骨形态发生蛋白12诱导骨髓间充质干细胞构建组织工程肌腱

背景：以往肌腱损伤的修复方法有端端吻合、自体肌腱移植、同种异体肌腱或人工肌腱移植等,但均有其各自缺点。 目的：探讨以家兔骨髓间充质干细胞为种子细胞,骨形态发生蛋白12诱导,聚羟基乙酸复合材料作为支架材料,预构组织工程化肌腱重建家兔跟腱缺损的可能性。 方法：家兔抽取股骨近端骨髓,分离所得细胞传代至第2代,加入10 μg/L骨形态发生蛋白12诱导分化,并与Ⅰ型胶原溶液按一定比例移植于预张的聚羟基乙酸缝线上预制组织工程化肌腱备用。将家兔制备成跟腱缺损模型,分别采用不同方法修复跟腱缺损：组织工程化肌腱修复,Ⅰ型胶原-聚羟基乙酸缝线修复,丝线行端端修复。修复12周对各组肌腱行形态学、力学及组织病理学观察。 结果与结论：修复12周家兔肌腱组织病理切片：组织工程化肌腱组可见大量梭形纤维母细胞顺应力学方向排列均匀分布于胶原中,纤维细胞明显增多,胶原致密;Ⅰ型胶原-聚羟基乙酸缝线组可见部分纤维组织增生伴少许肉芽组织形成,胶原纤维呈松散网丝状,细胞排列紊乱分布不均;丝线组可见纤维组织旁见大量肉芽组织形成。修复12周家兔肌腱生物力学强度：骨形态发生蛋白12+聚羟基乙酸重建肌腱的力学强度明显优于Ⅰ型胶原-聚羟基乙酸组,与丝线缝合组差异无显著性意义;但骨形态发生蛋白12+聚羟基乙酸重建肌腱的力学强度低于正常肌腱。提示以自体骨髓间充质干细胞作为种子细胞,骨形态发生蛋白12诱导,以聚羟基乙酸为支架,可望构建组织工程化肌腱。构建的组织工程肌腱具有一定的生物力学特性,能用于修复跟腱缺损。     

Tissue-engineered peripheral nerve grafting by differentiated bone marrow stromal cells

Bone marrow stromal cells are multipotential stem cells that contribute to the differentiation of tissues such as bone, cartilage, fat and muscle. In the experiment, we found that bone marrow stromal cells can be induced to differentiate into cells expressing characteristic markers of Schwann cells, such as S-100 and glial fibrillary acidic protein, promoting peripheral nerve regeneration. Tissue-engineered bioartificial nerve grafting of rats by differentiated bone marrow stromal cells was applied for bridging a 10 mm-long sciatic nerve defect. Twenty-eight inbred strains of female F344 rats weighing 160 approximately 200 g were randomly divided into four nerve grafting groups, with seven rats in each group. Differentiated bone marrow stromal cell-laden group: poly(lactic-co-glycolic) acid tubes with an intrinsic framework were seeded with syngeneic bone marrow stromal cells which were induced for 5 days; Schwann cell-laden group: poly(lactic-co-glycolic) acid tubes with an intrinsic framework were seeded with syngeneic Schwann cells; acellular group: poly(lactic-co-glycolic) acid tubes were only filled with an intrinsic framework; autografts group. Three months later, a series of examinations was performed, including electrophysiological methods, walking track analysis, immunohistological staining of nerves, immunostaining of S-100 and neurofilament, and axon counts. The outcome indicated that bone marrow stromal cells are able to differentiate into Schwann-like cells and Schwann-like cells could promote nerve regeneration. Bone marrow stromal cells may be potentially optional seed cells for peripheral nerve tissue engineering because of abilities of promoting axonal regeneration.     

自体骨髓干细胞移植治疗肝硬化的疗效观察

治疗终末期肝病,原位肝移植是最理想的手段,然而供体短缺、手术损伤大、术后的免疫排斥反应以及费用高昂等问题限制了肝移植技术的发展和临床应用。自体骨髓干细胞移植操作简便、有效、侵入性小且并发症少,具有重要的临床意义。我科2009年9月至2011年1月共收治严重肝硬化失代偿期患者8例,经自体骨髓干细胞移植,治疗效果良好     

人骨髓间质干细胞与新型可降解材料生物相容性的实验研究

目的：本研究利用人骨髓间质干细胞（MSC）的增殖与分化潜能作为指标，对可降解偏磷酸钙（dCMP）材料和羟基磷灰石（HA）材料的生物相容性进行体外研究。 方法： 通过扫描电镜观察MSC在dCMP表面粘附的情况，并利用ICP和IC分析dCMP和HA的降解产物元素含量，同时采用FACS、ALP活性检测及ARS等方法对降解产物的毒性效应进行检测。 结果： dCMP对MSC的增殖有促进作用，且不影响MSC的成骨分化进程及分化后的矿化功能；而HA对MSC的成骨分化进程无影响，但对MSC的增殖和成骨分化后的矿化功能均有抑制作用。 结论： dCMP的生物相容性较HA为佳，更适合作为骨替代材料。     

骨髓间充质干细胞移植治疗糖尿病足过程中血管内皮生长因子的表达

背景：目前已有研究证明骨髓间充质干细胞移植治疗糖尿病足能够获得良好的效果。 目的：评价骨髓间充质干细胞移植治疗糖尿病足溃疡的效果以及血管内皮生长因子的表达情况。 方法：应用文献检索的方法获取骨髓间充质干细胞移植治疗糖尿病足及血管内皮生长因子表达的相关研究文献,对符合研究标准的文献进行深入的数据分析,文章选取骨髓间充质干细胞移植治疗糖尿病足的实验研究和临床应用进行分析,实验研究中,建立大鼠糖尿病足溃疡模型,给予骨髓间充质干细胞移植治疗,观察创面溃疡的愈合情况,并分析血管内皮生长因子的表达情况。临床应用研究中,对应用骨髓间充质干细胞移植治疗糖尿病足的患者进行观察随访,观察创面溃疡的愈合情况以及不良反应发生情况。 结果与结论：实验研究显示骨髓间充质干细胞移植治疗糖尿病足创面溃疡的愈合快于常规治疗,但是与正常足溃疡愈合相比仍较慢,且移植治疗后血管内皮生长因子的表达升高,但是低于正常足溃疡对照的水平。临床应用研究显示糖尿病足溃疡患者经骨髓间充质干细胞移植治疗后,创面溃疡基本均可完全愈合,且无心、脑血管疾病、肝肾功能损伤以及出凝血时间改变等不良反应发生。     

T cell regeneration after allogenic bone marrow transplantation

Various T cell subsets were characterized by double immunofluorescent staining using monoclonal antibodies (MoAb) in blood, bone marrow (BM) and tissues of 29 patients after allogeneic BM transplantation (BMT). In an attempt to prevent graft versus host disease (GvHD), 15 patients received cyclosporin A (Cy A). In the remaining 14 patients the BM was pre-incubated with a MoAb, OKT3. The regeneration of T4⁺ subset was delayed and the level of T8⁺ cells was abnormally high even 1 year after engraftment. This did not have any predictive value for the appearance of complications such as GvHD or severe viral infections. The number of T8⁺ cells was lower in the group of patients who received Cy A than in the OKT3 group (0·7±0·2 vs 1·5±0·3×10⁹/1 at day 90). In contrast to normal individuals, the T4/T8 ratio in both blood and regenerating BM of BMT patients was <1. A sizeable subset of circulating T cells expressed the phenotype T8⁺,T10⁺,HNK-1⁺,DR⁺. Circulating cells of this phenotype were transiently very high (up to 50%) in patients with active GvHD or suffering from severe viral infection. This subpopulation of lymphocytes was not found in the epidermal infiltrate that accompanied GvHD where the predominant phenotype was T8⁺,T1^-,T10^-,HNK-1^-,DR^-. We conclude therefore that after BMT the number and phenotype of circulating T cells reflects the T cell distribution seen in the regenerating BM.     

碱性成纤维细胞生长因子基因修饰骨髓间充质干细胞移植修复急性肾损伤

BACKGROUND:It has been confirmed that basic fibroblast growth factor (bFGF) can promote the growth, proliferation, differentiation and functional expression of most cells derived from neuroderm and mesoderm. OBJECTIVE:To investigate the effect of bFGF-transfected bone marrow mesenchymal stem cell transplantation in rats with acute kidney injury. METHODS:bFGF genes were transfected into bone marrow mesenchymal stem cells via an adenovirus vector, and then expression of bFGF in transfected cells was identified using RT-PCR technology. Rat models of acute kidney injury were prepared by clipping bilateral renal pedicles, and then randomized into three groups (n=20): rats were given injection of bone marrow mesenchymal stem cell suspensions via tail vein as negative transfected group, those given injection of bFGF-transfected bone marrow mesenchymal stem cell suspensions via tail vein as bFGF-transfected group, and the others given injection of DMEM via tail vein as model group. Four weeks later, levels of serum creatinine and urea nitrogen were detected, expressions of connective tissue growth factor and growth factor in renal tissues were detected by Western blot assay, and morphology of renal tissues was observed using hematoxylin-eosin staining. RESULTS AND CONCLUSION:bFGF genes were successfully transfected into bone marrow mesenchymal stem cells. Compared with the model group, the levels of serum creatinine and urea nitrogen were significantly reduced in bFGF-transfected and negative transfected groups, especially in the bFGF-transfected group (P < 0.05), while expressions of connective tissue growth factor and transforming growth factor in renal tissues in bFGF-transfected and negative transfected groups were significantly weakened in these two groups (P < 0.05), but there were no significant differences between the bFGF-transfected group and negative transfected group (P > 0.05). Besides, renal tissues necrosis and inflammatory reactions were mitigated in the negative transfected group; renal tubules with normal outlines and no overt necrotic cells could be found in the bFGF-transfected group. These findings show that bFGF-transfected bone marrow mesenchymal stem cell transplantation plays a better role in acute kidney injury repair in rats.     

Morphology of the bone marrow after stem cell transplantation

In many haematological conditions the only curative option is stem cell (SCT) or bone marrow (BM) transplantation. Little information exists about BM morphology following non-ablative engraftment. During the pretransplantation period and depending on the kind of pretreatment, there may be hypoplasia, residual disease and varying degrees of fibrosis. In the post-transplantation period, after 1-3 weeks of transfusion-dependent pancytopenia, the first signs of successful engraftment are indicated by the recurrence of neutrophils, monocytes and erythrocytes in the peripheral blood. In the BM there is slow regeneration of erythropoiesis, followed by the other lineages of haematopoiesis and increase in reticulin fibres or even a resolution of fibrosis. Diagnostic problems arise when neoplastic lympho- or haematopoiesis are maintained following transplantation. Moreover, there may be a significant graft versus tumour response reaction or an already relapsing disease needing aggressive treatment. On the other hand, a conspicuous dyshaematopoiesis should not be mistaken as representing a myelodysplastic syndrome. The presence of granulomas being treatment-related or a manifestation of intercurrent granulomatous disease has to be considered. More advanced knowledge of the histological features of regenerating BM will certainly aid the recognition of relapsing disease and is needed for the adequate reporting of post-transplant alterations associated with a successful or failing engraftment.     

Tissue-engineered bone repair of goat-femur defects with osteogenically induced bone marrow stromal cells

Tissue engineering can generate bone tissue and has been shown to provide a better means of repairing weight-bearing bone defect. Previous studies, however, have heretofore been limited to the use of nonosteogenically induced bone marrow stromal cells (BMSCs) or the application of slow-degradation scaffolds. In this study, weight-bearing bone was engineered using osteogenically induced BMSCs. In addition, coral was used as a scaffold material, due to its proper degradation rate for the engineering and repair of a goat femur defect. A 25 mm long defect was created at the middle of the right femur in each of 10 goats. The rates of defect repair were compared in an experimental group of ten goats receiving implants containing osteogenically induced BMSCs and in the control group of goats (n = 10) receiving just coral cylinders. In the experimental group, bony union was observed by radiographic and gross view at 4 months, and engineered bone was further remodeled into newly formed cortexed bone at 8 months. There was increased gray density of radiographic rays in the repaired area, which was significantly different (p < 0.05) from that of the control group. H&E staining demonstrated that trabecular bone was formed at 4 months. Moreover, irregular osteon was observed at 8 months. Most importantly, the tissue-engineered bone segment revealed a similarity to the left-side normal femur in terms of bend load strength and bend rigidity, showing no significant difference (p > 0.05). In contrast, the coral cylinders of the control group showed no bone formation. Furthermore, almost complete resorption of the carrier had occurred, being evident at 2 months in the control group. H&E staining demonstrated that a small amount of residual coral particle was surrounded by fibrous tissue at 4 months whereas the residues disappeared at 8 months. Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and coral can ideally heal critical-sized segmental bone defects in the weight-bearing area of goats.     

经冠脉移植骨髓单个核细胞对猪梗死心肌的修复作用

目的观察经冠脉移植骨髓单个核细胞的定植、分化和对心功能的影响。方法小型猪冠状动脉前降支结扎90min后再灌注制备心肌梗死模型,分为骨髓细胞移植组(BM-MNCs组,n=6)和对照组(n=5),分别经冠脉移植PKH26标记的骨髓单个核细胞和培养基,术后6周进行病理学检查并行血流动力学和超声心动图检查心功能变化。结果在BM-MNCs组,心肌梗死后6周在缺血心肌内可找到移植细胞,其Ⅷ因子和desmin免疫组化染色均为阳性;血流动力学指标显示左室等容舒张期压力最大变化率和心排量与心肌梗死后90min比较明显改善(P<0.05);超声心动图显示每搏输出量和心排量比对照组明显改善(P<0.05);心肌梗死边缘区的小血管数目明显多于对照组(P<0.05)。结论经冠脉移植骨髓单个核细胞可定植于梗死区心肌,并可分化为肌源性细胞和血管内皮细胞,可促进小血管再生,有改善心功能的潜能。     

少量多次异基因骨髓移植的实验研究

目的：探讨少量多次异基因骨髓移植的ＧＶＨＤ反应。方法：采用不同少量异基因骨髓细胞的多次尾静脉注射。观察大小便，皮毛，体位及死亡率等ＧＶＨＤ反应一般表现。移植后不同天数进行体内混合淋巴细胞反应及嵌合体测定。外周血象及骨髓有核细胞按常规方法计数。半固体琼脂法进行骨髓ＣＦＵ－ＧＭ测定。肝、脾、小肠及皮肤病理学检查判断ＧＶＨＤ反应程度。外周血Ｌ６１５细胞及脾脏Ｌ６１５细胞比例判断ＧＶＬ作用。结果：一次性大剂量组ＧＭＨＤ反应表现明显，单纯多次异基因骨髓细胞移植的ＧＶＨＤ反应不明显，而少量多次骨髓移植组则ＧＶＨＤ反应强度界于两者之间，并且具有较好的造血恢复作用。在ＧＶＬ作用方面，一次性大剂量骨髓移植组产生较强的近期抗白血病作用，但多死于ＧＶＨＤ反应。少量多次骨髓移植１×１０６骨髓细胞及 １×１０６脾单个核细胞组抗白血病作用较强，３０ ｄ生存率达 ６０％，死亡原因主要为白血病。结论：少量多次骨髓移植可以减轻ＧＶＨＤ反应，保留部分ＧＶＬ作用。     

Engineering injectable bone using bone marrow stromal cell aggregates

With the increasing popularity of minimally invasive surgery, to develop an injectable bone would be highly preferable for the repair of bone nonunions and defects. However, the use of dissociated cells and exogenous carriers to construct injectable bone faces several drawbacks. To circumvent these limitations, we first harvested a cell sheet from rabbit bone marrow stromal cells using a continuous culture method and a scraping technique. The obtained sheet was then cut into fragments of multicellular aggregates, each of which was composed of a certain number of cells, extracellular matrix, and intercellular connections. The aggregates showed apparent mineralization properties, high alkaline phosphatase activity, increased osteocalcin content, and upregulated bone markers, implying their in vitro osteogenic potential. Then, serum-free medium (the control group), dissociated cell suspension (the cell group), and suspension of multicellular aggregates (the aggregate group) were injected subcutaneously on the back of the nude mice to evaluate ectopic bone formation. The results revealed that the aggregate group showed significantly larger and denser bone at the injection sites than the cell group, whereas bone formation did not occur in the control group. Additionally, when injecting them locally into the mandibular fracture gap of delayed healing in a rabbit model, we observed the most improved bone healing in the aggregate group. More cells survive and retain at the injection sites in the aggregate group than that in the cell group postoperatively. Our study indicates that the multicellular aggregates might be considered a promising strategy to generate injectable bone tissue and improve the efficacy of cell therapy.     

偏磷酸钙纳米粒子的细胞相容性

背景：偏磷酸钙具有优异的细胞相容性能和降解性能及细胞亲和性,人骨髓间充质干细胞可以在多孔偏磷酸钙孔洞内生长和增殖,但有关偏磷酸钙纳米粒子的研究较少。 目的：制备纳米级偏磷酸钙微粒,通过流式细胞术快速检测不同浓度纳米级偏磷酸钙微粒对人骨髓间充质干细胞凋亡的影响。 方法：采用湿法球磨法制备偏磷酸钙纳米粒子,通过扫描电镜和透射电镜观察纳米粒子的形貌,通过X射线衍射分析确定纳米粒子的晶体结构。将偏磷酸钙纳米粒子混入CYAGON OricellTM 基础培养基,使得偏磷酸钙纳米粒子的质量浓度分别为10,1,0.1 mg/L,将其与人骨髓间充质干细胞共培养7 d,通过流式细胞术分析偏磷酸钙纳米粒子质量浓度与细胞凋亡的关系。 结果与结论：采用湿法球磨法成功制备了偏磷酸钙纳米粒子,直径为10-30 nm,粒径分布较均匀,分散性较好,但晶体形状不规则;X射线衍射分析晶相检测其主晶相为β-Ca(PO3)2晶体。10 mg/L质量浓度组细胞G0/G1和G2/M比例高于1,0.1 mg/L质量浓度组(P < 0.01);10 mg/L质量浓度组细胞早期、中晚期、总细胞凋亡率高于1,0.1 mg/L质量浓度组(P < 0.01);说明偏磷酸钙纳米粒子对人骨髓间充质干细胞的增殖有影响,当其质量浓度从1 mg/L增加至10 mg/L后,细胞凋亡率显著增加。     

Remodeling of the thoracic aorta after bone marrow cell transplantation

Stem cells are characterized by their ability to differentiate into multiple cell lineages and display the paracrine effect. The aim of this work was to evaluate the effect of therapy with bone marrow cells (BMCs) on blood glucose, lipid metabolism and aortic wall remodeling in mice through the administration of a high fat diet and subsequent BMCs transplantation. C57BL/6 mice were fed a control diet (CO group) or an atherogenic diet (AT group). After 16 weeks, the AT group was divided into four groups: an AT 14 days group and AT 21 days group, that were given an injection of vehicle and sacrificed at 14 and 21 days after, respectively; AT-BMC 14 days group and AT-BMC 21 days group that was given an injection of BMCs and sacrificed at 14 and 21 days after. The CO group was sacrificed along with other groups. The BMCs transplant had reduced blood glucose, triglycerides and total cholesterol. The Qa (1/mm²) was quantitatively reduced in AT 14 days group, AT 21 days group and was high in AT-BMC 21 days group. The AT 21 days group exhibited increased tunica media and elastic system fibers. The immunolabeling for α-SMA and VEGF showed less immunolabeling in transplanted groups with BMCs. The immunostaining for PCNA seems to be more expressive in the group AT-BMC 21 days group. To conclude, our results support the concept that in mice, the injection of BMCs improve glucose levels, lipid metabolism and remodeling of the aortic wall in animals using atherogenic diet.     

转染IL-6基因骨髓基质细胞系对骨髓移植后免疫功能重建的促进作用

目的　探讨转染IL 6基因的骨髓基质细胞系对同基因骨髓移植 (BMT)后小鼠免疫功能重建的促进作用。方法　将IL 6cDNA片段连接到pcDNA3真核表达载体上。用脂质体将pcD NA3IL 6转入骨髓基质细胞系QXMSC1,ELISA法测定转染IL 6基因骨髓基质细胞QXMSC1IL 6培养上清中IL 6的含量 ,有限稀释挑选多个细胞克隆 ,选择表达量最高的转基因细胞系QXMSC1IL 6用于动物实验。BABL c小鼠经γ射线致死量照射后 ,输入同基因骨髓细胞 (10 7 只 )同时输入骨髓基质细胞QXMSC1IL 6 (5× 10 5 只 )。在骨髓移植后 30d、6 0d检测BMT小鼠淋巴细胞对LPS ,ConA增殖反应 ,T辅助细胞前体 (helpTlymphocyteprecursor,HTLp) ,杀伤性T细胞前体 (cytotoxicTlymphocyteprecursor,CTLp) ,迟发型超敏反应 (delayed typehypersensitivity ,DTH)及空斑形成细胞数 (plaqueformingcell,PFC) ,反映骨髓移植后小鼠免疫功能。结果　成功构建pcDNA3IL 6重组体。该细胞体外培养 2 4h分泌IL 6的含量为 11.15 (± 2 .4 1) μg 10 6 。QXMSC1IL 6细胞系能明显增强BMT后淋巴细胞对LPS、ConA反应性 ,小鼠对异基因小鼠脾细胞DTH反应增强 ,脾脏中HTLp ,CTLp及PFC数明显增加。转入外源IL 6cDNA基因的骨髓基质细胞系QXMSC1IL 6在体内能明显促进BMT后小鼠T、B淋巴     

Angiogenesis facilitated by autologous whole bone marrow stem cell transplantation for Buerger's disease

We hypothesized that angiogenesis can be triggered by autologous whole bone marrow stem cell transplantation. Twenty-seven patients (34 lower limbs) with Buerger's disease, who were not candidates for surgical revascularization or radiologic intervention, were enrolled in this study. Six sites of the tibia bone were fenestrated using a 2.5-mm-diameter screw under fluoroscopic guidance. Clinical status and outcome were determined using the "Recommended Standards for Reports." To mobilize endothelial progenitor cells (EPCs) from bone marrow, recombinant human granulocyte colony-stimulating factor (r-HuG-CSF) was injected subcutaneously as a dose of 75 microg, preoperatively. During the follow-up period (19.1 +/- 3.5 months), one limb showed a markedly improved outcome (+3), and 26 limbs showed a moderately improved outcome (+2). Thirteen limbs among 17 limbs with nonhealing ulcers healed. Postoperative angiograms were obtained for 22 limbs. Two limbs showed marked (+3), five limbs moderate (+2), and nine limbs slight (+1) collateral development. However, six limbs showed no collateral development (0). Peripheral blood and bone marrow samples were analyzed for CD34 and CD133 molecules to enumerate potential EPCs, and EPC numbers were found to be increased in peripheral blood and in bone marrow after r-HuG-CSF injection. In conclusion, the transplantation of autologous whole BMCs by fenestration of the tibia bone represents a simple, safe, and effective means of inducing therapeutic angiogenesis in patients with Buerger's disease.     

Histopathology of bone marrow reconstitution after allogeneic bone marrow transplantation

In order to study haematopoietic reconstitution in allogeneic bone marrow transplantation we investigated bone marrow histology in 61 biopsies of 37 patients, treated with HLA-compatible bone marrow grafts for leukaemia or severe aplastic anaemia. The biopsies were taken from the day of transplantation until 100 d after transplantation. Stromal changes, in particular oedema, fibrosis and granulomas, were found during the whole period of observation. These changes were more prominent in biopsies from leukaemia patients than from patients with aplastic anaemia. The cellularity in the biopsies increased until 28 d after bone marrow transplantation and was stable thereafter. Initially, only clusters of cells belonging to a single cell lineage were seen, suggesting that the first outgrowth of haematopoietic cells is by proliferation of committed precursor cells. Long-lasting abnormalities in localization of haematopoietic cells in the bone marrow space and of the myeloid: erythroid ratio were seen; dyserythropoiesis was common.