本文關鍵詞： 胚胎干細胞 神經(jīng)前體細胞 帕金森 畸胎瘤 兔脾成纖維細胞 LIF 多向分化　出處：《浙江大學》2008年博士論文　論文類型：學位論文

【摘要】： 帕金森病(Parikinson's disease,PD)是一種嚴重的神經(jīng)系統(tǒng)退行性疾病,病變主要累及中腦黑質(zhì)致密部,大量多巴胺能神經(jīng)元變性或進行性缺失導致紋狀體內(nèi)多巴胺水平降低,患者在臨床上表現(xiàn)為靜止性震顫、運動遲緩和肌強直。隨著人類壽命的延長,PD發(fā)病率已明顯上升,然而PD的藥物治療藥效不能持久,同時有副作用的危害,因此以細胞替代為出發(fā)點的干細胞移植療法為PD治療提供了一個新策略。在眾多候選細胞中,胚胎干細胞(ESCs)因其無限的自我更新能力和三胚層分化能力,成為PD治療的理想細胞來源。未分化的ESCs移植研究顯示,雖可引起模型動物的部分功能恢復,同時亦存在畸胎瘤的威脅。ESCs來源的神經(jīng)前體細胞既具有神經(jīng)細胞分化的能力,同時保留了一定的增殖特性,有可能在體內(nèi)持續(xù)呈現(xiàn)其神經(jīng)替代和保護作用。 本文采用無血清、低密度快速誘導ESCs向神經(jīng)前體細胞分化,短期內(nèi)可得到大量可用于移植的細胞。經(jīng)免疫細胞化學和RT-PCR分析表明,這些細胞為高純度的神經(jīng)前體細胞,其分化率接近100%。將所誘導細胞以10~5植入PD模型小鼠紋狀體后發(fā)現(xiàn),2周始紋狀體內(nèi)就有多巴胺能神經(jīng)元(酪氨酸羥化酶陽性)出現(xiàn),同時HPLC分析表明,2周時紋狀體多巴胺分泌水平顯著增加,與PBS移植組相比存在顯著差異(P＜0.05),4周時多巴胺水平有所降低,此外4周時18.75%PD模型鼠移植部位成瘤,經(jīng)組織學分析表明其中含有三胚層來源的多種細胞。以上數(shù)據(jù)說明,這種快速誘導體系可產(chǎn)生有一定功能的神經(jīng)前體細胞,但該高純度前體細胞仍有致瘤性,可能提示該細胞的發(fā)育學地位更加接近于ESCs,對其發(fā)育學表型的進一步探討,將為ES來源的移植級別神經(jīng)前體細胞的研究奠定基礎。 ESCs來源于著床前胚胎的內(nèi)細胞團,其常規(guī)建系和培養(yǎng)都是建立在胚胎成纖維細胞(MEF)飼養(yǎng)層和外源LIF的共同作用之下。而MEF體外傳代次數(shù)有限,通常在5-7代,其次,在無外源性UF的輔助作用時,單獨MEF培養(yǎng)不足以維持小鼠ES細胞的未分化和自我更新特征,另據(jù)最近的報道顯示MEF可能攜帶有逆轉(zhuǎn)錄病毒,從而存在較大的安全隱患。為此,我們從兔脾中分離到一類成纖維樣細胞(RSF),這類細胞增殖能力是MEF的2-3倍,且體外傳代能力長達13代。最為突出的是這類細胞可在無外源LIF添加的情況下維持小鼠ESCs的生長,目前已支持ESCs生長超過10代,且ESCs能維持其未分化表型,如AKP、SSEA-1、REX1的表達,完整的體內(nèi)外分化潛能,核型分析也表明其具有正常的二倍體核型,40XY。此外經(jīng)透射電鏡分析證實,我們所獲得的RSF細胞未攜帶逆轉(zhuǎn)錄病毒等動物病原體,提示RSF體系是微生物學安全的ESCs培養(yǎng)體系。由此我們可知:RSF來源廣泛,簡便易得,體外增殖能力強,同時不需外源LIF的輔助作用,還是微生物安全的培養(yǎng)體系,可望替代MEF成為ESCs新的培養(yǎng)體系,并且該體系價廉,易得,數(shù)量充足,有較大的推廣價值,必將有力推動ESCs的基礎和臨床應用研究。 成纖維細胞與間充質(zhì)干細胞(MSC)形態(tài)相似,且均為中胚層來源,而后者已顯示出多種分化潛能,故而為探討RSF的分化潛能,我們對其進行了成骨、成脂、平滑肌誘導和肝細胞誘導的初步研究。經(jīng)AKP和Von Kossa染色表明,RSF可誘導為成骨細胞,RT-PCR分析也進一步證實了其成骨潛能;油紅染色顯示,經(jīng)常規(guī)脂肪誘導和低血清bFGF誘導,均可引起高比例脂肪細胞分化;糖原染色證實RSF亦可跨胚層分化為肝細胞;此外RT-PCR顯示,RA誘導RSF細胞平滑肌分化后,有平滑肌特異標志物表達。 綜上所述,我們所獲得的RSF細胞是一類具有多功能性的細胞,不但顯示出強大的ESCs支持能力,并且具有多向分化潛能,包括分化為同胚層的成骨、脂肪細胞、平滑肌細胞,以及跨胚層分化為肝細胞,其分化譜及分化機制還有待進一步闡明。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R329

【參考文獻】

相關期刊論文 前1條

1 ;Differentiation of Mesenchymal Stem Cells Into Dopaminergic Neuron-like Cells in vitro[J];Biomedical and Environmental Sciences;2005年01期

，

本文編號：1447471