【摘要】：周圍神經(jīng)損傷是一種常見的臨床疾病。周圍神經(jīng)系統(tǒng)在損傷后是具有一定再生能力的,而且周圍神經(jīng)再生是一個涉及許多細胞活動的復雜的生物學過程,其損傷及再生的確切機制尚未完全闡明。有大量研究表明,雪旺細胞在周圍神經(jīng)再生的過程中扮演了極其重要的角色,也是造成中樞神經(jīng)系統(tǒng)與周圍神經(jīng)系統(tǒng)再生能力迥異的一個重要原因。在周圍神經(jīng)系統(tǒng)受到損傷后,損傷位置附近的雪旺細胞會經(jīng)歷脫髓鞘的過程,并去分化為類似發(fā)育過程中未成熟狀態(tài)的雪旺細胞,在這個狀態(tài)下,雪旺細胞又會再次進入細胞周期開始分裂增殖,并沿著再生的軸突向前遷移,最后包繞新生的軸突再次形成髓鞘,以達到修復神經(jīng)的目地。然而,目前關于神經(jīng)損傷后再生的相關的調(diào)節(jié)機制仍不完全清楚,而且該過程由于涉及到多種細胞類型以及大量的基因,因此為了找到在周圍神經(jīng)再生過程中的一些關鍵調(diào)控基因并進一步闡明相關的核心基因在神經(jīng)再生中的作用,我們便借助了生物信息學的方法,以期發(fā)現(xiàn)并研究損傷后再生過程中重要核心調(diào)控因子的功能,并為臨床藥物的研發(fā)和組織工程神經(jīng)的構建提供基礎。在本研究中,我們首先通過外科手術制作了大鼠坐骨神經(jīng)離斷模型,通過表達譜芯片技術分析坐骨神經(jīng)損傷后0,1,4,7和14天坐骨神經(jīng)近側殘端5 mm的神經(jīng)組織,并采用隨機方差模型對各個時間點的樣本進行差異基因及表達趨勢的顯著性分析,我們共檢測到6046個差異基因(P0.01, FDR0.05),經(jīng)KEGG分析,獲得關鍵信號通路及對整個信號通路基因表達變化影響最顯著的核心調(diào)控基因,結果顯示tenascin-C (TNC)在神經(jīng)損傷再生這一過程中處于調(diào)控網(wǎng)絡的核心地位,暗示了TNC在神經(jīng)再生過程中可能扮演著重要的角色。TNC是一種胞外基質(zhì)大分子蛋白,是tenascin家族最早被發(fā)現(xiàn)的成員,往往被發(fā)現(xiàn)在發(fā)育過程中和損傷部位表達豐富,而在成年個體的組織中表達較少。此外,TNC還被發(fā)現(xiàn)在腫瘤系統(tǒng)中有著促進多種腫瘤細胞遷移和侵襲的能力,并且在中樞神經(jīng)系統(tǒng)的相關研究中,TNC還被發(fā)現(xiàn)具有促進軸突生長的能力,然而在周圍神經(jīng)系統(tǒng)中目前關于TNC的研究還很少,并且結合TNC在前文生物信息學分析中顯示出的重要作用,在周圍神經(jīng)系統(tǒng)中對TNC進行深入的研究是有著很大的必要性的。在后續(xù)的深入研究中,我們發(fā)現(xiàn)了在坐骨神經(jīng)遭受到橫斷損傷后,坐骨神經(jīng)中近側斷端的TNC會在損傷4天后持續(xù)地維持在很高的表達水平,而在正常神經(jīng)組織中的表達卻很低,進一步的暗示了TNC在周圍神經(jīng)再生過程中的重要作用。之后我們又通過了免疫熒光的方法分別檢測了TNC與雪旺細胞標志物S100β,巨噬細胞標志物CD68和成纖維細胞標志物P4HB(prolyl-4-hydroxylase beta)的共定位情況,發(fā)現(xiàn)了在周圍神經(jīng)損傷后TNC幾乎不與雪旺細胞和巨噬細胞共定位,而與成纖維細胞則共定位良好,說明了成纖維細胞在坐骨神經(jīng)損傷后會大量表達TNC。由于各種創(chuàng)傷均會造成不同程度的細胞變性、壞死和組織缺損,必須通過細胞增殖和細胞間基質(zhì)的形成來進行組織修復。在此修復過程中,成纖維細胞起著十分重要的作用。以傷口愈合過程為例,成纖維細胞通過有絲分裂大量增殖,并合成和分泌大量的膠原纖維和基質(zhì)成分,參與損傷后組織的修復與重建。Alison Lloyd等人在2010年發(fā)現(xiàn)了在周圍神經(jīng)損傷后,成纖維細胞會聚集到損傷部位,通過EphB信號引導雪旺細胞軸向排列,以促進周圍神經(jīng)的再生�；谶@篇報道以及我們發(fā)現(xiàn)的神經(jīng)損傷后成纖維細胞會大量分泌TNC這一現(xiàn)象,我們提出了成纖維細胞在周圍神經(jīng)受到損傷后會大量表達TNC以影響雪旺細胞的某些生物學過程這個假設。為了驗證這一假設,我們首先在體外設計了一個基于transwell的成纖維細胞-雪旺細胞共培養(yǎng)系統(tǒng),我們發(fā)現(xiàn)了在transwell共培養(yǎng)系統(tǒng)的下層種入的成纖維細胞的數(shù)量會影響遷移過transwell小室的雪旺細胞的數(shù)量,二者是呈正比關系的,證明了成纖維細胞確實具有影響雪旺細胞遷移的能力,并且證明了這一共培養(yǎng)系統(tǒng)的有效性。之后為了驗證TNC是否是成纖維細胞影響雪旺細胞的一個媒介,我們設計了TNC的siRNA,并在RNA水平和蛋白水平驗證了siRNA的基因沉默效率,并分別通過了transwell實驗和細胞劃痕實驗證明了抑制成纖維細胞TNC的表達可以顯著降低與之共培養(yǎng)的雪旺細胞的遷移能力；之后我們又通過使用外源性的TNC處理單獨體外培養(yǎng)的雪旺細胞,發(fā)現(xiàn)了在培養(yǎng)液中加入1μ g/ml和10μ g/ml的TNC蛋白可以有效地促進雪旺細胞的遷移,結合上面的結果,說明了TNC是成纖維細胞影響雪旺細胞遷移的一個重要媒介。之后,我們還通過體內(nèi)實驗發(fā)現(xiàn),相比對照組,外源性的1μg/ml和10μg/ml TNC蛋白可以在坐骨神經(jīng)損傷后顯著促進雪旺細胞的遷移速度和遷移數(shù)量,而且對于軸突的生長也有有效的促進作用。除了考查TNC對于雪旺細胞遷移的影響之外,我們還考察了TNC對于雪旺細胞的另外兩個重要功能,即增殖和成髓鞘的影響,通過體外細胞培養(yǎng)實驗我們發(fā)現(xiàn)在培養(yǎng)液中加入1μ g/ml和10μ g/ml的TNC蛋白后,雪旺細胞的增殖速度與對照組沒有出現(xiàn)顯著差異,說明了TNC對于雪旺細胞的增殖沒有影響；之后為了考查TNC對于雪旺細胞成髓鞘能力的影響,我們使用了體外雪旺細胞-神經(jīng)元的共培養(yǎng)成髓鞘模型,我們發(fā)現(xiàn)在誘導成髓鞘的過程中加入1 μ g/ml的TNC蛋白對于雪旺細胞的成髓鞘過程會出現(xiàn)明顯的抑制。整聯(lián)蛋白是一種跨膜的異質(zhì)二聚體,它由α和β兩個非共價結合的跨膜亞基。細胞外球形結構域是一個露出細胞膜外約20 nm的頭部,頭部可同細胞外基質(zhì)蛋白結合。整聯(lián)蛋白的兩個亞基,α和β鏈都是糖基化的,并通過非共價鍵結合在一起。整聯(lián)蛋白是一種介導細胞和其外環(huán)境(主要是細胞外基質(zhì))之間的連接的跨膜受體。在信號轉導中,整聯(lián)蛋白將細胞外基質(zhì)的化學成分與力學狀態(tài)等有關信息傳入細胞并且參與了細胞通信、細胞周期以及細胞運動等多項細胞功能的調(diào)節(jié)。在周圍神經(jīng)損傷和再生的過程中,雪旺細胞發(fā)揮了非常重要的作用,而整聯(lián)蛋白也被發(fā)現(xiàn)具有影響著雪旺細胞的多種生物學行為(包括遷移,增殖及成髓鞘)的能力。我們前期的芯片數(shù)據(jù)分析預測了整聯(lián)蛋白β 1很可能作為TNC的下游基因在再生過程中起著關鍵的作用。因此為了研究TNC與整聯(lián)蛋白β1是否存在著上下游的關系,我們首先設計了整聯(lián)蛋白β1的siRNA并分別在mRNA水平和蛋白水平驗證了其基因沉默效率,之后我們發(fā)現(xiàn)在使用siRNA干擾體外培養(yǎng)雪旺細胞的整聯(lián)蛋白β1后,TNC促進雪旺細胞的遷移的效果消失了；除此之外,我們還直接在蛋白水平使用抗體封閉了雪旺細胞表面的整聯(lián)蛋白β1,與siRNA干擾的結果相似,在封閉了整聯(lián)蛋白β 1的功能后,TNC促進雪旺細胞的遷移的效果也消失了；而之后為了更為直觀的研究TNC是否能直接的與雪旺細胞表面的整聯(lián)蛋白β1相結合,我們又使用了一個免疫共沉淀實驗顯示了TNC與整聯(lián)蛋白β 1的直接結合,綜合上面的結果,我們可以得出TNC是通過結合到雪旺細胞表面的整聯(lián)蛋白β1進而影響雪旺細胞的遷移這一結論。接下來,我們檢測了整聯(lián)蛋白β1下游與遷移相關的Rho GTPase家族的幾個重要效應分子,發(fā)現(xiàn)了TNC可以誘導RhoA和Racl而非Cdc42的激活,且對其總表達量無影響。而之后的實驗也發(fā)現(xiàn)Racl的抑制劑而非RhoA的抑制劑可以顯著降低由TNC誘導的雪旺細胞遷移,進一步確定了TNC是通過結合雪旺細胞表面受體整聯(lián)蛋白β1,進一步使下游的Racl激活,以促進雪旺細胞的遷移。綜合以上結果表明,TNC在坐骨神經(jīng)再生過程中是成纖維細胞調(diào)節(jié)施萬細胞遷移的一個重要調(diào)節(jié)因子,是二者存在密切相互作用的一個新證據(jù)。成纖維細胞相對于坐骨神經(jīng)的其它組成細胞(如雪旺細胞,神經(jīng)元,巨噬細胞等)目前還缺乏相對系統(tǒng)的研究。而近幾年,已經(jīng)有越來越多的從事周圍神經(jīng)再生領域研究的學者將研究的注意力放到成纖維細胞上,尤以Alison Lloyd等人在2010年首次對于成纖維細胞和雪旺細胞的相互作用做出了深入的研究,讓我們對于周圍神經(jīng)系統(tǒng)中成纖維細胞的功能有了一個全新的認識。隨著對于周圍神經(jīng)成纖維細胞研究的繼續(xù)深入,成纖維細胞在周圍神經(jīng)再生過程中發(fā)揮的作用將會越來越多的被探索出來,極可能是未來周圍神經(jīng)再生領域研究的一個重要方向。
[Abstract]:Peripheral nerve injury is a common clinical disease. Peripheral nerve regeneration is a complex biological process involving many cellular activities. The exact mechanism of injury and regeneration of peripheral nerve has not been fully elucidated. Schwann cells in the vicinity of the injured peripheral nervous system undergo demyelination and dedifferentiate into Schwann fines similar to those in the immature state of development. In this state, Schwann cells enter the cell cycle again and begin to divide and proliferate, migrate forward along the regenerated axon, and finally form myelin sheath around the regenerated axon to repair the nerve. In order to find some key regulatory genes involved in peripheral nerve regeneration and further elucidate the role of related core genes in nerve regeneration, we have resorted to bioinformatics methods in order to discover and study the important core modulations in the process of regeneration after injury. In this study, we first established a rat model of sciatic nerve amputation by surgical operation, and then analyzed the 5 mm nerve tissue of the proximal sciatic nerve stump at 0, 1, 4, 7 and 14 days after sciatic nerve injury by expression spectrum chip technique. Random variance model was used to analyze the significance of different genes and their expression trend. We detected 6046 different genes (P 0.01, FDR 0.05). KEGG analysis showed that tenascin-C (TNC) had the most significant effect on the change of gene expression in the whole signal pathway. Nerve regeneration is at the core of the regulatory network, suggesting that TNC may play an important role in nerve regeneration. In addition, TNC has been found to promote the migration and invasion of a variety of tumor cells in the tumor system. In related studies of the central nervous system, TNC has also been found to promote axonal growth. However, there are few studies on TNC in the peripheral nervous system, and TNC binding precedes it. In subsequent in-depth studies, we found that TNC in the proximal sciatic nerve was maintained at a very high level 4 days after sciatic nerve transection. After that, we detected TNC and Schwann cell marker S100beta, macrophage marker CD68 and fibroblast marker P4HB (prolyl-4-hydroxylase b) by immunofluorescence. The co-localization of ETA showed that TNC almost did not co-localize with Schwann cells and macrophages after peripheral nerve injury, but co-localized well with fibroblasts, suggesting that fibroblasts would express TNC in large quantities after sciatic nerve injury. Fibroblasts play an important role in tissue repair by cell proliferation and intercellular matrix formation. In wound healing, for example, fibroblasts proliferate through mitosis, and synthesize and secrete a large number of collagen fibers and matrix components, which participate in tissue repair and repair after injury. Alison Lloyd et al. found in 2010 that after peripheral nerve injury, fibroblasts aggregated to the injured site and directed Schwann cells to arrange axially through EphB signals to promote peripheral nerve regeneration. Based on this report and our findings, fibroblasts secrete a large amount of TNC after nerve injury, we propose that To verify this hypothesis, we first designed a transwell-based fibroblast-Schwann cell co-culture system in vitro. We found that the underlying layer of the Transwell co-culture system The number of fibroblasts seeded into the cells affected the number of Schwann cells that migrated through the Transwell chamber, which was in direct proportion to the number of fibroblasts. This proved that fibroblasts did have the ability to affect the migration of Schwann cells and the effectiveness of the co-culture system. We designed siRNA for TNC as a mediator, and validated the gene silencing efficiency of siRNA at RNA and protein levels. Transwell and cell scratch experiments showed that inhibition of TNC expression in fibroblasts significantly reduced the migration ability of Schwann cells co-cultured with TNC; and then we made it possible to inhibit the expression of TNC in fibroblasts. In vitro cultured Schwann cells were treated with exogenous TNC. It was found that the addition of 1 and 10 ug/ml TNC proteins in the culture medium could effectively promote the migration of Schwann cells. Combined with the above results, TNC was an important mediator of fibroblasts affecting the migration of Schwann cells. Compared with the control group, exogenous 1 ug/ml and 10 ug/ml TNC protein could significantly promote the migration rate and quantity of Schwann cells after sciatic nerve injury, and also promote the growth of axons. In addition to examining the effect of TNC on the migration of Schwann cells, we also investigated the effect of TNC on Schwann cells. Two other important functions, proliferation and myelination, we found that the proliferation rate of Schwann cells was not significantly different from that of the control group after adding 1 and 10 ug/ml TNC protein in the culture medium, which indicated that TNC had no effect on the proliferation of Schwann cells. We used the Schwann cell-neuron co-culture myelination model in vitro to investigate the effect of 1 ug/ml TNC protein on the myelination of Schwann cells. Two non-covalently bound transmembrane subunits of beta. An extracellular spherical domain is a head that exposes an extracellular membrane about 20 nm and binds to extracellular matrix proteins. Both subunits of the integrin, alpha and beta chains, are glycosylated and bind through non-covalent bonds. The integrin is a mediator of cells and their external environment (mainly In signal transduction, integrins transmit information about the chemical composition and mechanical state of the extracellular matrix into cells and participate in the regulation of cellular communication, cell cycle and cell movement. In the process of peripheral nerve injury and regeneration, Schwann is fine. Cells play a very important role, and integrins have been found to influence a variety of biological behaviors of Schwann cells, including migration, proliferation and myelination. Our previous data analysis on chip data predicted that integrin beta 1 may play a key role in the regeneration process as a downstream gene of TNC. To investigate the relationship between TNC and integrin beta 1, we first designed siRNA of integrin beta 1 and validated its gene silencing efficiency at mRNA and protein levels, respectively. Then we found that TNC promoted the migration of Schwann cells by interfering with integrin beta 1 of Schwann cells cultured in vitro with siRNA. In addition, we blocked integrin beta 1 on the surface of Schwann cells directly at the protein level with antibodies, similar to the results of siRNA interference. After blocking the function of integrin beta 1, the effect of TNC on promoting Schwann cell migration disappeared; and then, for a more intuitive study of whether TNC can directly interact with Schwann cells. Combining the above results, we can conclude that TNC affects the migration of Schwann cells by binding to integrin beta 1 on the surface of Schwann cells. Several important effector molecules of the Rho GTPase family associated with migration downstream of integrin beta 1 were measured and TNC was found to induce the activation of RhoA and Racl rather than Cdc42 without affecting the total expression of RhoA. TNC is further activated by binding to Schwann cell surface receptor integrin beta 1 to promote the migration of Schwann cells. These results suggest that TNC is an important regulator of Schwann cell migration by fibroblasts during sciatic nerve regeneration, and there is a close interaction between TNC and Schwann cell migration. There is a new evidence that fibroblasts are not systematically studied in relation to other components of the sciatic nerve (such as Schwann cells, neurons, macrophages, etc.). In recent years, more and more researchers in the field of peripheral nerve regeneration have focused their attention on fibroblasts, especially Alison Lloyd. The first in-depth study of the interaction between fibroblasts and Schwann cells in 2010 has led to a new understanding of the function of fibroblasts in the peripheral nervous system. The role will be more and more explored, which is likely to be an important direction in the field of peripheral nerve regeneration in the future.
【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R741

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