贻贝粘附蛋白改性的树脂—牙本质粘接界面的抗蛋白水解性及粘接耐久性的研究

发布时间：2018-08-13 16:58

【摘要】：目的当代牙本质粘接体系中,无论是酸蚀-冲洗类粘接剂抑或是自酸蚀粘接剂,所形成的混合层底部始终存在一层裸露的胶原纤维。裸露的胶原纤维在牙体内源性的胶原酶的攻击下发生降解,继而引发树脂-牙本质的粘接强度及粘接持久性下降。本实验的目的在于探究贻贝粘附蛋白对胶原酶活性,牙本质胶原降解以及牙本质粘接微拉伸强度的影响,以期提高牙本质粘接的耐久性。方法评价贻贝粘附蛋白对胶原酶活性的影响:本实验应用Sensolyte?MMP通用型分光光度试剂盒检测胶原酶活性抑制程度。1mg/ml贻贝粘附蛋白与100U/ml VII型胶原酶(基质金属蛋白酶的代表类型之一)为实验组;GM6001(一种已知的人工合成的基质金属蛋白酶抑制剂)与100U/ml VII型胶原酶为阳性对照组;蒸馏水与100U/ml VII型胶原酶为阴性对照组。每组分5个亚组(n=5),充分反应后加入检测基底液,于412nm波长下进行分光光度检测,比较酶活性的抑制程度。评价贻贝粘附蛋白对牙本质胶原酶解的抑制性:将30片人牙本质片随机分成三组(n=10):贻贝粘附蛋白组,GM6001组,蒸馏水组。各组牙本质片脱矿后分别于胶原酶溶液中温育7天,取上清液检测羟脯氨酸含量,评估胶原降解程度。评价贻贝粘附蛋白对树脂-牙本质粘接强度的影响:取60个新鲜离体牙,暴露冠部牙本质后,采用酸蚀-冲洗类粘接剂进行树脂-牙本质粘接。根据酸蚀冲洗后预处理方式不同,随机分成三组:贻贝粘附蛋白组,GM6001组,蒸馏水组。将粘接完成的试件制备成树脂-牙本质粘接条,然后根据是否进行冷热循环(5°C和55°C,2500个循环)及酶解反应(3周,37°C)再将各组分成2个亚组,分别进行树脂-牙本质粘接的即刻微拉伸检测和老化处理后的微拉伸检测。扫描电镜观察牙本质段的拉伸断面。另取两个完整的未拉断的树脂-牙本质粘接试件进行粘接界面的扫描电镜检测。结果贻贝粘附蛋白组,GM6001组,蒸馏水组的胶原酶活性(nmol/min/mg)分别为20.22±0.93,29.11±1.17和31.39±0.52,三组间均具有统计学差异(p0.01)。各组牙本质胶原降解量(羟脯氨酸释放量μg/m L)分别为2.70±0.53,4.00±1.19和5.40±1.00,三组间均具有统计学差异(p0.01)。三组即刻微拉伸强度结果无显著差异,但冷热循环及酶解实验后,三组微拉伸结果(MPa)呈现统计学差异,分别为11.39±2.52,10.77±3.16和5.83±2.02(p0.001)。扫描电镜检测结果与上述结果指标相一致。即刻粘接界面扫描结果显示:贻贝粘附蛋白组的混合层中罕见裸露的胶原纤维,而GM6001组以及蒸馏水组可见少量胶原纤维。酶解反应后的粘接界面扫描结果显示:贻贝粘附蛋白组以及GM6001组暴露的胶原纤维较蒸馏水组少,且蒸馏水组中的树脂突牙本质小管中存在较大的间隙。各组微拉伸后的断面扫描电镜检测结果显示:贻贝粘附蛋白组与GM6001组的拉伸断面大多位于混合层顶端,牙本质小管被树脂突阻塞,而蒸馏水组的断面多位于混合层基底部,大量牙本质小管呈现空虚状态。结论贻贝粘附蛋白能够抑制胶原酶活性,抑制牙本质胶原纤维的降解,有望提高树脂-牙本质粘接的耐久性。
[Abstract]:Objective A layer of exposed collagen fibers always exists at the bottom of the mixed layer in the contemporary dentin bonding system, whether it is etch-rinse adhesive or self-etch adhesive. The purpose of this study was to investigate the effects of mussel adhesion protein on the activity of collagenase, the degradation of dentin collagen and the micro-tensile strength of dentin adhesion, so as to improve the durability of dentin adhesion. Inhibition of collagenase activity was detected by kit test. 1 mg/ml Mussel Adhesion Protein and 100U/ml type VII collagenase (one of the representative types of matrix metalloproteinase) were used as experimental group; GM6001 (a known synthetic inhibitor of matrix metalloproteinase) and 100U/ml type VII collagenase were used as positive control group; distilled water and 100U/ml type VII collagenase were used as distilled water and 100U/ml type VII collagenase were used as positive control group. Each group was divided into five subgroups (n=5). After full reaction, the basal fluid was added and the inhibition degree of enzyme activity was measured by spectrophotometry at 412 nm. To evaluate the inhibitory effect of mussel adhesion protein on dentin collagenase hydrolysis, 30 pieces of human dentin tablets were randomly divided into three groups (n=10): mussel adhesion protein group, GM6001 group, distillation. After demineralization, the dentin tablets were incubated in collagenase solution for 7 days. The content of hydroxyproline in the supernatant was measured to evaluate the degree of collagen degradation. According to the different pretreatment methods after etching and rinsing, they were randomly divided into three groups: mussel adhesive protein group, GM6001 group and distilled water group.The bonded specimens were prepared into resin-dentin bonding strips, and then divided into two subgroups according to whether the bonded specimens were subjected to cold and heat cycles (5 C and 55 C, 2500 cycles) and enzymatic hydrolysis (3 weeks, 37 C). The tensile section of dentin segment was observed by scanning electron microscopy. Two intact and unbroken resin-dentin bonding specimens were examined by scanning electron microscopy. Results The collagen of mussel adhesive protein group, GM6001 group and distilled water group were detected by scanning electron microscopy. The activity of enzymes (nmol / min / mg) was 20.22 (+ 0.93), 29.11 (+ 1.17) and 31.39 (+ 0.52), respectively. there were statistical differences among the three groups (p0.01). the degradation of dentin collagen (hydroxyproline release amount (ug / ml) was 2.70 (+ 0.53), 4.00 (+ 1.19) and 5.40 (+ 1.00), respectively. There was no significant difference among the three groups (p0.01). The results of scanning electron microscopy (SEM) were consistent with the above results. Immediate bonding interface scan showed that collagen fibers rarely exposed in the mixed layer of mussel adhesive protein group. A small amount of collagen fibers were found in GM6001 group and distilled water group. Scanning results showed that collagen fibers were less exposed in mussel adhesin group and GM6001 group than in distilled water group, and there were larger gaps in resin-processed dentin tubules in distilled water group. The results showed that most of the tensile sections of the mussel adhesin group and GM6001 group were located at the top of the mixed layer, and the dentinal tubules were obstructed by resin process, while those of the distilled water group were mostly located at the base of the mixed layer, and a large number of dentinal tubules were empty. It is expected that the durability of resin dentin bonding will be improved.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.1

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