【作者】 刘进；

【导师】 肖明振；

【作者基本信息】 第四军医大学 ， 口腔内科学， 2009， 博士

【摘要】 组织工程通常的方法是把种子细胞播种于生物可降解的支架材料中,利用临时充填在其中的多孔性细胞外基质塑型,期望细胞能够长入和聚集。然而,支架结构与其希望模拟的体内组织相比依然相当原始,此外,细胞向支架内部迁移不足,或者在支架内不能保持,细胞的增殖速率和支架的降解速率不一致,宿主的免疫反应和炎性反应都是存在的问题。与传统方法相比,有学者提出了一种新的以细胞层为基础的组织工程技术,这种技术作为一种新的选择,利用温敏性培养皿,经简单的温度变化,可以非损伤性的一起获得完整的培养细胞层及其所沉积的细胞外基质,从而避免了使用蛋白水解酶。利用获得相同或者不同的细胞层相互层叠,不使用支架材料来获得细胞密度高和功能化的组织,这种以温度敏感的培养皿为基础发展出技术目前被称为细胞层工程(Cell Sheet Engineering)。将聚N-异丙基丙烯酰胺(PNIPAm)接枝聚合培养皿表面,利用PNIPAm的温度依赖性的可溶/不溶性变化,在37℃培养细胞,降低温度至低临界溶解温度(约32℃)之下,则细胞从培养皿表面脱附,于是可以获得温度敏感性的细胞培养皿。本课题利用等离子处理技术和紫外线接枝的方法,在普通培养皿的表面接枝具有温度敏感性的PNIPAm,借助于表面分析技术FT-IR、XPS和AFM、SEM微观形貌观察来证实是否接枝成功,探讨了制备温敏性培养皿的可行性。以细胞毒性试验对材料的毒性做出评价,进一步观察了在PNIPAm接枝的培养皿上不同细胞的贴壁、生长、增殖状况和细胞层降温脱附行为。第一部分温敏性培养皿的制备本试验将低温等离子体预处理和紫外线照射接枝结合起来,在细胞培养皿(PS)上引入PNIPAm。以接触角变化程度作为衡量指标,采用正交设计,对影响接枝的多个因素的参数进行多个水平的分析和筛选优化,结果表明在等离子体复合参数0.08,等离子体预处理时间2min,N-异丙基丙烯酰胺单体浓度30%,紫外线照射时间15min条件的接枝反应,接触角的变化值较大。第二部分温敏性培养皿的表面分析本试验将第一部分试验制备的PNIPAm接枝表面采用FT-IR、XPS方法进行了表面分析,结果证实了PS表面有PNIPAm接枝,并且接枝物与常规方法制备的PNIPAm化学组成相似;AFM、SEM微观形貌观察,接枝和未接枝的PS表面存在明显差别。这些结果说明采用等离子体预处理和紫外线照射接枝的方法适合对PS表面用PNIPAm进行接枝改性,从而获得了具有温度敏感性的PS表面。第三部分温敏性培养皿的细胞培养研究本试验采用直接接触方式,以MMT法计算L929细胞的相对增殖率,观察了温敏性培养皿材料和普通PS培养板之间的毒性比较,结果证实前者无毒性或有轻微毒性,结果评价认为材料合格,可以用于实验室细胞培养及相关用途。进一步利用两种不同的细胞永生化的成牙本质细胞系(OLC)和人牙周膜细胞(HPLC)在接枝有PNIAPm的培养皿上进行培养,观察到细胞可以正常贴壁,生长并增殖,并且在形成连续的细胞层之后,通过降低温度,获得了完整的细胞层结构,结合前两部分实验结果,证实了利用等离子体预处理和紫外线照射接枝制备温敏性培养皿的可行性,为后续研究提供了实验基础。更多还原

【Abstract】 Tissue engineering techniques rely largely on scaffold-based approaches, often using biodegradable and biologically derived polymersto provide a porous, space-filling, temporary extracellularmatrix (ECM) of desired macroscopic form within which cells grow and integrate. However, such scaffold architectures are primitive compared to those they intend to duplicate in vivo. Additionally, insufficient cell migration into and retention within scaffolds, different rates of cell proliferation compared to scaffold degradation,and host inflammatory reactions remain problems.Further improvements in scaffold design may overcome some of these problems.In contrast to conventional methods, a novel cell sheet-based technique has been proposed .This technique intend to achieve high cell density and function by layering confluent viable cell sheets without the use of acellular scaffolds that limit mass transport. Using thermo-sensitive cell culture surfaces, confluently cultured cells spontaneously detach as intact sheets simply by reducing the culture temperature. Covalently grafted poly (N- isopropylacrylamide) (PNIPAm),which exhibits reversible temperature- dependent soluble/insoluble changes (i.e., lower critical solution temperature of 32℃) in aqueous solution, facilitates both cell adhesion and cell detachment, eliminating use of cell-harvesting enzymes that dissociate cell cultures into dispersed cells.This paper was mainly about the surface modification of cell culture dish by conbining plasma pretreatment and UV-incued PNIPAm graft polymerization, in orer to aquire the thermo-sensitive cell culture dish. Moreover, the surface was characterized and analysed by FT-IR、XPS、AFM and SEM. Cytotoxicity of the dish was also evaluated.In the end ,two types of cells (OLC and HPLC)were cultured and cell sheets detachment can be observed by reducing the culture tempareture.1. The preparation of thermo-sensitive cell culcure dishThe normal cell culture dishes (Polystyrene) were subjected to plasma pretreatment and UV-incued PNIPAm graft polymerization,without photo-initiator. The changement of contactact angle was used as the indication of the surface graft modifaction and orthogonal experimental design was used to arrange the experiment groups. Four different levels of four factors were analysed,intending to find an optimized combinations.The optimized combinations were W/FM 0.08GJ/Kg,plasma pretreatment time 2min,NIPAm monomo concentration 30%,UV irradiation time 15min,under which conditions the changement of contactact angle were maximal.2. The analysis and characterization of thermo-sensitive cell culcure dish surfaceThe surface of prepared cell culture dish was characterized and analysed by FT-IR、XPS、AFM and SEM. The IR spectrum showed the function group-amide I peak appeared on the grafting surface,which was characteristic function group of PNIAPm.The XPS analysis showed the presence of atom C ,N ,O on the grafting surface ,which was the chemical components of NIAPm.The AFM and SEM showed the similar appearance of grating surface ,apparently different to normal suiface.Obvious difference were confirmed between the grafting and normal surface of cell culture dish,which proved the results that PNIAPm had been successfully grafted onto the surface of cell culture dish.3. The cytotoxic evaluation and observation of cell culcure on the thermo-sensitive cell culcure dishThe cytotoxicity of thermo-sensitive cell culcure dish was evaluated by direct contact with MTT-based method.The relative cell groth rate of L929 cell represented the proliferation of cells on materials with respect to control over time. The result showed that cytotoxicity of thermo-sensitive cell culcure dish was none or little,which can be used for lab use.Additional cell culture was performed with two types of cell (OLC and HPLC)and cell sheets detachment were observed by reducing the culture tempareture after the confluently growth of the two types of cells.更多还原