【作者】 姜威；

【导师】 韩泽民；

【作者基本信息】 河北联合大学 ， 口腔临床医学， 2011， 硕士

【摘要】 【目的】通过大鼠拔牙后即刻种植体洞型的骨损伤模型,利用组织学和免疫组织化学的方法,结合统计学分析,观察即刻种植窝预备区域早期骨愈合过程中降钙素基因相关肽(calcitonin gene-related peptide, CGRP)的表达特点。探讨降钙素基因相关肽在牙槽骨愈合和改建时的表达规律,为把降钙素基因相关肽引入到牙槽外科和口腔种植基础及临床医学研究中进行初步探索。【方法】16只Wistar成年雄性大鼠随机分到4个组中,每组4只,所有动物在同一天制备即刻种植窝预备牙槽骨损伤模型,1至4组依次分别在模型制备后4、7、14、21天取标本。即刻种植窝预备牙槽骨损伤模型制备:大鼠称重后以0.4%戊巴比妥钠(1ml/100g)通过腹腔注射麻醉大鼠,麻醉成功后用橡皮套将大鼠四肢及上颌下颌固定在实验手术台上,用普通镊子由助手协助显露上颌第一磨牙区域,手术区常规消毒、铺巾,在无菌的条件下用小尖头探针分离牙龈,用牙镊拔出左上颌第一磨牙然后用小螺纹钻预备深度和直径合适的种植体窝,术后用消毒小棉球压迫止血。种植窝制备后满4、7、14、21天后在模型制备区域取一小段牙槽骨组织块,所有标本经10%甲醛溶液固定24小时后取出,中性EDTA脱钙液脱钙,然后脱蜡、包埋,切片,免疫组织化学染色、HE染色(观察骨愈合区组织形态),最后光镜下免疫阳性细胞计数,采集图像,应用Image-Pro plus 6.0图像分析系统测量累积光密度值,统计学分析。【结果】1.模型制备成功:16只动物在试验期间种植洞形制备顺利,术区周围组织未见明显肿胀感染现象。2.HE观察结果显示,4天、7天时,骨损伤修复区纤维性组织较多;整个骨损伤修复区域细胞的增殖在4天,7天时最为活跃,细胞核染色较深,14天,21天时,细胞核小、染色相对较浅;四个时期在骨修复区边缘均可见到前成骨样细胞及破骨细胞,中心区域未曾发现。3.骨组织免疫组织化学染色显示,CGRP免疫组织化学染色阳性的细胞,胞浆着色;CGRP在成熟的骨细胞中未见着色,在骨修复区边缘的类成骨细样细胞着色;在模型建立后第7天取标本组CGRP主要在骨修复改建中心区的成纤维细胞中着色,与其它不同时间段种植区骨愈合组织相比,此期CGRP在骨修复改建中心区域染色阳性细胞数最多,染色强度最强,4组经秩和检验CGRP阳性细胞数和IOD值均是在第七天时最高,具有统计学意义(P<0.01)。【结论】1. CGRP在即刻种植术骨愈合区域存在高表达现象,该现象发生在成骨样细胞高增殖状态(骨细胞形成的关键时期)。CGRP在正常的牙槽骨及颌骨未见明显表达,从相反角度证明了,只有在骨修复改建期细胞功能活跃时CGRP才呈现阳性表达。推测CGRP可能是促进骨愈合和抑制骨吸收的一个重要因子。2. CGRP主要在纤维性骨痂向骨性骨痂过度时呈高峰态表达,而CGRP的表达高峰期也是成纤维细胞被诱导表达其成骨作用的关键时期,提示CGRP有可能是诱导成纤维细胞表达其成骨作用的一个重要因子。3.CGRP主要在前成骨细胞和被诱导表达成骨作用的成纤维细胞胞质中染色呈强阳性,考虑CGRP的表达可能是前成骨细胞形成和增殖活动活跃的一个标志,也可能是成纤维细胞被诱导开始表达成骨作用的一个标志。更多还原

【Abstract】 [Objective] Established the rat alveolar bone injured models of the oral immediately implant bed preparation , which aim was to observe the characteristics of calcitonin gene related peptide expression in the alveolar bone healing process by HE and immunohistochemistry assay. We discussed the experession of calcitonin gene-related peptidein alveolar bone healings and remodeling , and investigated the peptide action mechanism on alveolar surgery and oral implant.[Methods] 16 healthy adult male Wistar rats were randomly divided into 4 groups , there were 4 each group. Established the rat alveolar bone injured models of the oral Immediately implant bed preparation , and collected specimens group after 4、7、14、21days. The bone injured models of immediate implantation fossa preparation : Rats were weighed and anesthetized by 0.4% sodium pentobarbital intraperitoneal injection at a dose of 1 ml per gramme . Under sterile conditions, pulled out the first molar by dental tweezers , and then prepared implants nest in proper depth and diameter with a small impact drilling . Taken a short alveolar bone (4 mm×4 mm×4 mm) in the model preparation area as specimens respectively after 4、7、14、21days. all specimens were fixed 24 hours by 10% formaldehyde solution , And decalcified by neutral EDTA , dewaxed , embedded, sliced,SP stained, HE stained . Finally ,counted immunoreactive cells under the light microscope , captured images, measured cumulative optical density by Image-Pro plus 6.0 system, done statistical analysis .[Results] 1. Established Models successfully: 16 rats were well during the procedure of implanting hole shape, and the surgical operation areas had no significant swelling and infection .2. HE staining showed that more fibrous tissuse in bone repair areas at the 4th day and the 7th day; The repair bone cell were most activtive in bone repair areas at the 4th day and the 7th day, which nuclei were deeply stained; There had osteoblasts and osteoclasts apppeared at the edge of bone repair areas. But no in the central areas .3. Osseous tissue immunohistochemistry staining show : masculine cells’endochylema were stained ; the ripe osseous cells weren’t stained , but the osteoblast were stained in the margin of osseous repair area; 7 days after established models , the fibroblast were stained in the center of osseous repair area , the positive cell amount was the most and stain intensity was the the most strong in this stage ,which compared to the bone healing tissue of implantation area in other different time . Rank sum test of 4 groups , the amount of CGRP positive cells and IOD came to the peak in the seventh day , and it was have the covariance learns meaning .[Conclusions] 1.There was higher CGRP expression in bone healing area , meanwhile osteoblast-like cell higher proliferation (the critical period of bone cell formation). CGRP alveolar bone were significantly downregulated in the normal, proved the opposite point of view , which CGRP showed positive active only when the bone remodeling cells’function was active. It may be an important factor to promote bone formation and to inhibit bone resorption.2.The peak expression of CGRP appeared at the phase ,which the fibrous callus change to the bony callus, and the phase was also important at the fibroblasts were induced to express bone formation .It suggested that CGRP may be an important factor for inducing fibroblasts to express the bone formation function .3. CGRP showed strong positive experssion in the cytoplasm of former osteoblasts and fibroblasts of expressing bone formation function . we consideried the positive expression of CGRP not only a sign of the former osteoblasts formation and proliferation , but also inducing fibroblast expressing bone formation function .更多还原