糖化温敏凝胶的制备及其与HepG2细胞相互作用研究

利用自由基聚合法合成了半乳糖糖化温敏凝胶(P(NIPAAm-co-GAC))和壳聚糖糖化温敏凝胶(P(NIPAAm-co-CSA)),对其温度响应性和溶胀性能进行了研究,结果表明,两种糖化温敏凝胶在水中和细胞培养基中均显示较好的温度响应性,以及比聚(N-异丙基丙烯酰胺)温敏凝胶(PNIPAAm)更好的溶胀性能.进一步研究人肝肿瘤细胞(HepG2)在凝胶表面的细胞行为发现,HepG2在P(NIPAAm-co-GAC)、PNIPAAm凝胶表面吸附量及活性较高,表现出良好的生长趋势,而在P(NIPAAm-co-CSA)凝胶表面吸附量和活性很低,其增殖受到抑制;通过降低环境温度,能使培养在P(NIPAAm-co-GAC)和PNIPAAm凝胶表面的HepG2细胞发生自动脱附,避免了酶解法对细胞功能造成的损伤,并且细胞片层比单个细胞表现出更快的脱附速率;研究细胞转载行为表明,通过温度诱导得到的细胞片层,其生物活性远远大于通过酶解法得到的细胞的生物活性.

SYNTHESIS OF GLYCOSYLATED THERMO-RESPONSIVE HYDROGELS AND THEIR INTERACTIONS WITH HepG2 CELLS

A series of galactose glycosylated thermoresponsive hydrogel (P(NIPAAm-co-GAC)) and chitosan glycosylated thermoresponsive hydrogel (P(NIPAAm-co-CSA) ) were synthesized by free radical polymerization with different ratios. The temperature responsibility of the copolymerized hydrogels was investigated. The lower critical solution temperature (LCST) of P(NIPAAm-co-GAC) hydrogels was 30 ～ 35℃ in distilled water and cell culture media. However, the LCST of P(NIPAAm-co-CSA) hydrogels was 30 ～ 35℃ in distilled water and lower than 30℃in cell culture media, respectively. Furthermore, behaviors of HepG2 cell cultured on the P(NIPAAm-co-GAC),P(NIPAAm- co-CSA) and poly (N-isopropylacrylamide) (PNIPAAm) hydrogels were investigated. Cell morphologies on different hydrogels scaffolds were photographed under a phase contrast microscope equipped with a digital camera. Investigation of the growth behaviors and viability of HepG2 cells on different hydrogels indicated that HepG2 cell grew very well on the surface of P(NIPAAm-co-GAC) and PNIPAAm hydrogels. However, the growth of HepG2 cell was inhibited on the surface of P(NIPAAm-co-CSA) hydrogels. The 3-(4,5-dimethy thioazol-2-yl)-2,5-di-phenytetrazoliumromide (MTT) assay was used to quantify the cell viability of HepG2 on different hydrogels.Cell viability on P(NIPAAm-co-GAC) hydrogels was 10 times higher than that on P(NIPAAm-co-CSA) hydrogels after 6 day's incubation. Moreover, the viability of HepG2 ceil increased with increasing the amount of introduced galactose. On the contrary, the viability of HepG2 cell decreased with increasing the amount of introduced chitosan.Investigation of the temperature induced detachment indicated that the ceil could be spontaneously detached from the surface of poly(NIPAAm-co-GAC) hydrogels by lowing temperature, which could avoid the damage to the cells caused by enzymatic digestion. The detachment rate of cell sheets was much faster than that of single ceils, which was indicated by the detachment rate of 64% for cell sheets and 32% for single cells after 10 min at low temperature. Investigation of cells transshipment indicated that the viability of cells obtained by temperature induced detachment was much higher than that obtained by enzymatic digestion. In conclusion, the introduction of chitosan into PNIPAAm was not favorable for the growth of HepG2, but the incorporation of galactose greatly facilitated its growth. Further more, intact cell sheets could be obtained by controlling the environmental temperature. All these results provided a novel method and experimental basis for developing materials used in tissue engineering.