【作者】 张莉；

【导师】 顾汉卿；

【作者基本信息】 天津医科大学 ， 生物材料与组织工程， 2007， 硕士

【摘要】 N-异丙基丙烯酰胺水凝胶是一种温度敏感性水凝胶。在某一临界温度可发生体积相变。作为一种新型的智能型生物材料其受到广泛的关注，在药物释放、分离领域、酶的吸附、细胞培养等方面有重要的应用前景。组织工程的迅猛发展已经取得了大量的成果，但是某些方面依然存在挑战。组织工程中种子细胞是一切的基础。在细胞培养过程中，细胞传代时，传统的酶解法对细胞的功能、遗传特性有损伤，这个问题一直困扰着科技人员。Yamada等利用N-异丙基丙烯酰胺（NIPAAm）的温度敏感性，使细胞在临界温度之上时贴附在材料表面生长，温度在临界温度以下时细胞脱附，改变了传统的酶解脱附细胞的方法。本研究采用辐射法用NIPAAm和聚丙烯酰胺（AAm）凝胶制备用于组织工程的膜材，探索一种新的智能材料用于细胞培养，使其通过温度变化达到可人工控制细胞脱附的目的。1．本文利用辐射法合成了不同辐射强度，不同单体浓度的聚N-异丙基丙烯酰胺均聚物，测定了其最低临界温度（LCST）为33℃左右。并观察了辐射强度、单体浓度对粘均分子量的影响。结果表明，随辐射强度及单体浓度的增大，均聚物的分子量增大。并采用直接接触法研究了PNIPAAm均聚物的细胞毒性，结果表明其细胞毒性为0级。2．采用辐射法合成了不同单体组成比的聚丙烯酰胺-co-N-异丙基丙烯酰胺共聚凝胶。分别用DSC法及溶涨法考察了其温度敏感性。结果表明随共聚物中AAm组分的增大，其LCST增大，当AAm与NIPAAm的摩尔比为1时，其LCST超过100℃。另外随AAm组分的增大，其溶胀率也增大。3．采用辐射法合成了不同单体组成的P（AAm-g-NIPAAm）水凝胶。并对其组成和结构及温敏性进行了表征。通过DSC及721分光光度计得到其LCST均为31℃左右。溶胀率随AAm的增多而增大。其核磁共振图谱及红外光谱图显示了NIPAAm的特征峰-CONH-及-CH（CH₃）₂。利用接触角测定证明了接枝凝胶在温度发生变化时出现亲水-疏水的转变。4．细胞培养实验中，在P（AAm-g-NIPAAm）凝胶膜表面上种植了人脐静脉上皮细胞。倒置纤维镜下的照片显示贴附生长情况良好，细胞大面积贴附，铺展生长。在降低环境温度到25℃后，人工振荡的辅助条件下细胞自动脱附，保护了细胞功能的完好，避免了酶解脱附细胞造成细胞功能的损伤。从材料的生物活性展现细胞培养工程和生物医学工程领域的应用前景，并为进一步的研究奠定基础。更多还原

【Abstract】 Poly（N-isopropylacrylamide） hydrogel is a thermo-sensitive hydrogel which can changevolume at a critical temperature. As a new type of intelligent material, which has importantapplication in drug release, separate field, enzymatic adsorption, cell cultivation and so on, ithas been paid much attention. The rapid development of Tissue Engineering has a lot ofachievements. But there also exists many challenges in some aspects. Seed cell is thefoundation of tissue engineering. Conventional enzymatic degradation has damages on functionand heredity of cell during cell passage. This problem has always feazed researchers. Yamadaetc. makes use of the thermo-sensitivity of N-isopropylacrylamide synthesizing athermo-sensitive material on which cell can place themselves and grow above the lower criticalsolution temperature（LCSY）, and desorp when temperature is below LCST. This changes themethod of conventional enzymatic degradation which used to make cell fallen. In this article,P（AAm-g-NIPAAm） which is prepared to be used as a new intelligent material on tissueengineering is synthesized from NIPAAm and PAAm hydrogel by radiation. We will make surewhether it can be used on cell culture and can make cell desorp automatically by decreasetemperature to below its LCST in this article.1. Poly（N-isopropylacrylamide） of different radiation intensity and monomer concentration issynthesized by radiation. We measure its LCST and it is about 33℃. Influence of radiationintensity and monomer concentration on its viscosity average molecular weight is alsoobserved. And the results show that molecular weight of PNIPAAm increases with increaseof radiation dose and monomer concentration. We also researched cell toxicity of PNIPAAmby the method of direct touch and get the conclusion that cell toxicity of PNIPAAm is grade0.2. P（AAm-co-NIPAAm） hydrogel of different monomer proportion is synthesized by radiation.Its thermo-sensitivity is researched by DSC and the method of swelling ratio respectively.The results show that LCST increase with the increase of content of AAm in thecomposition of monomer. The LCST of P（AAm-co-NIPAAm） exceeds 100℃when molarration of AAm and NIPAAm is 1:1. In addition, swelling ratio of P（AAm-co-NIPAAm）increases with the increase of content of AAm in the composition of monomer.3. P（AAm-g-NIPAAm） hydrogel of different monomer proportion is synthesized by radiation.Its composition, structure and thermo-sensitivity are studied. Its LCST is 31℃despite ofdifferent monomer proportion which is obtained by DSC and 721 spectrophotometerrespectively. Its swelling ratio increases with increase of AAm in the monomer proportion.characteristic peak -CONH- and-CH（CH₃）₂ of NIPAAm appear on infrared spectrum and ¹H NMR. We also measured its contact angle and the results show transition of hydrophilicand hydrophobic happens when temperature changes.4. In experiment of cell culture, we plant epithelia on the film of P（AAm-g-NIPAAm）hydrogel. We can see cell grow well on the material from Inverted microscope. Whenmaking environment temperature fall to 25℃, cell desorp from material automatically withsurging slightly. We can protect functions of cell from damaging by enzymatic degradationby this method.更多还原