【作者】 刘刚；

【导师】 胡蕴玉；

【作者基本信息】 中国人民解放军第四军医大学 ， 外科学， 2003， 博士

【摘要】 细胞在材料表面的粘附是贴壁依赖型细胞生长的前提，细胞只有在表面以一定的粘附力发生粘附并铺展后，细胞才能生长。作为骨组织工程细胞外基质替代物的人工合成的可降解高分子聚合物材料绝大多数亲水性差，细胞吸附力弱，需要进行必要的表面改性和表面修饰，以提高表面生物活性，利于细胞在材料上的粘附，进而影响细胞的增殖和分化。研究表明，细胞-材料粘附主要由细胞膜上的一个特殊受体整合素介导。而大多数蛋白如纤维连接蛋白、骨桥蛋白、Ⅰ型胶原、骨涎蛋白、玻基结合素等分子结构上包含精-甘-天冬氨酸(Arg-Gly-Asp，RGD)序列，后者可以特异性结合整合素，从而介导细胞粘附。因此将这些蛋白包被、固定到材料表面，观察骨组织工程种子细胞MSCs细胞的粘附、生长特性是本研究的中心环节，并从以下方面进行探讨：(1)采用不同原代细胞分离方法，研究其对MSCs细胞的生物学特性影响。(2)检测基因胜肽胶对MSCs细胞粘附、增殖及分化的影响。(3)分别采用Ⅰ型胶原及纤维粘连蛋白(fibronectin，FN)包被聚乙醇酸-乳酸共聚物(Poly(1actide-co-glycolide)，PLGA)膜及多孔块型PLGA材料，观察细胞在单层或三维培养状态下，Ⅰ型胶原及FN对MSCs细胞粘附、增殖及向成骨细胞分化效应及能力。(4)检测Ⅰ型胶原对MSCs细胞骨架肌动蛋白及细胞内游离钙离子的影响，了解Ⅰ型胶原促进MSCs细胞粘附的内在机制。(5)观察经Ⅰ型胶原包被的多孔块型PLGA材料体内成骨效果，尤其是材料中心部位的成骨情况。 第四军医大学博士学位论文一为研制具有细胞粘附能力、高效诱骨效应的新型活化人工骨提供实验依据和新的解决途径。 研究结果显示：（1）密度梯度高心法接种后24小时可见少数细胞贴壁，3天后，细胞增殖活跃。红细胞裂解法较密度梯度离心法细胞贴壁较慢，36小时才可见零星细胞贴壁，两种方法细胞形态相似。活细胞百分比，红细胞裂解法为（gi士 4）％，密度梯度离心治（83士 5）％，有明显差异。传代培养：2～6代范围内，红细胞裂解法较梯度离心法细胞增殖更旺盛（分别是按种时细胞数的4．67／4．10倍人其他细胞生物学行为如细胞贴壁率，克隆形成率两者无明显差异。（2）电镜下 PLGA膜有均匀一致的文理结构，表面结构均～，无明显突起及裂痕；多孔块型PLGA材料可见相互贯通的大孔结构，大孔的形貌较圆滑，较少尖锐的棱角，孔隙平均尺寸约为400 v你在大孔的周围布满了相互贯通的微孔结构，孔隙形貌多为近似的圆形，孔隙平均尺寸约为5 u m。同样结构均一，符合实验要求。u）基因胜肽胶可促进 MSCs细胞的早期粘附：细胞接种后3小时，实验组（1．5士0．18）X m’，与对照组（0．47士0．22）X10’有显著性差异（P＜0．01），与空白对照组（1．04士0．21）X10’有差异O钥．05人 接种6小时及12小时，实验组与空白对照组均无差异，不能继续促进粘附：其对细胞增殖、细胞碱性磷酸酶活性也均无明显促进作用。N）1型胶原、纤维连接蛋白均能促进细胞粘附：单层培养状态下，纤维粘连蛋白于细胞接种6小时后、I型胶原于细胞接种后12小时开始促进细胞粘附；三维培养体系，I型胶原、纤维粘连蛋白于细胞接种6小时即促进细胞粘附，纤维粘连蛋自作用更明显。电镜照片显示，经1型胶原、纤维粘连蛋白修饰后，细胞分布均匀，铺展良好。G）I型胶原、纤维粘连蛋白促进细胞增殖，细胞接种后 3、6、gd三个检测时间点，实验组细胞均明显高于对照组。与 1型胶原相比，纤维粘连蛋白刺激作用更强。①）I型胶原、纤维粘连蛋白尚能诱导MSCS细胞向成骨细胞分化，不仅表达成骨细胞标志物OCN、ALP、OPN mRNA，而且碱性磷酸酶活性明显增高，碱性磷酸酶及钙结节 7 第四军医大学博士学位论文一染色均强阳性，I型胶原组MSCS细胞碱性磷酸酶活性较FN组更高，有显著性差异；同时，兔疫组化染色表明，经纤维粘连蛋白作用的MSCs1型胶原表达阳性。（7）参照EIAmin方法，细胞骨架肌动蛋白（actinj的结构可根据不同细胞类型加以描述。根据细胞形态，将细胞分为1、11两种类型。1型细胞为多角形或长校形，actin表达较少并相对聚集；11型细胞细胞圆形，铺展良好，。cdn形成丝状结构，表达丰富。I型胶原包被后细胞接种6小时，实验组1型细胞占56％，11型细胞为44％，而对照组1型细胞则占79％，11型细胞为21％；接种12小时，实验组I型细胞占门％，大部分为11型细胞，为而对照组 1型细胞只占 45％。提示，材料经 1型胶原修饰后，细胞多数为圆形，铺展良好，细胞骨架肌动蛋白构建活跃。（8）在1型胶原存在的条件下生长的细胞内荧光较强，而对照组细胞内仅可见少许荧光；在细胞内钙离子浓度曲线图上同样实验组单个细胞内钙离子浓度较高，说明1型胶原可增加细胞内游离钙离于浓度。（8）通过样本连续切片，图像分析系统分析各组动物新骨形成‘情况，显示应用多孔PLGA块状材料经1型胶原修饰后体内成骨明显优于对照组，表现在：1．早期成?更多还原

【Abstract】 Cell adhesion to surface of the substrate is essential to development of the anchorage-dependent cells.Only after adhering to surface followed by spreading can cells develop and proliferate.Most synthetic polymers used as orthopaedic matrix substitute present hydrophobicity,which may correlates to the low degree of cell attachment.Modification with cell adhesion protein/peptides can be benificial to the cell adhesion on polymers and then affect the cell proliferation and differentiation.Cell attachment to substrate is primarily mediated by integrins,a widely expressed family of heterodimeric surface receptors.Most extrcellular matrix proteins such as fibronectin,osteopontin,collagen type I , bone sialoprotein and vitronectin contain an Arg-Gly-Asp (RGD) sequence which is specific to the fixation of cell membrane receptors like integrin.The main aim of this research is to measure,assess adhesion,proliferation of rabbit marrow stromal cells(MSCs) on the polymers coated by fibronectin, collagen type I or biotiegen,which includes:(1)Biologic characteristics of rabbit MSCs were observed by two types of separating method in primary culture.(2)Adhesion,proliferation and differentiation of MSCs cultured on polymers coated with biotiegen were assessed.(3)also, adhesion,proliferation and differentiation of MSCs were assessed on PLGA film orporous PLGA substrates coated with fibronectin, or collagen type I respectively.(4)Bone formation was observed on the porous PLGA substrates coated with collagen type I in vivo.This research aims to give new way to make novel synthetic bone with cell adhesion and high bone induction capabilities.Results obtained included:(1) MSCs began to attach to culture flask after 24h cell inoculation by centrifugation method.Cell proliferation was active after 3d.and MSCs began to attach to culture flask only after 36h cell inoculation by erythrocyte splitting method. Cell morphology was similar no matter what method was used.Percentage of live cells was significantly higher in erythrocyte splitting method that was (91 ±4)%than in centrifugation method that was (83±5) % .Proliferative ability of MSCs seperated by erythrocyte splitting method at 2 to 6 generation was much higher than by another method.The maximal cell number was 4.67 vs 4.10 times that of the initial cell number at cell inoculation. There was no difference in other biologic characteristic of MSCs between the two separation method,such as cell anchorage ratio and clone formation ratio.(2)PLGA film presented uniformity frame with no protuberance and fissure under scanning electron microscopy(SEM).Big aperture with smooth wall and average 400 μ m in size running-through each other was observed in porous PLGA substrate,around the big aperture there were many round micropores about 5μm size.All of the structure were equal and uniform,which satisfied the further research work.(3)MSCs adhesion at earlier time was promoted by biotiegenrAfter 3h ,cell number was (1.5 ±0.18) × 105 in the PLGA film coated with biotiegen group,which was significantly higher than that in PLGA film group (P<0.01) and higher than that in coverslip group (P<0.05) ,which cell number was (1.04± 0.21) × 105 .After 6h and 12h biotiegen could not promote cell adhesion,and cell proliferation and alkaline phosphatase(ALP) activity were not promoteddramatically during 9 days.(4) Cell adhesion was promoted by fibronectin or collagen type I . Cell adhesion dramatically increased after 6h by fibronectin and after 12h by collagen type I in monolayer culture. Fibronectin or collagen type I promoted cell adhesion after 6h under three dimension condition. Fibronectin had more ability to promote cell adhsion than collagen type I under such circumstances,SEM micrographs showed that MSCs all had a spread-out appearance with uniform distribution on polymer substrates precoated with fibronectin or collagen type I .(5) Fibronectin or collagen type I also promoted cell proliferation within 9 days culture under three dimension condi更多还原