Study of platelet-rich fibrin promoting endothelial cell differentiation and angiogenesis induced by transplantation of adipose-derived stem cells

Diabetic patients are characterized by long wound healing time, and adipose stem cells (ADSCs) can secrete growth factors to promote angiogenesis and improve diabetic wound healing. In this research, we attempted to interrogate the impact of platelet-rich fibrin (PRF) on ADSCs in diabetic wound healing. ADSCs were harvested from human adipose tissues and identified through flow cytometry. After pretreatment with cultured medium supplemented with different concentrations of PRF (2.5%, 5%, and 7.5%), proliferation and differentiation capacity of ADSCs were assessed by CCK-8 assay, qRT-PCR and immunofluorescence (IF), respectively. Tube formation assay measured angiogenesis. Western blot analysis analyzed expression of endothelial markers and the extracellular signal-regulated kinase (ERK) and serine/threonine kinase (Akt) pathways in PRF-induced ADSCs. The CCK-8 experiment indicated that PRF enhanced proliferation of ADSCs in dose-dependent manner, relative to normal control group. The expression of endothelial markers and the capacity of tube formation were significantly promoted by 7.5% PRF. The release of growth factors containing vascular endothelial grow factor (VEGF) and insulin-like growth factor-1 (IGF-1) from PRF was increased with the extension of detection time. When the receptors of VEGF or/and IGF-1 were neutralized, ADSCs differentiation into endothelial cells were obviously inhibited. Additionally, PRF stimulated ERK and Akt pathways, and the inhibitors of ERK and Akt attenuated PRF-induced differentiation of ADSCs into endothelial cells. In conclusion, PRF promoted endothelial cell differentiation and angiogenesis induced by ADSCs in diabetic wound healing, which appears to give guidance for treating patients.     

Expression of CD105 and CD34 receptors controls BMP‐induced in vitro mineralization of mouse adipose‐derived stem cells but does not predict their in vivo bone‐forming potential

Adipose‐derived stem cells (ADSCs) can be excellent alternative to bone marrow derived stem cells for enhancing fracture repair since ADSCs can be isolated comparatively in large numbers from discarded lipoaspirates. However, osteogenic potential of ADSCs in vivo is very controversial. We hypothesized that adipose‐derived stem cells (ADSCs) that respond maximally to bone morphogenetic proteins (BMPs) in vitro would possess maximum bone‐forming potential. Four purified populations of mouse ADSCs: CD105⁺CD34⁺, CD105^?CD34^?, CD105⁺CD34^? and CD105^?CD34⁺ were obtained using fluorescence‐activated cell sorting (FACS) and their BMP‐responsiveness was determined in vitro. CD105⁺CD34^? population showed the strongest response to BMPs in terms of robust increase in mineralization. Expression of CD105 correlated with high BMP‐responsive phenotype and larger cell size while expression of CD34 correlated with low BMP‐responsive phenotype and smaller cell size. CD105⁺CD34^? population displayed higher gene expression of Alk1 or Alk6 receptors in comparison with other populations. However, CD105⁺CD34^? ADSCs failed to induce ectopic bone formation in vivo after they were transplanted into syngeneic mice, indicating that in vitro BMP‐responsiveness is not a good indicator to predict in vivo bone forming potential of ADSCs. Therefore greater precautions should be executed during selection of competent ADSCs for bone repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:625–632, 2015.

     

SOX-9和转化生长因子-β1共同诱导大鼠脂肪干细胞向软骨细胞分化

目的探讨应用SOX-9和TGF-β1基因转染大鼠ADSCs(Adipose stromal cells,ADSCs)后,目的基因分泌情况及向软骨细胞的分化效果,为构建新型的组织工程软骨种子细胞提供思路。方法6周龄健康雄性Wistar大鼠2只,体重约150g。扩增、提取质粒pcDNA3.1-TGF-β1、pcDNA3.1-SOX-9。分离、纯化Wistar大鼠ADSCs,倒置相差显微镜观察原代和传代ADSCs形态学改变,免疫荧光法检测细胞表面标志。将SOX-9和TGF-β1基因单独或共转染第3代ADSCs,按转染情况分为5组:对照组、转染空载体组、转染SOX-9组、转染TGF-β1组、SOX-9与TGF-β1共转染组。对转染后细胞进行筛选,MTT法测定筛选后细胞的增殖活性,RT-PCR和Westernblot法检测筛选后细胞的表达。结果免疫荧光法检测ADSCs的CD29、CD44呈阳性反应,CD34、CD106呈阴性反应。MTT法测定各组细胞在490nm波长处的吸光度(A)值,未转染组、空载体组与SOX-9组、TGF-β1组、SOX-9与TGF-β1共转染组间差异显著(P<0.01)。RT-PCR和Western blot检测目的基因和蛋白的表达量,SOX-9表达以转染SOX-9组最多;TGF-β1表达以SOX-9与TGF-β1共转染组组最多;II型胶原表达以SOX-9与TGF-β1共转染组最多。结论SOX-9和TGF-β1基因共同转染ADSCs修复软骨缺损是一个较有前景的发展方向,对组织工程软骨应用于临床具有重要意义。     

兔脂肪基质细胞的分离培养及其成骨活性的研究

目的探讨兔脂肪组织来源脂肪基质细胞的分离培养方法及其成骨活性．方法取成年新西兰大白兔腹股沟皮下脂肪组织1～2mL,采用机械切割及Ⅰ型胶原酶消化法从脂肪组织中分离获得脂肪基质细胞,原代培养及传代以后,以诱导培养基（内含10mmol／Lβ-甘油磷酸钠、10～8mol／L地塞米松、50mg／L维生素C）进行诱导培养．实验评估：应用形态学观察、碱性磷酸酶钙-钴法染色检测碱性磷酸酶,Von Kossa钙化结节染色检测钙化结节的形成等方法来鉴定诱导分化所得细胞的成骨活性．结果行诱导培养的脂肪基质细胞呈多层成长,形态多为梭形,细胞外基质有白色钙化结节形成;细胞增殖速度减慢,生长周期变长．碱性磷酸酶钙钻法染色及Von Kossa钙化结节染色后均表现为阳性,未诱导培养的脂肪基质细胞则表现为阴性．结论初步建立了一套南脂肪组织分离培养脂肪基质细胞的方法,并证明该细胞在体外诱导培养条件下具备向成骨细胞分化的能力．     

脂肪基质细胞移植治疗杜氏型肌营养不良的临床观察

目的：观察脂肪基质细胞（ADSCs）移植治疗杜氏型肌营养不良（DMD）前后血清酶学变化及临床效果。方法：选择2009-06～2011-06莱芜市人民医院神经内科DMD患者,其中男性48例,女性3例;年龄1～20岁,平均年龄13.3岁。阳性家族史22例。经莱芜市人民医院伦理委员会同意,并征求患者本人及家人签字同意后,进行自体ADSCs移植治疗,设自身治疗前后血清酶学变化对照方法进行疗效观察,应用四肢肌内注射的方式进行ADSCs移植。结果：ADSCs移植治疗12个月肌力增加者占患者总数的64.8%。血清肌酸激酶（CK）降低率80.4%,血清乳酸脱氢酶（LDH）降低率78.4%。结论：ADSCs移植治疗DMD,对肌肉组织有一定的修复作用,使DMD患者的运动功能得到改善,肌力有所提高,血清酶学较移植前下降。ADSCs移植无不良反应,患者依从性好,是治疗DMD的新手段。     

脂肪干细胞复合PLGA对骨质疏松骨折愈合后生物力学的影响

目的探讨骨质疏松骨折后脂肪干细胞(ADSCs)复合聚乳酸共聚物(PLGA)对骨折愈合后生物力学的影响。方法建立大鼠骨质疏松骨折模型,将成功建立模型的27只大鼠分为治疗组(n=18)和空白对照组(n=9),治疗组大鼠的右侧股骨骨折肢为脂肪干细胞复合PLGA治疗组(ADSCs/PLGA组),左侧为单纯脂肪干细胞组(ADSCs组),于治疗后2、4、8周分别处死大鼠,取股骨测量新生骨痂的横截面积及相关生物力学指标。结果骨折后2周,与空白对照组和ADSCs组比较,ADSCs/PLGA组骨痂横截面积最大,空白组最小,各组间两两比较差异有统计学意义(P<0.05),骨折后4周各组间骨痂横截面积比较差异无统计学意义。各组剪切强度、弹性模量随着时间的推移,逐渐升高,各时间点均以ADSCs/PLGA组测定值最高,空白组最低,各组间比较差异均有统计学意义(P<0.01);剪切应变随着时间推移,逐渐降低,各时间点均是ADSCs/PLGA组测定值最低,空白组最高,各组间比较差异均有统计学意义(P<0.01)。最大载荷测定值在各时间点各组间比较为ADSCs/PLGA组最高,空白组最低,各组间比较差异均有统计学意义(P<0.01)。骨折后4、8周,最大应力测定值ADSCs/PLGA组最高,空白组最低,各组之间比较差异有统计学意义(P<0.01)。结论脂肪干细胞复合PLGA可以提高骨质疏松骨折后骨质愈合的质量,为临床骨质疏松骨折的治疗提供实验依据。     

脂肪干细胞中慢病毒-血管内皮生长因子165过表达的研究

【摘要】背景与目的：目前,越来越多的研究探讨干细胞成为基因治疗载体的最可能性,但是有关于脂肪干细胞的研究还很少,因此我们拟构建慢病毒-VEGF165的过表达载体并转染脂肪组织来源的干细胞以产生能用于基因治疗的种子细胞。 方法：将克隆EHS1001-6895048 5313912通过PCR方法突变成VEGF165的转录本片段(NM_001025368)。采用三质粒系统（pGC-FU、pHelp1.0和pHelp2.0）包装过表达慢病毒载体。载体构建成功并行滴度测定后转染ADSCs。使用免疫荧光、ELISA和western blot分析鉴定VEGF165在ADSCs中的表达。 结果：使用DNA测序及将质粒转染293T细胞后的表达检测证明所构建的载体为VEGF165与GFP融合表达载体。RT-qPCR检测后计算得到的病毒滴度达到2×108TU/ml。免疫荧光证实大约95%的ADSCs能表达绿色荧光蛋白,ELISA检测出在转染VEGF165后的ADSCs培养基里VEGF浓度为850.86～1202.13pg/ml(平均 923±31.22pg/ml)而在只转染GFP的ADSCs培养基里并不能检测到(p<0.001),并且这一结果也得到western blot检测结果的证实。 结论：本研究通过慢病毒- VEGF165转染ADSCs获得稳定表达VEGF165的脂肪组织来源干细胞为广泛地进行血管再生研究,特别是糖尿病性勃起障碍奠定基础。     

Zoledronate inhibits the differentiation potential of adipose-derived stem cells into osteoblasts in repairing jaw necrosis

ObjectiveTo explore the inhibitory effects of zoledronate (ZOL) on adipose-derived stem cells (ADSCs) into osteoblasts for repairing jaw necrosis.MethodsADSCs were induced to differentiate into osteoblasts. The differentiation characteristics of osteoblasts was observed under inverted microscope by alizarin red staining. The transwell assay was performed to evaluate the migration of ADSCs co-cultured with osteoblasts and divided into ZOL group treated with ZOL and N-ZOL group without ZOL treatment. The differentiation and proliferation characteristics of ADSCs differentiated osteoblasts were observed respectively. The expression of CTSK (Cathepsin K) and FGFR3 (Fibroblast growth factor receptor 3) in osteoblasts were analyzed by immunofluorescence and western blot.ResultsThe differentiation degree and proliferation of ADSCs to osteoblasts in N-ZOL group were both higher than those in ZOL group. The migratory cell number in ADSCs differentiation in ZOL group was higher than that of N-ZOL group. The protein expression of CTSK and FGFR3 in ADSCs differentiated to osteoblasts in ZOL group was higher than that in N-ZOL group.ConclusionThe differentiation of ADSCs into osteoblasts is significantly inhibited by ZOL. Due to this reason, it may be difficult to achieve good results by ZOL induced ADSCs into osteoblasts in repairing jaw necrosis.     

PRF及所含三因子对大鼠脂肪干细胞迁移的影响

目的 研究富血小板纤维蛋白(platelet-rich fibrin,PRF)及所含三因子TGF-β1、PDGF-AB、 VEGF对培养的大鼠脂肪干细胞(adipose tissue-derived stem cell,ADSCs)迁移的影响,并初步探讨其机制。方法 无菌切除SD大鼠腹股沟处的白色脂肪组织,采用酶消化法原代培养SD大鼠ADSCs,多向诱导分化法鉴定ADSCs,一次离心法提取制备PRF膜。采用划痕实验和Transwell实验检测细胞的迁移能力,实时荧光定量PCR分析迁移相关因子MT1-MMP和MMP-2 mRNA 的表达情况。采用SPSS13.0软件包对数据进行统计学分析。结果 Transwell实验显示,PRF组的迁移细胞数显著高于阴性对照组（P＜0.05）和 抑制剂组（P＜0.05）;不同浓度的TGFβ1、PDGF-AB、VEGF组的组内迁移细胞数的差异显著（P﹤0.05）。经PCR检测,PRF组细胞基因MMP2和MT1-MMP较对照组的表达显著上调（P＜0.05）,TGF-β1、 PDGF-AB和 VEGF的细胞基因MMP2和MT1-MMP较对照组表达均上调,组间差异显著（P＜0.05）。结论 PRF促进了ADSCs的迁移,PRF所分泌的三因子均可不同程度地促进ADSCs的迁移,并呈一定的量-效关系,其迁移能力的增加可能与MT1-MMP和MMP-2 mRNA的上调密切相关。