Adipose tissue-derived stem cells as a therapeutic tool for cardiovascular disease

Adipose tissue-deried stem cells( ADSCs) are adult stem cells that can be easily harvested from subcutaneous adipose tissue. Many studies have demonstrated that ADSCs differentiate into vascular endothelial cells(VECs), vascular smooth muscle cells(VSMCs), and cardiomyocytes in vitro and in vivo. However, ADSCs may fuse with tissue-resident cells and obtain the corresponding characteristics of those cells. If fusion occurs, ADSCs may express markers of VECs, VSMCs, and cardiomyocytes without direct differentiation into these cell types. ADSCs also produce a variety of paracrine factors such as vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1 that have proangiogenic and/or antiapoptotic activities. Thus, ADSCs have the potential to regenerate the cardiovascular system via direct differentiation into VECs, VSMCs, and cardiomyocytes, fusion with tissueresident cells, and the production of paracrine factors. Numerous animal studies have demonstrated the efficacy of ADSC implantation in the treatment of acute myocardial infarction(AMI), ischemic cardiomyopathy(ICM), dilated cardiomyopathy, hindlimb ischemia, and stroke. Clinical studies regarding the use of autologous ADSCs for treating patients with AMI and ICM have recently been initiated. ADSC implantation has been reported as safe and effective so far. Therefore, ADSCs appear to be useful for the treatment of cardiovascular disease. However, the tumorigenic potential of ADSCs requires careful evaluation before their safe clinical application.

High-Fat Diets-Induced Metabolic Alterations Alter the Differentiation Potential of Adipose Tissue-Derived Stem Cells

Background: Adipose tissue-derived stem cells (ASC) possess the ability to differentiate into adipocytes or endothelial cells to help in the adipogenesis, vasculogenesis and vascular repair. This study aims at determining the impact of high-fat diets (HFD)-induced type 2 diabetes (T2D) on the differentiation potential of ASC. Results: C57BL/6J male mice were fed a vegetal (VD) or an animal (AD) HFD. Isolation of ACS from mice showing different levels of metabolic alterations reveals that advanced T2D did not affect the number of cells per gram of tissue. Rather, a higher proportion of inflammatory CD36+ cells was identified in HFD fed mice. Despite a marked decreased expression of adipogenic genes (aP2, C/EBPα and PPARγ2), ASC from HFD groups had a higher adipogenic potential and a lower endothelial differentiation potential in vitro compared to control. ASC from the VD group had enhanced cyclin B1 expression and had more adipogenic potential compared to AD group. Conclusion: Our results demonstrate that the metabolic modifications, linked to the nature of fatty acids in diets, modulate the differentiation potential of ASC with increased adipogenesis to the detriment of the endothelial pathway. Results highlight the importance of evaluating the ASC differentiation behavior in a context of autologous cell-based therapy for the repair of vascular tissues in diabetic patients.

Optimization of adipose tissue-derived mesenchymal stromal cells transplantation for bone marrow repopulation following irradiation

BACKGROUND Bone marrow(BM)suppression is one of the most common side effects of radiotherapy and the primary cause of death following exposure to irradiation.Despite concerted efforts,there is no definitive treatment method available.Recent studies have reported using mesenchymal stromal cells(MSCs),but their therapeutic effects are contested.AIM We administered and examined the effects of various amounts of adipose-derived MSCs(ADSCs)in mice with radiation-induced BM suppression.METHODS Mice were divided into three groups:Normal control group,irradiated(RT)group,and stem cell-treated group following whole-body irradiation(WBI).Mouse ADSCs(mADSCs)were transplanted into the peritoneal cavity either once or three times at 5×10^(5) cells/200μL.The white blood cell count and the levels of,plasma cytokines,BM mRNA,and BM surface markers were compared between the three groups.Human BM-derived CD34+hematopoietic progenitor cells were co-cultured with human ADSCs(hADSCs)or incubated in the presence of hADSCs conditioned media to investigate the effect on human cells in vitro.RESULTS The survival rate of mice that received one transplant of mADSCs was higher than that of mice that received three transplants.Multiple transplantations of ADSCs delayed the repopulation of BM hematopoietic stem cells.Anti-inflammatory effects and M2 polarization by intraperitoneal ADSCs might suppress erythropoiesis and induce myelopoiesis in sub-lethally RT mice.CONCLUSION The results suggested that an optimal amount of MSCs could improve survival rates post-WBI.

Unmodified autologous stem cells at point of care for chronic myocardial infarction

BACKGROUND Numerous studies investigated cell-based therapies for myocardial infarction(MI).The conflicting results of these studies have established the need for developing innovative approaches for applying cell-based therapy for MI.Experimental studies on animal models demonstrated the potential of fresh,uncultured,unmodified,autologous adipose-derived regenerative cells(UAADRCs)for treating acute MI.In contrast,studies on the treatment of chronic MI(CMI;>4 wk post-MI)with UA-ADRCs have not been published so far.Among several methods for delivering cells to the myocardium,retrograde delivery into a temporarily blocked coronary vein has recently been demonstrated as an effective option.AIM To test the hypothesis that in experimentally-induced chronic myocardial infarction(CMI;>4 wk post-MI)in pigs,retrograde delivery of fresh,uncultured,unmodified,autologous adipose-derived regenerative cells(UA-ADRCs)into a temporarily blocked coronary vein improves cardiac function and structure.METHODS The left anterior descending(LAD)coronary artery of pigs was blocked for 180 min at time point T0.Then,either 18×106 UA-ADRCs prepared at“point of care”or saline as control were retrogradely delivered via an over-the-wire balloon catheter placed in the temporarily blocked LAD vein 4 wk after T0(T1).Effects of cells or saline were assessed by cardiac magnetic resonance(CMR)imaging,late gadolinium enhancement CMR imaging,and post mortem histologic analysis 10 wk after T0(T2).RESULTS Unlike the delivery of saline,delivery of UA-ADRCs demonstrated statistically significant improvements in cardiac function and structure at T2 compared to T1(all values given as mean±SE):Increased mean LVEF(UA-ADRCs group:34.3%±2.9%at T1 vs 40.4±2.6%at T2,P=0.037;saline group:37.8%±2.6%at T1 vs 36.2%±2.4%at T2,P>0.999),increased mean cardiac output(UA-ADRCs group:2.7±0.2 L/min at T1 vs 3.8±0.2 L/min at T2,P=0.002;saline group:3.4±0.3 L/min at T1 vs 3.6±0.3 L/min at T2,P=0.798),increased mean mass of the left ventricle(UA-ADRCs group:55.3±5.0 g at T1 vs 71.3±4.5 g at T2,P<0.001;saline group:63.2±3.4 g at T1 vs 68.4±4.0 g at T2,P=0.321)and reduced mean relative amount of scar volume of the left ventricular wall(UA-ADRCs group:20.9%±2.3%at T1 vs 16.6%±1.2%at T2,P=0.042;saline group:17.6%±1.4%at T1 vs 22.7%±1.8%at T2,P=0.022).CONCLUSION Retrograde cell delivery of UA-ADRCs in a porcine model for the study of CMI significantly improved myocardial function,increased myocardial mass and reduced the formation of scar tissue.

Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector

Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to construct a lentiviral VEGF165 expression vector and then infect the ADSCs to produce therapeutic seed cells.Methods EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF165 transcript （NM_001025368）. Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T cells.And then the ADSCs （multiplicity of infection=20） were transfected with the vectors after titer determination. Stable expression of VEGF165 in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis.Results DNA sequencing and 293T transfection verified VEGF165 was linked to the GFP fused vector. The virus titer is up to 2x10a determined by quantitative PCR. VEGF165 transduced cells could show green fluorescence confirmed by immunofluorescence staining （almost 95%）. ELISA analyses could detect out the density of VEGF was 850.86-1202.13pg/ml （mean （923.00±31.22） pg/ml） in the supernatant of VEGF16s-transduced cells but not detected in the GFP-transduced cells （P 〈0.001） and the Western blotting analyses also confirmed VEGF165 expression in VEGF165-transduced cells.Conclusions The VEGF165 over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.

Rho-associated coiled kinase inhibitor Y-27632 promotes neuronal-like differentiation of adult human adipose tissue-derived stem cells

Background Y-27632 is a specific inhibitor of Rho-associated coiled kinase （ROCK） and has been shown to promote the survival and induce the differentiation of a variety of cells types. However, the effects of Y-27632 on adult human adipose tissue-derived stem cells （ADSCs） are unclear. This study aimed to investigate the effects of Y-27632 on the neuronal-like differentiation of ADSCs. Methods ADSCs were isolated from women undergoing plastic surgery and cultured. ADSCs were treated with different doses of Y-27632 and observed morphological changes under microscope. The expression of nestin, neuron specific enolase （NSE） and microtubule-associated protein-2 （MAP-2） in ADSCs treated with Y-27632 was detected by immunocytochemistry and Western blotting analysis. Results Y-27632 had the potency to induce neuronal-like differentiation in ADSCs in a dose-dependent manner. Moreover, the differentiation induced by Y-27632 was recovered upon drug withdraw. ADSCs treated with Y-27632 expressed neuronal markers such as NSE, MAP-2 and nestin while untreated ADSCs did not express these markers. Conclusion Selective ROCK inhibitor Y-27632 could potentiate the neuronal-like differentiation of ADSCs, suggesting that Y-27632 could be utilized to induce the differentiation of ADSCs to neurons and facilitate the clinical application of ADSCs in tissue engineering.

Transplantation of neural progenitor cells differentiated from adipose tissue-derived stem cells for treatment of sciatic nerve injury

Objectives: Currently, the clinical repair of sciatic nerve injury remains difficult.Previous studies have confirmed that transplantation of adipose tissue-derived stem cells promotes nerve regeneration and restoration at peripheral nerve injury sites. Methods: In this study, adipose tissue-derived stem cells were induced to differentiate into neural progenitor cells, transfected with a green fluorescent protein-containing lentivirus, and then transplanted into the lesions of rats with sciatic nerve compression injury. Results: Fluorescence microscopy revealed that the transplanted cells survived,migrated, and differentiated in rats. At two weeks post-operation, a large number of transplanted cells had migrated to the injured lesions; at six weeks post-operation, transplanted cells were visible around the injured nerve and several cells were observed to express a Schwann cell marker. Sciatic function index and electrophysiological outcomes of the transplantation group were better than those of the control group. Cell transplantation promoted the recovery of motor nerve conduction velocity and compound muscle action potential amplitude, and reduced gastrocnemius muscle atrophy.Conclusions: Our experimental findings indicate that neural progenitor cells,differentiated from adipose tissue-derived stem cells, are potential seed stem cells that can be transplanted into lesions to treat sciatic nerve injury. This provides a theoretical basis for their use in clinical applications.

Chemically induced hypoxia by dimethyloxalylglycine(DMOG)-loaded nanoporous silica nanoparticles supports endothelial tube formation by sustained VEGF release from adipose tissue-derived stem cells

Inadequate vascularization leading to insufficient oxygen and nutrient supply in deeper layers of bioartificial tissues remains a limitation in current tissue engineering approaches to which prevascularization offers a promising solution.Hypoxia triggering pre-vascularization by enhanced vascular endothelial growth factor(VEGF)expression can be induced chemically by dimethyloxalylglycine(DMOG).Nanoporous silica nanoparticles(NPSNPs,or mesoporous silica nanoparticles,MSNs)enable sustained delivery of molecules and potentially release DMOG allowing a durable capillarization of a construct.Here we evaluated the effects of soluble DMOG and DMOG-loaded NPSNPs on VEGF secretion of adipose tissue-derived stem cells(ASC)and on tube formation by human umbilical vein endothelial cells(HUVEC)-ASC co-cultures.Repeated doses of 100 mM and 500 mM soluble DMOG on ASC resulted in 3-to 7-fold increased VEGF levels on day 9(P<0.0001).Same doses of DMOG-NPSNPs enhanced VEGF secretion 7.7-fold(P<0.0001)which could be maintained until day 12 with 500 mM DMOG-NPSNPs.In fibrin-based tube formation assays,100 mM DMOG-NPSNPs had inhibitory effects whereas 50 mM significantly increased tube length,area and number of junctions transiently for 4 days.Thus,DMOG-NPSNPs supported endothelial tube formation by upregulated VEGF secretion from ASC and thus display a promising tool for prevascularization of tissue-engineered constructs.Further studies will evaluate their effect in hydrogels under perfusion.

Recent advances in andrology-related stem cell research

Stem cells hold great promise for regenerative medicine because of their ability to self-renew and to differentiate into various cell types. Although embryonic stem cells （BSC） have greater differentiation potential than adult stem cells, the former is lagging in reaching clinical applications because of ethical concerns and governmental restrictions. Bone marrow stem cells （BMSC） are the best-studied adult stem cells （ASC） and have the potential to treat a wide variety of diseases, including erectile dysfunction （ED） and male infertility. More recently discovered adipose tissuederived stem cells （ADSC） are virtually identical to bone marrow stem cells in differentiation and therapeutic potential, but are easier and safer to obtain, can be harvested in larger quantities, and have the associated benefit of reducing obesity. Therefore, ADSC appear to be a better choice for future clinical applications. We have previously shown that ESC could restore the erectile function of neurogenic ED in rats, and we now have evidence that ADSC could do so as well. We are also investigating whether ADSC can differentiate into Leydig, Sertoli and male germ cells. The eventual goal is to use ADSC to treat male infertility and testosterone deficiency. （Asian JAndrol 2008 Mar; 10： 171-175）

Stem cell therapy for erectile dysfunction

Erectile dysfunction(ED)is an important health problem that has commonly been clinically treated using phosphodiesterase type 5 inhibitors(PDE5Is).However,PDE5Is are less effective when the structure of the cavernous body has been severely injured,and thus regeneration is required.Stem cell therapy has been investigated as a possible means for regenerating the injured cavernous body.Stem cells are classified into embryonic stem cells and adult stem cells(ASCs),and the intracavernous injection of ASCs has been explored as a therapy in animal ED models.Bone marrowderived mesenchymal stem cells and adipose tissuederived stem cells are major sources of ASCs used for the treatment of ED,and accumulated evidence now suggests that ASCs are useful in the restoration of erectile function and the regeneration of the cavernous body.However,the mechanisms by which ASCs recover erectile function remain controversial.Some studies indicated that ASCs were differentiated into the vascular endothelial cells,vascular smooth muscle cells,and nerve cells that originally resided in the cavernous body,whereas other studies have suggested that ASCs improved erectile function via the secretion of anti-apoptotic and/or proangiogenic cytokines ratherthan differentiation into other cell types.In this paper,we reviewed the characteristics of stem cells used for the treatment of ED,and the possible mechanisms by which these cells exert their effects.We also discussed the problems to be solved before implementation in the clinical setting.

Nanotechnology-assisted adipose-derived stem cell （ADSC） therapy for erectile dysfunction of cavernous nerve injury： In vivo cell tracking, optimized injection dosage, and functional evaluation

Stem cell therapy is a potentially promising option for erectile dysfunction; however, its risk of tumorigenicity is a clinical hurdle and the risk is positively related to the number of injected cells. Our previous study showed that nanotechnology improved adipose-derived stem cell （ADSC） therapy for erectile dysfunction of cavernous nerve injury （CNI） by attracting cells in the corpus cavernosum. These results indicated the possibility of using a reduced dosage of ADSCs for intracavernous injection. In this exploratory study, we used lower dosage （2 × 105 cells） of ADSCs for intracavernous injection （ICI） and the nanotechnology approach. Intracavernous pressure and mean arterial pressure were measured at day 28 to assess erectile function. The low-dose ADSC therapy group showed favorable treatment effects, and nanotechnology further improved these effects. In vivo imaging of ICI cells revealed that the fluorescein signals of NanoShuttle-bound ADSCs （NanoADSCs） were much stronger than those of ADSCs at days 0, 1, and 3. Both immunofluorescence and Western blot analysis showed a significant increase in smooth muscle, endothelium, and nerve tissue in the ADSC group compared to that in the CNI group; further improvement was achieved with assisted nanotechnology. These findings demonstrate that nanotechnology can be used to further improve the effect of small dosage of ADSCs to improve erectile function. Abundant NanoADSCs remain in the corpus cavernosum in vivo for at least 3 days. The mechanism of erectile function improvement may be related to the regeneration of the smooth muscle, endothelium, and nerve tissues.

兔脂肪干细胞(ADSCs)与聚羟基乙酸/壳聚糖(PLGA/CS)支架材料生物相容性研究

目的探讨兔脂肪来源于细胞（adipose-derived stemcells，ADSCs）作为种子细胞复合新型聚羟基乙酸（polylaetic-co—glycolicacid，PLGA）／壳聚糖（chitosan，CS）支架材料的生物相容性，为进一步构建组织工程化脂肪组织提供实验依据。方法取雄性新西兰大白兔腹股沟脂肪组织，I型胶原酶消化分离出ADSCs进行培养，贴壁细胞至3～5代，评价其多向分化能力。将干细胞收集重悬后，以l×10^7／mL的密度接种于多孔PLGA／CS支架，形成细胞一支架材料复合物。培养7天后，扫描电子显微镜观察细胞在支架材料上的黏附生长和基质分泌情况，评价细胞与支架材料的生物相容性；Dil荧光标记检测细胞在支架材料上的分布；Hochest33258检测细胞在支架上的生长情况。接种1和7天后，分别对细胞一材料复合物行AnnexinV／PI双染色法检测材料对细胞的毒性作用。用成脂诱导条件培养基诱导分化ADSCs及ADSCs支架材料复合物，7天后，尼罗红荧光染色液检测ADSCs在不同环境下的成脂分化能力。结果原代培养的ADSCs呈成纤维细胞样外观，在相应诱导条件下能够分化为脂肪细胞和骨细胞。细胞接种于PLGA／CS支架材料上第8天分裂增殖达到高峰，扫描电子显微镜下提示ADSCs在支架表面贴附生长良好，并向孔隙内壁充分延伸，细胞周围形成丰富的基质成分。活死双染结合共聚焦显微镜显示材料对细胞活性无影响，尼罗红染色可见成脂诱导后的ADSCs细胞质内有红色脂滴颗粒形成。结论多孔PLGA／CS支架与兔ADSCs具有良好的生物相容性，可作为构建组织工程脂肪组织的支架材料。

脂肪干细胞(ADSCs)在瘢痕防治中的基础研究进展

瘢痕组织是人体创伤过程中的一种自然产物,它不但影响外观,甚至还有可能因为挛缩而引起功能障碍。瘢痕的发生机制尚未完全清楚,目前人体炎症介质过度分泌、细胞因子释放失调、成纤维细胞增殖和降解不平衡以及胶原过度沉积被认为是瘢痕形成的主要因素。瘢痕的治疗方案主要包括抗瘢痕药物的应用、手术切除、皮瓣转移、皮肤软组织扩张、CO2激光等,都具有一定的效果,但亦有一定的局限性。近年来,脂肪干细胞（adi pose derived stem cells,ADSCs）因其特性被大家所重视,现就ADSCs的主要生理特性作一综述。

The CD133/1⁺cell subset from human subcutaneous adult fat retains hemogenic potential

Research has shown that cells from adult fat tissue can effect long-term blood reconstitution. Fat-derived multipotentiality was ascribed to CD34+ perivascular populations from its prominent microvasculature, that represent mostly non-hemogenic, mesenchymal cells, although this tissue contains a CD34+45+ subset committed to a hemogenic fate. Here, in order to analyze cell subsets presenting hemogenic capabilities within fat, CD133/1+ and pericytes, the latter defined by CD140b (PDGFRb, Platelet-Derived Growth Factor Receptor Beta) expression, were immunomagnetically selected from stromal-vascular fractions (SVF). In Vitro Colony Forming Unit (CFU) assays were negative for CD140b+ pericytes and positive for CD133/1+ cells when a prolonged CFU assay was performed, revealing fat as another store of primitive progenitors that retain hemogenic potential.

Current Status of ADSCs-Enriched Fat Grafts in Plastic Surgery

Autologous fat grafting is an increasingly popular technique in plastic surgery for volume augmentation and rejuvenation.However,the unpredictability of long-term volume retention limits its clinical application.Various animal studies have documented the positive effects of adipose tissue-derived stem cells(ADSCs)on the acceleration of lipofilling.However,the results have been inconsistent,and there is an insufficient number of high-quality clinical studies to formulate evidence-based recommendations for ADSC-enriched fat grafts.Moreover,related technical standards,such as the final count of harvested ADSCs and the enrichment ratio,have not yet been established.This systematic review included all clinical trials on ADSC-enriched fat grafts in plastic surgery from PubMed in the past 10 years,as well as all registered clinical trials on ClinicalTrials.Gov.To examine the current landscape of ADSCs harvest,we summarize the current applications of ADSCs in the field of plastic surgery and discuss the current barriers to universal clinical use.

IGF-1和动态微环境对脂肪干细胞向心肌细胞分化作用的研究

体外组织工程模型中,生物化学和机械信号对心肌再生起着很重要的促进作用,对人胰岛素样生长因子(IGF-1)和三维动态微环境对脂肪干细胞向心肌细胞分化过程中的促进作用进行了研究.带有IGF-1基因的质粒整合到胶原-壳聚糖支架中,脂肪干细胞接种到整合质粒的支架内,未整合质粒的支架作为对照组,心肌细胞培养基作为分化培养基,转瓶生物反应器提供动态微环境.经2周分化培养后,检测质粒在支架内释放及表达情况、细胞在支架内的活性以及心肌功能性蛋白和基因的表达.结果表明:动态微环境能促进质粒DNA的释放和转染;IGF-1可促进脂肪干细胞在胶原-壳聚糖支架内增殖以及向心肌细胞分化;动态微环境可加强IGF-1的促增殖分化作用.因此,IGF-1和动态微环境能独立或相互促进脂肪干细胞在胶原-壳聚糖支架内活性,动态微环境还可强化IGF-1对脂肪干细胞的促分化作用.对体外构建工程化心肌组织进行心肌再生研究有着重要的指导意义.

人脂肪干细胞复合胶原/壳聚糖支架在生物反应器中扩增的研究

研究表明脂肪组织是多潜能干细胞的又一新来源,脂肪组织来源的干细胞可被用于细胞治疗及组织工程。然而,传统的培养方法很难满足临床需求。为获得大量的脂肪干细胞以满足临床需求,现将脂肪干细胞接种到胶原/壳聚糖支架上,比较细胞在静态环境和在转瓶中动态扩增的情况。通过CCK-8检测细胞增殖情况;并分析葡萄糖和乳酸的代谢情况;14d后扫描电镜观察细胞在支架内的生长情况;流式细胞仪检测干细胞相关表面标记表达;RT-PCR检测干细胞相关转录因子的表达;并检测扩增后的干细胞的多向分化潜能。结果表明:在动态微环境中扩增14d后,与静态条件下相比,支架内的细胞具有更强的增殖活性和更好的多向分化潜能。所扩增的细胞能够保持原有干细胞的特性。结论:所设计的支架-转瓶培养系统是一个简便有效的扩增脂肪干细胞的方法。

心肌样微环境诱导脂肪干细胞分化的研究

微环境在促进干细胞分化过程中起着重要的作用,研究心肌样微环境介导脂肪干细胞向心肌细胞分化有重要意义.将脂肪干细胞与心肌细胞直接或通过细胞培养小室间接共培养,检测脂肪干细胞的分化情况.对于直接共培养体系,采用绿色荧光蛋白CFSE对脂肪干细胞进行标记,然后与心肌细胞以1:5混合后进行直接共培养,2周后,通过流式细胞仪分选分化的脂肪干细胞,并检测其分化情况.检测方法包括:扫描电镜和透射电镜观察细胞的超微结构;免疫细胞化学检测心肌特异性肌球蛋白重链(MHC)、肌钙蛋白(TnⅠ)和连接蛋白(Cx43);Western blot定量分析;RT-PCR检测心脏特异性转录因子mRNA的表达.结果表明,分化的脂肪干细胞呈现心肌样超微结构,并表达心肌特异性蛋白和转录因子,并且直接共培养体系中分化的脂肪干细胞其表达率明显高于间接共培养体系中的表达率.因此,在心肌样微环境中,除了可溶性细胞因子对分化起作用以外,心肌细胞产生的机械牵拉对脂肪干细胞向心肌细胞分化也起着重要的作用.

TGF-β1基因修饰诱导脂肪干细胞成软骨分化的实验研究

[目的]本研究通过选择对软骨细胞ECM合成具有促进作用的TGF-β1转染脂肪干细胞(ADSCs),观察转染后目的基因的稳定表达和对软骨细胞外基质(ECM)的两种重要组分:Ⅱ型胶原(typeⅡcollage,ColⅡ)和ag-grecan的合成,为构建新型的组织工程软骨提供实验依据。[方法]TGF-β1基因转染脂肪干细胞及阳性细胞克隆株的筛选;RT-PCR检测TGF-β1、纤连蛋白(fibronectin,FN)、ColⅡ、Aggrecan的表达;Western-blot检测TGF-β1。[结果]RT-PCR结果显示:实验组(TGF-β1稳定转染组)的TGF-β1、FN、ColⅡ、Aggrecan的表达较空染组和正常对照组均明显增多(P<0.01);Western blot检测实验组(TGF-β1稳定转染组)TGF-β1蛋白的表达较空染组和正常对照组均明显增多(P<0.01)。[结论]成功分离培养脂肪干细胞;以脂质体介导法将TGF-β1目的基因成功转染ADSCs,转染后成软骨分化的特异性细胞外基质增多,表明转染TGF-β1基因可以促使ADSCs向软骨细胞方向分化,从而为软骨组织工程提供新的思路和方法。

核心结合因子(Cbfa1／Runx2)重组腺病毒载体的构建、鉴定及在脂肪来源干细胞中的表达

目的:验证核心结合因子(Cbfa1/Runx2)的重组腺病毒载体可高效感染脂肪组织来源干细胞并表达蛋白。方法:分离、扩增脂肪组织来源干细胞并诱导其向骨细胞、脂肪细胞分化。将Cbfa1/Runx2cDNA的全长序列亚克隆到穿梭质粒pAdTrack上,在BJ5183细菌内和pAdEasy1同源重组,筛选阳性克隆,命名为Ad-Cb-fa1/Runx2。经酶切、PCR鉴定,线性化后以脂质体法转染293T细胞进行包装、扩增,利用报告基因GFP对病毒滴度和感染效率进行监测。通过RT-PCR、间接免疫荧光检测Ad-Cbfa1/Runx2感染脂肪组织来源干细胞后Cbfa1/Runx2基因的表达。结果:脂肪组织来源干细胞细胞数目两天增长一倍。在特定诱导条件下,可向成脂细胞、成骨细胞分化。酶切及PCR鉴定证实Cbfa1/Runx2基因重组腺病毒载体构建成功。Ad-Cbfa1/Runx2对脂肪组织来源干细胞的转染效率为81.39%。转染3天后,PCR、间接免疫荧光检测到脂肪组织来源干细胞中Cbfa1/Runx2蛋白的表达。结论:成功分离、培养了脂肪组织来源干细胞,构建了含有小鼠Cbfa1/Runx2基因的重组腺病毒载体,证明了Ad-Cbfa1/Runx可高效感染脂肪组织来源干细胞并表达Cbfa1/Runx2蛋白。