【摘要】：肝功能衰竭是臨床上極具挑戰(zhàn)性的一種病癥，雖然肝移植可挽救許多肝病末期的患者的生命，但是由于肝臟供體的短缺等原因，許多患者在等待肝移植的過(guò)程中死亡。20世紀(jì)逐漸發(fā)展的生物人工肝支持系統(tǒng)，主要是用來(lái)為肝衰竭患者提供體外肝臟功能支持的方法，經(jīng)過(guò)多年的發(fā)展，生物人工肝技術(shù)已經(jīng)逐漸成熟，成為肝衰竭患者治療的新方法之一。 本課題旨在研究生物人工肝中肝細(xì)胞培養(yǎng)所需的微載體的制備工藝，制備出性能良好的多孔微載體，并培養(yǎng)肝細(xì)胞，驗(yàn)證其良好的生物相容性，為臨床上肝病的治療打下基礎(chǔ)。 1.以殼聚糖為基質(zhì)，通過(guò)共混法引入海藻酸鈉或N-羧丙酰殼聚糖鈉，采用乳液冷凍干燥法，并結(jié)合正戊醇和醋酸銨作致孔劑來(lái)制備海藻酸鈉/殼聚糖多孔微載體和N-羧丙酰殼聚糖鈉/殼聚糖微載體，，在該微載體上培養(yǎng)人肝細(xì)胞L-02。 2.用掃描電子顯微鏡觀察微載體的微觀結(jié)構(gòu)形貌，以吸水率、體外降解率、MTT比色等指標(biāo)，綜合評(píng)價(jià)海藻酸鈉/殼聚糖微載體和N-羧丙酰殼聚糖鈉/殼聚糖微載體的性能及生物活性。 3.對(duì)于N-羧丙酰殼聚糖鈉/殼聚糖微載體，選擇的致孔劑不同，所得的微載體的表觀形貌也不同。以正戊醇為致孔劑，凍干得到的微載體孔徑為3-55m，孔隙率為88%；而以正戊醇和醋酸銨兩種致孔劑得到的支架孔徑為15-55m，孔隙率為94%。兩種方法得到的支架硬度高，溶脹性良好，吸水率高，兩種空白N-羧丙酰殼聚糖鈉/殼聚糖支架在體外可完全降解。光學(xué)顯微鏡觀察L-02人肝細(xì)胞在N-羧丙酰殼聚糖鈉/殼聚糖支架上生長(zhǎng)良好。 4.對(duì)于海藻酸鈉/殼聚糖微載體，以正戊醇為致孔劑，凍干得到的微載體孔徑為3-50m，孔隙率為89%；以正戊醇和醋酸銨兩種致孔劑制孔得到的微載體孔徑為15-55m，孔隙率為95%。且兩種方法得到的微載體硬度高，溶脹性良好，吸水率高，空白海藻酸鈉/殼聚糖微載體在體外可完全降解。光學(xué)顯微鏡觀察L-02人肝細(xì)胞在海藻酸鈉/殼聚糖微載體上生長(zhǎng)良好。 綜上所述，本課題所制備的多孔微載體具有良好的生物學(xué)性能，可滿足生物人工肝對(duì)肝細(xì)胞培養(yǎng)的要求。
[Abstract]:Liver failure is a very challenging disease in clinic. Although liver transplantation can save the lives of many patients at the end of liver disease, it is due to the shortage of liver donors and so on. Many patients died while waiting for liver transplantation. The biological artificial liver support system developed gradually in the 20th century, mainly used to provide in vitro liver function support for patients with liver failure, after years of development. Biological artificial liver technology has gradually matured and become one of the new methods for the treatment of liver failure patients. The purpose of this paper is to study the preparation of microcarriers for hepatocyte culture in bioartificial liver, to prepare porous microcarriers with good performance, and to culture liver cells to verify their good biocompatibility. For the clinical treatment of liver disease lay the foundation. 1. Sodium alginate or sodium N-carboxypropionyl chitosan was introduced by blending with chitosan as the matrix, and the emulsion freeze-drying method was used. Sodium alginate / chitosan porous microcarriers and N-carboxypropionyl chitosan / chitosan microcarriers were prepared by using n-pentanol and ammonium acetate as pore-inducing agents. L-02.2 human hepatocytes were cultured on the microcarriers. The microstructure and morphology of microcarriers were observed by scanning electron microscope (SEM). The indexes of water absorption, in vitro degradation rate and MTT colorimetric analysis were used. The properties and biological activities of sodium alginate / chitosan microcarriers and N-carboxypropionyl chitosan / chitosan microcarriers were comprehensively evaluated. For N-carboxypropionyl chitosan / chitosan microcarriers, the morphology of the microcarriers was different with different pore-forming agents. Using n-pentanol as the pore-inducing agent, the pore size of the microcarrier was 3-55m and the porosity was 88m, while the pore size of the scaffold was 15-55m and the porosity of the scaffold was 9455 m. The scaffolds obtained by the two methods have high hardness, good swelling and high water absorption. The two blank N-carboxypropionyl chitosan / chitosan scaffolds can be completely degraded in vitro. L-02 human hepatocytes grew well on N-carboxypropionyl chitosan / chitosan scaffolds under optical microscope. For sodium alginate / chitosan microcarrier, the pore size of freeze-dried microcarrier was 3-50 m, the porosity was 89 m, and the pore size of microcarrier was 15-55 m with 95% porosity by using n-pentanol and ammonium acetate as pore-forming agent. The microcarriers obtained by the two methods have high hardness, good swelling and high water absorption. The blank sodium alginate / chitosan microcarriers can be completely degraded in vitro. L-02 human hepatocytes grew well on sodium alginate / chitosan microcarriers under optical microscope. In conclusion, the porous microcarriers prepared in this paper have good biological properties and can meet the requirements of bioartificial liver for hepatocyte culture.
【學(xué)位授予單位】：上海理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.08

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