【作者】 薛明；

【导师】 艾红军；

【作者基本信息】 中国医科大学 ， 口腔临床医学， 2004， 博士

【摘要】 实验目的 由慢性根尖周炎、牙周炎引起的牙槽骨缺损性疾病在临床上颇为常见。成骨细胞(Osteoblast)位于骨组织表面，是骨形成以及损伤后修复重建过程中的重要功能细胞，成骨细胞在骨形成和改建过程中可以产生骨基质并分泌多种生物活性物质，如胶原纤维和无定形有机基质，调节与影响着自身和破骨细胞的功能。而临床上在由于感染引起的骨破坏性疾病中，大部分成骨细胞的消失并不是成骨细胞向骨细胞、骨基质和骨膜方向转化，而是发生了细胞凋亡。所以现行的牙槽骨缺损性疾病的治疗方法难以获得理想的疗效。因而，探讨成骨细胞增殖分化及凋亡的特性，从而研究一种促进成骨细胞增殖分化；阻止其凋亡的生物制剂，可以为临床上骨缺损性疾病的生物学治疗开辟新思路。近年来，随着组织工程学在医学各学科的研究不断发展，骨替代产品应用于骨缺损性疾病的治疗已经成为可能，因而研究一种理想的骨诱导剂应用于临床具有重要意义。 纤维粘连蛋白(Fibronectin，FN)是细胞外基质(Extracellular matrix，ECM)中糖蛋白的最重要成分，主要分布于疏松结缔组织，除成纤维细胞外，上皮细胞、血管内皮细胞、成骨细胞、软骨细胞和巨噬细胞也可生成。血浆型FN以单体形式存在，通过N-端的70×10~3Kda的蛋白质与细胞表面特定区域发生聚合，单体之间通过二硫键桥彼此连接形成FN多聚体，FN的聚合是发挥其生物学功能的先决条件。FN单体是一种由相似的两个肽链亚单位(A和B链)通过羧基端的二硫键结构成为分子量约220×10~3Kda的多功能二聚体糖蛋白。由于FN对细胞的生长、增殖分化、粘附移动、损伤修复等过程起到重要的作用，因而受到医学各学科研究领域的重视。以往的实验证明：FN是成骨细胞增殖分化及凋亡的至关重要因子。本实验应用外源性FN作用于体外培养的成骨细胞，旨在研究FN对成骨细胞增殖分化及凋亡的影响，探讨应用FN治疗骨缺损性疾病的可行性。实验方法 1.成骨细胞的分离纯化 取生后Zd的Wistar乳鼠拉颈处死后投人盛有75%乙醇的容器中消毒，剪开头顶部皮肤，取颅盖骨放人盛有缓冲液的培养皿中，用缓冲液洗净，加人0.25%胰蛋白酶溶液，在37℃恒温消化巧min。再用lm群血n型胶原酶溶液在37℃条件下消化90 min，移于离心管中离心(1 O00r/min，5min)用199培养液将沉淀物洗两次，吹散细胞沉淀，制成细胞悬液，接种于培养瓶内。差速勃附法纯化细胞。 2.成骨细胞的培养及鉴定 调整上述已纯化的细胞悬液细胞密度至1护个/nil，接种于25nil培养瓶内，置于37T、5%COZ培养箱中，2d后换液，除去未贴壁细胞，并适时传代，另在一小培养皿中加人盖玻片，接种少量细胞悬液，置于37℃、5%C02培养箱中培养，用I型胶原免疫组化方法作细胞鉴定。 3.成骨细胞的形态学观察 应用倒置相差显微镜动态观察成骨细胞的生长特性并拍照记录。将培养的成骨细胞用胰酶消化，离心，PBS冲洗，0 .3%的戊二醛和四氧化饿双重固定，乙醇逐级脱水，醋酸异戊醋置换，临界点干燥，喷金镀膜，S一520型扫描电镜观察;培养形成单层细胞，经胶原酶消化，分散离心获得成骨细胞团块，加人4%戊二醛预固定，饿酸染色，Hi鱿achi一600型透射电镜下观察并拍照纪录。 4.Alam二cBlueTM法测定细胞增殖率 取培养的成骨细胞第3代，调整细胞浓度至2 x 104/血，以每孔100闪接种于96孔板，每组8孔，培养条件为37℃，5%COZ饱和湿度。在含10%胎牛血清的199液中培养48h，换成浓度为10林岁而，20林岁血，30林岁时，40林群耐，50林扩而的牛血浆FN，无血清培养72h，在最后4h每孔加入20闪的AIamarBlu(，TM，37℃、5%CoZ培养箱中继续孵育至结束，酶标仪540run、620nm波长下分别测定各孔吸光度，按说明书要求计算各组均数并作统计分析。 5.EUSA方法检测碱性磷酸酶(ALP)活性 细胞接种至24孔板，24h后换成浓度为ro林岁nil，20林岁nil，30林岁nil，40林岁耐，50林扩血的牛血浆FN，无血清培养72h。吸出培养液，加0.1%TritonX一100 100林l，置于4℃过夜，然后加人pNPP，37℃孵育30h，3N氢氧化钠终止反应。将各孔液体分别移人%孔板，用酶标仪读取OD值(波长405nm)。 6.流式细胞术检测FN对成骨细胞细胞周期及凋亡的影响 细胞以1 xlos/血接种至25d培养瓶，在含15%胎牛血清的199培养液中培养3d。换为无血清199培养液。24h后加人浓度为10林岁耐，20林岁血，30林岁血，40林岁nil，50林岁d的牛血浆FN，无血清培养72h，0.25%胰酶消化各组细胞。pBS洗2次，加人配制好的pl染液(pl smg、RNase Zmg、生理盐水65耐、构缘酸钠loomg加蒸馏水100而，调PH值7.2一7.6)，37℃孵育30min，用FACS流式细胞仪检测，Ce断t软件收集10 000个细胞，CellQuest软件获取并分析数据，根据G0/G:期、S期、GZ/M期细胞数。采用sPss 1 0 .0统计软件进行数据分析。 7.westem blotting法检测FN作用后成骨细胞Bcl一2及Bax基因的蛋白表达变化 处理后的细胞，PBS洗2次，离心，加人300闪的裂解buffer。超声粉碎后，12000甲而分，4℃，更多还原

【Abstract】 Chronic periapical periodontitis and periodontitis are two common diseases of stamatology, Which can cause severe damage or deform of bone tissue. Osteoblasts are loc ated at the surface of bone tissue, They are functional cell, play an important role in bone ?forming or reparing stages. In many cases of bone tissue damage or deform, The loss of osteoblasts is not attribute to forming new bone tissue or matrix , But occurring apoptosis. That is the reason why present therapy methods may not obtain satisfying results, therefore, A new biological therapy method should be investigated to solve such kind of problem. How can we take adventage of the biological function of some biological active factor in order to increase the proliferation and differentiation of osteoblasts and decrease the rate of apoptosis at the same time,become a focus question in stomatology. With the development of bone tissue engineering, The application of biological therapy of bone damage or deform has clinical significance.Fibronectin ( FN) is a heterodimeric extracellular matrix (ECM) glycopro-tein which has been shown to regulate adhesion,migration, proliferation, differentiation and apoptosis of mesenchymal cells. Present study has been proved that FN is a survival factor for differentiated osteoblasts, and also plays a unique role in osteoblasts apoptosis. therefore, The clinical application of FN as a new biological agent is a promising and valuble therapy method.The purpose of this study is to establish an ideal and effective cultivation system including the methods of isolation, purification , cultivation and identification of rat calvaria osteoblasts in vitro and to investigate the effects of bovine plasma fibronectin on the proliferation, differentiation and apoptosis of rat osteoblasts in vitro in order to offer an experimental basis for the clinical application.Methods1. Isolation , purification , cultivation and identification of Osteoblasts Calvariae from 2d - old new bone rats were removed aseptically. The peri-osteal layers on both sides were carefully stripped off with tweezers under D -Hanks. Then, the bone specimens were treated by trypsinization for 20 min at 37 X,, and the supernatant was discarded. After that the specimens were digested by collagenase for 90 min, the supernatant was collected and centrifuged. The collagenase - release cells were plated in 199 medium supplemented with 15% fetal bovine serum (FBS). The medium was changed twice per week until the cells reached confluence. The differential adherence method was used to purify the osteoblasts. The immunohistochemical method of type I collagen was used to identify the osteoblasts.2. The morphologic observation of osteoblastsObserve shapes and structures of cultured osteoblasts with phase contrast microscope, Scan electron microscope ( SEM) and transmission electron microscope (TEM).3. Effects of bovine plasma FN on the proliferation of rat osteoblasts Osteoblasts were collected using 0. 25% trypsin and replated at a density of5 104cells/ml in 96 - well flat - bottomed plates. The medium was changed to serum - free medium after 24 h. The next day FN with different concentration (0 ug/ml, 10 ug/ml, 20 ug/ml, 30 ug/ml, 40 ug/ml, 50 ug/ml) was administrated. The proliferative response to FN was determined by the percents of reduced AlamarBlue4. Effects of bovine plasma FN on the differentiation of rat osteoblasts Osteoblasts were collected using 0. 25% trypsin and replated at a density of5 x 104cells/ml in 96 - well flat - bottomed plates. The medium was changed to serum -free medium after 24h. The next day FN with different concentration (0 ug/ml, 10 ug/ml, 20 ug/ml, 30 ug/ml, 40ug/ml, 50ug/ml) was administrated. The activity of alkaline phosphatase (ALP) was measured by Elisa method.5. Analysis the cell cycle and apoptosis effects by FNOsteoblasts were plated in flasks at a density of 1 x 105cells/ml, and allowed to attach for 3d. After that the medium was changed to serum - free medium. FN with different concen更多还原