【摘要】：各種原因引起的視神經(jīng)損傷性疾病在臨床上較為常見,其中部分疾病會造成視神經(jīng)進(jìn)行性、不可逆性損傷,最終導(dǎo)致視力喪失。視神經(jīng)細(xì)胞是一種終末分化細(xì)胞,再生能力差,導(dǎo)致?lián)p傷的視網(wǎng)膜神經(jīng)難以修復(fù)再生,目前臨床尚無有效方法治愈視神經(jīng)疾病。近年來,應(yīng)用干細(xì)胞治療視網(wǎng)膜神經(jīng)疾病這一方法極具前景,牙髓干細(xì)胞(dental pulp stem cells,DPSCs)由于其與視神經(jīng)細(xì)胞同樣來源于神經(jīng)嵴,且較神經(jīng)干細(xì)胞更易獲得,而有望成為修復(fù)重建神經(jīng)組織病損的種子細(xì)胞。目前研究認(rèn)為,Notch及Wnt/β-catenin信號通路是脊椎動物視網(wǎng)膜神經(jīng)發(fā)育所必需的。作為一種廣泛表達(dá)于多個物種且在進(jìn)化過程中高度保守的基因,Notch基因主要參與對細(xì)胞分化的抑制信號。Wnt/β-catenin信號的激活貫穿視網(wǎng)膜及視神經(jīng)發(fā)育的整個過程,提示其在神經(jīng)發(fā)育中起重要作用。分泌性卷曲蛋白2(secreted frizzled related protein 2,SFRP2)是一種Wnt信號通路抑制劑,同時也間接地參與了除Wnt信號以外的多個信號通路,如Notch及BMP信號通路等。SFRP2可與金屬蛋白酶ADAM10結(jié)合,抑制Notch信號通路。已有研究表明SFRP2在神經(jīng)形成過程中高表達(dá),我們推測其可能通過抑制在干細(xì)胞增殖過程中發(fā)揮重要作用的Notch及Wnt信號通路,促進(jìn)神經(jīng)嵴來源的DPSCs向神經(jīng)樣細(xì)胞分化。為了驗證上述假設(shè),本課題借助多種細(xì)胞生物學(xué)和分子生物學(xué)手段,通過重組腺病毒載SFRP2基因體外轉(zhuǎn)染人牙髓干細(xì)胞(human dental pulp stem cells,h DPSCs),同時利用h DPSCs同視網(wǎng)膜色素上皮(retinal pigment epithelium,RPE)細(xì)胞共培養(yǎng)法誘導(dǎo)h DPSCs的神經(jīng)向分化,明確SFRP2在h DPSCs向神經(jīng)樣細(xì)胞分化過程中的作用,初步探討視神經(jīng)修復(fù)再生機(jī)制及臨床應(yīng)用前景。方法:從完整拔除的健康第三磨牙內(nèi)分離牙髓組織,酶消化復(fù)合組織塊法原代培養(yǎng)h DPSCs。取第3代h DPSCs,MTT法繪制其生長曲線,并通過成脂及成骨誘導(dǎo)檢測其多向分化潛能。通過流式細(xì)胞術(shù)和細(xì)胞毒性實驗(MTT法)篩選合適的病毒感染復(fù)數(shù)(multiplicity of infection,MOI)。采用800 particles/cell的Ad-SFRP2轉(zhuǎn)染h DPSCs,RT-PCR檢測SFRP2的表達(dá);并將轉(zhuǎn)染Ad-SFRP2的h DPSCs與人RPE細(xì)胞共培養(yǎng)誘導(dǎo)其神經(jīng)向分化,q PCR檢測神經(jīng)相關(guān)基因GFAP、Nestin、MAP2、HES1、β-3tublin及ATOH7的表達(dá)。結(jié)果:倒置顯微鏡下見體外培養(yǎng)的第3代h DPSCs貼壁生長,形態(tài)規(guī)則,呈長梭形,核圓,位于中央。甲苯胺藍(lán)染色可見細(xì)胞成集落生長,分別進(jìn)行成脂和成骨誘導(dǎo),35天油紅O染色及21天茜素紅染色可見脂滴及鈣結(jié)節(jié)。生長曲線顯示第4天細(xì)胞由滯留期進(jìn)入對數(shù)生長期,第9天細(xì)胞進(jìn)入平臺期。轉(zhuǎn)染Ad-EGFP的h DPSCs在轉(zhuǎn)染后8 h鏡下即可見綠色熒光蛋白的表達(dá),轉(zhuǎn)染Ad-SFRP2的h DPSCs在轉(zhuǎn)染后72 h提取總m RNA,RT-PCR檢測到SFRP2基因的表達(dá)。共培養(yǎng)及Ad-SFRP2轉(zhuǎn)染結(jié)果表明,GFAP的表達(dá)早期表現(xiàn)為共培養(yǎng)誘導(dǎo)組高于轉(zhuǎn)染組,晚期則表現(xiàn)轉(zhuǎn)染組稍高表達(dá);Nestin早期在共培養(yǎng)誘導(dǎo)組及轉(zhuǎn)染組中表現(xiàn)為一定的上調(diào)趨勢,晚期表達(dá)則呈下調(diào)趨勢;β-3tublin的表達(dá)在共培養(yǎng)誘導(dǎo)組及轉(zhuǎn)染組中呈持續(xù)性下調(diào);MAP2在共培養(yǎng)誘導(dǎo)組晚期呈現(xiàn)較高表達(dá);HES1在共培養(yǎng)誘導(dǎo)組早期呈現(xiàn)較高表達(dá),晚期則表達(dá)降低,在轉(zhuǎn)染組較非轉(zhuǎn)染組呈下降趨勢;ATOH7的表達(dá)在各組均無統(tǒng)計學(xué)意義。結(jié)論:SFRP2可以促進(jìn)牙髓干細(xì)胞的神經(jīng)向分化,但具體機(jī)制有待于進(jìn)一步實驗闡明。
[Abstract]:The optic nerve injury disease caused by various causes is more common in the clinic, in which some of the diseases can cause progressive and irreversible damage of the optic nerve and eventually lead to loss of vision. As the nerve cell is a terminal differentiation cell, the regeneration ability is poor, and the injured retinal nerve is difficult to repair and regenerate, and the present invention has no effective method to cure the optic nerve diseases. in recent year, that method of using stem cell to treat the retinal nerve disease is very promising, and the dental pulp stem cells (DPSCs) are also more accessible to the neural stem cells due to the fact that it is derived from the neural stem cells in the same way as the visual nerve cells, And is expected to be a seed cell for repairing the damaged nerve tissue. In that present study, the Notch and Wnt/ n-cata signal pathway is necessary for the development of the retinal nerve in the vertebrate. As a gene that is widely expressed in multiple species and highly conserved in the evolution process, the Notch gene is mainly involved in the inhibition of cell differentiation. The activation of the Wnt/ P-cata signal extends through the whole process of the development of the retina and the optic nerve, suggesting that it plays an important role in the development of the nerve. The secreted frizzled protein 2 (SFRP2) is a Wnt signaling pathway inhibitor, and is also indirectly involved in a plurality of signal paths other than the Wnt signal, such as a Notch and a BMP signal path, and the like. The SFRP2 can be combined with the metal protease ADAM10 to inhibit the Notch signaling pathway. It has been shown that SFRP2 is highly expressed in the process of neural formation, and we speculate that it is possible to promote the differentiation of DPSCs from the source of neural stem cells to neural-like cells by inhibiting the Notch and Wnt signaling pathways that play an important role in stem cell proliferation. In order to verify the above-mentioned hypothesis, human dental pulp stem cells (h DPSCs) were transfected with the recombinant adenovirus-loaded SFRP2 gene in vitro by means of a variety of cell biology and molecular biology methods. The nerve-to-differentiation of h-DPSCs was induced by the co-culture of RPE cells, and the role of SFRP2 in the differentiation of h-DPSCs into the neural-like cells was determined. Methods: The pulp tissue was separated from the intact third molar, and the primary cultured h-DPSCs were cultured by enzyme-digested composite tissue-block method. The growth curves of the third generation h DPSCs and the MTT method were obtained, and their multi-directional differentiation potential was detected by lipogenesis and osteogenesis. The appropriate number of viral infections (MOI) was selected by flow cytometry and cytotoxicity assay (MTT method). The expression of SFRP2 was detected by using an Ad-SFRP2 of 800 parts/ cell, and the expression of SFRP2 was detected by RT-PCR. The expression of GFAP, Nestin, MAP2, HES1, HCO3-3 and ATOH7 was detected by co-culture of h-DPSCs transfected with Ad-SFRP2 and human RPE cells. Results: The growth and morphology of the third generation h DPSCs cultured in vitro under the inverted microscope were in the center. In the staining of toluidine blue, the growth of the cells was observed, and the lipid and calcium nodules were observed by the staining and osteogenesis induction, the 35-day oil-red O-staining and the 21-day fluorescein-red staining. The growth curve shows that the 4-day cell is in the logarithmic growth phase from the retention period, and the 9-day cell enters the plateau phase. The expression of the green fluorescent protein was observed at 8 h after the transfection of Ad-EGFP, and the expression of the gene of SFRP2 was detected by RT-PCR at 72 h after the transfection of Ad-SFRP2. The results of co-culture and the transfection of Ad-SFRP2 showed that the expression of GFAP was higher than that of the transfected group in the early stage. The expression of 1-3 tubuin was down-regulated in the co-culture induction group and the transfected group; MAP2 showed a high expression in the late stage of the co-culture induction group; the expression of HES1 in the early stage of the co-culture induction group was higher, and the expression of the later stage decreased, and the expression of the non-transfected group in the transfection group was decreased. The expression of ATOH7 was not statistically significant in each group. Conclusion: SFRP2 can promote the differentiation of the nerve of the dental pulp stem cells, but the specific mechanism is to be further clarified.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R782

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