EphB4通过骨膜蛋白促进CTLA4修饰的MSCs有效成骨的机制研究

发布时间：2017-12-27 14:47

本文关键词：EphB4通过骨膜蛋白促进CTLA4修饰的MSCs有效成骨的机制研究　出处：《第三军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景临床上,大段骨缺损的治疗一直是骨科医生面临的一大难题,而利用自体间充质干细胞(mesenchymal stem cells,MSCs)作为种子细胞所构建的组织工程骨(Tissue engineering bone,TEB)在治疗该类疾病中取得了良好疗效。然而,自体MSCs的获得要受患者的个体差异及体外培养时间的影响,不能很好的满足临床随取随用的需求。因此,探索异体MSCs能否作为TEB的理想种子细胞成了该领域的研究热点。目前,针对异体MSCs的免疫原性一直没有定论,但有报道指出,在移植过程中,异体MSCs仍然会激活宿主的免疫系统,造成移植失败。基于此,我们在前期研究中将细胞毒性T淋巴细胞相关抗原4免疫球蛋白(cytotoxic T lymphocyte associated antigen-4immuoglobulin,CTLA4-Ig)基因导入MSCs中,发现其在异体移植时具有良好的成骨效果,但具体机制不明。近年研究发现,促红细胞生成素产生的肝细胞受体(Erythropoietin-producing hepatocyte receptors,Ephs)和其配体肝配蛋白(Eph receptor interacting proteins,ephrin)在骨组织的新陈代谢中扮演了重要角色,其中以EphB4/ephrin B2轴的调控作用最为关键。EphB4/ephrin B2轴具有双向信号转导作用,分别调控着成骨细胞的骨生成作用和破骨细胞的骨吸收作用。该信号的激活对于骨重建区成骨细胞及其前体细胞的进一步分化和成熟至关重要。同时,组织微环境对于组织的再生重建同样十分重要。组织微环境主要由细胞与胞外基质(extracellular matrix,ECM)构成,而细胞与ECM的相互作用可以调控细胞的增殖、迁移和分化。在骨组织中,骨膜蛋白(periostin,POSTN)被证实参与了骨组织ECM的构成,是行使细胞─ECM相互作用的关键蛋白,可通过结合整合素(Integrin)来介导细胞内外间生物信号的传导功能,促进骨组织的再生。综上所述,我们推测免疫系统的激活将严重影响骨组织中EphB4/ephrin B2轴的调节作用,同时破坏POSTN与细胞之间的生物信号传导,而CTLA4的存在能够逆转这种趋势。本次研究通过利用CTLA4修饰的MSCs来构建TEB,一方面采用不同的动物移植模型来验证成骨修复效果,另一方面在体外利用免疫激活共培养体系验证其对于MSCs-CTLA4中EphB4和POSTN表达的影响,并揭示EphB4与POSTN之间的关系,最后探索Eph B4通过POSTN促进MSCs-CTLA4成骨分化的机制,旨在为进一步阐明MSCs-CTLA4在异体移植中有效成骨的具体机制提供理论依据。第一部分Eph B4在CTLA4修饰的MSCs异位成骨中的作用方法:1.密度梯度离心法分离MSCs,采用DBM双面接种法构建TEB,然后将TEB置于成骨诱导培养基中诱导14天。在植入小鼠体内前更换常规培养基,利用CTLA4腺病毒载体进行感染,构建表达CTLA4的TEB。2.建立小鼠股骨旁TEB异位移植模型,Micro-CT分析异位成骨情况。后续制备移植区域组织切片,HE及Masson染色观察组织形态,免疫组化染色观察CTLA4及Eph B4的表达情况。3.体外利用植物血凝素(phytohaemagglutinin,PHA)刺激外周血单核细胞(peripheral blood mononuclear cell,PBMCs)来建立免疫激活微环境,然后将MSCs或MSCs-CTLA4置于其中共培养,建立免疫激活共培养体系。4.ELISA测定共培养体系中IL-2和IFN-γ的表达情况。Q-PCR和Western blotting实验测定免疫激活共培养前后MSCs及MSCs-CTLA4中Eph B4的表达情况。5.ephrinB2-FC激活EphB4后,利用Q-PCR、免疫荧光、特殊染色法测定相关成骨标志物表达情况。6.为了探索EphB4促进MSCs成骨分化的具体机制,我们构建了Eph B4过表达及si RNA载体作为对照组,利用Western blotting实验测定Eph B4激活后相应信号蛋白的表达情况。结果:1.普通光镜观察结果显示,接种了MSCs的DBM可见细胞形成膜样结构并附着于DBM支架上;荧光显微镜观察结果显示在MSCs-CTLA4构建的DBM中可见细胞发出绿色荧光,证明携带CTLA4基因的腺病毒感染TEB成功。2.小鼠股骨旁TEB异位移植区组织切片中,免疫组化测定CTLA4的阳性表达仅在DBM+MSCs-CTLA4组中可见,其余组均为阴性染色。免疫荧光染色结果显示Eph B4在DBM+MSCs-CTLA4组中的表达量显著高于其余组。HE、Masson染色和Micro-CT三维重建结果均显示相对于其他组,DBM+MSCs-CTLA4组中可见明显的新生骨组织区。相关骨生成数据分析结果显示DBM+MSCs-CTLA4组在新生骨量与骨体积比(BV/TV),骨小梁数量(Tb.N)和骨小梁厚度(Tb.Th)中显著高于其余三组,而在骨小梁分离度(Tb.Sp)中显著低于其余三组(P0.05)。3.未激活状态下的PBMCs在培养基中呈分散排布,当加入PHA刺激后,PBMCs中的T淋巴细胞母细胞化,成团聚集在一起。光镜下显示MSCs-CTLA4共培养组中PBMCs的聚集程度显著低于MSCs共培养组。ELISA检测结果显示IL-2和IFN-γ在MSCs-CTLA4共培养组中的表达水平显著低于MSCs共培养组(P0.05)。4.Western blotting结果显示在免疫激活微环境中,Eph B4在MSCs中的表达水平显著下调,而在MSCs-CTLA4中则维持了正常水平,Q-PCR结果趋势与其一致(P0.05)。5.各组细胞经ephrinB2-FC刺激后,Q-PCR检测结果显示转录因子2(Runt-related transcription factor 2,Runx2)、碱性磷酸酶(Alkaline phosphatase,ALP)、骨钙素(Osteocalcin,OCN)和I型胶原(Collagen I,COL1)在正常培养情况下均有显著增高,而在免疫激活微环境下仅在MSCs-CTLA4组中能观察到该现象。OCN荧光染色、ALP及茜素红染色结果显示在免疫激活共培养后,ephrin B2-FC的刺激仅能上调MSCs-CTLA4中OCN、ALP和钙结节的表达强度,积分光密度值(Integrated option density,IOD)分析结果也与观察趋势一致(P0.05)。6.Western blotting结果显示MSCs在经ephrin B2-FC刺激后,其β-catenin表达逐渐升高,且伴随Runx2和COL1的表达升高。我们进一步利用Eph B4过表达和siRNA腺病毒载体来设立对照组,在激活Eph B4后的检测结果显示MSCs组、空载病毒组和Eph B4过表达组中糖原合成酶激酶3β(Glycogen synthase kinase 3β,GSK-3β)的磷酸化水平增高,同时活性β-catenin的表达水平也随之增高,伴随Runx2和COL1的表达也增高。而上述趋势在Eph B4-si RNA组中则未观察到。同时在免疫激活共培养后,上述效应也只能在MSCs-CTLA4中观察到,而在MSCs组中也未能观察到。第二部分Eph B4信号上调MSCs中骨膜蛋白的表达方法:1.制作小鼠股骨旁TEB异位移植区组织切片,免疫组化测定POSTN表达情况。2.免疫细胞化学测定POSTN在MSCs中表达定位情况,同时Western blotting、Q-PCR、ELISA测定Eph B4激活后POSTN的蛋白、基因表达及体外分泌情况。3.利用NVP-BHG-712、Integrinαvβ3封闭抗体、Eph B4过表达及si RNA载体来设立对照组,Q-PCR及特殊染色法检测Eph B4激活后各组细胞的成骨分化情况,Western blotting检测相关信号分子表达情况。结果:1.免疫组化染色结果显示MSCs-CTLA4组中POSTN的染色显著强于其余组,IOD分析结果与肉眼观察一致(P0.05)。2.免疫细胞化学染色结果显示POSTN定位于MSCs胞质中。各组经ephrinB2-FC刺激后,MSCs组和EphB4过表达组中POSTN的蛋白,mRNA和培养基中的蛋白分泌水平都显著高于其余组(P0.05),而Eph B4-siRNA组和NVP-BHG-712组则与空白组表达水平相近。3.Q-PCR结果显示Runx2、Osterix、COL1和ALP的m RNA水平在ephrin B2-FC刺激组、Eph B4过表达组和POSTN刺激组中都有明显增高(P0.05),而在BHG712阻断剂组、Integrinαvβ3封闭组和Eph B4-siRNA组中上调趋势不明显。ALP及茜素红染色及IOD分析结果与上述各成骨标志物的mRNA表达趋势一致。Western blotting结果显示MSCs在ephrin B2-FC刺激下,单纯刺激组和Eph B4过表达组GSK-3β的磷酸化水平均增高,伴随β-catenin、Runx2和COL1的表达增高,而Eph B4-siRNA组、Integrinαvβ3封闭组和BHG712阻断剂组未观察到此现象。第三部分骨膜蛋白在CTLA4修饰的MSCs成骨修复中的作用及机制研究方法:1.建立兔桡骨缺损TEB原位移植模型,X-ray及Micro-CT分析成骨修复情况。后续制备移植区域组织切片,HE及Masson染色观察组织形态,免疫组化染色观察POSTN及β-catenin的表达情况。2.免疫细胞化学测定POSTN在MSCs中表达定位情况,Q-PCR和Western blotting实验测定免疫激活共培养前后MSCs及MSCs-CTLA4中POSTN的表达情况。3.ALP及茜素红染色测定MSCs及MSCs-CTLA4在免疫激活共培养前后对于POSTN成骨诱导效应的反应。4.Western blotting实验测定POSTN促进MSCs成骨分化的具体机制结果:1.X-ray及Micro-CT结果均显示8周后DBM+MSCs-CTLA4组修复区新生骨塑形明显优于其余组,且横断面显示MSCs-CTLA4组修复区桡骨髓腔已完全再通,而其余两组仍有不同程度的阻塞。相关骨生成数据分析结果显示DBM+MSCs-CTLA4组中BMD,BV及BV/TV均高于其余两组(P0.05)。免疫组化测定POSTN及β-catenin的表达强度在DBM+MSCs-CTLA4组中显著高于其余两组,IOD分析结果同观察一致(P0.05)。2.免疫细胞化学染色结果显示MSCs及MSCs-CTLA4中的POSTN阳性染色均定位于胞质中。Western blotting及Q-PCR结果显示在免疫激活共培养条件下,MSCs中POSTN的表达相对普通培养条件来说显著降低(P0.05),而MSCs-CTLA4组中POSTN的表达则保持在正常水平。3.ALP及茜素红染色结果显示,POSTN的刺激可以显著提高MSCs及MSCs-CTLA4中两者的染色程度,而Integrinαvβ3封闭组可见该效应被抑制。同时在免疫激活共培养后,POSTN上调ALP及钙结节生成的效应仅在MSCs-CTLA4组中可见,而在MSCs组中未观察到此现象。IOD分析结果与观察结果一致(P0.05)。4.Western blotting结果显示POSTN的刺激可上调MSCs组和MSCs-CTLA4组中低密度脂蛋白受体相关蛋白(lipoprotein-related protein,LRP)6的磷酸化水平,同时p-GSK-3β的水平也随之增高,伴随β-catenin和Runx2的表达上调,而Integrinαvβ3抑制剂组则未见此效应发生。在免疫激活共培养后,该效应只在MSCs-CTLA4组中可见,而在MSCs组中未观察到此现象。全文结论1.MSCs-CTLA4构建的TEB在小鼠股骨旁异位移植模型中能够有效成骨,且Eph B4表达显著高于其余组。体外免疫激活微环境可下调MSCs中Eph B4的表达水平,而CTLA4的存在能够维持MSCs中EphB4的表达。同时EphB4可通过与Wnt信号通路发生间接交联反应来促进MSCs的成骨分化。2.EphB4的激活可以上调MSCs中POSTN的表达水平。3.MSCs-CTLA4构建的TEB在兔桡骨缺损原位移植模型中成骨修复效果显著,且移植区修复区POSTN的表达量显著高于对照组。体外免疫激活微环境可下调MSCs中POSTN的表达水平,且能抑制POSTN对于MSCs的成骨诱导效应,而CTLA4的存在能够逆转该趋势。4.POSTN可与Wnt信号通路发生直接交联反应,通过激活Wnt/LRP6/GSK-3β/β-catenin通路来促进MSCs的成骨分化。
[Abstract]:Background: clinical, treatment of large bone defects has been a major problem faced by the Department of orthopedics, autologous mesenchymal stem cells (mesenchymal stem cells, MSCs) as seed cells in bone tissue engineering constructs (Tissue engineering bone, TEB) has a good effect in the treatment of such diseases. However, the acquisition of autologous MSCs is affected by individual differences in the patient and the time of culture in vitro, which can not meet the needs of clinical follow-up. Therefore, it is a hot topic in this field to explore whether allogeneic MSCs can be used as ideal seed cell of TEB. At present, the immunogenicity of allogeneic MSCs has not been conclusive. However, it has been reported that allograft MSCs still activates the host immune system and causes graft failure during transplantation. Based on this, we will study in the early stage of cytotoxic T lymphocyte antigen 4 immunoglobulin (cytotoxic T lymphocyte associated antigen-4immuoglobulin, CTLA4-Ig) gene into MSCs, we find that it has in the allograft bone effect is good, but the specific mechanism is unknown. In recent years, the study found that hepatocyte receptor erythropoietin produced (Erythropoietin-producing hepatocyte receptors, Ephs) and its ligand (Eph protein receptor interacting with liver proteins, ephrin) plays an important role in bone tissue in the The new supersedes the old., regulation of EphB4/ephrin B2 axis is the key. The EphB4/ephrin B2 axis has bi-directional signal transduction, which regulates the osteogenesis of osteoblasts and the bone resorption of osteoclasts. The activation of this signal is essential for further differentiation and maturation of osteoblasts and their precursor cells in the bone reconstruction area. At the same time, the organization of microenvironment is also important for the regeneration and reconstruction of the tissue. The tissue microenvironment is mainly composed of extracellular matrix (ECM), and the interaction between cell and ECM can regulate cell proliferation, migration and differentiation. In bone tissue, Periostin (POSTN) has been proved to be involved in the constitution of ECM in bone tissue, and it is the key protein to exercise cell ECM interaction. It can mediate intracellular signal transduction between cells and promote the regeneration of bone tissue by combining integrin (Integrin). In conclusion, we speculate that activation of the immune system will seriously affect the regulation of EphB4/ephrin B2 axis in bone tissue, and destroy the biological signal transduction between POSTN and cells. The presence of CTLA4 can reverse this trend. This study through the use of modified CTLA4 MSCs to build TEB, using a different animal transplantation model to verify the bone repairing effect, on the other hand, the co culture system to verify its influence on the expression of EphB4 and POSTN in MSCs-CTLA4, in vitro by use of immune activation and reveal the relationship between EphB4 and POSTN, and finally explore the Eph B4 through the promotion of POSTN MSCs-CTLA4 into the mechanism of osteoblast differentiation, in order to provide theoretical basis for the further elucidation of the mechanisms of MSCs-CTLA4 in allograft bone effectively. The first part is the role of Eph B4 in CTLA4 modified MSCs ectopic osteogenesis. Methods: 1. density gradient centrifugation was used to separate MSCs, TEB was constructed by DBM double inoculation method, and TEB was placed in osteogenic induction medium for 14 days. The conventional culture medium was replaced before the mice were implanted, and the CTLA4 adenovirus vector was used to infect and construct the TEB expressing CTLA4. 2. the model of TEB heterotopic transplantation of the para femur in mice was established, and the ectopic osteogenesis was analyzed by Micro-CT. The tissue section of the transplanted region was prepared, the tissue morphology was observed by HE and Masson staining, and the expression of CTLA4 and Eph B4 was observed by immunohistochemical staining. 3. in vitro, peripheral blood mononuclear cell (PBMCs) was stimulated by phytohaemagglutinin (PHA) to establish immune activated microenvironment, then MSCs or MSCs-CTLA4 were placed in co culture, and an immune activated co culture system was established. 4.ELISA was used to determine the expression of IL-2 and IFN- gamma in the co culture system. The expression of Eph B4 in MSCs and MSCs-CTLA4 before and after immuno activation co culture was measured by Q-PCR and Western blotting. After 5.ephrinB2-FC activation of EphB4, the expression of related bone markers was measured by Q-PCR, immunofluorescence and special staining. 6., in order to explore the specific mechanism of EphB4 promoting MSCs osteogenic differentiation, we constructed Eph B4 overexpression and Si RNA vector as control group, and detected the expression of corresponding signal proteins after Eph B4 activation by Western blotting test. Results: 1. ordinary optical microscope showed that the inoculation of DBM cells visible the formation of MSCs membrane like structure and attached to the DBM scaffold; fluorescence microscope observation results show that the construction of MSCs-CTLA4 DBM cells showed green fluorescence, carrying CTLA4 gene of adenovirus infection TEB. 2. in the tissue sections of the TEB heterotopic transplantation area of the femur, the positive expression of CTLA4 was detected in the DBM+MSCs-CTLA4 group, while the rest of the groups were negative staining. The results of immunofluorescence staining showed that the expression of Eph B4 in the DBM+MSCs-CTLA4 group was significantly higher than that in the other groups. The results of HE, Masson staining and Micro-CT 3D reconstruction showed that compared with the other groups, the new bone tissue was visible in the DBM+MSCs-CTLA4 group. Correlation analysis of bone formation data showed that the DBM+MSCs-CTLA4 group was significantly higher in the new bone mass and bone volume ratio (BV/TV), trabecular bone mass (Tb.N) and small bone Liang Houdu (Tb.Th) than those in the other three groups, while in trabecular bone separation (Tb.Sp) was significantly lower than that in the other three groups (P0.05). 3. PBMCs in the inactivated state was dispersed in the medium, and when PHA was stimulated, the T lymphocyte blends in PBMCs and clustered together. The degree of aggregation of PBMCs in the MSCs-CTLA4 co culture group was significantly lower than that in the MSCs co culture group. The results of ELISA test showed that the expression level of IL-2 and IFN- y in the co culture group of MSCs-CTLA4 was significantly lower than that in the co culture group.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R68

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