携载重组胶原蛋白的卡波姆凝胶喷剂制备及对大鼠口腔溃疡愈合作用观察

Preparation of carbomer gel spray containing recombinant collagen and its healing effect on oral ulcers in rats

目的 制备携载重组胶原蛋白的卡波姆凝胶喷剂,并观察其对大鼠口腔溃疡的愈合作用。方法 选取可用于口服制剂的卡波姆971P为主要材料,添加不同质量浓度的甘油、磷酸二氢钾(KH2PO4)、Ⅲ型重组胶原蛋白(rhColⅢ)等制备凝胶喷剂,观察凝胶喷剂在不同pH下的喷出性能、不同缓冲剂对凝胶喷剂粘度变化的影响、不同pH卡波姆凝胶对胶原蛋白溶解度的影响。使用醋酸灼烧法建立大鼠口腔溃疡模型,模型建立成功后随机分为空白对照组、卡波姆组(采用不含胶原蛋白的卡波姆凝胶喷剂治疗)、胶原蛋白组(采用含rhColⅢ的卡波姆凝胶喷剂治疗),比较各组大鼠溃疡愈合率及黏膜组织HE染色结果。结果 卡波姆粘度在2 500~3 000 mpa·s时具有良好的喷出性能。随着离子溶度的升高,卡波姆粘度不断下降(P均<0.05)。当凝胶pH是4时,卡波姆中有大量白色团块(未溶解的胶原蛋白),继续延长搅拌时间白色团块未减少;往凝胶中继续缓慢少量添加KOH并不断搅拌,随着pH不断升高,白色团块不断减小,当pH升高到5左右,白色团块消失。空白对照组、卡波姆组、胶原蛋白组制模后第3天溃疡愈合率分别为38.7%、45.6%、53.1%,空白对照组与胶原蛋白组相比,P<0.05;空白对照组、卡波姆组、胶原蛋白组制模后第5天溃疡愈合率分别为62.9%、72.4%、88.5%,胶原蛋白组分别与空白对照组、卡波姆组比较,P均<0.05。制模后第3天,各组口腔黏膜上皮处均有缺损,空白对照组缺损最大,上皮层和固有层中单核细胞、多形核细胞等炎症细胞数量较多,新生血管数量较少;相比于空白对照组,卡波姆组炎症细胞数量减少,新生血管数量较多;而相比于其他两组,胶原蛋白组炎症细胞相对最少,新生血管数量较多,且缺损面积减少;制模后第5天,空白对照组黏膜上皮尚有缺损,炎症细胞数量较多;相比于空白对照组,卡波姆组上皮缺损和炎症细胞数量减少,新生血管数量较多;阳性对照组相比于其他两组,炎症细胞数量最少,新生血管数量明显增多,上皮缺损已完全愈合。结论 携载重组胶原蛋白的卡波姆喷剂具有良好的喷出性能和粘附性能,该喷剂能显著促进大鼠口腔溃疡的愈合。

Objective To prepare the carbomer gel spray containing recombinant collagen and its healing effect on oral ulcers in rats.Methods Carbomer 971P,which could be used for oral preparation,was selected as the main materi-al,and different mass concentrations of glycerol,potassium dihydrogen phosphate(KH2PO4),and type III recombinant collagen(rhColⅢ)were added to prepare gel sprays,to observe the spraying performance of gel sprays at different pH,the effect of different buffers on the change of the viscosity of the sprays,and the effect of carbomer solutions at different pH on the solubility of collagen.The oral ulcer model of rats was established using the acetic acid cautery method.After the models were successfully established,the rats were randomly divided into the blank control group,carbomer group(treated with rhColⅢ-free carbomer spray),and collagen group(treated with carbomer spray-containing rhColⅢ);the ulcer healing rate and HE staining result of mucosal tissues were compared between the groups.Results The carbomer viscosity had good ejection properties at 2500-3000 mpa·s.With the increase of ionic solubility,the viscosity of carbomer decreased continuously(P<0.05).When the pH of the gel was 4,carbomer gel had large white clusters(undissolved col-lagen),and the white clusters did not decrease over the stirring time;we continued to add KOH slowly in small quantities to the gel and stirred,the white clusters continued to decrease with the rising pH value,and the white clusters disap-peared when the pH increased about 5.The ulcer healing rates of the blank control group,carbomer group and collagen group were 38.7%,45.6%and 53.1%on the third day after moulding,respectively,and significant difference was found between the blank control group and collagen group(P<0.05);the ulcer healing rates of the blank control group,carbomer group and collagen group were 62.9%,72.4%and 88.5%on the 5th day,respectively,and significant differ-ence was found between the the collagen group and blank control group,and between the the collagen group and carbomer group(both P<0.05).On day 3 after modelling,there were defects in the epithelium of the oral mucosa in all groups,with the largest defects in the blank control group,a larger number of inflammatory cells such as monocytes and polymor-phonuclear cells on epithelial layer and lamina propria and a smaller number of neovasculature;in the carbomer group,the number of inflammatory cells was reduced and neovasculature was higher in comparison with those of the blank control group;compared with the other two groups,the collagen group had relatively the fewest inflammatory cells,a large num-ber of new blood vessels,and a reduced defect area.On day 5,the blank control group still had defects in the mucosal epithelium and a large number of inflammatory cells;compared with the blank control group,the carbomer group had a re-duced number of epithelial defects and inflammatory cells,and a larger number of neovessels;and the positive control group had the smallest number of inflammatory cells,a significant increase in the number of neovessels,and the epitheli-al defects were completely healed in comparison with the other two groups.Conclusion Carbomer spray loaded with re-combinant collagen had good ejection and adhesion properties,and the spray significantly promoted the healing of oral ul-cers in rats.