【作者】 李光辉；

【导师】 刘彦普； 张浚睿；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2013， 博士

【摘要】 颅骨缺损是颅颌面外科及整形外科面临的常见问题，其形成原因有脑外伤、颅内肿瘤及脑血管意外手术等。大面积颅骨缺损的患者容易罹患一系列神经症状，如头痛、眩晕、易激惹、记忆力下降、抑郁等，临床上称之为颅骨缺损综合征，严重影响患者的生活质量。并且，失去颅骨保护的脑组织易于受到外力的伤害，缺损颅骨还会给患者带来严重的美观问题。因此，如何修复缺损的颅骨成为临床工作者必需解决的一个重要问题。传统的治疗颅骨缺损方法包括自体骨及同种异体骨移植等，存在明显的缺陷。组织工程骨技术的发展为颅骨缺损修复提供了新的思路。组织工程骨的概念包括了种子细胞、支架材料和细胞因子三要素。其中，遴选优秀的种子细胞是构建组织工程骨的前提和基础。骨髓来源的间充质干细胞（BM-MSC）是目前应用最为广泛的种子细胞，其成骨能力在一系列体外及体内实验研究中已得到证实。但是，BM-MSC也存在一些缺点，尤其是促进新血管发生的作用不明显，不利于修复大面积的骨缺损。外周血CD34+（PB-CD34+）细胞是包含造血干细胞（HSC）和内皮祖细胞（EPC）等细胞成分的混合细胞群。近年来，PB-CD34+促进新生血管发生的作用已被多个实验所证实。并且，PB-CD34+细胞还可以向成骨细胞分化并促进新骨的形成。因此我们推测，可否将PB-CD34+细胞与BM-MSC进行共培养，构建以PB-CD34+细胞为辅助细胞，BM-MSC为目的细胞的共培养体系？共培养所得细胞的生物学特性，尤其是体外及体内成骨能力如何？共培养细胞能否做为一种新的种子细胞应用于临床？为了回答这些问题，本研究首先建立PB-CD34+细胞与BM-MSC共培养体系，并通过一系列的体外、体内实验验证共培养细胞能否做为一种新型的种子细胞用于组织工程骨的构建。研究目的1.分离PB-CD34+细胞及BM-MSC，建立以PB-CD34+细胞为辅助细胞，BM-MSC为目的细胞的共培养体系。2.探讨共培养细胞的生物学特性，尤其是体外及体内成骨能力，并将共培养细胞与单纯BM-MSC的成骨能力做一对比。3.应用共培养细胞膜片复合羟基磷灰石（HA）支架材料修复兔颅骨极限缺损模型，探讨共培养细胞的临床应用前景。研究方法1.应用流式细胞仪分选兔PB-CD34+细胞；应用密度梯度离心结合贴壁培养法分离BM-MSC，并利用流式细胞技术检测所得细胞的表面分子表达，克隆集落形成、MTT法检测细胞增殖，并进行成骨、成脂及成神经诱导，从而鉴定所得细胞为BM-MSC；最后以直接接触方式对PB-CD34+细胞及BM-MSC进行共培养，建立共培养体系，并检测共培养细胞的构成。2.对共培养细胞的生物学特性进行分析：检测细胞周期及凋亡，检测细胞体外成骨能力，对细胞进行成膜诱导，将细胞膜片复合HA进行裸鼠体内移植，检测细胞体内成骨能力，以上检测项目均设计与单纯BM-MSC进行比较、对照。3.建立兔颅骨极限缺损模型。分别利用共培养细胞及单纯BM-MSC细胞膜片复合HA进行修复。术后8周处死动物，分别通过影像学、组织学及分子生物学检测手段比较两种方法的修复效果。实验结果1.应用流式细胞仪分选可以得到PB-CD34+细胞，分选率为0.8%。分离所得的BM-MSC，以流式细胞仪鉴定其高表达间充质干细胞标志物，不表达造血系干细胞标志物；通过克隆形成实验及MTT法检测细胞增殖能力，证实分离细胞具有自我更新能力；通过成骨、成脂及成神经分化实验，证实了分离细胞具有多向分化能力。共培养体系建立，该体系中既包括大量的BM-MSC，也包括贴壁生长的PB-CD34+细胞。2.将共培养细胞的生物学特性与单纯BM-MSC相比较：流式细胞仪检测细胞周期及凋亡发现，共培养细胞具有更高的增殖能力和相对少的早期凋亡率；体外及体内成骨能力检测发现，共培养细胞的成骨能力更强。3.移植共培养细胞及单纯BM-MSC细胞膜片复合HA修复兔颅骨极限缺损模型发现：活体常规CT及术后所取颅骨标本micro-CT扫描显示，共培养细胞组实验动物的修复效果较好；组织学检测及定量分析显示，共培养细胞组新生的骨小梁体积及新生骨修复缺损比率均高于单纯BM-MSC组；实时定量PCR及Westernblot检测发现，共培养细胞组缺损修复区成骨及促血管生成相关因子的表达量高于单纯BM-MSC组。结论1.通过将PB-CD34+细胞与BM-MSC以直接接触方式共培养，可以建立一种特殊的共培养细胞体系。2.共培养细胞具有比单纯BM-MSC更强的体外及体内成骨能力。3.应用共培养细胞修复兔颅骨极限缺损模型的效果优于单纯BM-MSC，共培养细胞有很好的临床应用前景。更多还原

【Abstract】 The calvarial skull defect caused by trauma, intracranial tumors and cerebral vascularaccident surgery is a common problem in craniofacial surgery and plastic surgery.Patientswith large area skull defect are susceptible to a range of neurological deficits, such asheadaches, dizziness, memory decline, irritable, depressed, clinically referred to skulldefect syndrome, severe impacting patients’ quality of life. Moreover, loss of the skullprotecting, the brain is vulnerable to external forces. Skull defects also cause seriousaesthetic problems for patients. Therefore, how to repair skull defect becomes an important issue for clinical workers.Traditional methods of treatment of skull defects, including bone autograft andallograft bone transplantation, held obvious shortcomings. Development of bone tissueengineering provided a new way for repairing skull defect. Bone tissue engineeringincluded the seed cells, scaffold and cell factor. The selection of excellent seed cells fortissue engineering was the premise and basis. Bone marrow-derived mesenchymal stemcells (BM-MSC) was the most widely used as seed cells and its osteogenic ability wereconfirmed by a series of in vitro and in vivo studies. However, BM-MSC also held anumber of disadvantages, in particular failure to promote new blood vessels, in case of thedetriment of repairing large bone defects.Peripheral blood CD34+(PB-CD34+) cell contained hematopoietic stem cells (HSC)and endothelial progenitor cells (EPC). In recent years, the PB-CD34+cells’ role inpromoting neovascularization had been confirmed by multiple experiments. PB-CD34+cells could also provide the osteoblast differentiation and promote the formation of newbone. Therefore, we assumed, will the PB-CD34+cells co-cultured with the BM-MSC asa co-culture system? What is the biological characteristics of co-cultured cells, particularlythe in vitro and in vivo osteogenetic ability? Can co-cultured cells serve as a new seedcells for clinical use? In order to answer these questions, this study established PB-CD34+cells together with BM-MSC co-culture systems, and through a series of in vitro and invivo experimental verification the capability of co-cultured cells as a new type of seedcells for bone tissue engineering.Objectives1. To separate PB-CD34+cells and BM-MSC, establishment of a PB-CD34+cells andBM-MSC co-culture system.2. To explore all the biological characteristics of co-cultured cells, in particular in vitroand in vivo osteogenic ability, and do comparison between co-cultured cells andBM-MSC. 3. To explore the prospects of clinical application of co-cultured cells, co-cultured cellsheet composite with hydroxyapatite (HA) scaffold was used to repair a rabbitcalvarial critical-size defect model.Methods1. Application flow cytometry points selected rabbit PB-CD34+cell; application densitygradient centrifugal combined posted wall training law separation BM-MSC, and usesflow cytometry technology detection proceeds cell of surface molecular expression,clone set falls formed, and MTT law detection cell proliferation, and for osteogenesisand adipogenesis differentiation induced, to identification proceeds cell for BM-MSC;finally to directly contact way on PB-CD34+cell and the BM-MSC for co-culture,establish co-culture system, and detect the constitute of co-culture cells.2. Analysis of biological characteristics of cultured cells: detect cell cycle and apoptosis,cells osteogenic differentiation in vitro, induce cell sheet, transplant cell sheetcomposited with HA in nude mice, to detect osteogenic differentiation in vivo, moretest items are designed to compare, in contrast to simple BM-MSC.3. A rabbit calvarial critical-size defect model was established. Respectively the use ofco-cultured cells and BM-MSC cell sheet composite to repair the skull defect. Afterthe execution of animals, respectively by radiographic, histological and molecularbiology test were made to compare the two methods.Results1. PB-CD34+cells were separated using flow cytometry, and the separation rates was0.8%. The separated the BM-MSC, using flow cytometry to identify their highexpression of mesenchymal stem cell markers, without expression of hematopoieticstem cell markers. Through clone experiments and MTT assay for detection offorming cell proliferation, we confirmed the separated cells with self-renewalcapacity; by osteoblasts, adipogenic differentiation experiment we confirmed theisolation cells had multipotent differentiation of capacity. Establishment of co-culture system, the system includes a large number of BM-MSC, including adherentPB-CD34+cells.2. All biological characteristics of cultured cells compared with BM-MSC: flowcytometry cell cycle and apoptosis of found, a total of proliferation of co-culturedcells had a higher capacity and a relatively small rate of early apoptotic; and the invitro and in vivo osteogenic ability and osteogenic potential of co-cultured cells werestronger than BM-MSC.3. Transplantation co-culture cell and BM-MSC cell sheet composited with HA repair arabbit calvarial critical-size defect model: Live general CT and the micro-CT scandisplayed, co-cultured cell group experimental animal of repair effect was better;histological detection and the quantitative analysis displayed, co-cultured cell groupof new bone volume and bone repair ratio were better than BM-MSC group; real-timequantitative PCR and the Western blot detection found, co-cultured cells stimulatedand upregulated the expression of osteogenic and angiogenesis-related factors betterthan BM-MSC group.Conclusion1. Co-cultured PB-CD34+cells with BM-MSC by direct contact, we can establish aspecial cell culture system.2. Co-cultured cells demonstrated higher osteogenic differentiation capacity thanBM-MSC in vitro and in vivo.3. Co-cultured cells improve bone formation in a rabbit calvarial defect model betterthan BM-MSC, holding an promising clinical application in future.更多还原