DEVELOPMENT OF DENTINE REGENERATION THERAPY BY GENE DELIVARY SYSTEM USING NATURAL BIOACTIVE MATERIAL, CHITOSAN

壳聚糖具有多种生物活性。我们发现壳聚糖单体通过BMP-2的表达促进骨形成细胞NOS-1中碱性磷酸酶活性的加速。随后，我们利用cDNA微阵列和实时荧光定量PCR技术研究了壳聚糖单体是否激活了NOS-1细胞的信号转导通路，结果显示，与各种信号相关分子相关的10个基因在实验组中的表达量在3天后比对照组高出[≥] 2.0倍。实时荧光定量PCR分析表明，壳聚糖单体诱导四个信号转导基因的表达增加。MAPKs抗体芯片显示这些分子均被磷酸化。结论：超低浓度的壳聚糖单体可以通过mRNA水平调节成骨细胞的活性，壳聚糖单体直接影响信号转导通路，为壳聚糖在基因诱导中的应用提供了依据，即壳聚糖对DNA电穿孔细胞的保护作用。我们研究了电穿孔法将BMP-2基因导入NOS-1细胞。以细胞内GFP的荧光强度判断诱导成功的标志。在电穿孔过程中添加壳聚糖单体，使诱导细胞的增殖能力明显增强。这些发现表明，在电穿孔过程中应用壳聚糖将有助于通过体内牙本质再生维持牙髓伤口愈合中的细胞活力。

Chitosan has many bioactive effects. We discovered that chitosan monomer promotes the acceleration of alkaline phosphatase activity in osteablastic NOS-1 cells through BMP-2 expression. After that, we tried to demonstrate whether chitosan monomer activated pathways of signal transduction in NOS-1 cells using cDNA microarray and real-time PCR analyses.cDNA microarray analysis revealed that 10 genes conserning to various signaling-related molecules were expressed at 【greater than or equal】 2.0-fold higher ratio levels in the experimental group when compared with the control group after 3 days. Real-time PCR analysis showed that chitosan monomer induced an increase in the expression of four signal transduction genes. Furthrmore, MAPKs antibo lies array indicated that these molecules were phosphorylated. We concluded that a super-low cincentration of chitosan monomer could modulate the activity of osteoblastic cells through mRNA levels and that chitosan monomer directly affects signal transduction pathways.The aforementioned data conduct the application of chitosan to the induction of gene, which means the protection effect of chitosan during electroporation of DNA into the cells. We investigated the induction of BMP-2 gene into NOS-1 cells using electroporation. The marker of successful induction was judged with the fluorescence of GFP inside the cells. The supplementation of chitosan monomer during the electroporation produced that the proliferation of the induced cells were much higher in the chitosan group. These findings suggest that the application of chitosan during electroporation would be useful for maintaining cell vitality in pulp wound healing through dentine regeneration in vivo.