骨髓间充质干细胞和内皮祖细胞移植促进血流重建的比较

目的:比较骨髓间充质细胞(Bone Marrow Mesenchymal Stem Cells,BM/MSC)和骨髓源内皮祖细胞(Bone Marrow Endothelialprogenitor cells,BM/EPC)移植促进血流重建的效果,为进一步优化骨髓干细胞移植治疗肢体缺血提供理论基础。方法:获取Lewis大鼠骨髓单个核细胞,在体外培养分化为MSC和EPC。采用Lewis大鼠建立单侧后肢缺血模型。在模型建立后3天,将0.8mlD-Hanks液注入大鼠缺血侧后肢,为对照组(n=6);将8×106个骨髓MSC植入大鼠缺血侧后肢,为MSC组(n=6);将体外培养的8×106个EPC植入大鼠缺血侧后肢,为EPC组(n=6)。细胞移植后3周行缺血大鼠后肢动脉造影,检测缺血侧后肢侧支血管数;获取缺血侧后肢腓肠肌,分别行CD31和α-SMA免疫组化染色,计算毛细血管密度和小动脉密度。结果:MSC组与EPC组侧支血管数无显著性差异,二者均高于对照组;EPC组毛细血管密度明显高于MSC组,二者均高于对照组;MSC组与EPC组小动脉密度无显著性差异,二者均高于对照组。结论:骨髓间充质干细胞移植和内皮祖细胞移植均能够明显促进血流重建,而且骨髓间充质干细胞在治疗肢体缺血性疾病中的优势应该受到重视。     

Stem cell therapy for the treatment of Leydig cell dysfunction in primary hypogonadism

The production of testosterone occurs within the Leydig cells of the testes. When production fails at this level from either congenital, acquired, or systemic disorders,the result is primary hypogonadism. While numerous testosterone formulations have been developed, none are yet fully capable of replicating the physiological patterns of testosterone secretion. Multiple stem cell therapies to restore androgenic function of the testes are under investigation. Leydig cells derived from bone marrow, adipose tissue, umbilical cord, and the testes have shown promise for future therapy for primary hypogonadism. In particular, the discovery and utilization of a group of progenitor stem cells within the testes, known as stem Leydig cells(SLCs), has led not only to a better understanding of testicular development, but of treatment as well. When combining this with an understanding of the mechanisms that lead to Leydig cell dysfunction, researchers and physicians will be able to develop stem cell therapies that target the specific step in the steroidogenic process that is deficient. The current preclinical studies highlight the complex nature of regenerating this steroidogenic process and the problems remain unresolved. In summary, there appears to be two current directions for stem cell therapy in male primary hypogonadism. The first method involves differentiating adult Leydig cells from stem cells of various origins from bone marrow, adipose, or embryonic sources. The second method involves isolating, identifying, and transplanting stem Leydig cells into testicular tissue. Theoretically, in-vivo re-activation of SLCs in men with primary hypogonadism due to age would be another alternative method to treat hypogonadism while eliminating the need for transplantation.     

Advancements in adipose-derived stem cell therapy for skin fibrosis

Pathological scarring and scleroderma, which are the most common conditions of skin fibrosis, pathologically manifest as fibroblast proliferation and extracellular matrix (ECM) hyperplasia. Fibroblast proliferation and ECM hyperplasia lead to fibrotic tissue remodeling, causing an exaggerated and prolonged wound-healing response. The pathogenesis of these diseases has not been fully clarified and is unfortunately accompanied by exceptionally high medical needs and poor treatment effects. Currently, a promising and relatively low-cost treatment has emerged-adipose-derived stem cell (ASC) therapy as a branch of stem cell therapy, including ASCs and their derivatives-purified ASC, stromal vascular fraction, ASC-conditioned medium, ASC exosomes, etc., which are rich in sources and easy to obtain. ASCs have been widely used in therapeutic settings for patients, primarily for the defection of soft tissues, such as breast enhancement and facial contouring. In the field of skin regeneration, ASC therapy has become a hot research topic because it is beneficial for reversing skin fibrosis. The ability of ASCs to control profibrotic factors as well as anti-inflammatory and immunomodulatory actions will be discussed in this review, as well as their new applications in the treatment of skin fibrosis. Although the long-term effect of ASC therapy is still unclear, ASCs have emerged as one of the most promising systemic antifibrotic therapies under development.     

两种细胞来源的21-三体综合征诱导多能干细胞系的建立及比较

21-三体综合征是染色体异常导致的疾病,通过重编程21-三体综合征患儿两种组织来源的细胞成为多能干细胞,比较两种组织来源的细胞建立21-三体综合征诱导多能干细胞（T21-iPSCs）系的效率,为进一步研究21-三体综合征发病机制提供细胞模型,并为选择高效制备T21-iPSCs的组织类型提供理论依据。该实验利用慢病毒介导4种转录因子（Oct4、Sox2、Klf4、c-Myc）分别诱导人21-三体综合征的羊水细胞和胎儿皮肤成纤维细胞,建立诱导多能干细胞系（Trisomy 21 human amniotic fl uid induced pluripotent stem cells,T21 hAF-iPSCs;Trisomy 21 human dermal fi broblast induced pluripotent stem cells,T21 hDF-iPSCs）,T21 hAF-iPSCs及T21 hDF-iPSCs在蛋白和mRNA水平上均表达人胚胎干细胞的多能性分子标记,如Oct4、Nanog等,具有在体外及体内分化三个胚层的能力,其在培养过程中能维持异常核型并能维持自我更新状态。结果发现,利用羊水细胞建立T21-iPSCs效率高于皮肤成纤维细胞,羊水细胞可能是制备T21-iPSCs的理想细胞类型。     

适宜电刺激促进脂肪干细胞向神经方向分化

目的:研究不同强度电刺激对脂肪干细胞(adipose tissue-derived stem cells,ADSC)向神经元方向分化的作用。方法:在细胞电刺激室内对ADSCs进行电刺激(10Hz,1h),强度选用五个强度,分别为0 V/cm、0.5 V/cm、1.0 V/cm、3.0 V/cm、5.0 V/cm;电刺激后,对细胞进行流式凋亡检测及CCK-8活性检测,明确不同电刺激对ADSCs的影响;同时,应用免疫荧光染色、Western Blotting评估各组细胞神经特异性标志物microtubule-associated protein 2(MAP-2)与β-tubulin的表达情况。应用RT-PCR检测MAP-2、β-tubulin和neurofilaments 200(NF-200)的mRNA水平,评价不同强度电刺激对ADSCs其向神经方向分化的影响。结果:1V/cm的电刺激,未引起明显的细胞凋亡,同时促进了细胞增殖。此外,1V/cm电刺激后,细胞中的MAP-2和β-tubulin的免疫荧光染色强度及蛋白含量显著提高;MAP-2、β-tubulin和NF-200的mRNA及蛋白量显著提高。3V/cm及更高强度的电刺激可导致凋亡细胞数目显著增加。结论:强度为1V/cm的电刺激可促进脂肪干细胞的增殖,并促进其向神经方向分化,为神经损伤的治疗提供了新的可行方法。     

大鼠脂肪基质细胞成脂分化及慢病毒感染的实验研究

目的诱导大鼠脂肪基质细胞成脂分化,观察慢病毒感染效果。方法大鼠脂肪基质细胞培养至第3代后,间接免疫荧光法鉴定细胞表面抗原CD44,并采用MTT法绘制生长曲线;第3代基质细胞成脂诱导分化为脂肪细胞,油红O染色法鉴定,并行慢病毒感染。结果第3代脂肪基质细胞表面抗原CD44表达呈阳性;细胞生长曲线呈"S"形。细胞经成脂诱导分化剂诱导10d后,胞内有大量脂滴形成,脂滴大小不等。油红O染色显示脂滴被染成红色;携带GFP报告基因的慢病毒可感染成熟脂肪细胞,感染率约为80%,且细胞被感染后状态良好。结果 慢病毒可高效感染由大鼠脂肪基质细胞成脂诱导分化成的脂肪细胞,为研究脂肪细胞的基因功能、相关疾病的基因治疗提供了一个有效工具。     

Multiple sources of non-embryonic multipotent stem cells: processed lipoaspirates and dermis as promising alternatives to bone-marrow-derived cell therapies

A body of evidence points to the existence of stem cell stores in adult tissues, in addition to the well-known hematopoietic stem cells from bone marrow. Many reports describe the ability of these multipotent cells (developmentally non-compromised with their organs of origin) to give rise to many different cell types in response to specific stimuli. This apparent plasticity provides new perspectives in tissue engineering and suggests the usefulness of these cells in future protocols of autologous transplantation, gene therapy, and tissue reconstitution in a number of pathological processes. Lipoaspirates and dermis represent accessible sources for obtaining such cells, with minimal discomfort to the donor, and might be promising candidates for cell therapy procedures once their features are experimentally accessed. The intention of the present work has been to gather reports on the phenotypic characteristics, profile, and plastic potential of these stem cells.     

Incorporation of DMSO and dextran-40 into a gelatin/alginate hydrogel for controlled assembled cell cryopreservation

A new cell cryopreservation strategy for cell-assembling constructs was proposed. With this strategy, different concentrations of dimethysulfoxide (DMSO) and dextran-40 were directly incorporated into the cell/gelatin/alginate systems, prototyped according to a predesigned structure, cryopreserved at −80 °C for 10 days and followed a thawing process at 17 °C. The rheological properties, bonding water contents and melting points of the gelatin/alginate hydrogel systems were changed with the addition of different amounts of DMSO. The microscopy analysis, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT) and hematoxylin and eosin (HE) staining indicated that the cell numbers were progressively in a selected DMSO concentration range. With DMSO 5% (v/v) alone, the metabolic rate in the construct attained (81.3 ± 5.7)%. A synergistic effect was achieved with the combination of the DMSO/gelatin/alginate and dextran-40/gelatin/alginate hydrogel systems. These results indicated that the inclusion of DMSO and dextran-40 in the hydrogel could effectively enhance the cell preservation effects. This cryopreservation strategy holds the ability to be widely used in organ manufacturing techniques.     

Cellular Reprogramming of Human Peripheral Blood Cells

Breakthroughs in cell fate conversion have made it possible to generate large quantities of patient-specific cells for regenerative medicine. Due to multiple advantages of peripheral blood cells over fibroblasts from skin biopsy, the use of blood mononuclear cells （MNCs） instead of skin fibroblasts will expedite reprogramming research and broaden the application of reprogramming technology. This review discusses current progress and challenges of generating induced pluripotent stem cells （iPSCs） from peripheral blood MNCs and of in vitro and in vivo conversion of blood cells into cells of therapeutic value, such as mesenchymal stem cells, neural cells and hepatocytes. An optimized design of lentiviral vectors is necessary to achieve high reprogramming efficiency of peripheral blood cells. More recently, non-integrating vectors such as Sendai virus and episomal vectors have been successfully employed in generating integration-free iPSCs and somatic stem cells.