TWI555841B - Carbonized materials and methods of promoting cell differentiation using carbonized materials - Google Patents
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本發明係與細胞分化有關;特別是指一種碳化材料及利用碳化材料促進細胞分化之方法。 The present invention relates to cell differentiation; in particular, to a carbonized material and a method for promoting cell differentiation using a carbonized material.
在神經科學的醫療研究突飛猛進的現代,已發現有多種腦神經病變的原因。以帕金森氏症為例,其病因是由於人類中樞神經系統中本應釋放神經傳導物質-多巴胺的神經元受損或死亡;而多巴胺對於腦中基底核在運動功能的調控是不可或缺的,故多巴胺的數量減少會使得人類的運動協調功能受損。 In the modern advancement of neuroscience medical research, a variety of causes of cranial neuropathy have been discovered. In the case of Parkinson's disease, the cause is due to damage or death of neurons in the human central nervous system that should release neurotransmitter-dopamine. Dopamine is indispensable for the regulation of motor function in the basal ganglia of the brain. Therefore, the reduction in the number of dopamine will impair human motor coordination.
罹病初期可透過多巴胺藥物彌補患者供應不足的多巴胺,而能有效控制用運動功能受損的問題。但藥物治療無法控制嚴重病患之症狀,甚至僅會帶來副作用,此時便需要外科手術的介入。其中,細胞替代療法(cell replacement therapy)是通過移植正常細胞來取代受損細胞,或藉此為受損神經細胞的再生提供一個橋樑,使神經系統的傳導通路完整或再生,以恢復神經傳導功能。 In the early stage of rickets, dopamine can be used to make up for the lack of dopamine in patients, and it can effectively control the problem of impaired motor function. However, drug treatment cannot control the symptoms of serious patients, and even only causes side effects. At this time, surgical intervention is required. Among them, cell replacement therapy replaces damaged cells by transplanting normal cells, or provides a bridge for the regeneration of damaged nerve cells, and completes or regenerates the conduction pathway of the nervous system to restore nerve conduction function. .
而臨床上常取得的正常神經細胞係由人類神經幹細胞(human neural stem cells;hNSC)分化而來。在過去,人類神經幹細胞係培養於平面的培養皿上,其分化率僅約2%;即使在添加分化因子(differentiation factor;DF)後,其分化率最高也僅能提升至約18%,分化效率著實不高。是以,雖然細胞替代療法避免了藥物治療帶來的副作用,但細胞分化的效率確實有待提升。 The normal neural cell line that is often obtained clinically is differentiated from human neural stem cells (hNSC). In the past, human neural stem cell lines were cultured on flat culture dishes with a differentiation rate of only about 2%; even after the addition of differentiation factor (DF), the differentiation rate was only increased to about 18%, differentiation. Efficiency is not high. Therefore, although cell replacement therapy avoids the side effects of drug treatment, the efficiency of cell differentiation does need to be improved.
有鑑於此,本發明之目的在於提供一種碳化材料及利用碳化材料促進細胞分化之方法,能藉由該碳化材料的立體堆疊結構特性模擬生物體內的環境,即,形成一立體支架以促進細胞分化。 In view of the above, an object of the present invention is to provide a carbonized material and a method for promoting cell differentiation by using a carbonized material, which can simulate the environment in a living body by the three-dimensional stacked structure of the carbonized material, that is, form a stereo scaffold to promote cell differentiation. .
緣以達成上述目的,本發明所提供利用碳化材料促進細胞分化之方法包含以下步驟:A、提供一纖維織布,其中該該纖維織布包含一聚丙烯腈纖維;B、高溫碳化該纖維織布以產生一碳化材料;其中該纖維織布係在壓力大於100托耳且小於一大氣壓,且溫度介於500至1200℃的環境下被碳化;高溫碳化的時間介於1至25分鐘,且碳化過程中供應一惰性氣體於碳化環境;該碳化材料包含有複數個碳纖維,其中各該碳纖維包含有一本體與複數個碳柱,該些碳柱係形成於該本體表面,且該些碳柱以各種方向相互堆疊;以及C、將一細胞置於該碳化材料上,其中該細胞具有分化能力,可分化為具有一特定功能之一分化細胞;藉此,該細胞於分化過程中透過依附該些碳柱,以促進其分化功能,加速該細胞分化為具有該特定功能之該分化細胞。 In order to achieve the above object, the method for promoting cell differentiation by using a carbonized material according to the present invention comprises the following steps: A, providing a fiber woven fabric, wherein the fiber woven fabric comprises a polyacrylonitrile fiber; B, high temperature carbonizing the fiber woven fabric Clothing to produce a carbonized material; wherein the fiber woven fabric is carbonized at a pressure greater than 100 Torr and less than one atmosphere and at a temperature between 500 and 1200 ° C; the high temperature carbonization time is between 1 and 25 minutes, and An inert gas is supplied to the carbonization environment during the carbonization process; the carbonization material comprises a plurality of carbon fibers, wherein each of the carbon fibers comprises a body and a plurality of carbon columns, the carbon columns are formed on the surface of the body, and the carbon columns are Various directions are stacked on each other; and C, a cell is placed on the carbonized material, wherein the cell has the ability to differentiate and can differentiate into a differentiated cell having a specific function; thereby, the cell is attached to the differentiation process during differentiation A carbon column is used to promote its differentiation function, accelerating the differentiation of the cell into the differentiated cells having this specific function.
本發明另提供一種作為促進細胞分化之基質的碳化材料,係以以下步驟所製成:A、提供一纖維織布,其中該該纖維織布包含一聚丙烯腈纖維;B、高溫碳化該纖維織布以產生一碳化材料;其中該纖維織布係在壓力大於100托耳且小於一大氣壓,且溫度介於500至1200℃的環境下被碳化;高溫碳化的時間介於1至25分鐘,且碳化過程中供應一惰性氣體於碳化環境;其中,該碳化材料包含有複數個碳纖維,且各該碳纖維包含有一本體與複數個碳柱,該些碳柱係形成於該本體表面,且該些碳柱以各種方向相互堆疊。 The present invention further provides a carbonized material as a substrate for promoting cell differentiation, which is produced by the following steps: A, providing a fiber woven fabric, wherein the fiber woven fabric comprises a polyacrylonitrile fiber; B, high temperature carbonizing the fiber Woven to produce a carbonized material; wherein the fiber woven fabric is carbonized at a pressure greater than 100 Torr and less than one atmosphere and at a temperature between 500 and 1200 ° C; the carbonization time is between 1 and 25 minutes. And supplying an inert gas to the carbonization environment during the carbonization process; wherein the carbonization material comprises a plurality of carbon fibers, and each of the carbon fibers comprises a body and a plurality of carbon columns, and the carbon columns are formed on the surface of the body, and the The carbon columns are stacked on each other in various directions.
本發明之效果在於,該纖維織布碳化所形成之碳化材料提供了醫療方面的新用途,且能有效提升細胞分化的效率。 The effect of the present invention is that the carbonized material formed by the carbonization of the fiber woven fabric provides a new medical use and can effectively improve the efficiency of cell differentiation.
100‧‧‧碳化材料 100‧‧‧Carbide
10‧‧‧碳纖維 10‧‧‧Carbon fiber
12‧‧‧本體 12‧‧‧Ontology
14‧‧‧碳柱 14‧‧‧ carbon column
20‧‧‧細胞 20‧‧‧ cells
22‧‧‧軸突 22‧‧‧ axons
24‧‧‧多巴胺 24‧‧‧Dopamine
D1‧‧‧高度 D1‧‧‧ Height
D2‧‧‧直徑 D2‧‧‧ diameter
θ‧‧‧接觸角 Θ‧‧‧contact angle
圖1係本發明一較佳實施例利用碳化材料促進細胞分化之方法之流程圖。 1 is a flow chart of a method for promoting cell differentiation using a carbonized material in accordance with a preferred embodiment of the present invention.
圖2係本發明上述較佳實施例之碳化材料之立體圖,揭示碳化材料於電子顯微鏡下的成像。 2 is a perspective view of a carbonized material of the above preferred embodiment of the present invention, showing the imaging of the carbonized material under an electron microscope.
圖3係本發明上述較佳實施例之碳化材料之立體圖,揭示碳柱於電子顯微鏡下的成像。 Figure 3 is a perspective view of the carbonized material of the above preferred embodiment of the present invention, revealing the imaging of the carbon column under an electron microscope.
圖4係本發明上述較佳實施例之碳化材料之示意圖。 Figure 4 is a schematic illustration of the carbonized material of the above preferred embodiment of the present invention.
圖5係神經幹細胞附著於習用之細胞培養基質上進行分化之立體圖。 Fig. 5 is a perspective view showing the differentiation of neural stem cells attached to a conventional cell culture medium.
圖6係本發明上述較佳實施例之碳化材料之示意圖,揭示其碳柱之立體結構可促進神經幹細胞的分化。 Fig. 6 is a schematic view showing the carbonized material of the above preferred embodiment of the present invention, revealing that the three-dimensional structure of the carbon column promotes differentiation of neural stem cells.
為能更清楚地說明本發明,茲舉較佳實施例並配合圖示詳細說明如後,請參圖1所示,為本發明一較佳實施例利用一碳化材料100促進細胞分化之方法的流程圖。 In order to explain the present invention more clearly, the preferred embodiment will be described in detail with reference to the accompanying drawings. Referring to FIG. 1 , a method for promoting cell differentiation using a carbonized material 100 is illustrated in a preferred embodiment of the present invention. flow chart.
該碳化材料100係一碳纖維織物(carbon fiber fabric),本案申請人已於中華民國專利公告號第I445661公開其碳化方法及使用之碳化裝置,製程約略如下:首先,提供一纖維織布,其中該該纖維織布包含一聚丙烯腈纖維;接著,高溫碳化該纖維織布以產生該碳化材料100。其中該纖維織布係在壓力大於100托耳且小於一大氣壓,且溫度介於500至1200℃的環境下被碳化;高溫碳化的時間介於1至25 分鐘。此外,碳化過程中供應一惰性氣體(如氮氣)於碳化環境中,以避免該纖維織布與空氣中的氧氣接觸而產生氧化反應。其他詳細製程容此不加贅述。 The carbonized material 100 is a carbon fiber fabric. The carbonization method and the carbonization device used by the applicant have been disclosed in the Chinese Patent Publication No. I445661. The process is roughly as follows: First, a fiber woven fabric is provided, wherein The fiber woven fabric comprises a polyacrylonitrile fiber; then, the fiber woven fabric is carbonized at a high temperature to produce the carbonized material 100. Wherein the fiber woven fabric is carbonized under a pressure of more than 100 Torr and less than one atmosphere, and the temperature is between 500 and 1200 ° C; the time of high temperature carbonization is between 1 and 25 minute. In addition, an inert gas (such as nitrogen) is supplied to the carbonization environment during the carbonization process to prevent the fiber woven fabric from coming into contact with oxygen in the air to generate an oxidation reaction. Other detailed procedures are not mentioned here.
請配合圖2所示,該碳化材料100包含有複數個縱橫交錯的碳纖維束,各該碳纖維束又以複數個線狀的碳纖維10集合而成。其特徵在於,若透過電子顯微鏡並以高倍數觀察各該碳纖維10,可觀察到各該碳纖維10除包含有一線狀的本體12,尚包含有複數個碳柱14;該些碳柱14位於該本體12表面,並以各種方向堆疊(圖3參照)。 As shown in FIG. 2, the carbonized material 100 includes a plurality of cross-hatched carbon fiber bundles, and each of the carbon fiber bundles is assembled from a plurality of linear carbon fibers 10. It is characterized in that, when the carbon fibers 10 are observed at a high magnification by an electron microscope, it can be observed that each of the carbon fibers 10 includes a plurality of carbon columns 14 in addition to a linear body 12; the carbon columns 14 are located therein. The body 12 is surfaced and stacked in various directions (see Figure 3 for reference).
請配合圖4所示,為了更清楚定義該些碳柱14的尺寸,以及該些碳柱14與細胞的相對關係,圖中的該些碳柱14以軸向垂直表面的矩陣排列表示。其中,各該碳柱14的高度D1約為0.001至5mm;且各該碳柱14的直徑D2約為1至4μm。 Please refer to FIG. 4, in order to more clearly define the size of the carbon columns 14, and the relative relationship of the carbon columns 14 to the cells, the carbon columns 14 in the figure are represented by a matrix arrangement of axial vertical surfaces. The height D1 of each of the carbon columns 14 is about 0.001 to 5 mm; and the diameter D2 of each of the carbon columns 14 is about 1 to 4 μm.
最後,將一具有分化能力的細胞20置於該碳化材料100上,於此實施例中,該細胞20為神經幹細胞,但並不以神經幹細胞為限制,而亦可以為類神經細胞等同樣可以分化為一神經細胞的前驅細胞。神經細胞的特定功能係長出至少一軸突,並透過該軸突末稍釋放多巴胺,以將訊息自該細胞傳遞至其他細胞。 Finally, a cell 20 having differentiation ability is placed on the carbonized material 100. In this embodiment, the cell 20 is a neural stem cell, but is not limited by a neural stem cell, but may also be a neuron-like cell. Differentiate into a precursor cell of a nerve cell. The specific function of the nerve cells grows at least one axon and releases dopamine through the axon to transfer information from the cell to other cells.
請配合圖5所示,神經幹細胞在過去都是培養於平面的細胞培養基質1上以進行分化,但分化率低。換言之,該細胞20長出軸突22的速度較慢,且釋放多巴胺24以傳遞訊息的功能較低。此外,由於該細胞20係整體貼附於基質1上,故該細胞20所貼附之基質1的表面與該細胞20最高點所構成的接觸角θ小,並不利於細胞的生長、移動及分化。 As shown in Fig. 5, neural stem cells have been cultured on a flat cell culture substrate 1 for differentiation in the past, but the differentiation rate is low. In other words, the cell 20 develops a slower rate of axonal 22 and releases dopamine 24 to deliver a lower level of function. In addition, since the cell 20 is attached to the substrate 1 as a whole, the contact angle θ formed by the surface of the substrate 1 to which the cell 20 is attached and the highest point of the cell 20 is small, which is not conducive to cell growth and movement. Differentiation.
請配合圖6所示,相較於過去將細胞20置於 基質1,本實施例碳化材料100之該碳柱14供該細胞20於分化過程中附著,而能有利於該細胞20之軸突22的形成,同時促進軸突22末端釋出多巴胺24。此外,本發明之該碳化材料100具有導電的特性,詳而言之,該碳化材料100中的碳原子是以sp2混成軌域相互鍵結,故具有容易吸引電子的特性。由於多巴胺24釋放電子,故釋出多巴胺24的軸突22末稍能被該碳柱14吸引,而能牢固的附著於該碳柱14上。該細胞20藉由軸突22攀附於該些碳柱14上,使得細胞不單只是貼附於該本體12表面,而能使該接觸角θ增大,有利於細胞的生長、移動及分化。 Referring to FIG. 6, the carbon column 14 of the carbonized material 100 of the present embodiment is attached to the cell 20 during the differentiation process, and the axon of the cell 20 can be favored. The formation of 22 simultaneously promotes the release of dopamine 24 at the ends of axons 22. Further, the carbonized material 100 of the present invention has an electrically conductive property. In detail, the carbon atoms in the carbonized material 100 are bonded to each other by the sp 2 mixed orbital region, and thus have characteristics of easily attracting electrons. Since the dopamine 24 releases electrons, the axon 22 releasing the dopamine 24 is slightly attracted to the carbon column 14 and can be firmly attached to the carbon column 14. The cells 20 are attached to the carbon columns 14 by the axons 22, so that the cells not only attach to the surface of the body 12, but also increase the contact angle θ, which is favorable for cell growth, movement and differentiation.
此外,該細胞20在分化過程中除了型態上的改變及多巴胺的釋放外,其細胞20內的酪氨酸羥化酶(tyrosine hydroxylase)濃度亦會提升。酪氨酸羥化酶是將細胞20內之酪氨酸轉變為多巴胺24的重要酵素,故酪氨酸羥化酶在細胞內的濃度及活性可作為神經幹細胞分化的指標。和貼附在基質1的細胞20相比,附著於該碳化材料100進行分化的細胞20具有較高的酪氨酸羥化酶濃度及活性。由此可知,該碳化材料100確實促進了細胞20的型態改變以及與分化相關的細胞內訊息傳遞,而有利於細胞20的分化。 In addition, the cell 20 has a higher concentration of tyrosine hydroxylase in the cell 20 in addition to a change in morphology and release of dopamine during differentiation. Tyrosine hydroxylase is an important enzyme for converting tyrosine in cell 20 into dopamine 24. Therefore, the concentration and activity of tyrosine hydroxylase in cells can be used as an indicator of neural stem cell differentiation. The cells 20 adhering to the carbonized material 100 for differentiation have higher tyrosine hydroxylase concentration and activity than the cells 20 attached to the matrix 1. It can be seen that the carbonized material 100 does promote the type change of the cells 20 and the intracellular signal transmission associated with differentiation, and facilitates the differentiation of the cells 20.
綜上所述,本發明利用碳化材料作為促進細胞分化之基質,係利用該碳化材料100的立體結構及導電的特性,促進該細胞20於其上的生長及分化。 In summary, the present invention utilizes a carbonized material as a matrix for promoting cell differentiation, and utilizes the three-dimensional structure and conductive properties of the carbonized material 100 to promote growth and differentiation of the cell 20 thereon.
本實施例碳化材料100可進一步應用於一培養皿上,其中該培養皿具有一底板與一內部空間,且該底板具有一上表面面對該內部空間。該碳化材料100係結合於該上表面,故當細胞20種於該培養皿之底板的上表面時,便可附著於該碳化材料100上生長,藉此促進該細胞20的分化。 The carbonized material 100 of the present embodiment can be further applied to a culture dish, wherein the culture dish has a bottom plate and an inner space, and the bottom plate has an upper surface facing the inner space. The carbonized material 100 is bonded to the upper surface, so that when the cells 20 are placed on the upper surface of the bottom plate of the culture dish, they can be attached to the carbonized material 100 to grow, thereby promoting differentiation of the cells 20.
上述包含該碳化材料100之亦可用於重建因疾病或受傷所造成的神經缺損。傷口處的組織和血液中存在組織再生所需的幹細胞,故若要修復神經缺損,需要在神經缺損處搭建生物支架,以供細胞附著、增殖及分化。然而,利用習用的生物支架僅能支持一定程度的組織再生,且該生物支架本身並不具有誘導組織再生及分化的能力。若以本發明碳化材料100做為搭建於神經缺損處的生物支架,即可加速傷口處幹細胞的生長及分化,而能達到更佳的神經重建效果。 The inclusion of the carbonized material 100 described above can also be used to reconstruct a nerve defect caused by a disease or injury. There are stem cells required for tissue regeneration in the tissue and blood at the wound. Therefore, if nerve defects are to be repaired, it is necessary to construct a biological scaffold at the nerve defect for cell attachment, proliferation and differentiation. However, the use of conventional biological scaffolds can only support a certain degree of tissue regeneration, and the bioscaffold itself does not have the ability to induce tissue regeneration and differentiation. If the carbonized material 100 of the present invention is used as a biological stent built on a nerve defect, the growth and differentiation of stem cells at the wound can be accelerated, and a better nerve reconstruction effect can be achieved.
以上所述僅為本發明較佳可行實施例而已,舉凡應用本發明說明書及申請專利範圍所為之等效變化,理應包含在本發明之專利範圍內。 The above is only a preferred embodiment of the present invention, and equivalent changes to the scope of the present invention and the scope of the patent application are intended to be included in the scope of the present invention.
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TWI445661B (en) * | 2008-04-16 | 2014-07-21 | Linkwin Technology Co Ltd | Continuous negative pressure carbonization apparatus and method of the same |
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