【作者】 杨东明；

【导师】 许建中；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 研究背景及意义:严重创伤、感染、肿瘤、骨病等原因所致的骨缺损在临床治疗中非常常见,治疗方法主要有自体骨移植、异体或异种骨移植、生物材料填充等。自体骨移植是治疗骨缺损最有效的方法及金标准,但自体骨往往不能满足较大骨缺损对植骨量的要求,同时会造成供骨区新的创伤;而异体或异种骨移植以及生物材料等均难以获得满意的疗效。近年来组织工程骨的研究为骨缺损修复带来了新的希望。骨髓间充质干细胞（bone marrow derived mesenchymal stem cells,MSCs）被认为是骨组织工程最佳的种子细胞。因为这种细胞取材方便,可通过穿刺获得,创伤小,取材后并发症少,细胞培养增殖快,在体内外成骨诱导环境下可分化为骨组织,而且具有多向分化潜能。尽管自体MSCs或成骨细胞用于动物及临床个体化治疗已经取得了良好的结果,但MSCs在骨髓中含量很少,并随着年龄的增长减少;现有方法体外迅速扩增困难;而且一些自身免疫性疾病患者,其MSCs增殖能力明显减弱;且随着培养代数增加可能出现“反分化”现象和致瘤性,从而失去了细胞应有的功能和安全性。因此,自体骨髓间充质干细胞的应用会受来源和数量限制,难以满足临床“随取随用”的要求。建立同种异体MSCs种子细胞库是解决这些问题的捷径,也是组织工程从实验室走向产业化的重要前提。近年来,国内外研究发现MSCs具有免疫调节作用,已往低等级动物模型也证明同种异体种子细胞及其构建的组织工程肌腱、软骨、骨组织植入体内免疫反应轻微,不足以影响工程化组织植入体内后的修复功能,但对经成骨诱导和经炎前因子处理后骨髓间充质干细胞的免疫原性及免疫调节机制知之甚少。本课题主要研究经成骨诱导和炎前因子处理后骨髓间充质干细胞的免疫原性,探讨其是否具有免疫调节机制,为同种异体MSCs产业化应用及最终走向临床打下基础。实验目的:（1）从人骨髓中分离培养hMSCs并在体外诱导成骨细胞,对其细胞生物学特性进行观察;（2）应用流式、RT-PCR和WB研究hMSCs、DOC免疫原性及经IFN-γ处理后其免疫原性的变化;（3）应用混合淋巴细胞反应（MLR）研究hMSCs、DOC及经IFN-γ处理后在不同数量、不同刺激条件、不同天数下对外周血单个核细胞（PBMCs）增殖的影响;应用ELISA研究hMSCs、DOC及经IFN-γ处理后细胞因子分泌情况,探讨hMSCs免疫调节机制,从而了解以hMSCs为种子细胞构建的同种异体组织工程移植的可行性。实验方法:1.人骨髓间充质干细胞生物学特性（1）无菌条件下在志愿者髂嵴处穿刺抽取20ml骨髓,经密度梯度离心分离纯化hMSCs,培养在含15%胎牛血清的F12-DMEM培养液里。（2）比较hMSCs及DOC细胞生长曲线、细胞周期。（3）FACS检测第3代hMSCs、DOC（诱导后第6、12、18天）细胞的CD14、CD29、CD34,CD44、CD45、CD105,CD166表达。（4）用四环素法、茜素红法;Ⅰ型胶原、骨钙素免疫组化染色鉴定DOC。2.人骨髓间充质干细胞免疫原性实验研究（1）以未分化的骨髓间充质干细胞（hMSCs）为对照,采用流式细胞技术分别检测hMSCs、hMSCs+IFN-γ和诱导后第6、12、18天的DOC、DOC+WN-γ的CD40、CD40L、CD80、CD86、HLA-Ⅰ、HLA-Ⅱ等免疫相关分子的表达;（2）采用RT-PCR技术分别检测hMSCs、hMSCs+IFN-γ和诱导后第6、12、18天的DOC、DOC+WN-γ的B7-H1、B7-H4、HLA-B、HLA-DRA、HLA-DRB等免疫相关基因表达情况;（3）采用WB技术分别检测hMSCs、hMSCs+IFN-γ和诱导后第6、12、18天的DOC、DOC+IFN-γ的B7-H1、B7-H4等免疫相关因子表达情况;3.人骨髓间充质干细胞免疫调节机制实验研究（1）应用混合淋巴细胞反应（MLR）观察（1）不同数量级hMSCs、hMSCs+IFN-γ和诱导后第18天的DOC、DOC+WN-γ对经同种异体PBMCs刺激的PBMCs增殖的影响;（2）不同数量级hMSCs、hMSCs+IFN-γ和诱导后第18天的DOC、DOC+IFN-γ对经有丝分裂原（PHA）刺激的PBMCs增殖的影响;（3）不同天数hMSCs、hMSCs+IFN-γ和诱导后第18天的DOC、DOC+IFN-γ对经同种异体PBMCs和PHA刺激的PBMCs增殖的影响;（4）应用ELISA检测hMSCs、hMSCs+IFN-γ和诱导后第18天的DOC、DOC+WN-γ其培养上清IL-2、IL-4、IL-10、TNF-α、TGF-β1和PGE2的表达情况。实验结果:1.人骨髓间充质干细胞生物学特性（1）细胞呈纺锤形或三角形,类似纤维细胞,旋涡样排列;原代hMSCs接种后4d,可观察到成纤维细胞样的细胞集落,9至12d长满单层培养瓶底,传代细胞4至7d长满单层;hMSCs及DOC细胞贴壁率无明显差别,生长曲线hMSCs倍增时间为38.1h,DOC为39.7h。hMSCs和DOC细胞周期比较,hMSCs细胞周期中S+G₂比例较多,G₁比例较少。表明:hMSCs生长增殖速度较DOC快。（2）第3代细胞FACS检测结果显示:分离培养的细胞CD29、CD44、CD105、CD166表达强阳性,而CD14、CD34、CD45阴性。（3）成骨诱导16d,四环素、茜素红染色见到团块状细胞中有钙盐沉积;Ⅰ型胶原、骨钙素免疫细胞化学检测见到胞浆内有大量的棕黄色颗粒。2.人骨髓间充质干细胞免疫原性实验研究（1）FACS检测结果显示:与hMSCs组比较,hMSCs+IFN-γ组、DOC+IFN-γ组HLA-Ⅰ表达上调（P＜0.05）,HLA-Ⅱ表达明显上调（P＜0.01）。（2）RT-PCR结果显示:与hMSCs组比较,DOC组表达无统计学差异,hMSCs+IFN-γ组、DOC+WN-γ组B7-H1、B7-H4、HLA-B、HLA-DRA、HLA-DRB表达明显上调（P＜0.01）。（3）WB结果显示:与hMSCs组比较,DOC18天组、DOC+IFN-γ组B7-H1表达明显上调（P＜0.01）;DOC12天组、hMSCs+WN-γ组B7-H4表达上调（P＜0.05）;hMSCs+WN-γ组、DOC+IFN-γ组B7-H4表达明显上调（P＜0.01）。3.人骨髓间充质干细胞免疫调节机制实验研究（1）应用混合淋巴细胞反应（MLR）观察①实验组数量级hMSCs、hMSCs+WN-γ和诱导后第18天的DOC、DOC+FN-γ对经同种异体PBMCs刺激的PBMCs增殖均有抑制作用,其抑制作用与细胞数量成正相关,其中10⁵组与10³组相比有统计学意义,（P＜0.01）。②不同数量级hMSCs和诱导后第18天的DOC对经有丝分裂原（PHA）刺激的PBMCs增殖均有抑制作用,其抑制作用与细胞数量成正相关。与阳性对照组相比,在10³数量级,hMSCs+IFN-γ组、DOC+IFN-γ组对经有丝分裂原（PHA）刺激的PBMCs增殖无抑制作用;hMSCs+IFN-γ和诱导后第18天的DOC+IFN-γ对经有丝分裂原（PHA）刺激的PBMCs增殖均有抑制作用,其抑制作用与细胞数量成正相关。③与阳性对照组相比,不同天数hMSCs和诱导后第18天的DOC对经同种异体PBMCs和有丝分裂原（PHA）刺激的PBMCs增殖均有抑制作用,第6天抑制作用最强;与阳性对照组相比,在第4、6、8天,hMSCs+IFN-γ和诱导后第18天的DOC+IFN-γ对经同种异体PBMCs和有丝分裂原（PHA）刺激的PBMCs增殖均有抑制作用,第8天抑制作用最强;在第2天,hMSCs+IFN-γ和DOC+IFN-γ对经同种异体PBMCs和有丝分裂原（PHA）刺激的PBMCs增殖均无抑制作用。（2）应用ELISA检测hMSCs、hMSCs+IFN-γ和诱导后第18天的DOC、DOC+IFN-γ培养上清,均能分泌IL-2、IL-4、IL-10、TNF-α、TGF-β1和PGE2;与未经IFN-γ刺激组相比,IFN-γ刺激组各细胞因子分泌水平均显著增高（P＜0.01）。结论:（1）采用密度梯度离心分离人骨髓获得的细胞在形态、表面标志、多向分化能力方面都符合hMSCs的特征,在成骨诱导条件下可向成骨细胞分化,表达Ⅰ型胶原和骨钙素,是骨组织工程较理想的种子细胞。（2）体外实验表明MSCs、DOC保持低免疫原性,在炎前细胞因子（IFN-γ）刺激下其免疫原性增强。（3） hMSCs、hMSCs+IFN-γ和诱导后第18天的DOC、DOC+IFN-γ均可抑制同种异体PBMSc或PHA刺激的PBMCs增殖,其抑制作用与细胞数量成正相关,其机制可能是通过调控具有免疫调节作用细胞因子的分泌水平来调控免疫反应。总之,hMSCs经成骨诱导和炎前因子处理后其免疫原性可能增高,但仍具有免疫调节作用,从而为同种异体MSCs产业化应用及最终走向临床提供理论依据。更多还原

【Abstract】 Background:Bone defect caused by severe trauma,infection,tumors,osteonosus and so on is very common clinically,treated mainly with autogenous bone transplantation, allogeneic or xenogenic bone grafts,biomaterial replacement and other approaches. Autogenous bone grafting is the gold standard and the most effective methods for treating osseous defects,but autologous bone grafts often don’t satisfy the amount of bone tissue needed for reconstruction and would bring patients new wound,the other ways have also no satisfied effect.In recent years tissue engineering bone brings new hope for bone defect repairing.MSCs（bone marrow derived mesenchymal stem cells） are considered as the optimal seed cells for bone tissue engineering.MSCs can be derived conveniently because they can be obtained through puncture only with small wounds and fewer complications, and be culturabel with rapid proliferation.Osteogenic induction in vivo and in vitro environment not only can differentiate into bone tissue,but also has more potential to differentiate.In addition,MSCs are also easily separated and cultured.In recent years,autologous osteoblast or MSCs for the individual animal and clinical treatment has produced good results.But MSCs content in the bone marrow is low,and decreasing with age and difficult in rapid amplification in vitro.Moreover,some autoimmune diseases significantly weaken its proliferation ability in MSCs.With increased culturing algebra "anti-differentiation" phenomenon and tumor growth may appear, accompanied with the loss of cell function and the safety.Therefore,autologous MSCs is limited owing to the use of sources and quantitative restrictions and can not meet "off the shelf" requirement.Establishing allogenic seed cell bank of MSCs is the shortcuts to solve these problems.Previous studies in lower-class animals showed that grafting in engineered tendon,cartilage and bone constructed using allogeneic seeding cells elicit no visible immunologic reaction and may influence on repairing bone defect,but people know few about the immunogenicity and immunoregulation of hMSCs after Osteogenic differentiated and treated by IFN-γ.The aim of the study is to investigate the the immunogenicity and immunoregulation of hMSCs after Osteogenic differentiated and treated by IFN-γ,it will prepare for the further application of allogeneic hMSCs.Objective:（1）To observed hMSCs isolated and cultured in vitro and induced osteoblast,and its characteristics in cell biology were;（2）To investigate the immunogenicity of hMSCs、Osteogenic cells differentiated from mesenchymal stem cells（DOC） and treated after IFN-γby FACS、RT-PCR and WB;（3）To explore the immunomodulatory mechanisms of hMSCs、DOC and treated after IFN-γby MLR under different conditions on peripheral blood mononuclear and cytokine secretion by ELISA.Material and methods:1.The biological characteristics of hMSCs.（1） Under aseptic conditions,about 20ml bone marrow was aspirated from ailiac crest of volunteer. hMSCs obtained by density gradient centrifugation and purification from bone marrow, were cultured with 15%fetal bovine serum in the culture medium F12-DMEM.（2） The growth curve and the growth cycle of hMSCs and DOC cells are detected and compared.（3） （The expression of CD14,CD29,CD34,CD44,CD45,CD105,CD166 in the third generation cell are dectcted by FACS.4） hMSCs differentiated into osteoblast were identified using tetracycline,alizarin red;collagenⅠand osteocalcin immunohistochemical staining.2.Experiment on immunogenicity of hMSCs、DOC、hMSCs+IFN-γ、DOC+IFN-γ.（1） Undifferentiated hMSCs was used as control group,investigated the expression of CD40、CD40L、CD80、CD86、HLA-Ⅰ、HLA-Ⅱof hMSCs、hMSCs+IFN-γ;DOC、DOC+IFN-γin day6、day12、day18.（2）Gene expression of B7-H1、B7-H4、HLA-B、HLA-DRA、HLA-DRB of hMSCs、hMSCs+IFN-γ;DOC、DOC+IFN-γin day6、day12、day18 were detected by RT-PCR.（3） Gene expression of B7-H1、B7-H4 of hMSCs、hMSCs+IFN-γ; DOC、DOC+IFN-γin day6、day12、day 18 were detected by WB.3.Experiment on immunoregulation of hMSCs、DOC、hMSCs+IFN-γ、DOC+IFN-γ. （1） By mixed lymphocyte reaction（MLR）the following were observed:①Effect on PBMCs proliferation stimulated by allograft PBMCs of different magnitude of hMSCs、hMSCs+IFN-γ;DOC、DOC+IFN-γin dayl8;②Effect on PBMCs proliferation of hMSCs、 hMSCs+IFN-γ;DOC、DOC+IFN-γin day18 by mitogen-stimulated;③Effect on PBMCs proliferation stimulated by allograft PBMCs and mitogen of different days of 10⁴hMSCs、hMSCs+IFN-γ;DOC、DOC+IFN-γin day18;（2） To detected the expression of IL-2、IL-4、IL-10、TNF-α、TGF-β1 and PGE2 in their culture supernatant by ELISA.Results:1.The biological characteristics of human MSCs.（1）The characteristics of hMSCs:cells appearance presented cambiform or triangular-shaped,fibroblasts-like and vortex-like.Four days of hMSCs of original generation,the fibroblast-like cell colony was observed,and hMSCs have fulfilled the bottom of the monolayer cultural bottle in 9-12 days,while the passage cells having done in 4-7 days;There was no obvious difference in adherence of DOC and hMSCs.Doubling time in Growth curve of hMSCs was 38.1h,and DOC was 39.7h.Compared with the cell cycle of DOC,hMSCs accounted for larger proportion in S+G2 phase of cell cycle,relatively small proportion in G1 phase.These suggested that hMSCs proliferated and growed faster than DOC.（2） The third generation was showed by FACS that expression of CD29,CD44,CD105 and CD166 was strong positive in the cultured cells,and expression of CD14,CD34 and CD45 is negative.（3） On 16^thd after osteogenic induction,there were a lot of calcium deposition in the massive cells with Alizarin red and tetracycline staining;there were lots of brown-positive cells by osteocalcin and collagenⅠimmunocytochemical staining.2.Experiment on immunogenicity of hMSCs、hMSCs+IFN-γ、DOC、DOC+IFN-γ. （1） Undifferentiated hMSCs was used as control group,results by FACS showed that expression of HLA-Ⅰincreased in hMSCs+IFN-γgroup and DOC+IFN-γgroup（P＜0.05）; morever,expression of HLA-Ⅱincreased significantly（P＜0.01）.（2） Undifferentiated hMSCs was used as control group,results by RT-PCR showed that expression of B7-H1、B7-H4、HLA-B、HLA-DRA、HLA-DRB significantly increased expression in hMSCs+ IFN-γgroup and DOC+IFN-group（P＜0.01）.（3） Undifferentiated hMSCs was used as control group,results by WB showed that expression of B7-H1、B7-H4 significantly increased expression in hMSCs+ IFN-γgroup and DOC+IFN-group（P＜0.01）.3.Experiment on immunoregulation of hMSCs、hMSCs+IFN-γ、DOC、DOC+IFN-γ. （1） By mixed lymphocyte reaction（MLR）the following were founded:①Different magnitude of hMSCs、hMSCs+IFN-γ;DOC、DOC+IFN-γin day18 can inhibit the proliferation of PBMCs stimulated by allograft PBMCs.The inhibition effects correlated with cell quantity positively,there is statistics evaluation between 10⁵ magnitude and 10³ magnitude（P＜0.01）.②Different magnitude of hMSCs、DOC can inhibited the proliferation of PBMCs stimulated by mitogen.Besides the 10³ magnitude,Different magnitude of hMSCs+IFN-γ、DOC+IFN-γin day18 can inhibited the proliferation of PBMCs stimulated by mitogen,The inhibition effects correlated with cell quantity positively,PBMC proliferation.Inhibition effects correlated with cell quantity positively.③In different days,10⁴ magnitude hMSCs and DOC can inhibited the proliferation of PBMCs stimulated by allograft PBMCs and mitogen.The lowest peak appear in sixth day;Besides the second day,10⁴ magnitude hMSCs+IFN-γand DOC+IFN-γcan inhibited the proliferation of PBMCs.（2） hMSCs、hMSCs+IFN-γ;DOC、DOC+IFN-γin day18 could secrete IL-2、IL-4、IL-10、TNF-αin low level;TGF-β1、and PGE2 in high level; compared to the group without the stimulation of IFN-γ,all of cytokine increase of group stimulated after IFN-γand some cytokine have statistics evaluation（P＜0.01）.Conclusions:（1） The cells obtained from dissociating red marrow by density gradient centrifugation have matched the hMSCs features in aspects of appearance,cell surface marker and multi-directional differentiation ability,it can be differentiated into osteoblast, and express collagenⅠand osteocalcin under the inducible conditions.（2） In vitro experiments show the progeny of hMSCs and DOC remains low immuogenicity,but the proinflammatory cytokine stimulation enhance the immunogenicity.（3） Besides PHA group,Different magnitude of hMSCs,hMSCs+IFN-γ;DOC、DOC+IFN-γin day18 can inhibited the proliferation of PBMCs stimulated by allograft PBMCs.The inhibition effects correlated with cell quantity positively,which can regulate immune responses by probably secreting cytokines level for immune regulatory role.In short,allogeneic hMSCs may be good and effective choice for tissue engineering.更多还原