Promotion of minTBP-1-PRGDN on the attachment,proliferation and collagen I synthesis of human keratocyte on titanium

AIM: To investigate the influence of minTBP-1-PRGDN on the attachment,proliferation and collagen I synthesis of human keratocyte on titanium(Ti) surface. · METHODS: The chimeric peptide RKLPDAPRGDN(minTBP-1-PRGDN) was synthesized by connecting RKLPDA(minTBP-1) to the N-terminal of PRGDN,the influence of minTBP-1-PRGDN on the attachment,proliferation and collagen I synthesis of human keratocyte on Ti surface were tested using PRGDN and minTBP-1 as controls. The keratocytes attached to the surface of Ti were either stained with FITC-labeled phalloidin and viewed with fluorescence microscope or quantified with alamar Blue method. The proliferation of keratocytes on Ti were quantified with 3-(4,5-dim-ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptaking methods. The secretion of type I collagen was determined using an ELISA kit. ·RESULTS: The results showed that minTBP-1-PRGDN at a concentration of 100ng/mL was the most potent peptide to enhance the attachment of human keratocytes to the surface of Ti(1.40±0.03 folds,P =0.003),to promote the proliferation(1.26 ±0.05 folds,P =0.014) and the synthesis of type I collagen(1.530 ±0.128,P =0.008). MinTBP-1 at the same concentration could only promote the attachment(1.13±0.04 folds,P =0.020) and proliferation(1.15±0.06 folds,P =0.021),while PRGDN had no significant influence(P 】0.05). ·CONCLUSION: Our data show that the novel chimericpeptide minTBP-1-PRGDN could promote the attachment,proliferation and type I collagen synthesis of human keratocytes on the surface of titanium.

碱烧伤诱导兔部分性角膜缘干细胞失代偿模型

目的:探讨诱导兔部分性角膜缘干细胞失代偿(limbal stem cell deficiency,LSCD)动物模型的一种新方法。方法:分别采用C57小鼠和新西兰白兔制作完全性和部分性角膜缘干细胞失代偿动物模型。小鼠(n=30)采用1mol/L氢氧化钾溶液浸泡的滤纸片(直径3mm)置于左眼中央角膜表面30s,随后用生理盐水冲洗。白兔(n=19)切除瞬膜(第三眼睑)后,采用1mol/L氢氧化钾溶液浸泡的滤纸片(直径5mm)置于左眼颞上方角膜表面30s,随后用生理盐水冲洗。烧伤眼术后采用0.5%盐酸左氧氟沙星滴眼液4次/d。烧伤前、烧伤后第1、2、4wk,2mo采用裂隙灯显微镜观察、摄像,记录角膜溃疡、穿孔等并发症。术后2mo采用印迹细胞学检测角膜杯状细胞分布,根据裂隙灯显微镜检查所见和角膜印迹细胞学检查判断LSCD严重程度。术后2mo处死动物,角结膜切片观察角膜新生血管、杯状细胞分布。意外死亡动物不计入总数,计算并比较完全性LSCD和部分性LSCD的模型诱导成功率。结果:30只小鼠中6只意外死亡,2只于烧伤后出现角膜穿孔,其余22只发生完全性LSCD,诱导成功率92%,烧伤后2mo小鼠角膜可见新生血管广泛分布于角膜浅层及深基质层,病理切片可见角膜新生血管。19只白兔,7只意外死亡,其余12只发生不同程度LSCD(部分性LSCD,平均累及1.17±0.39个象限),未发生角膜穿孔情况,诱导成功率100%(P=0.543)。正常角膜区域无杯状细胞,LSCD区域角膜上皮印迹细胞学PAS染色可见杯状细胞,平均密度58.60±12.58个细胞/HP。结论:中央角膜碱烧伤可以诱导产生完全性LSCD,部分动物会因为角膜溃疡穿孔而导致模型诱导失败,LSCD往往比较严重,且合并深层角膜新生血管。颞上方角膜碱烧伤可以诱导产生部分性LSCD,合并较少的角膜病变,角膜新生血管位于浅层。