小鼠胚胎干细胞来源神经前体细胞的移植研究及兔脾成纤维样细胞的多功能性研究

发布时间：2018-01-20 08:03

本文关键词： 胚胎干细胞神经前体细胞帕金森畸胎瘤兔脾成纤维细胞 LIF 多向分化　出处：《浙江大学》2008年博士论文　论文类型：学位论文

【摘要】： 帕金森病(Parikinson's disease,PD)是一种严重的神经系统退行性疾病,病变主要累及中脑黑质致密部,大量多巴胺能神经元变性或进行性缺失导致纹状体内多巴胺水平降低,患者在临床上表现为静止性震颤、运动迟缓和肌强直。随着人类寿命的延长,PD发病率已明显上升,然而PD的药物治疗药效不能持久,同时有副作用的危害,因此以细胞替代为出发点的干细胞移植疗法为PD治疗提供了一个新策略。在众多候选细胞中,胚胎干细胞(ESCs)因其无限的自我更新能力和三胚层分化能力,成为PD治疗的理想细胞来源。未分化的ESCs移植研究显示,虽可引起模型动物的部分功能恢复,同时亦存在畸胎瘤的威胁。ESCs来源的神经前体细胞既具有神经细胞分化的能力,同时保留了一定的增殖特性,有可能在体内持续呈现其神经替代和保护作用。本文采用无血清、低密度快速诱导ESCs向神经前体细胞分化,短期内可得到大量可用于移植的细胞。经免疫细胞化学和RT-PCR分析表明,这些细胞为高纯度的神经前体细胞,其分化率接近100%。将所诱导细胞以10~5植入PD模型小鼠纹状体后发现,2周始纹状体内就有多巴胺能神经元(酪氨酸羟化酶阳性)出现,同时HPLC分析表明,2周时纹状体多巴胺分泌水平显著增加,与PBS移植组相比存在显著差异(P＜0.05),4周时多巴胺水平有所降低,此外4周时18.75%PD模型鼠移植部位成瘤,经组织学分析表明其中含有三胚层来源的多种细胞。以上数据说明,这种快速诱导体系可产生有一定功能的神经前体细胞,但该高纯度前体细胞仍有致瘤性,可能提示该细胞的发育学地位更加接近于ESCs,对其发育学表型的进一步探讨,将为ES来源的移植级别神经前体细胞的研究奠定基础。 ESCs来源于着床前胚胎的内细胞团,其常规建系和培养都是建立在胚胎成纤维细胞(MEF)饲养层和外源LIF的共同作用之下。而MEF体外传代次数有限,通常在5-7代,其次,在无外源性UF的辅助作用时,单独MEF培养不足以维持小鼠ES细胞的未分化和自我更新特征,另据最近的报道显示MEF可能携带有逆转录病毒,从而存在较大的安全隐患。为此,我们从兔脾中分离到一类成纤维样细胞(RSF),这类细胞增殖能力是MEF的2-3倍,且体外传代能力长达13代。最为突出的是这类细胞可在无外源LIF添加的情况下维持小鼠ESCs的生长,目前已支持ESCs生长超过10代,且ESCs能维持其未分化表型,如AKP、SSEA-1、REX1的表达,完整的体内外分化潜能,核型分析也表明其具有正常的二倍体核型,40XY。此外经透射电镜分析证实,我们所获得的RSF细胞未携带逆转录病毒等动物病原体,提示RSF体系是微生物学安全的ESCs培养体系。由此我们可知:RSF来源广泛,简便易得,体外增殖能力强,同时不需外源LIF的辅助作用,还是微生物安全的培养体系,可望替代MEF成为ESCs新的培养体系,并且该体系价廉,易得,数量充足,有较大的推广价值,必将有力推动ESCs的基础和临床应用研究。成纤维细胞与间充质干细胞(MSC)形态相似,且均为中胚层来源,而后者已显示出多种分化潜能,故而为探讨RSF的分化潜能,我们对其进行了成骨、成脂、平滑肌诱导和肝细胞诱导的初步研究。经AKP和Von Kossa染色表明,RSF可诱导为成骨细胞,RT-PCR分析也进一步证实了其成骨潜能;油红染色显示,经常规脂肪诱导和低血清bFGF诱导,均可引起高比例脂肪细胞分化;糖原染色证实RSF亦可跨胚层分化为肝细胞;此外RT-PCR显示,RA诱导RSF细胞平滑肌分化后,有平滑肌特异标志物表达。综上所述,我们所获得的RSF细胞是一类具有多功能性的细胞,不但显示出强大的ESCs支持能力,并且具有多向分化潜能,包括分化为同胚层的成骨、脂肪细胞、平滑肌细胞,以及跨胚层分化为肝细胞,其分化谱及分化机制还有待进一步阐明。
[Abstract]:Parkinson's disease (Parikinson's disease PD) is a severe neurodegenerative disease, the lesions involved the substantia nigra, a progressive degeneration of dopaminergic neurons or lack of the level of dopamine in the striatum decreased in patients with clinical manifestations of tremor, bradykinesia and rigidity. With the extension of human life the incidence of PD has increased significantly, but the drug treatment efficacy of PD cannot harm lasting, also can have side effects, so cell replacement as the starting point of stem cell transplantation therapy provides a new strategy for the treatment of PD. In a number of candidate cells, embryonic stem cells (ESCs) because of its infinite self the self-renewal and differentiation of three germ layers, become the ideal cell source. PD treatment on transplantation of undifferentiated ESCs showed partial functional recovery can be caused by animal models, at the same time there is also a teratoma The neural precursor cells threatening.ESCs have both the ability of neuronal differentiation, while maintaining certain proliferation characteristics. They may continue to present their neural replacement and protection in vivo.
The serum free, low density ESCs rapid induction into neural precursor cell differentiation, the short term can be a large number of transplanted cells. Through the analysis of RT-PCR and immunocytochemistry showed that these cells were of high purity of the neural precursor cells, the differentiation rate is close to 100%. to induce cells to 10~5 mice implanted with PD after 2 weeks was found in striatum, striatum on dopaminergic neurons (tyrosine hydroxylase positive), and HPLC analysis showed that 2 weeks of striatal dopamine secretion increased significantly, compared with PBS transplantation group, there is significant difference (P < 0.05), 4 weeks when dopamine levels decreased in 4 weeks 18.75%PD rat model of tumor transplantation site, histologic analysis showed that many cells containing three germ layers. The above data shows that this system can produce a rapid induction of functional neural precursor cells, but the High purity precursor cells still have tumorigenicity, suggesting that the developmental status of the cells is closer to ESCs. Further exploration of their developmental phenotypes will lay the foundation for the study of ES derived neural precursor cells.
ESCs derived from the preimplantation embryo inner cell mass, the conventional establishment and culture are built in embryonic fibroblasts (MEF) under the action of the feeder layer and exogenous LIF and MEF cultured in vitro. The number is limited, usually in 5-7 generation, secondly, in the auxiliary function without exogenous UF, alone MEF training is not enough to maintain the undifferentiated mouse ES cell self-renewal and other characteristics, according to recent reports show that MEF may carry a retrovirus, so there is a big security risk. Therefore, we isolated from rabbit spleen to a class of fibroblast like cells (RSF), the ability of cell proliferation is 2-3 times MEF in vitro, and the ability for 13 generations. The most prominent is that cells can maintain the growth of ESCs in mice without exogenous LIF cases, now supports ESCs growth for more than 10 generations, and ESCs can maintain the undifferentiated phenotype, such as AKP, SSEA-1, REX1 expression, end The in vivo differentiation potential, karyotype analysis also showed that it has a normal karyotype, 40XY. addition by transmission electron microscopy confirmed, we obtained RSF cells carrying the retrovirus and other animal pathogens, suggesting that RSF system is the microbiological safety culture system of ESCs. From this we know: RSF wide source, simple and easy to obtain, proliferation strong ability in vitro, and without auxiliary effect of exogenous LIF, or microbial safety culture system, is expected to replace MEF as the new ESCs culture system, and the system is cheap, easy to get sufficient quantity, has great promotion value, will greatly promote the research on the basis of ESCs and clinical application.
Fibroblast and mesenchymal stem cells (MSC) were similar in morphology, and mesoderm, the latter has shown multiple differentiation potential, so as to explore the differentiation potential of RSF, we carried on the osteogenic, adipogenic, preliminary study on the induction of liver cells and smooth muscle induced by AKP and Von Kossa. Staining showed that RSF can be induced into osteoblasts, RT-PCR analysis also further confirmed their osteogenic potential; oil red staining showed that the induced regular fat induction and low serum bFGF, can cause a high proportion of fat cell differentiation; glycogen staining confirmed that RSF can differentiate into liver cells; in addition RT-PCR, RA induced by RSF smooth muscle cell differentiation, with smooth muscle specific markers.
In summary, we have obtained the RSF cell is a kind of multi functional cells, not only shows a strong ESCs support ability, and have the potential of multi-directional differentiation, including differentiation of bone, with layers of fat cells, smooth muscle cells, and the differentiation of liver cells, the differentiation spectrum and differentiation mechanism has yet to be further clarified.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

【参考文献】