【作者】 王立勋；

【导师】 黄煌洲； 姜建元；

【作者基本信息】 复旦大学 ， 外科学， 2008， 博士

【摘要】 第一部分大鼠骨髓间质干细胞的分离培养和鉴定实验一大鼠骨髓间质干细胞的分离和培养目的:分离和培养大鼠的骨髓间质干细胞。掌握原代细胞的培养技术,了解干细胞的生物学特性。材料和方法:四月龄的SD大鼠,沿用Friedenstein的方法,利用间质干细胞粘附于组织培养板的特性,首先利用梯度离心分离出单核细胞,再利用干细胞粘附于塑料培养板的特点,获得较纯的贴壁生长的骨髓间质干细胞。结果:细胞贴壁较快,呈克隆生长,细胞形态为均一的长梭形。在接种24小时后可见成纤维状的细胞以散在的方式贴壁,2—3天就可见一些集落形成,7—10天后集落迅速增多,并且逐渐长大融合成片。随着集落生长的不断扩大而融合为单层。传代培养后细胞不再以成集落的方式生长,而是呈均匀分布生长。原代培养结束时细胞数为10⁵左右,P10代细胞数约为10¹²左右。结论:大鼠骨髓间质干细胞的分离和培养技术成熟,细胞增殖能力旺盛。实验二大鼠骨髓间质干细胞的鉴定目的:通过表面抗原检测确定培养细胞的正确性。为下一步实验确定良好的种子细胞。材料和方法:通过流式细胞仪检测大鼠骨髓问质干细胞的表面抗原,包括CD29,CD44,CD90,CD105,CD34,CD45,CD11b,组织相容性复合体Ⅰ、Ⅱ。结果:与对照组相比,大鼠骨髓间质干细胞表面抗原CD29,CD44,CD90,CD105显示阳性,而表面抗原CD34,CD45,CD11b为阴性,组织相容性复合体Ⅰ、Ⅱ均为阴性。结论:大鼠骨髓间质干细胞表面标志为非单一性,表达间质细胞、内皮细胞和表皮细胞的表面标志,一般认为整合素家族成员CD29,粘附分子CD44、CD105以及胸腺和外周T淋巴细胞标志CD90等是骨髓问质干细胞的重要标志物。第二部分携带hBMP2基因的慢病毒转染骨體间质干细胞的定向分化目的:观察慢病毒载体诱导rMSCs后的变化情况,检测rMSCs是否按预期方向向成骨细胞转化。材料和方法:含有hBMP2基因的慢病毒载体诱导rMSCs,诱导第0、3、6、9、12、15、18、21天测定碱性磷酸酶的活性及细胞染色情况;采用钙的定量测定沉积钙的浓度;免疫组化分析Ⅰ型胶原蛋白表达情况。结果:诱导后的rMSCs,随着大鼠骨髓间质干细胞诱导时间的延长,其碱性磷酸酶的活性逐渐增加,染色良好,而未经病毒诱导的干细胞,培养21天,其碱性磷酸酶的活性无明显的变化;诱导组Ca²⁺的浓度持续升高,到第15天后Ca²⁺的浓度达到最大,此后达到平稳期,未诱导组钙沉积无明显变化;诱导组Ⅰ型胶原蛋白表达明显增加,染色呈阳性,而未诱导组无明显变化。结论:通过免疫组化和组织化学技术证明,诱导的rMSCs细胞能够产生工型胶原、碱性磷酸酶,并具有矿化能力,证实在慢病毒载体的诱导下rMSCs向成骨细胞分化。第三部分成骨细胞—β-磷酸三钙复合体的构建目的:观察大鼠成骨细胞及MSCs在β-磷酸三钙表面的贴附及生长情况。材料和方法:四块多孔β-磷酸三钙（直径8mm,厚1mm）,随机分成实验组和对照组。实验组β-磷酸三钙在负压条件下用重组人纤维连接蛋白（100μg/ml）浸渍过夜。再将两组β-磷酸三钙分别与rMSCs和大鼠成骨细胞共同培养10天,扫描电镜下观察比较实验组与对照组细胞在β-磷酸三钙表面的贴附数量和形态。结果:实验组细胞的贴附数量显著高于对照组（P＜0.01）。成骨细胞实验组分泌大量胶原纤维和形成钙化结节。结论:纤维连接蛋白对β-磷酸三钙的表面修饰能明显提高大鼠成骨细胞和rMSCs的贴附率,有利于成骨细胞的生长及成骨表型。第四部分自体成骨细胞—β-磷酸三钙修复大鼠颅骨极量骨缺损目的:评价携带hBMP2基因的慢病毒转染rMSCs与β-磷酸三钙复合构建的组织工程化骨对骨缺损的修复效果。材料和方法:250～350 g雄性SD大鼠42只,均在全麻及无菌条件下取其左侧股骨及胫骨,行MSCs培养及成骨诱导。实验细胞在培养14d后消化接种于经纤维连接蛋白表面修饰的含孔磷酸钙陶瓷上继续培养10d。所有大鼠均造成颅骨极量缺损（直径8mm）模型,用自体细胞行骨缺损修复。随机分为Lev-BMP2转染细胞+β-TCP组（n=10）;rMSC+β-TCP组（n=10）;β-TCP组（n=10）;Lev-BMP2转染细胞组（n=4）;rMSC组（n=4）;空白对照组（n=4）。于第4、8、12、20周从形态学及组织学角度观察比较颅骨缺损修复愈合情况。结果:无论X线摄片及组织切片均表明,实验组颅骨极量缺损修复优于同期对照组。在各时间点Lev-BMP2转染细胞+β-TCP组和rMSC+T-TCP组在植骨区均有新骨形成,而β-TCP组仅见边缘区成骨。结论:Lev-BMP2转染BMSCs复合β-TCP构建的组织工程化骨可以修复大鼠颅骨极量骨缺损。更多还原

【Abstract】 PartⅠisolation culture and identification of rat Mesenchymal stem cells（rMSCs）Experiment 1 isolation and culture of rat Mesenchymal stem cells（rMSCs） Objective:to isolate and culture rat Mesenchymal stem cells（rMSCs）.To grasp the techniques of rMSCs culture and investigate the biological characters of rMSCs. Materials and Methods:4-month-old SD rats were used,follow the methods of Friedenstein,because of the characters of rMSCs adhereing to tissue culture flask,（as rMSCs is adherent to tissue petri dish,） monocytes were isolated by gradient centrifugation first;then pure adherent rMSCs were obtained by the property that rMSCs is adherent to plastic petri dish.Results:rMSCs are rapidly adherent,grow in clone,the morphology of cell are homogeneous fusiform,within 24hs fibrous like cells were seen sporadic adherent,some cell clony were seen in 2-3 days,and they increased rapidly in 7-10 days,gradually grow into mixture. As the cell clony grown,they became monolayer at last.After passage,rMSCs no longer growed in clony but in homogeneous distribution,rMSCs number of primary culture is about 10⁵,and passage 10 is about 10¹².Conclusions:isolation and culture of rMSCs are feasible,and have powerful ability of cell proliferation.Experiment 2 identification of rat bone marrow Mesenchymal stem cells （rMSCs）Objective:surface antigen was used to identify the property of cultured cells. to prepare the seed cells of next step.methods and materials:to detect the surface antigen of rat bone marrow MSCs with flow cytometry,include CD29,CD44,CD90,CD105,CD34,CD45,CD11b.results:in contrast to the control group, the surface antigen CD29,CD44,CD90,CD105 of rat bone marrow rMSCs were positive; surface antigen CD34,CD45,CD11b were negative,and major MHC-Ⅰ、Ⅱ（major histocompatibility complex-Ⅰ、Ⅱ） were negative too.Conclusions:rat bone marrow rMSCs surface markers are multiplicity,which express the surface markers of interstitial cells,endotheliocytes and epidermic cells,generally CD29（the member of integrin family）,adhesion molecule CD44,CD105 and CD166,surface marker CD90 of T lymphocytes in thoracic gland and periphery circulation and so on were important markers of bone marrow rMSCs.partⅡdirectional differentiation of rMSCs infected by lentiviral vector with hBMP-2Objectives:to observe the change of rMSCs induced by lentivral vector,to detect if rMSCs transform to osteoblast as expectation.Materials and methods:after lentiviral vector induced rMSCs,in the 0,3,6,9,12,15,18 and 21 day,alkaline phosphatase（ALP）activity is evaluated;to determin calcium ion concentration with quantitative assay;immunohistochemistry is used to detect the expression of typeⅠcollagen.Results:as the induced time prolonging,in rMSCs after induction, alkaline phosphatase（ALP） activity is gradually increasing,but ALP activity of rMSCs that were not induced had no obvious change after 21 days culture.After induced,Calciumion concentration is continuous increasing,reached climax after 15 days,then get in stationary phase.Calcium deposition had no marked change in the group that were not induced;typeⅠcollagen expression increased obviously in induced rMSCs and staining is positive,in contrast,which had no change in the control.Conclusions:with immunohistochemistry and histochemistry methods we revealed that induced rMSCs can produce typeⅠcollagen,ALP and had the ability of mineralization,which confirmed rMSCs can be induced to differentiate into osteoblast by lentivral vector.partⅢosteoblast cultivated with TCP in vitroObjective:To investigate osteoblast and MSCs grewing and adhension in calciumphosphate bioceram.Materials and methods:Four porous ceramics 8mm×1mm divided into experimental and control groups randomly.The experimental group was soaked in 100g/ml recombinant human fibronectin under negative pressure over night.Then both groups were cultivated in vitro simultaneously with osteoblasts derived from bone marrow MSCs and MSCs of rats respectively for ten days.The morphogenetic pattern and number of the anchored cells were observed under scanning electron microscope.Results:There was statistically significant difference of cell numbers between the experimental and the control groups P＜0.01. Many secretory collagen fiber bundles and mineralized nods were observed in the experimental group under high magnifier.Conclusions:It seems that the fibronectin surface coating is beneficial to osteoblast anchorage and its osteogenic pheno type.PartⅣRepair of critical size rat calvarial defects with autogenous osteoblasts loading onβ-TCPObjective:To evaluate osteogenetic effectiveness of lentiviral vector mediated hBMP-2 gene（Lev-hBMP-2） transfected bone marrow derived mesenchymal stem cells for repair of bone defect mixed withβ-TCP in the repair of critical size rat calvarial defects.Materials and Methods:Forty two male adult SD rats（weight: 250-350g） were obtained bone marrow from the left femurs and tibias under general anesthesia and sterile condition.BMSCs were cultured and transfected by Lev-hBMP-2 in vitro.The BMSCs were collected from bone marrow of rats,cultured and transfected by Lev-hBMP-2 in vitro for 14 days.The cells were then seeded and subcultured for another 10 days inβ-TCP that had been subjected to surface porous calcium phosphate ceramic that had been subjected to surface modification via soaking in recombinant human fibronectin.The cells and cell-ceramic compound were used to repair critical size（diameterof 8 mm） calvarial defects in the corresponding rat in the experimental group.The rat were divided into six groups at randomly:1.Lev-hBMP-2 transfected BMSCs+β-TCP（n=10）;2.BMSCs+β-TCP（n=10）; 3.β-TCP（n=10）;4.Lev-hBMP-2 transfected BMSCs（n=4）;5.BMSCs（n=4）;6. control（n=4）.The skull defect reconstruction was observed histomorphometrically at 4th、8th、12th、20th week after operation.Results:tissue slicing showed that the whole defected areas can found new bone grewing during different stages in the experimental group while osteogenic tissue only appeared in the margin of ceramic in the control group. Conclusions:The tissue-engineered bone made by Lev-hBMP-2 transfected BMSCs mixed withβ-TCP can repair critical size rat calvarial defects更多还原