牙龈卟啉单胞菌精氨酸特异性牙龈素基因疫苗预防比格犬种植体周围炎的实验研究

Experimental research on Arginine-gingipain A gene vaccine from Porphyromonas gingivalis that prevents periimplantitis in Beagle dogs

目的构建牙龈卟啉单胞菌精氨酸特异性牙龈素基因疫苗p VAX1-rgp A,并对成年犬进行免疫接种,观察该疫苗在防治种植体周围炎发生发展中的作用。方法构建真核表达质粒p VAX1-rgp A。拔除15只成年犬下颌双侧第二、三前磨牙,随机即刻植入种植体。3个月后,实验犬随机均分成A、B、C组,分别接种重组质粒p VAX1-rgp A、热失活牙龈卟啉单胞菌、空白质粒p VAX1,连续接种3次。接种开始前、接种结束2周后采用酶联免疫吸附试验检测血清IgG和唾液分泌型IgA(s IgA)含量。随机选取一侧采用丝线结扎法构建种植体周围炎,并检测种植体周探诊深度(PD)和探诊出血指数(BOP)。结扎6周后处死所有动物,制作50μm厚的硬组织切片,亚甲基蓝染色后观察种植体周骨丧失程度。结果 A、B组动物免疫后,IgG、s IgA抗体较未免疫前明显增高(P<0.05),同时较C组升高(P<0.05),但A、B组间差异无统计学意义(P>0.05)。丝线结扎第4和6周时,C组结扎侧的PD明显高于A、B组(P<0.05),A、B组间差异无明显统计学意义(P>0.05)。A组结扎侧骨丧失量明显小于其他两组(P<0.05)。硬组织切片可见,各组种植体周骨丧失区均有大量的炎症细胞和细菌存在,C组结扎侧最严重。结论精氨酸特异性牙龈素(rgp A)基因疫苗产生的IgG和s IgA,能有效减弱犬种植体周围炎的骨丧失量。

Objective This study aims to use Arginine-gingipain A gene vaccine （pVAX1-rgpA） to immunize adult Beagle dogs and to evaluate its effect during peri-implantitis progression and development. Methods Plasmid pVAX1-rgpA was constructed. The second and third bilateral mandible premolars of 15 adult Beagle dogs were extracted, and the implants were placed immediately. After 3 months, the animals were randomly divided into groups A, B, and C. Afterward, the animals were immunized thrice with plasmid pVAX1-rgpA, with heat-killed Porphyromonas gingivalis, or pVAX1, respectively. IgG in the serum and secretory IgA （sIgA） in saliva were quantitatively analyzed by enzyme-linked immunosorbent assay before and after 2 weeks of immunization. Peri-implantitis was induced with cotton ligatures fixed around the neck of implants. Probing depth （PD） and bleeding on probing were recorded. All animals were sacrificed after ligaturation for 6 weeks. Decalcified sections with thickness of 50 μm were prepared and dyed with methylene blue to observe the bone phenotype around implants. Results Levels of serum IgG and sIgA in saliva were higher in groups A and B after immunization than before the process （P〈0.05） and higher than those in group C （P〈0.05）. However, no difference was observed between groups A and B （P〉0.05）. At 4 and 6 weeks after ligaturation, PD of the ligatured side in group C was higher than that in groups A and B （P〈0.05）. On the other hand, no difference was identified between groups A and B （P〉0.05）. Bone loss in group A was sig-nificantly lower than that of the other groups （P〈0.05）. Abundant inflammatory cells and bacteria were present in the bone loss area around the implants in the three groups, as identified through hard tissue section observation. However, group C presented the most number of inflammatory cells and bacteria in the bone loss area around the implants. Conclusion IgG and sIgA can be generated by immunity with rgpA DNA vaccine, which can significantly slow down bone loss during expe-rimental peri-implantitis in dogs.