基于TiO_2/Ti复合膜的人工细胞制备及体外研究
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摘要
人工细胞是免疫隔离技术与细胞移植技术相结合的产物,它能解决组织器官移植中存在的两大难题——供体来源稀缺和机体免疫排斥反应。目前,制备人工细胞的材料以水凝胶类高分子材料为主,不仅存在吸水膨胀、机械强度差、会降解等缺点,而且材料降解后会导致移植细胞携带的病毒侵入人体,从而使宿主受到伤害,甚至带来不可预期的后果。鉴于此,本文首次提出了采用TiO2/Ti复合膜制备人工细胞的方法,通过免疫隔离效果实验研究了该方法的可行性及最佳制备条件,并由此制备出人工胰岛细胞和人工下丘脑神经细胞。研究内容及成果如下:
为了制备出适用于人工细胞的选择性半透膜,以多孔Ti为基底,采用溶胶―凝胶法,通过浸渍提拉制成TiO2/Ti复合膜。该膜用作人工细胞膜的制备条件如下:基底的平均孔径为1-2μm时,烧结温度低于800℃,且涂膜次数必须超过3次。但当基底的平均孔径小于1μm时,则需将烧结温度控制在600-700℃,且涂膜次数必须少于3次才能得到理想的TiO2/Ti复合膜。
为了保证被包囊细胞的存活生长,将下丘脑神经细胞接种于TiO2/Ti复合膜后进行体外培养。实验结果表明,复合膜的生物活性随金红石型TiO2含量的提高而增强,即被包囊细胞的存活率随复合膜中金红石型TiO2含量的增高而增大。
首次制备出基于TiO2/Ti复合膜的人工下丘脑神经细胞。它能根据外界环境变化自行调整β-内啡肽的分泌量,以保证细胞内外液中β-内啡肽的水平处于一个稳定的平衡状态,而且其分泌物能有效抑制纳络酮催促的吗啡依赖性离体大鼠回肠所产生的戒断性收缩,故阿片依赖有望通过人工下丘脑神经细胞移植方法治愈。
首次制备出基于TiO2/Ti复合膜的人工胰岛细胞,它能根据外界葡萄糖浓度自行调整胰岛素的释放量,故可用于细胞移植治疗糖尿病。这将成为治愈糖尿病最有前景的治疗方案之一。
综上所述,通过控制工艺条件可以制备出具有良好免疫隔离效果的TiO2/Ti复合膜。采用该材料制备的人工细胞具有不可降解、生物活性好、机械强度高等优点,可有力促进人工细胞移植尤其是异种细胞移植的临床应用发展。
The artificial cells were developed by combination of immunoisolation and cell transplantation. It can resolve the two problems of organ transplantation―limited donor source and immunological rejection. At present, the artificial cell is mostly made from gelatinous natural or synthetic polymer material. The defects in such material prevent atificial cells from applying to clinic, such as water swelling, poor mechanical strength, residual toxicity monomer and so on, especially, some virus of transplanted cells maybe invade into human body and result in unexpected effects. In view of this, the artificial cells based on TiO2/Ti composite membrane were firstly presented. The feasibility and preparation conditions were investigated by the experiment of immunoisolation effect. The artificial hypothalamic neurons and artificial islet cells were firstly made from TiO2/Ti composite membranes. The contents and productions of research were summarized as follows.
The TiO2/Ti composite membranes with support of porous Ti were prepared by sol-gel method. The prepared condition of the composition membrane in application to artifical cell as follows. When the average pore diameter of porous Ti support was between 1-2μm, the better sintering temperature and coating times of TiO2/Ti composition, which have good effect on immunoisolation, were less than 800℃and exceeded 3 times, respectively. If the average pore diameter of porous Ti support was less than 1μm, the sintering temperature must be controlled at 600-700℃, and the coating times must be less than 3times.
The hypothalamic neurons planted in TiO2/Ti composite membrane and cultured in vitro. The results showed that the bioactivity of TiO2/Ti composite membranes was enhanced by increasing the content of rutile TiO2.
The artifical hypothalamic neurons based on TiO2/Ti composite membranes were firstly prepared. The encapsulated hypothalamic neurons not only survived and grew, but also could adjust theβ-endorphin secretion by themselves, in order to remain a stable balance ofβ-endorphin level between in intracellular fluid and in extracellular fluid. The secretion of artifical neurons could effectively inhibit the naloxone-precipitated withdrawal contracture in isolated ilea of rates, which were exposed to morphine. From these results, it was presumed that opiate dependence could be cured by artificial hypothalamic neurons transplantation.
The artifical islet cells based on TiO2/Ti composite membranes were firstly prepared by transition fit. The kind of artifical islet cells could automatic adjust the release of insulin based on the concentration of glucose in extracelluar fluid. It is a promising treatment of diabetes.
Summing up above results, the TiO2/Ti composite membranes, which have good immunoisolation effect, can be prepared by adjusting the condition of preparation, such as sintering temperature, coating times. The artifical cell based on such material have the virtue of undecomposition, good bioactivity and high mechanical strength and can promote the development of artifical cell transplantation, especially in the development of clinic application of xenotransplantation.
为了制备出适用于人工细胞的选择性半透膜,以多孔Ti为基底,采用溶胶―凝胶法,通过浸渍提拉制成TiO2/Ti复合膜。该膜用作人工细胞膜的制备条件如下:基底的平均孔径为1-2μm时,烧结温度低于800℃,且涂膜次数必须超过3次。但当基底的平均孔径小于1μm时,则需将烧结温度控制在600-700℃,且涂膜次数必须少于3次才能得到理想的TiO2/Ti复合膜。
为了保证被包囊细胞的存活生长,将下丘脑神经细胞接种于TiO2/Ti复合膜后进行体外培养。实验结果表明,复合膜的生物活性随金红石型TiO2含量的提高而增强,即被包囊细胞的存活率随复合膜中金红石型TiO2含量的增高而增大。
首次制备出基于TiO2/Ti复合膜的人工下丘脑神经细胞。它能根据外界环境变化自行调整β-内啡肽的分泌量,以保证细胞内外液中β-内啡肽的水平处于一个稳定的平衡状态,而且其分泌物能有效抑制纳络酮催促的吗啡依赖性离体大鼠回肠所产生的戒断性收缩,故阿片依赖有望通过人工下丘脑神经细胞移植方法治愈。
首次制备出基于TiO2/Ti复合膜的人工胰岛细胞,它能根据外界葡萄糖浓度自行调整胰岛素的释放量,故可用于细胞移植治疗糖尿病。这将成为治愈糖尿病最有前景的治疗方案之一。
综上所述,通过控制工艺条件可以制备出具有良好免疫隔离效果的TiO2/Ti复合膜。采用该材料制备的人工细胞具有不可降解、生物活性好、机械强度高等优点,可有力促进人工细胞移植尤其是异种细胞移植的临床应用发展。
The artificial cells were developed by combination of immunoisolation and cell transplantation. It can resolve the two problems of organ transplantation―limited donor source and immunological rejection. At present, the artificial cell is mostly made from gelatinous natural or synthetic polymer material. The defects in such material prevent atificial cells from applying to clinic, such as water swelling, poor mechanical strength, residual toxicity monomer and so on, especially, some virus of transplanted cells maybe invade into human body and result in unexpected effects. In view of this, the artificial cells based on TiO2/Ti composite membrane were firstly presented. The feasibility and preparation conditions were investigated by the experiment of immunoisolation effect. The artificial hypothalamic neurons and artificial islet cells were firstly made from TiO2/Ti composite membranes. The contents and productions of research were summarized as follows.
The TiO2/Ti composite membranes with support of porous Ti were prepared by sol-gel method. The prepared condition of the composition membrane in application to artifical cell as follows. When the average pore diameter of porous Ti support was between 1-2μm, the better sintering temperature and coating times of TiO2/Ti composition, which have good effect on immunoisolation, were less than 800℃and exceeded 3 times, respectively. If the average pore diameter of porous Ti support was less than 1μm, the sintering temperature must be controlled at 600-700℃, and the coating times must be less than 3times.
The hypothalamic neurons planted in TiO2/Ti composite membrane and cultured in vitro. The results showed that the bioactivity of TiO2/Ti composite membranes was enhanced by increasing the content of rutile TiO2.
The artifical hypothalamic neurons based on TiO2/Ti composite membranes were firstly prepared. The encapsulated hypothalamic neurons not only survived and grew, but also could adjust theβ-endorphin secretion by themselves, in order to remain a stable balance ofβ-endorphin level between in intracellular fluid and in extracellular fluid. The secretion of artifical neurons could effectively inhibit the naloxone-precipitated withdrawal contracture in isolated ilea of rates, which were exposed to morphine. From these results, it was presumed that opiate dependence could be cured by artificial hypothalamic neurons transplantation.
The artifical islet cells based on TiO2/Ti composite membranes were firstly prepared by transition fit. The kind of artifical islet cells could automatic adjust the release of insulin based on the concentration of glucose in extracelluar fluid. It is a promising treatment of diabetes.
Summing up above results, the TiO2/Ti composite membranes, which have good immunoisolation effect, can be prepared by adjusting the condition of preparation, such as sintering temperature, coating times. The artifical cell based on such material have the virtue of undecomposition, good bioactivity and high mechanical strength and can promote the development of artifical cell transplantation, especially in the development of clinic application of xenotransplantation.
引文
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