【作者】 余水长；

【导师】 夏春林；

【作者基本信息】 苏州大学 ， 人体解剖与组织胚胎学， 2014， 博士

【摘要】 间充质干细胞(mesenchymal stem cells, MSCs)是一种具有自我更新和向中胚层来源细胞分化能力的多能干细胞。MSCs具有低免疫原性，细胞移植无免疫排斥反应，对肿瘤有靶向性和抑瘤性等，使其在肿瘤治疗和损伤修复等研究中成为热点。MSCs的来源取自成人骨髓最为常见，但取材时需要骨髓穿刺，且人骨髓中的MSCs含量极低，细胞数量和扩增、分化能力随年龄增长均显著下降，使其临床应用受到限制。近年来，从胎盘羊膜间质中获取羊膜间充质干细胞（amniotic mesenchymal stem cells,AMSCs）成为MSCs的新来源，具有材料供应丰富、取材方便、操作安全、污染机率少，细胞增殖能力强、扩增速度快等优势，为干细胞移植治疗开辟新的途径。人脑胶质瘤是中枢神经系统中最常见的原发性恶性肿瘤，侵袭性强，发病早期即向周围正常脑组织内呈指状浸润性生长，术后易复发，患者预后差，死亡率高。近年来研究表明，MSCs具有向人脑胶质瘤的特异性定向迁移能力，除了部分迁移到胶质瘤内，主要是呈“胶囊样”分布于脑胶质瘤体与正常脑实质的边界，并有部分细胞能“追踪”瘤体外散在的胶质瘤细胞，可成为胶质瘤基因治疗的理想载体。MSCs本身对于脑胶质瘤具有抑瘤效应。体内外研究表明MSCs可抑制胶质瘤细胞增殖，可能与诱导细胞凋亡、阻碍细胞周期等有关。但胎盘来源的AMSCs对人脑胶质瘤是否具有趋瘤和抑瘤效应，机制如何，有待进一步研究。本实验旨在建立有效的AMSCs体外培养扩增体系，经诱导分化探讨AMSCs的细胞生物学特性；通过体外实验，观察AMSCs定向迁移至胶质瘤病变区域的能力及对肿瘤细胞生长的作用；建立裸鼠人脑胶质瘤模型，瘤内注射AMSCs，观察移植后AMSCs的存活、迁移及对肿瘤生长的作用，初步探讨AMSCs在体内外趋瘤抑瘤效应的可能机制。第一部分AMSCs的分离培养和细胞生物学特性鉴定目的：建立有效的AMSCs体外培养扩增体系，通过诱导分化探讨AMSCs的细胞生物学特性。方法：取健康产妇正常剖宫产足月胎儿的新鲜胎盘组织，钝性分离羊膜层，取羊膜组织消化培养，倒置显微镜观察AMSCs的细胞形态；流式细胞仪检测细胞表型；采用特定诱导条件将AMSCs分别向软骨细胞、骨细胞、脂肪细胞和神经组织方向诱导分化，采用特异性染色对诱导后细胞进行鉴定。RT-PCR法分析软骨细胞、骨细胞、脂肪细胞及神经组织特异性基因在诱导前后AMSCs中的表达。结果：（1）倒置显微镜观察AMSCs均呈典型的成纤维细胞样贴壁生长，流式细胞仪分析显示，AMSCs高表达CD73、CD90和CD105，不表达CD14、CD34、CD45和HLA-DR。（2）AMSCs经成骨诱导后，细胞均由长梭形向立方形转变，von Kossa染色可见细胞呈集落生长并出现钙结节；经成软骨诱导2w后，AMSCs形态逐渐变得扁平，甲苯胺蓝染色可见细胞被染成蓝色；经成脂肪诱导2w后，细胞内有明显的脂滴出现，油红O染色阳性；经成神经诱导24h后，AMSCs呈神经胶质细胞样或/和神经元样改变，多数细胞呈现GFAP免疫荧光标记阳性。（3）RT-PCR结果显示：AMSCs向软骨细胞、骨细胞、脂肪细胞诱导后，表达PLIN（脂肪细胞）、ACAN（软骨细胞）和RUNX2（骨细胞）特异性基因；向神经组织诱导前AMSCs即表达nestin mRNA、GFAP mRNA、mushashi-1mRNA以及β-tubulin III mRNA，诱导2d，除了表达以上基因外，还有NF mRNA表达，诱导5d，仅Nestin mRNA的表达有所下降。结论：从人胎盘羊膜组织中经过特定的分离消化培养易于获得AMSCs，且增殖能力强和传代稳定。研究结果表明AMSCs具有MSCs干细胞标记及细胞生物学特性，具有多向分化能力。第二部分AMSCs对胶质瘤细胞生长抑制作用的体外研究目的：观察AMSCs向人脑胶质瘤的定向迁移能力及对肿瘤细胞生长的作用，初步探讨AMSCs体外趋瘤抑瘤效应的可能作用机制。方法：在Transwell培养下室分别接种不同密度的人脑胶质瘤细胞系U251细胞，观察接种于上室的AMSCs的定向迁移能力。将生长良好的P3代AMSCs培养上清转移到微型浓缩器中，离心制备上清浓缩蛋白，加入U251细胞培养基作用后，采用Transwell侵袭实验检测U251细胞侵袭力的变化，透射电镜观察U251细胞形态学变化，AnnexinV-FITC-PI双染法检测细胞早期凋亡情况，RT-PCR法分析肿瘤细胞Casepase-3、Bax和Bcl-2的mRNA表达情况。结果：（1）Transwell迁移实验结果表明U251细胞可在体外共培养时增强AMSCs的定向迁移能力，且其效应与肿瘤细胞接种密度呈依赖性。（2）经AMSCs上清浓缩蛋白作用后，Transwell侵袭实验结果表明U251细胞侵袭力降低。（3）透射电镜下可见细胞核固缩，核内染色质密集、趋边凝聚明显，质膜脱落及凋亡小体等典型的凋亡形态特征。（4）Annexin V-FITC-PI双染结果显示：U251细胞在AMSCs上清浓缩蛋白作用24h时后的凋亡率为9.34±4.27％，而在48h后凋亡率为42.93±11.54％，二者之间有显著性差别(P<0.05)，提示细胞凋亡率随着作用时间的增加而升高。（5）RT-PCR检测结果表明：AMSCs上清浓缩蛋白作用24h后，U251细胞Casepase-3、Bax的mRNA表达水平明显高于对照组，在48h时进一步升高，实验组与对照组比较有显著性差异(P<0.05)；Bcl-2mRNA表达水平在24h和48h均低于对照组，实验组与对照组比较有显著性差异(P<0.05)。结论：胶质瘤微环境中分泌的各种趋化因子可增强AMSCs定向迁移能力，与接种的胶质瘤细胞密度呈依赖性。AMSCs可抑制胶质瘤细胞的侵袭能力。AMSCs可在体外抑制胶质瘤细胞的增殖，并诱导其凋亡，其机理可能是Bcl-2/Bax比值下降，激活caspase-3，最终导致胶质瘤细胞凋亡。第三部分AMSCs对胶质瘤生长抑制作用的体内研究目的：裸鼠瘤内注射AMSCs，观察AMSCs的存活、迁移以及移植对实体肿瘤的作用，初步探讨AMSCs趋瘤抑瘤的可能机制。方法：采用雄性裸鼠腋窝皮下注射U251细胞制作人脑胶质瘤模型，随机分为3组。对照组正常饲养，不予任何处理；PBS组瘤内注射0.2ml PBS缓冲液；AMSCs移植组瘤内注射BrdU标记的0.2ml AMSCs悬液，每天测量肿瘤大小。AMSCs移植14d后处死，取出肿瘤组织，HE染色观察肿瘤病理特征改变；免疫荧光染色观察AMSCs存活、迁移情况；透射电镜观察肿瘤细胞超微结构改变；RT-PCR法分析肿瘤组织Casepase-3、Bcl-2、Bax的mRNA表达情况。结果：（1）裸鼠皮下人脑胶质瘤造模成功率高，肿瘤接种3d后在腋窝皮下可见肿块，7d后皮下肿瘤可达5mm左右，肿瘤大小随时间的延长而增大。对照组和PBS组肿瘤的生长基本一致，AMSCs移植组肿瘤生长出现明显抑制，与对照组和PBS组相比有显著性差异（P<0.05），实验表明瘤内注射AMSCs可显著抑制裸鼠胶质瘤的生长。（2）光镜观察可见肿瘤细胞排列致密，毛细血管增生明显，部分可见坏死，周边可见肿瘤呈浸润性生长，侵入肌组织之间。病理性核分裂相多见，呈异型性。（3）免疫荧光染色显示瘤内有较多的BrdU阳性细胞。（4）透射电镜下可观察到肿瘤细胞核浆比高，核固缩、染色质趋边凝聚及凋亡小体等典型的凋亡形态特征。（5）RT-PCR检测结果表明AMSCs移植组肿瘤组织Casepase-3、Bax的mRNA表达水平明显高于对照组(P<0.05)，Bcl-2mRNA水平显著低于对照组(P<0.05)。结论：裸鼠胶质瘤模型瘤内移植AMSCs可在瘤内存活、迁移，通过诱导细胞凋亡等过程抑制胶质瘤的生长，对脑胶质瘤的临床治疗，可能成为更为有效的途径。更多还原

【Abstract】 Mesenchymal stem cells (MSCs) is a kind of mesoderm-derived stem cells with greatpotential of self-renewal and multilineage differentiation. MSCs, with less immunogenicity,tumor targeting and tumor suppression, have become the focus on research in the injuryrepair and tumor therapy. Previous research mainly concentrated on bone marrow-derivedMSCs which can only be obtained by bone marrow aspiration and distribute sparsely withincrease of age. Recently, amniotic membrane of placenta becomes one of the new sourcesof MSCs (amniotic mesenchymal stem cells, AMSCs) because it is rich in all kinds of stemcells, easily procured with rarely pollution, easily performed with good proliferationactivity, which may render these cells as good potential sources for future therapeuticapplications.Glioma is the most common primary malignant tumor of the central nervous system.It possesses extraordinary migratory ability and readily penetrate adjacent normal tissue atthe early period. Tumor cells migrating away from the primary tumor site often form tumormicrosatellites at distal sites. Recent research shows that MSCs possess great migratoryability and glioma tropism, and can be largely distributed at the border zone between tumorand normal parenchyma. They develop a capsule-like structure and also infiltrated into thetumor bed relatively uniformly. Moreover, some MSCs can chase down‘the outgrowingglioma cells. Thus, MSCs may serve as a promising vector for gene-targeted gliomatherapy. MSCs can also exert anti-tumor effect on glioma, via the induction of apoptosis ofglioma cells. Further study should be focused on the mechanism in glioma tropism andanti-tumor effect and what about the relevanted mechanism, pending further study.In our study, the effective amplification system for AMSCs was set up, and theneurobiology characteristics of AMSCs were explored by induction. The migratory ability,glioma tropism and anti-tumor effect of AMSCs were observed in a series of experimentsin vitro. Moreover, the AMSCs were adopted as seed cells, then their survival, migrationafter transplanted into glioma on nude mice were observed, and the possible mechanism inglioma tropism and anti-tumor effect was investigated. Part I Isolation, culture and identification of AMSCsObjective: To establish an effective culture system for AMSCs in vitro, and toexplore the neurobiology characteristics of AMSCs by induction.Methods: The amnion and chorion layers were bluntly separated. The AMSCsisolated from minced amnion and chorion tissues were cultured in vitro. Flow Cytometrywas used to detect the surface marker of AMSCs. The morphology was observed underinverted microscope. Then under specific induction conditions, AMSCs were induced tochondrocytes, osteocytes, adipocytes and neurocytes, and specific dye was adopted todetermine their differentiation potential. The specific genes expression of adipocytes,chondrocytes, and osteocytes and neural-specific protein were detected by RT-PCR afterthe AMSCs were treated by different induction conditions for different mesodermallineages.Results: The AMSC showed plastic adherence and typical fibroblastic morphology bylight microscopy, and the Flow Cytometry analysis showed that AMSCs highly expressedCD73、CD90、CD105, negative for CD14、CD34、CD45、HLA-DR; Exposure to osteogenicinductive medium resulted in secretion of extracellular calcium crystals, identified by vonKossa staining, indicating osteogenic differentiation. When cells were cultured inadipogenic inductive medium, intracytoplasmic lipid vacuoles were observed, andconfirmed by oil red O staining. After been cultured in chondrogenic inductive medium,chondrogenic differentiation was identified demonstrated by toluidine blue staining. Whencells were cultured in neural inductive medium, glial cell-like or/and neuron-likestructures were observed, and, most of the cells showed GFAP positive byimmunofluorescence. RT-PCR results showed, after exposed to different inducingconditions, the specific genes expression of PLIN (adipose cells), ACAN(chondrocytes)and RUNX2(osteocytes). AMSCs showed the specific genes expression of nestin, GFAP,mushashi-1and β-tubulin III before expored to neural tissue induing condition. NF wasexpressed positively after induction for2days. The expression level of nestin decreased5days after induction.Conclusions: Human placenta amnion was proved to be rich in AMSCs, which could be obtained from the amnion by specific separation digestion. The results of FlowCytometry and differentiation induction indicated that the cultured AMSCs maintained thesurface markers, cytobiological properties and the multi-directional differentiationpotential as MSCs.Part Ⅱ Experimental research on the inhibitory effect of AMSCson glioma cells in vitroObjective: To observe the directional migration ability of AMSCs on human gliomacells and its effect on glioma cell growth, and to explore the mechanism on its gliomatropism and anti-tumor effect in vitro.Methods: A transwell cell migration assay was used to investigate the impact ofU251cells on migratory ability of AMSCs cells after different density of U251cells wereplaced in the lower transwell chamber. Using a microconcentrator, the protein in theAMSCs medium supernatant were concentrated and were added into the U251cell culturemedium. Subsequently, the invasion of U251cells was detected by transwell matrigelinvasion assay; the ultrastructure morphological changes of glioma cells were observedunder transmission electron microscope; the cell apoptosis was detected byAnnexinV-FITC-PI double staining; the mRNA expression of Casepase-3, Bax and Bcl-2was detected by RT-PCR.Results: Transwell assay results showed that the migration of AMSCs was stimulatedin a dose-dependent manner varying with U251cell density. AMSCs could inhibit theU251cells invasiveness in Matrigel in vitro. Under electron microscope, cell shrinkagewith apoptosis and high dense masses scattered in nucleuses were observed and pyknosisof nucleuses and apoptotic bodies could be found. Annexin V-FITC-PI double stainingshowed an apoptosis rate of9.34±4.27%after24h treated by AMSCs, while42.93±11.54%after48h, which display time-dependent manner. RT-PCR showed that comparedwith control, the AMSCs treated group expressed lower mRNA levels of Casepase-3andBax, but higher mRNA levels of Bcl-2, which also displayed a time-dependent manner. Allof the differences were statistically significant (P<0.05).Conclusions: The soluble chemotatic factors released from glioma cells couldmediate the activation of AMSCs migration in a dose-dependent manner. AMSCs can inhibit glioma cells growth and invasiveness and induce apoptosis of glioma cells in vitrowhich possible related to the decreased ratio of Bcl-2/Bax would ultimately led to theactivation of caspases-3and an induction of apoptosis.Part Ⅲ Experimental research on the inhibitory effect of AMSCson glioma in vivoObjective: To observe the effect of AMSCs survival, migration and transplantation onglioma in vivo, and to explore the mechanism of inhibition effect of AMSCs on gliomafurtherly.Methods: The models were used to induce human glioma in nude mice, which weresubsequently randomly divided into blank, control and experimental groups for treatmentwith empty, PBS and BrdU-labeled AMSCs respectively. Two weeks after transplantation,glioma tissues were processed for hematoxylin-eosin staining and immunofluorescence toobserve survival and spatial distribution of the transplanted AMSCs. The ultrastructuremorphological changes of glioma cells were observed under transmission electronmicroscope. The mRNA expression of Casepase-3, Bax and Bcl-2was detected byRT-PCR in tumor tissue.Results: After the mice were implanted intratumorally with empty, PBS and AMSCs,the tumor volume was measured. PBS-treated group was consistent with contrl, whileAMSCs-treated group significantly reduced tumor size on14d as compared to control(P<0.05). Under light microscope, it revealed that the tumor cells arranged densely withinvasive growth into striated muscle. Nuclear atypia, hemorrhage, necrosis and capillaryaccrementition were visible. BrdU-labeled AMSCs were found uniformly distributed in theglioma under fluorescence microscope. Under electron microscope, cell shrinkage and highdense masses scattered in nucleuses were observed and pyknosis of nucleuses andapoptotic bodies could be found. RT-PCR showed that compared with control, the AMSCstreated group expressed lower mRNA levels of Casepase-3and Bax, but higher mRNAlevels of Bcl-2, which also display a time-dependent manner. All of the differences werestatistically significant (P<0.05).Conclusions: Intratumoral injection of AMSCs may inhibit glioma growth byinducing an apoptosis of glioma in vivo, which demonstrates that that AMSCs transplantation may be applied as a new effective therapeutic approach for the treatment ofgliomas.更多还原