with stable yields. Both the fundamental MSC characteristics and immunomodulatory properties of these human umbilical cord-derived cells have been confirmed, ensuring their usability in further studies. Our findings revealed that the hUC-MSCs differently influenced cancer cells of hematopoietic and non-hematopoietic origin through multiple mechanisms. They produced contact-independent inhibition on growth, migration and survival of A549and BEL7402tumor cells, showing a potential to induce dormancy of these tumor cells. Tumor dormancy-associated gene EphA5and Wnt/β-Catenin pathway were involved in malignant phenotype suppression on the above mentioned tumor cells induced by hUC-MSC conditioned medium. In addition, the hUC-MSCs restrained the development of tumor colony and the mechanism might include cell fusion between hUC-MSCs and tumor cells. Specifically, we observed a dual impact of hUC-MSCs on leukemia cells. They inhibited the proliferation of Jurkat and HL-60leukemia cells and also prohibited their death. It could be inferred that such proliferation inhibition could confer leukemia cells a better survival because proliferating cells are more vulnerable to apoptotic stimuli. This mechanism would preserve the self-renewal ability of cancer cells and thus sustain the malignant process. The hUC-MSCs also induced drug resistance of Jurkat leukemia cell line. Jaggedl, one of the Notch ligands, contributes to the hUC-MSC-induced survival and the self-maintenance of Jurkat cells, making it a potential target for the treatment of human T cell leukemia. Interestingly, forced IDO expression in hUC-MSCs abolished their anti-apoptotic effect on leukemia cells of T cell origin and non-lymphoid origin, accompanied by the improvement of their abilities to inhibit leukemia cell growth. These results suggested that equipping MSCs with IDO could be one of the reasonable strategies to reverse their cancer-supportive effect unfavorable for clinical applications. Taken together, our data provide new insights into how hUC-MSCs modulate the malignant phenotype of different cancer cells in vitro and advance understanding of the role of MSCs in carcinogenesis. It established a suitable model to evaluate the interactions between hUC-MSCs and other types of cancer cells. Importantly, it shed light on clinical therapeutic trials using hUC-MSCs for lung cancer and hepatocarcinoma suppression, but also cautioned the potential risk when applying them in the context of hematological malignancies.
成体干细胞是存在于胎儿和成人不同组织内的多潜能干细胞,其类型众多、获取相对容易,所受伦理争议较少,为肿瘤等人类重大疾病的治疗提供了崭新的手段。间充质干细胞(mesenchymal stem cells, MSCs)是成体干细胞家族的典型代表和重要成员,这一类成体干细胞主要存在于全身结缔组织或器官间质,能够进一步向间质系细胞分化,可用于不同类型组织器官的修复。因此,人们最初对MSCs的关注主要集中在其多向分化能力上。
随着干细胞研究和应用的不断深入,MSCs在肿瘤治疗中的潜在价值也逐渐为人们所认识。研究发现,MSCs不仅可直接抑制某些肿瘤的体内外生长,而且易于接受外源基因修饰,并具有靶向迁移到肿瘤组织、损伤组织、以及慢性炎性反应部位的特性,同时也是一类兼具造血支持作用和免疫抑制功能的低免疫原性细胞,既可被用作实体瘤基因治疗的载体细胞,又能以"MSCs/造血干细胞共移植”的方式实现对血液系统恶性肿瘤的有效治疗。正因为如此,间充质干细胞在肿瘤治疗中的作用才得到了越来越多研究者的认同,并已被用于多种肿瘤的基础和临床治疗研究,有着广阔的应用前景。但值得注意的是,MSCs在输入体内后,除发挥其应有的治疗作用外,还面临与宿主体内的肿瘤细胞发生相互作用的复杂处境。因此,MSCs在肿瘤发生发展中的作用以及二者关系的研究已成为细胞治疗领域的重要课题。然而,综合现有的报道来看,MSCs对特定类型肿瘤细胞恶性生物学行为的影响目前尚不明确,相关问题亟待进一步研究。
人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells, hUC-MSCs)是间充质干细胞家族的新生代表,具有组织来源原始、生物性能稳定、增殖分化能力强、采集方式对供者无侵害等优点,是实体瘤和白血病等疾病细胞治疗的理想种子细胞。已有研究将hUC-MSCs作为负载外源基因的载体细胞用于实体瘤动物模型的基因治疗,临床上也开始使用hUC-MSCs来治疗高危难治性血液病患者的急性移植物抗宿主病(acute graft-versus-host disease, aGVHD),并取得了良好的效果。尽管如此,hUC-MSCs在肿瘤治疗中的应用研究目前仍处于探索阶段,人们对于这一类间充质干细胞各方面的认识还相对有限,其本身对肿瘤细胞的直接作用和影响尚待研究。
在上述背景下,本论文首先对人脐带组织中的间充质样细胞进行了原代分离和培养,对其做包括细胞表面标志、增殖分化以及免疫调节能力等在内的MSC性质鉴定,在成功制各hUC-MSCs的基础上,又进一步研究了hUC-MSCs对肺癌、肝癌、白血病等不同类型肿瘤细胞恶性表型的调控作用,探讨了与hUC-MSCs对特定肿瘤细胞作用效应相关的机制和解决方案,以期进一步拓宽人们对人脐带间充质干细胞生物学性质的认识,并为这一类间充质干细胞的临床开发利用提供借鉴,具有理论和实际意义。
研究内容及方法
1.通过组织块贴壁法原代分离人脐带组织细胞,进行间充质干细胞的基本性质鉴定,并考察其是否具有免疫调节功能。具体内容包括：在倒置显微镜下观察所获细胞形态,MTT比色法测定细胞增殖情况,流式细胞术检测细胞免疫表型,对所获细胞向成脂、成骨以及成软骨细胞方向的分化能力进行评价,ELISA法测定hUC-MSCs对健康成年人外周血单个核细胞(peripheral blood mononuclear cells, PBMC)受OKT3刺激后γ-干扰素(interferon-γ, IFN-γ)分泌能力的影响,多色荧光标记流式细胞术检测PBMC中的特定T淋巴细胞亚群在hUC-MSCs作用下的变化。
2.收集人脐带间充质干细胞(hUC-MSCs)培养上清,按不同比例与肿瘤细胞培养基RPMI1640混合,制成hUC-MSCs条件培养基,分别作用于人肺癌细胞株A549和人肝癌细胞株BEL7402。通过流式细胞术测定PI掺入情况,判断hUC-MSCs对以上实体瘤细胞周期分布的影响,同时通过Transwell小室实验测定hUC-MSCs对这两种实体瘤细胞体外迁移能力的影响,并采用AnnexinV/PI双染流式细胞术检测肿瘤细胞在hUC-MSCs作用下的存活情况,利用实时荧光定量PCR检测肿瘤休眠相关基因的转录,通过Western blotting测定EphA5、Bcl-2、Caspase-7、β-Catenin和c-Myc等分子蛋白表达的变化。
3.用亲脂性荧光染料DiD标记hUC-MSCs,同时以另一种亲脂性荧光染料DiO标记实体瘤细胞A549和BEL7402,然后分别进行这两种肿瘤细胞与hUC-MSCs的直接接触培养,光镜和共聚焦显微镜观察共培养细胞的形态和位相关系,流式细胞术确认hUC-MSCs与肿瘤细胞的自发融合。
4.分别通过RT-PCR、免疫荧光以及流式细胞术检测Notch信号通路分子在hUC-MSCs和Jurkat白血病细胞的表达；将人白血病细胞株Jurkat接种于已贴壁的单层hUC-MSCs之上,进行体外共培养；通过抗体中和实验以及外源重组蛋白激活实验,结合CD45-APC/Annexin V-FITC/PI三色荧光标记流式细胞术,判断Jagged1分子在hUC-MSCs介导Jurkat白血病细胞耐药中的作用。
5.构建携带人吲哚胺-2,3-双加氧酶(indoleamine2,3-dioxygenase, IDO)基因cDNA的重组腺病毒穿梭质粒,与骨架质粒共转染HEK293细胞,包装出重组腺病毒并感染hUC-MSCs,获得IDO基因修饰型hUC-MSCs,通过CD45-APC/Annexin V-FITC/PI三色荧光标记流式细胞术,分别检测Jurkat或HL-60白血病细胞与IDO修饰或未修饰的hUC-MSCs共培养后的自发凋亡情况,并利用CFSE稀释法测定各组hUC-MSCs对上述两种白血病细胞分裂增殖能力的影响,综合评价IDO基因修饰策略在纠正hUC-MSCs保护白血病细胞这一不利性质中的作用。
结果
1.利用组织块法可在2-3周内从人脐带组织中分离出贴壁细胞,该方法的成功率高、分离效果稳定。所获细胞易于体外扩增,经多次传代后仍保持稳定的MSCs形态特征,均一地表达基质细胞相关的表面标记CD90、CD73、CD105、 CD44、CD29,不表达造血干细胞的表面标记CD34、人类白细胞共同抗原CD45、单核细胞表面标记CD14以及MHC-Ⅱ类分子HLA-DR,并具有向成脂、成骨和成软骨细胞方向分化的能力,符合间充质干细胞的国际通用鉴定标准。
2. hUC-MSCs具有免疫调节特性,可抑制OKT3刺激下PBMC的IFN-γ分泌,促进了PBMC中调节性T淋巴细胞(regulatory T cells, Treg)的生成,并使CD4+和CD8+T淋巴细胞的比值出现下调。
3. hUC-MSCs条件培养基对肺癌细胞A549和肝癌细胞BEL7402的细胞周期均产生了阻滞作用,A549细胞被阻滞于Go/G1期,BEL7402细胞被阻滞于S期。与此同时,hUC-MSCs条件培养基还抑制了A549和BEL7402实体瘤细胞的体外迁移,并诱导了这两种肿瘤细胞的凋亡。以上结果表明,hUC-MSCs能够以非直接接触的方式阻抑A549和BEL7402肿瘤细胞的恶性生物学行为。
4.在抑制A549和BEL7402实体瘤细胞恶性表型的同时,hUC-MSCs亦促进了肿瘤休眠基因EphA5在这两种实体瘤细胞中的转录。蛋白水平的检测显示,A549和BEL7402细胞均存在Wnt/β-catenin信号通路的过度活化,该通路的关键分子(3-catenin及其下游的原癌基因c-Myc在这两种实体瘤细胞中有高水平的表达,而在hUC-MSCs条件培养基作用下,A549和BEL7402细胞内的β-Catenin和c-Myc、以及抗凋亡蛋白Bcl-2和非活化型凋亡相关蛋白酶Caspase-7的表达均出现了显著下降,说明hUC-MSCs至少可通过抑制上述信号通路分子的表达而实现其抗A549肺癌细胞和BEL7402肝癌细胞的体外作用,同时也提示在hUC-MSCs的培养上清中可能存在Wnt信号通路的抑制因子。
5. hUC-MSCs在与实体瘤细胞A549或BEL7402直接接触培养时,紧密包围于肿瘤克隆团四周和侵润至克隆团内部,使肿瘤克隆团内部结构松动进而收缩脱落,并出现肿瘤细胞与间充质干细胞的相互融合,这种自发的细胞融合可能也是hUC-MSCs抑制A549和BEL7402实体瘤细胞的作用机制之一。
6. hUC-MSCs同样可抑制白血病细胞株Jurkat和HL-60的增殖,但这种增殖抑制并未引起白血病细胞的凋亡。相反,hUC-MSCs促进了与之共培养的Jurkat和HL-60白血病细胞的体外存活,还降低了Jurkat细胞对地塞米松的敏感性,有诱导白血病细胞耐药的作用。
7. hUC-MSCs表达Notch信号通路的配体分子Jagged1和Delta-like4,有进一步活化邻近细胞内Notch信号的能力。人白血病细胞株Jurkat组成性地表达Jagged1及其Notch1受体、以及Notch通路下游靶分子Hes-1,具有Notch通路自激活或相互激活的能力。与hUC-MSCs共培养后,Jurkat细胞的"Notch1/Jagged1'作用对、以及CD28分子的表达均出现上调。在共培养体系中加入Jagged1中和抗体能够减轻hUC-MSCs对药物所致Jurkat白血病细胞凋亡的抑制作用。对于单独培养的Jurkat细胞,Jagged1中和抗体和重组Jagged1蛋白则分别引起了地塞米松作用下Jurkat细胞凋亡率的增加和减少。
8.腺病毒表达系统成功介导了IDO基因和GFP报告基因在hUC-MSCs胞内的表达。经IDO基因修饰的hUC-MSCs失去了保护Jurkat或HL-60白血病细胞的能力,却显示出更强的抑制白血病细胞增殖的能力。
结论
1.采用组织块法能够稳定地从人脐带组织中分离间充质样细胞,所获得的细胞满足间充质干细胞鉴定的基本条件,并具有独特的免疫调节功能,可用于进一步的实验研究。
2.人脐带间充质干细胞(hUC-MSCs)对实体瘤细胞株A549(肺癌)和BEL7402(肝癌)的恶性生物学行为产生了全面的抑制,有诱导实体瘤细胞休眠的作用,有望作为新型的载体细胞用于肺癌和肝癌的生物治疗。
3.Wnt经典信号通路活化水平的下调、以及hUC-MSCs与实体瘤间的自发融合,是hUC-MSCs抑制A549和BEL7402实体瘤细胞的重要机制。
4. hUC-MSCs抑制了白血病细胞株Jurkat和HL-60的增殖,但却不会使这两种白血病细胞发生凋亡,反而会发挥抗白血病凋亡的作用。因此在将hUC-MSCs用于白血病的造血干细胞移植治疗时,应充分考虑并尽量避免hUC-MSCs保护白血病细胞的这一不利属性对白血病治疗的可能影响。
5. hUC-MSCs诱导了急性T淋巴细胞白血病细胞株Jurkat的体外耐药,Notch信号通路的配体之一Jagged1参与了hUC-MSCs对Jurkat细胞耐药的诱导,并与Jurkat细胞自身活性的维持有关,是治疗急性T淋巴细胞白血病的潜在分子靶点。
6.外源IDO基因修饰逆转了hUC-MSCs对白血病细胞Jurkat和HL-60的保护效应,是提高间充质干细胞的临床应用安全性的有效途径。
综上,本文综合研究了人脐带间充质干细胞对肺癌、肝癌实体瘤细胞以及白血病非实体瘤细胞体外生物学行为的直接影响,确认了hUC-MSCs对上述不同类型肿瘤细胞恶性表型的差异性调控作用,为hUC-MSCs在肿瘤治疗中的开发利用提供了有价值的实验参考,同时也建立了一套较为完善的评价体系,为hUC-MSCs与其它类型肿瘤细胞相互作用的体内外研究奠定了基础。
Background/Aims
Adult stem cells are undifferentiated cells that can be easily isolated from infants, children, and adults. They have the ability to divide indefinitely and produce different kinds of cells that maintain the body's tissues and organs. Because the production of adult stem cells does not require the destruction of an embryo, the use of adult stem cells in research and therapy is not ethically controversial, which paves a new way for the treatment of major human diseases such as cancer. As one of the representatives of adult stem cells and ideal therapeutic agents for the cell-based therapies, mesenchymal stem cells (MSCs) have been applied in basic research and clinical treatment of various malignant diseases. These cells are of stromal origin and may differentiate into a variety of mesenchymal lineage cells, which makes them applicable for damaged tissue repair or regenerative medicine. Besides, MSCs are capable of suppressing the growth of certain tumors, migrating into tumors and chronic inflammatory sites, amenable to genetic modification, and possess a profound immunosuppressive activity as well as hematopoiesis-supportive function. Such versatile properties have prompted their applications in the treatment of solid tumors and leukemia as gene delivery vehicle and immunosuppressant, respectively. Correspondingly, there have also emerged a great deal of interest in the direct interaction between MSCs and cancer cells over the past decade. However, the existing data on the functions of MSCs in cancer development remain controversial and further evaluation is greatly needed.
It has been reported that MSCs can be isolated from a great variety of tissues, including bone marrow, umbilical cord, placenta, adipose tissue, etc. Compared with bone marrow-derived MSCs (BM-MSCs), human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have several advantages such as better expandability, painless collection procedures, and lower risk of viral contamination. These advantages enable them to be excellent sources for the hematopoietic and non-hematopoietic cancer treatments. In spite of this, little is known about the actual effects or the direct impact of hUC-MSCs on specific cancer cells.
In the present study, hUC-MSCs were prepared and evaluated for their fundamental MSC characteristics. Specifically, the immunomodulatory properties of hUC-MSCs were also verified. Thereafter, they were used to treat cancer cells of hematopoietic and non-hematopoietic origins, followed by the investigation of their influence on the malignant phenotype of these cancer cells as well as the underlying mechanisms. Overall, the findings in this study enrich the knowledge about umbilical cord-derived MSCs and provide some useful clues for the safe development of MSCs as therapeutic tools.
Methods
Human UC-MSC cultures were established from umbilical cords of healthy donors by plastic adherence method. The resulted cells were further expanded and assessed for their morphology, proliferating ability by MTT assay, immunophenotype by flow cytometry, and triple differentiation potentials. The immunomodulatory abilities of hUC-MSCs were determined by evaluating their impacts on lymphocyte activation and T cell subpopulations.
For the observation of the effects of hUC-MSCs on solid tumor cells, human lung cancer cell line A549and human hepatocarcinoma cell line BEL7402were, respectively, cultured in the mixture of tumor cell culture medium and hUC-MSC supernatant at different proportions. As controls, tumor cells were grew in the mixture of tumor cell culture medium and hUC-MSC growth medium not containing FBS. After cultured for72h, tumor cells per well were collected and detected by flow cytometry for cell cycle distribution and cell apoptosis. Tumor migration ability was assessed by using the Transwell chambers. The transcription levels of tumor dormancy-associated genes including EphA5, AMOT, OPN and CD73in either tumor cells were evaluated by RT-qPCR, and the expression of EphA5, Bcl-2, Caspase-7, β-Catenin and c-Myc at protein level were detected by Western blotting analysis. On the other hand, the co-culture experiments were performed by direct contact of DiD-labeled hUC-MSCs and DiO-labeled A549or BEL7402tumor cells, followed by confocal observation-and flow cytometry assay.
For the observation of the effect of hUC-MSCs on leukemia cells, human T cell leukemia cell line Jurkat or human promyelocytic leukemia cell line HL-60were cultured in direct contact with hUC-MSCs. In some experiments, hUC-MSCs were equipped with human IDO gene via the aid of adenovirus expression system before they were used as stromal cells for Jurkat and HL-60leukemia cells. Spontaneous or drug-induced apoptosis in leukemia cells was detected by flow cytometry after cells were triply stained with CD45-APC/AnnexinV-FITC/PI. Leukemia cell proliferation was examined by CFSE dilution assay. Specifically, expression of Notch pathway-related molecules in hUC-MSCs and Jurkat cell line was confirmed by RT-PCR, immunofluorescence staining and flow cytometry, respectively. Blocking of Jagged1with neutralizing antibody and extra stimulation of Notch signaling by recombinant Jagged1were performed to identify the role of Jagged1in the hUC-MSC-induced drug resistance of Jurkat leukemia cell line.
Results
Fibroblast-like cells were successfully isolated from human umbilical cord tissues using the direct plastic adherence method. The cells formed whirlpool-like arrays when a confluent monolayer had developed. The flow cytometry analysis demonstrated that the hUC-MSCs showed good homogeneity and expressed MSC markers CD73, CD90, CD105, CD44and CD29, but were negative for CD34, CD45, HLA-DR and CD14. The same cells showed multilineage differentiation potential, as assessed by culturing in adipogenic, osteogenic or chondrogenic medium. They were able to suppress the production of IFN-y by OKT3-activated PBMC, promote the differentiation CD4+CD25+CD127low Treg cells and down-regulate the ratio of CD4+T cells versus CD8+T cells.
The hUC-MSCs inhibited in vitro migration of A549and BEL7402tumor cells, arrested these tumor cells in the Go/G1or S phase of the cell cycle, and induced their apoptosis independently of direct cell contact. RT-qPCR analysis indicated that the hUC-MSCs elevated the transcription of EphA5gene in these tumor cells. Western blotting data showed that the expressions of Bcl-2, pro-form Caspase-7, β-Catenin and c-Myc were all downregulated in the tumor cells treated with the hUC-MSC conditioned medium. By direct contact with tumor cells, the hUC-MSCs surrounded and then invaded into tumor cell clones, resulting in the loosening of the tumor clones. Of note, cell fusion between hUC-MSCs and tumor cells occurred spontaneously during co-culture.
By contrast, hUC-MSCs were able to maintain the viability of the Jurkat or HL-60leukemia cells by preventing them from apoptosis. Compared with control cells cultured alone, Jurkat cells that were cultured on the monolayer of hUC-MSCs showed an improved cell morphology and underwent far less apoptosis induced by dexamethasone. It was also observed that forced IDO expression in hUC-MSCs abolished the anti-apoptotic effect of hUC-MSCs on these leukemia cells and enhanced their leukemia-growth-inhibitory effect. Both hUC-MSCs and Jurkat cells expressed Jagged1. Furthermore, co-culture with hUC-MSCs led to a significant upregulation of Jagged1, Notch1and CD28in Jurkat cells, indicating that the Notch1/Jagged1pair may play a role in the autonomous or reciprocal activation of Notch signaling in these leukemia cells. Blocking Jagged1using the neutralizing antibody restored drug-induced apoptosis in the Jurkat cells that were co-cultured with hUC-MSCs, and also increased the drug sensitivity of the Jurkat cells that were cultured alone, while direct incubation with exogenously recombinant Jagged1 produced the same protective effects in Jurkat cells as those induced by hUC-MSCs.
Conclusions
We established a tissue adherence method-based protocol to isolate MSCs from human umbilical cords with stable yields. Both the fundamental MSC characteristics and immunomodulatory properties of these human umbilical cord-derived cells have been confirmed, ensuring their usability in further studies. Our findings revealed that the hUC-MSCs differently influenced cancer cells of hematopoietic and non-hematopoietic origin through multiple mechanisms. They produced contact-independent inhibition on growth, migration and survival of A549and BEL7402tumor cells, showing a potential to induce dormancy of these tumor cells. Tumor dormancy-associated gene EphA5and Wnt/β-Catenin pathway were involved in malignant phenotype suppression on the above mentioned tumor cells induced by hUC-MSC conditioned medium. In addition, the hUC-MSCs restrained the development of tumor colony and the mechanism might include cell fusion between hUC-MSCs and tumor cells. Specifically, we observed a dual impact of hUC-MSCs on leukemia cells. They inhibited the proliferation of Jurkat and HL-60leukemia cells and also prohibited their death. It could be inferred that such proliferation inhibition could confer leukemia cells a better survival because proliferating cells are more vulnerable to apoptotic stimuli. This mechanism would preserve the self-renewal ability of cancer cells and thus sustain the malignant process. The hUC-MSCs also induced drug resistance of Jurkat leukemia cell line. Jaggedl, one of the Notch ligands, contributes to the hUC-MSC-induced survival and the self-maintenance of Jurkat cells, making it a potential target for the treatment of human T cell leukemia. Interestingly, forced IDO expression in hUC-MSCs abolished their anti-apoptotic effect on leukemia cells of T cell origin and non-lymphoid origin, accompanied by the improvement of their abilities to inhibit leukemia cell growth. These results suggested that equipping MSCs with IDO could be one of the reasonable strategies to reverse their cancer-supportive effect unfavorable for clinical applications. Taken together, our data provide new insights into how hUC-MSCs modulate the malignant phenotype of different cancer cells in vitro and advance understanding of the role of MSCs in carcinogenesis. It established a suitable model to evaluate the interactions between hUC-MSCs and other types of cancer cells. Importantly, it shed light on clinical therapeutic trials using hUC-MSCs for lung cancer and hepatocarcinoma suppression, but also cautioned the potential risk when applying them in the context of hematological malignancies.
引文
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