【摘要】：在眼科領域內(nèi)有許多難治性眼病如老年性黃斑變性、缺血性視神經(jīng)病變、青光眼等因各種原因最終導致視網(wǎng)膜神經(jīng)細胞損傷或喪失,從而造成不可逆的視力損傷甚至致盲。近年來視網(wǎng)膜干細胞(retinal stem cells, RSCs)的發(fā)現(xiàn)為治療致盲性眼病開辟了廣闊的應用前景。 RSCs具有多向分化潛能,目前研究認為通過體內(nèi)外實驗可將其誘導成為視網(wǎng)膜光感受器樣細胞、視網(wǎng)膜神經(jīng)節(jié)樣細胞等多種視網(wǎng)膜細胞類型,而新近研究認為,RSCs經(jīng)某些特定基因修飾后細胞將向特定類型的視網(wǎng)膜細胞分化,但目前其定向分化具體機制尚不明確。 生長相關蛋白43(growth associated protein-43, GAP-43)是位于脊椎動物神經(jīng)元細胞膜上的一種磷酸蛋白質(zhì),廣泛分布于神經(jīng)系統(tǒng)的神經(jīng)元胞體及軸突內(nèi)�，F(xiàn)已證明GAP-43基因與神經(jīng)系統(tǒng)及視網(wǎng)膜的發(fā)育、神經(jīng)元軸突再生及神經(jīng)突觸建立均具有重要的作用。但對于其在神經(jīng)干細胞(neural stem cell, NSCs)分化或其它種類的干細胞向神經(jīng)細胞分化這一過程中的具體機制仍不為所知。因此本實驗研究將GAP-43的研究與RSCs及NSCs的分化結合起來,希望探索出其在不同干細胞向神經(jīng)細胞分化的可能機制并進行比較分析。 首先,本實驗就GAP-43在新生大鼠視網(wǎng)膜中的表達進行檢測,應用免疫細胞化學染色法及RT-PCR法均檢測到新生大乳鼠的視網(wǎng)膜細胞表達GAP-43,故而確認了GAP-43在視網(wǎng)膜細胞發(fā)育成熟過程中起一定作用。其次,通過無血清培養(yǎng)法于體外成功分離、培養(yǎng)并擴增大鼠RSCs及NSCs,兩種干細胞均可在體外傳代培養(yǎng),并且第2代的RSCs及NSCs均表達神經(jīng)干細胞表面標志物nestin及增殖標志物BrdU,并且用RT-PCR方法檢測到RSCs表達Pax6。通過生長曲線可以看出,兩種干細胞相比較,RSCs的體外培養(yǎng)、增殖等特點均與NSCs相似,但其自我更新的能力低于NSCs。在誘導分化方面,兩種干細胞也表現(xiàn)出相似的特點,即均可在體外應用血清誘導分化為神經(jīng)細胞,誘導分化后的細胞具有典型的神經(jīng)細胞形態(tài),并表達神經(jīng)元標志物NSE、NF、βⅢ-tubulin及神經(jīng)膠質(zhì)細胞的標志物GFAP。此外,應用RT-PCR方法可檢測到RSCs分化后的細胞表達視網(wǎng)膜神經(jīng)節(jié)細胞標志物Thy1.1,提示RSCs分化后的細胞具有視網(wǎng)膜神經(jīng)節(jié)細胞的特性。通過應用免疫細胞化學染色可以看出,兩種干細胞在誘導分化后干細胞標志nestin表達陽性的細胞比例下降,提示其進入分化狀態(tài),比較兩種干細胞誘導分化后表達NSE及GFAP陽性細胞比例可以看出RSCs組均明顯低于NSCs組,結果提示RSCs相比NSCs其體外向神經(jīng)元、神經(jīng)膠質(zhì)細胞分化的程度較低。在兩種干細胞分化的這一過程中第3、7天應用免疫細胞化學染色及RT-PCR方法均檢測到GAP-43持續(xù)表達,提示GAP-43基因在兩種干細胞向神經(jīng)細胞分化中發(fā)揮著重要作用。 在接下來的實驗中,首先通過測序確定GAP-43表達載體序列的正確性,并通過脂質(zhì)體轉染將GAP-43基因成功導入RSCs及NSCs中,通過G418的篩選富集了轉染成功的干細胞,分別應用免疫細胞化學染色、RT-PCR及western blot方法證明了GAP-43基因轉染到干細胞內(nèi)并予表達。篩選一周后應用5%血清誘導其分化,表達外源性基因的干細胞在血清誘導的向神經(jīng)細胞分化過程中表現(xiàn)出易于分化的趨勢,具體表現(xiàn)為分化成神經(jīng)細胞的突起數(shù)量及長度增加、突起更加舒展,經(jīng)免疫細胞化學染色,兩種基因修飾的干細胞分化后表達神經(jīng)元標志物NSE的陽性細胞比例均明顯提高(差異具有顯著性,P0.05),表達神經(jīng)膠質(zhì)細胞標志物GFAP陽性細胞比例變化不明顯(P0.05)。應用實時熒光定量PCR可檢測到GAP-43基因修飾的細胞分化后表達標志物NSE、βⅢ-tubulin mRNA均有所增加(部分差異具有顯著性,P0.05),GFAP mRNA水平增加不明顯(P0.05)。實驗證明GAP-43的表達促進了RSCs及NSCs向神經(jīng)元細胞方向分化。從形態(tài)學分析,RSCs與NSCs相比后者對于GAP-43基因表達的影響更為明顯。 為了進一步研究GAP-43的作用,本研究通過基因轉染將pGCsi-U6/Neo/GFP/ GAP-43shRNA成功導入RSCs及NSCs中,基因轉染后應用G418篩選、純化。并分別應用免疫細胞化學染色、RT-PCR及western blot方法證明了pGCsi-U6/Neo/GFP/GAP-43 shRN A轉染到干細胞內(nèi)并使細胞分化過程中的GAP-43基因沉默。研究發(fā)現(xiàn),GAP-43表達抑制后干細胞在血清誘導的細胞分化過程中表現(xiàn)出形成神經(jīng)細胞的突起明顯受阻,同時分化后標志物NSE免疫細胞化學染色陽性細胞的比例及NSE、βⅢtubulin標志物mRNA相對含量均下降(部分差異具有顯著性,P0.05),GFAP陽性細胞計數(shù)及mRNA水平變化無顯著性(P0.05)。實驗結果提示GAP-43沉默抑制了RSCs及NSCs向神經(jīng)元細胞方向分化。從形態(tài)學分析,兩種干細胞相比較NSCs所受GAP-43基因沉默的影響大于RSCs。 綜上所述,表達GAP-43將促進血清誘導的RSCs及NSCs向神經(jīng)元細胞分化,相反,抑制該基因表達后RSCs及NSCs向神經(jīng)元細胞分化明顯受到抑制,因此,GAP-43基因在兩種干細胞向神經(jīng)元細胞分化過程中具有重要的正向導向性作用,此種調(diào)控作用在NSCs分化過程表現(xiàn)的更為明顯。本研究為進一步探討干細胞向神經(jīng)細胞定向分化機理奠定了基礎。
[Abstract]:In the field of Ophthalmology, there are many refractory eye diseases such as age-related macular degeneration, ischemic optic neuropathy, glaucoma, and so on, which eventually lead to retinal nerve cell damage or loss, resulting in irreversible visual impairment or even blindness. In recent years, the discovery of retinal stem cells (RSCs) for the treatment of blindness Eye disease has opened up a broad prospect of application.
RSCs has multiple differentiation potential. At present, it is believed that in vitro and in vivo experiments can be induced into retinal photoreceptor like cells, retinal ganglion like cells and many types of retinal cells. Recent studies suggest that RSCs can differentiate into a specific type of retinal cells after some specific gene modification, but it is currently determined. The specific mechanism for differentiation is not yet clear.
The growth related protein 43 (growth associated protein-43, GAP-43) is a phosphoric acid protein located on the membrane of the neuron cell of vertebrates. It is widely distributed in the neurons and axons of the nervous system. It has been proved that the GAP-43 gene is important to the development of the nervous system and the retina, the regeneration of the axons and the establishment of synapses in the neurons. The specific mechanism in the process of differentiation of neural stem cell (NSCs) or other kinds of stem cells to neural cells is still unknown. Therefore, this study combines the study of GAP-43 with the differentiation of RSCs and NSCs, and hopes to explore its differentiation into neural cells in different stem cells. The possible mechanism and comparative analysis.
First, we detected the expression of GAP-43 in the retina of newborn rats. The expression of GAP-43 in the retinal cells of newborn rats was detected by immunocytochemical staining and RT-PCR method. Therefore, it was confirmed that GAP-43 was used in the development of retinal cells. Secondly, it was successful in vitro by serum-free culture. Isolation, culture and amplification of RSCs and NSCs, two kinds of stem cells can be cultured in vitro, and the second generation of RSCs and NSCs all express the surface marker of neural stem cells nestin and the proliferation marker BrdU, and the RT-PCR method to detect RSCs expression Pax6. through the growth curve can be seen, two kinds of stem cells are compared, RSCs in vitro culture, The characteristics of proliferation are similar to that of NSCs, but the ability of self renewal is lower than that of NSCs. in inducing differentiation. The two kinds of stem cells also show similar characteristics, that is, they can be induced to differentiate into neural cells in vitro by using serum, and the differentiated cells have typical Neurocellular state and expression of neuron markers NSE, NF, and beta III -tubulin. And the marker of glial cells GFAP. in addition, RT-PCR method can be used to detect the expression of retinal ganglion cell marker Thy1.1 after RSCs differentiation, suggesting that the cells after RSCs differentiation have the characteristics of retinal ganglion cells. By using immunocytochemical staining, the two kinds of stem cells can be induced to be thin after differentiation. The proportion of cell marker nestin positive cells declined, suggesting that the cells entered the differentiation state. Comparing the expression of NSE and GFAP positive cells after differentiation of two kinds of stem cells, the proportion of NSE and GFAP positive cells showed that the RSCs group was significantly lower than that of the NSCs group. The results suggested that RSCs had a lower degree of differentiation of the neuroglial cells than the NSCs. In this process, the continuous expression of GAP-43 was detected by immunocytochemical staining and RT-PCR method on day 3,7, suggesting that the GAP-43 gene plays an important role in the differentiation of two kinds of stem cells to neural cells.
In the next experiment, the correctness of the GAP-43 expression vector sequence was determined by sequencing, and the GAP-43 gene was successfully transfected into RSCs and NSCs through liposome transfection. The successful transfected stem cells were enriched by G418 screening. The immunocytochemical staining, RT-PCR and Western blot methods were used to prove the GAP-43 gene transfection. After one week, 5% serum was used to induce differentiation. The stem cells expressing exogenous genes showed a tendency to differentiate easily during the differentiation of neural cells induced by serum. The specific expression was the increase in the number and length of the neurites that differentiated into nerve cells, the protruding and the immunocytochemical staining, two The proportion of the positive cells expressing the neuron marker NSE after the differentiation of the gene modified stem cells was significantly increased (the difference was significant, P0.05). The expression of GFAP positive cells in the expression of the neuroglial cell markers was not significant (P0.05). The application of real-time fluorescent quantitative PCR could detect the expression marker NS after the cell differentiation of the GAP-43 gene. E, beta III -tubulin mRNA increased (part difference was significant, P0.05), and GFAP mRNA level increased not significantly (P0.05). Experiments showed that the expression of GAP-43 promoted the differentiation of RSCs and NSCs into neuronal cell direction.
In order to further study the role of GAP-43, this study successfully transfected pGCsi-U6/Neo/GFP/ GAP-43shRNA into RSCs and NSCs by gene transfection. After gene transfection, the gene transfection was screened and purified by G418. Immunocytochemical staining was used, and RT-PCR and Western blot methods were used to prove that pGCsi-U6/ Neo/GFP/GAP-43 shRN was transfected into the stem cells and fined. The GAP-43 gene was silenced in the process of cell differentiation. It was found that the protuberances of the cells in the differentiation of serum induced by GAP-43 expression were obviously blocked, and the proportion of NSE immunocytochemical staining positive cells after differentiation and the relative content of NSE, and the relative content of the beta III tubulin marker mRNA were all decreased. Some of the differences were significant, P0.05), there was no significant change in GFAP positive cell count and mRNA level (P0.05). The experimental results suggest that GAP-43 silence inhibits the direction differentiation of RSCs and NSCs into neuron cells. From morphological analysis, the effect of GAP-43 gene silencing on NSCs is greater than that of NSCs, compared with the two types of stem cells.
To sum up, the expression of GAP-43 will promote the differentiation of serum induced RSCs and NSCs into neuron cells. On the contrary, the differentiation of RSCs and NSCs to neuronal cells is inhibited obviously after the inhibition of the gene expression. Therefore, the GAP-43 gene has an important positive direction in the process of differentiation of two kinds of stem cells to neuronal cells. This regulation is in N The differentiation process of SCs was more obvious. This study laid a foundation for further study on the mechanism of neural differentiation of stem cells.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R774.5

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2 陳偉;人骨髓間充質(zhì)干細胞集落培養(yǎng)及其生物學特性的研究[D];第一軍醫(yī)大學;2004年

3 徐洪璋;骨髓間充質(zhì)干細胞的相關研究[D];中國人民解放軍第一軍醫(yī)大學;2003年

4 劉丹丹;人骨髓間充質(zhì)干細胞向內(nèi)皮細胞體外誘導分化的實驗研究[D];吉林大學;2004年

5 傅斌;人前列腺干細胞分離培養(yǎng)及其生物學特性研究[D];江西醫(yī)學院;2004年

6 黃宇婷;骨髓來源干細胞異位向子宮內(nèi)膜方向分化的研究[D];復旦大學;2009年

7 李勇;大鼠骨髓間充質(zhì)干細胞生物學特性及BMP2在誘導其向神經(jīng)元樣細胞分化中的作用[D];第三軍醫(yī)大學;2004年

8 才曉君;大鼠骨髓間充質(zhì)干細胞體外定向誘導分化為心肌樣細胞的實驗研究[D];山東大學;2005年

9 李銀華;脂肪干細胞作為血友病A基因治療靶細胞可能性的研究[D];武漢大學;2005年

10 鄭志宏;用分子組織工程學方法研究轉化生長因子β_1對骨髓基質(zhì)干細胞生物活性的影響[D];第四軍醫(yī)大學;2002年

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