纯钛种植体表面改性促进成骨细胞粘附活性的分子调控网络研究

Promoting adhesion activity of osteoblast is key factor for dental implant to achieving improved osseointegration at the implant-bone interface. The present research project compares the gene expression changes of three surface modification groups including nanotube, nanotube loaded by Ca/P and nanotube loaded by Ca/P plus collagen, with the smooth surface control group. Then, the molecular regulatory network that improves osteoblast adhesion activity is constructed. .Osteoblast is in vitro co-cultured with three kinds of modified titanium surface (nanotube, nanotube loaded by Ca/P and nanotube loaded by Ca/P plus collagen), using the smooth surface pure titanium test pieces as control. RNA is extracted, Gene Chip detection to examine the gene expression changes of the four groups are performed. Then gene expression profiling of osteoblast at different titanium surface is acquired. After that, the Gene Chip data is input into Ingenuity Pathway Analysis (IPA), the biological network analyzing platform, and analyzed by several bioinformatics methods. The methods include: differences genetic screening, cluster analyses, gene ontology and pathway analysis. Differences genetic screening selects the certain genes which expressed differently at each surface modification group, compared with the control group. Cluster analyses group the differently expressed genes by expression patterns. Gene ontology tends to find the function of the specific genes in the gene annotation database, from three aspects (cellular component, molecular function and biological process). Pathway analysis reveals and scores the most dramatic changed present known pathways. After comparing the gene expression changes of three surface modification groups with the smooth surface control, candidate characteristic genes related to the osteoblast adhesion activity are screened out.. In addition, the candidate characteristic genes are first confirmed by Real-time polymerase chain reaction and Western blot. Those verified candidate characteristic genes are named as characteristic genes. After that, characteristic genes are further studied by function research, including RNA silence and over-expression technology. From the characteristic genes, those verified genes that play a critical role in osteoblast adhesion activity are named as pivot genes. Then pathways which are selected from the Pathway analysis and proved to be closely related to osteoblast adhesion activity are connected by pivot genes to construct the molecular regulatory network. Among the molecular regulatory network, genes which significantly improve the adhesion activity are called “modifying gene”, and served as target genes for dental implant surface modification. The deep understanding of the molecular regulatory network of genes will provide new thoughts for surface modification of dental titanium implant.

促进成骨细胞粘附活性是增强牙种植体骨结合性能的关键。项目拟研究纯钛牙种植体三类表面改性组（纳米管、纳米管负载钙磷、纳米管负载钙磷及胶原）、光滑对照组的基因表达变化，构建促进成骨细胞粘附活性的分子调控网络。将成骨细胞接种在三类改性组和对照组试样上，体外培养，提取RNA，应用基因芯片检测基因表达数据，在Ingenuity Pathway Analysis平台上分析数据：差异基因筛选、聚类分析、GO功能分类和Pathway分析。在改性组与对照组相比较的基础上，筛选促进成骨细胞粘附活性的候选特征性基因；经Real-time PCR、Western blot初步验证，获取特征性基因；再通过基因沉默和过表达功能验证，获取枢纽基因作为节点，联接Pathway分析选取的信号通路，构建促进成骨细胞粘附活性的分子调控网络。从分子调控网络中选取促进粘附作用显著的改性基因，为研究牙种植体表面改性提供新思路。

口腔种植材料研究的核心问题就在于通过有效的种植体表面改性，实现种植体与骨界面之间早期、稳定的骨结合。而其中促进成骨细胞在种植材料表面的粘附是增强骨结合性能的首要环节和关键步骤。本项目将成骨细胞接种在三类改性组（纳米管、纳米管负载钙磷、纳米管负载钙磷及胶原）和对照组试样上，体外培养。MTT检测和碱性磷酸酶（ALP）活性检测显示，三类改性表面均可促进成骨细胞增殖和分化，其中纳米管负载钙磷及胶原改性的促进作用最为明显。接着，采用扫描电镜观察成骨细胞在各组材料表面的粘附形态，通过激光共聚焦显微镜观察材料表面成骨细胞的细胞骨架形态和粘附斑蛋白染色，检测成骨细胞粘附活性情况。结果提示，纳米管、纳米管负载钙磷、纳米管负载钙磷及胶原组均可增进成骨细胞在材料表面的粘附，细胞铺展和延伸更加充分，细胞骨架肌动蛋白纤维呈各向随机排列的特点更加明显，同时，粘附斑蛋白染色较之对照组明显增强，以纳米管负载钙磷及胶原组最为显著。随后，提取RNA，应用基因芯片检测基因表达数据，进行差异基因筛选和GO功能分类等数据分析。Real-time PCR结果显示，改性组较之光滑表面对照组，16个粘附相关基因，包括Itgb1、FAK(PTK2)、paxillin(PXN)、talin、RELA、NFKBIA等，表达上调，而IKB、CTNNB1基因表达下调，其中FAK处于交汇点的位置，是粘附相关的枢纽基因。经Western blot验证，构建促进成骨细胞粘附活性的分子调控网络。表面形貌对于细胞生物学行为的调控是经由细胞对外界刺激诱导而产生的信号传递过程而实现的。本项目提示：参与成骨细胞粘附的可能有两条信号通路：粘着斑信号通路和NF-κB信号通路。我们推测，整合素和粘附斑激酶可能参与种植体表面形貌和化学、生物成分改性后的成骨细胞粘附过程。其可能的信号通路包括：整合素信号通路（整合素—粘附斑激酶FAK—踝蛋白、桩蛋白、粘附斑蛋白—ROCK1—MLCP（Myl12b））和NF-κB通路（NF-κB—粘附斑激酶FAK—CTNNB—NFKB—ICAM-1/VCAM-1）。而上述两条通路的交汇点枢纽就是粘附斑激酶FAK。依据研究结果，课题组进一步结合3D打印技术，共申请国家发明专利3项，实用新型专利1项，以期为研究牙种植体表面改性提供理论基础和临床新思路。