脂肪组织来源干细胞定向分化脂肪组织的体内外实验研究

目的 分离和培养脂肪组织来源干细胞（ASCs），鉴定其是否具有干细胞表面标志，研究携带GFP基因的ASCs向脂肪组织的体外定向诱导分化能力，同时判断种子细胞ASCs与Ⅰ型胶原支架混合培养后在体内构建组织工程化脂肪组织的可能性。方法 取GFP小鼠腹股沟部脂肪组织，使用酶消化法进行原代培养，流式细胞仪鉴定其表面干细胞标志，细胞传至第3代后使用脂肪分化培养基诱导2周，观察细胞形态及功能变化。将诱导分化后的细胞与支架材料混合培养后12h，将支架材料移植到裸鼠背部皮下，观察新生组织情况，并对新生组织使用HE及油红O染色进行鉴定。结果 原代培养的ASC形态类似于成纤维细胞，具有很强的增殖能力，能持续稳定表达表面干细胞标志。在脂肪分化培养基的作用下，胞浆内脂滴不断聚集，逐渐演变为成熟的脂肪细胞，油红O染色阳性。体内实验中在裸鼠皮下发现了0．5ml的新生组织块，常规病理及油红O染色均证实其为成熟脂肪组织块。结论 脂肪组织来源的干细胞ASC能在体外定向诱导分化为成熟脂肪细胞，且ASC能作用种子细胞与Ⅰ型胶原支架在体内成功构建脂肪组织。     

人脂肪细胞的可塑性

目的 探索体外培养环境下人成熟脂肪细胞的去分化现象,旨在挖掘其作为种子细胞的潜能,为组织工程研究开辟新思路.方法 自成人吸脂术后抽吸物提取成熟脂肪细胞及脂肪组织来源干细胞(adipose-derived stromal cells,ASCs),天花板贴壁培养法诱导成熟脂肪细胞去分化,观察细胞形态变化,获得去分化脂肪细胞(dedifferentiated adipocytes,DA).相同的条件下,MTT比色法比较DA、ASCs活性并绘制细胞生长曲线;流式细胞仪鉴定DA、ASCs表面分子的表达;油红O染色、茜素红染色、阿尔辛蓝染色分别鉴定DA、ASCs成脂分化、成骨、成软骨分化能力.结果 人成熟脂肪细胞在体外培养环境下能去分化为成纤维细胞状DA;MTT比色法测细胞活性:DA、ASCs均有很强的增殖能力,两者差异无统计学意义;流式细胞仪测定:DA、ASCs中HLA-ABC、CD29、CD44均为阳性,CD45、CD34、CD106均为阴性;成脂分化2周,油红O染色可见DA、ASCs内出现红色脂滴;成骨分化2周,茜素红染色可见DA、ASCs内红色钙盐沉积;成软骨分化2周,阿尔辛蓝染色可见DA、ASCs内软骨基质沉积.结论 成熟的脂肪细胞在体外培养条件下可成为DA,DA具有很强的增殖活性,表达部分干细胞特征性表面蛋白,有成骨、成软骨及强大的成脂分化能力,有望成为组织工程优秀的种子细胞.

Abstract：

Objective To explore the dedifferentiation phenomenon of human mature adipocytes cultured in vitro and to discuss the possibility of using dedifferentiation adipocytes ( DA ) as seed cells.Methods Mature adipocytes and ASCs were harvested from human fat aspirates. Mature adipocytes were cultured and induced to DA by ceiling adherent culture method. Cell morphology were observed during the whole process. Viabilities of DA and ASCs were compared by MTT chromatometry and cell growth curves were drawn based on it. Cell surface markers of DA and ASCs were detected by flow cytometry. The adipogenic,osteogenic and chondrogenic ability of DA and ASCs were assessed by oil red O staining,alizarin bordeaux staining and alcian blue staining, respectively. Regults Human mature adipocytes can dedifferentiate into fibroblast-shaped DA. MTT chromatometry assay demonstrated that DA and ASCs both had strong reproductive activity, with no significant difference between them. Flow cytometry assay demonstrated that both DA and ASCs expressed HLA-ABC, CD29 and CD44, while didn't express CD45,CD34 and CD106. After two weeks of adipogenic differentiation, lipid droplets could be displayed by oil red O staining in both DA and ASCs. After two weeks of osteogenic differentiation, calcium salts mineralization in DA and ASCs could be detected by alizarin bordeaux staining. After two weeks of chondrogenic differentiation, matrix of cartilage cells in DA and ASCs could be detected by alcian blue staining. Conclusions Mature adipocytes can be dedifferentiated into DA in vitro. DA has strong reproductive activity, as well as osteogenic, chondrogenic ability and strong adipogenic ability. It expresses some of the stem cell-related cell surface proteins and is a promising seed cell for adipose tissue engineering.     

Cell biological study of cultured cells derived from the fattyoffluid portions of liposuction aspirates]

OBJECTIVE: To explore an approach to isolate and culture the Adipose derived stem cells (ASCs) from the fatty and the fluid portions of liposuction aspirates, and to investigate the growth kinetics, morphology, differentiation capability, cell senescence, surface marker profiles of the ASCs. METHODS: The liposuction aspirates were divided into fatty portion and liquid portion. ASCs were isolated from each portion by collagenase digestion and directly centrifugate and cultured to observe the morphology and biology characters in vitro. Cell activity was studied by MTT chromatometry and analyzed statistically. Cell cycle was detected by flow cytometry. Cells were randomly selected from the 3rd, 4th, 6th, 8th generation cells to dectect senescence of ASCs by acridine orange staining. The cell surface markers were detected by flow cytometry and immunohistochemistry. Adipogenic and osteogenic lineage differentiation of ASCs was assessed by Oil Red O and alizarin bordeaux staining respectively. RESULTS: A large amount of ASCs could be islated and cultured both from the fatty portion and the liquid portion, including PLA cells and LAF cells which had fibroblastic characters with strong viability and proliferative activity. The statistical result indicated that the cell activity of PLA cells and LAF cells was very similar. ASCs from passage 3, 4, 6, 8 didn't show insenecence. CD29, CD44, CD34, which were the markers of mesenchymal stem cells, vWF, CD31, CD105, SMA were all expressed in ASCs. Adipogenic differentiation of ASCs was assessed by Oil Red O staining after 2 weeks. The cells contained many lipid-filled droplets. After 2 weeks' osteogenic induction, cells were positively stained by alizarin Bordeaux. CONCLUSIONS: The method can isolate ASCs by directly centrifugate from the fatty and the fluid portions of human liposuction aspirates. The way of culture is convenient and economical. ASCs isolated from the liquid and fatty portions of liposuction aspirates show identical in cells numbers and quality. LAF cells and PLA cells have similar characters in growth dynamics, morphology, cell senescence, surface marker profiles and differentiation ability, etc. Expression of the cell surface marker of stem cells is also observed in ASCs. ASCs can differentiate into adipose and osteogenesis directionally. The results suggest that the ASCs, which are isolated with minimum intervention, may be the ideal seed cells for adipose tissue engineering in future.     

人脂肪源细胞外囊泡联合脱细胞脂肪组织支架构建组织工程脂肪

目的探讨人脂肪源细胞外囊泡(human adipose tissue derived extracellular vesicle,hAT-EV)联合脱细胞脂肪组织支架(decellularized adipose tissues,DAT)构建组织工程脂肪的可能性,为临床上软组织缺损修复提供新方案。方法将吸脂手术患者自愿捐赠的脂肪组织分为2份,1份脂肪组织进行脱细胞处理,并采用组织学(HE、Masson染色)、扫描电镜观察,Ⅰ、Ⅳ型胶原及层粘连蛋白免疫组织化学染色和Western blot检测进行表征。另1份脂肪组织使用去外泌体的完全培养基孵化48 h,离心收集培养基上清并使用超高速离心法获取hATEV。使用透射电镜观察其形态,纳米颗粒跟踪分析仪NanoSight分析hAT-EV粒径分布范围,Western blot检测分析hAT-EV膜表面蛋白。将PKH26荧光标记的hAT-EV与脂肪干细胞(adipose derived stem cells,ADSCs)共培养后,共聚焦荧光显微镜观察hAT-EV能否进入ADSCs;将hAT-EV与ADSCs共培养15 d,油红O染色评估其成脂效果。将DAT组织剪碎并注射至8只6周龄雌性C57小鼠背部两侧,左侧每周注射0.2 mL hAT-EV作为实验组,右侧每周注射0.2 mL PBS作为对照组。12周后处死小鼠,取两侧DAT新生物进行湿重称量,并通过HE染色和围脂滴蛋白免疫荧光染色评估hAT-EV在体内诱导脂肪新生的能力。结果脂肪组织经脱细胞后,HE、Masson染色示DAT主要由排列松散的胶原构成,未见细胞核;扫描电镜示DAT中未发现细胞和细胞碎片,同时可见到粗大的胶原纤维束;免疫组织化学染色及Western blot检测示,DAT中保留了Ⅰ、Ⅳ型胶原和层粘连蛋白。经鉴定,hAT-EV呈双层包膜的球形,高表达CD63、凋亡诱导因子6相互作用蛋白抗体、肿瘤易感基因101,97.9%粒径分布范围为32.67~220.20 nm,峰值为91.28 nm。共聚焦荧光显微镜和油红O染色示,hAT-EV被ADSCs摄取并诱导其成脂分化。大鼠体内实验显示,实验组新生脂肪组织湿重显著高于对照组(t=2.278,P=0.048);HE染色示,实验组脂滴结构较对照组多,对照组胶原含量高于实验组;围脂滴蛋白免疫荧光染色示,实验组DAT新生物中脂肪组织比例高于对照组(t=4.648,P=0.017)。结论DAT搭载hAT-EV可作为一种诱导脂肪组织新生的新方法,在软组织缺损修复中具有潜在应用前景。     

脂肪干细胞对D-半乳糖致裸鼠皮肤老化的拮抗作用及对皮肤恢复功能的影响

目的:本文通过研究脂肪干细胞(Adipose stem cells,ASCs)在D-半乳糖诱导裸鼠皮肤老化的拮抗作用及其对皮肤恢复功能的影响,旨在探讨抗衰老机制并为临床上抗衰老疗法提供新的思路。方法:选择SPF级裸鼠40只,随机分为4组:对照组、D-半乳糖+磷酸盐缓冲液(PBS)组、D-半乳糖+ASCs组与D-半乳糖+氨基胍(AG)组,每组10只。对D-半乳糖+PBS组,D-半乳糖+ASCs组和D-半乳糖+AG组的裸鼠的背部进行皮下注射1 000mg/kg D-半乳糖,每天1次,持续8周。2周之后,3组小鼠背部分别注射PBS缓冲液(0.5ml)、ASCs(1×10^6/ml)和AG(100mg/kg),均注射至真皮层,每天1次,4周后处死。取每组皮肤组织,采用硫代巴比妥酸法测定丙二醛(MDA),黄嘌呤氧化酶法测定超氧化物歧化酶(SOD),ELISA法测定晚期糖基化终末产物(AGEs)、总胶原蛋白、Ⅰ型胶原蛋白及Ⅲ型胶原蛋白水平。通过HE染色和马松染色比较各组皮肤组织真皮层厚度及胶原分布比,采用免疫组化测定各组CD31及血管内皮生长因子(VEGF)表达情况。结果:四组裸鼠的体重与模型构建前并无显著差别。对照组裸鼠表现为充满活力,反应敏捷,并且皮肤富有弹性,粪便无明显臭味;衰老小鼠模型皮肤逐渐变弱,变薄,失去弹性,并患有便秘,且排泄物发臭;而经ASCs或AG处理的小鼠裸鼠在处理后情况有所改善。相较于对照组,D-半乳糖+PBS组裸鼠SOD、总胶原蛋白、CD31、VEGF、Ⅰ型胶原蛋白及Ⅲ型胶原蛋白水平显著降低,MDA及AGEs表达量显著增加,而通过ASCs或AG处理的裸鼠各指标水平均有所改善。HE及马松染色结果显示,相较于对照组,D-半乳糖+PBS组裸鼠真皮层相对较薄,胶原分布比明显降低,而经ASCs或AG处理的裸鼠鼠真皮层有所增厚,胶原分布比显著上升。结论:ASCs移植对D-半乳糖诱发衰老的裸鼠的自由基与糖基化水平具有良好的拮抗作用,为ASCs的移植抵抗肌肤衰老的理论提供了实验依据。     

从脂肪抽出物的脂类和液体部分提取脂肪来源干细胞的细胞生物学研究

目的 探索从抽吸物中脂质和液体部分分离、体外培养脂肪组织来源干细胞的新方法,并通过其生长动力学、形态学、分化能力、细胞衰老和表面标记物轮廓5个方面的特征进行鉴定比较.方法 抽脂术后抽吸物分解为脂质和液体部分.脂质和液体部分分别用酶消化法和直接离心过滤法分离、培养ASCs,观察其在体外培养的形态学和生物学特点;MTT比色法测细胞活性,统计学分析;流式细胞仪测定细胞周期;随机选取3、4、6、8代做丫啶橙染色检测细胞的衰老;用流式细胞仪、免疫组织化学染色法鉴定其表面分子表达;成脂、成骨定向诱导分化,油红O染色、茜素红染色定性.结果 从吸脂抽吸物的脂质和液体两部分中都能培养出大量的ASCs,分别为PLA和LAF,呈成纤维细胞样贴壁生长,MTY测定细胞活性及细胞周期研究发现PLA、LAF这两种细胞的活力与增殖能力是非常相似的;丫啶橙染色3、4、6、8代细胞无明显衰老;流式细胞仪检测显示干细胞标志的CD29、CIM4、CD34的表达均呈阳性;免疫化学染色发现Ⅷ因子、CD31、CD105、SMA表达阳性;成脂诱导分化2周后,细胞内可见有大量脂滴,油红O染色可见胞浆内有大量红染颗粒.成骨诱导2周后,细胞可见白色矿化钙盐沉积,茜素红染色可见成骨细胞红染.结论 本实验建立了一种自人体脂肪抽吸物中脂质和液体部分分离和培养ASCs的新方法,经济简便实用,从成人脂肪抽吸物液体部分中也可以分离得到大量的可为脂肪组织工程所利用的ASCs,其细胞量与脂质来源的ASCs的量基本相同.贴壁的LAF与PLA细胞在细胞的生长动力学、形态学、细胞衰老、表面标志物和分化能力等方面具有非常相似的特性,都具有很强的增殖活性且衰老率较低,能稳定表达干细胞表面标志并能实现定向成脂、成骨多向诱导分化.这种经过最小限度人工干预的ASCs可能是将来脂肪组织工程比较理想的种子细胞之一.     

人脂肪来源干细胞复合I型胶原支架构建工程化脂肪组织的实验研究

目的 探讨构建组织工程化脂肪组织的可行性,为临床修复软组织缺损寻找一种新方法.方法 以酶消化法从人脂肪抽吸术抽吸物脂质部分获取人脂肪来源干细胞作为种子细胞,并行Dil体外荧光标记,以Ⅰ型胶原支架为载体材料,将细胞成脂诱导后,以1×107/ml细胞密度与支架复合后接种于裸鼠左侧背部皮下,未诱导组不对细胞进行任何诱导,以相同方式接种于裸鼠右侧背部皮下,空白对照组将Ⅰ型胶原空白支架接种于裸鼠颈部正中皮下,每组各6只实验鼠;于第12周取材,通过大体和荧光显微镜观察、湿重测定、组织学检测和油红0染色定性判断体内成脂能力.结果 原代培养的脂肪来源干细胞,经成脂诱导能演变为成熟脂肪细胞,油红0染色阳性.诱导组裸鼠皮下均发现新生组织块,新生物平均湿重为0.020 g,常规病理切片及油红0染色均证实其为成熟脂肪组织,Dil荧光显色阳性证实其为外源性;未诱导组4只裸鼠皮下发现新生组织块,新生物平均湿重为0.014 g,常规病理切片及油红0染色证实其含有部分成熟脂肪组织,Dil荧光显色阳性证实其为外源性.两组新生物湿重比较差异有统计学意义(P<0.01);空白对照组未见新生组织形成.结论 用酶消化法从人脂肪抽吸术抽吸物脂质部分提取的细胞为脂肪组织来源干细胞,该细胞能作为种子细胞经成脂诱导后.与Ⅰ型胶原支架在体内成功构建脂肪组织.     

基质血管成分与体外扩增的脂肪来源干细胞促进移植脂肪成活的对比研究

目的:比较血管基质成分(st romal vascul ar fract i on,SVF)与体外扩增的脂肪来源干细胞(adi pose-deri ved st emcel l s,ASCs)对移植脂肪成活的促进作用。方法:用手术方法切取家兔腹股沟脂肪,进行ASCs体外分离培养;取第3代ASCs分别进行成脂、成骨诱导实验,CD29和CD31流式鉴定;制备SVF,进行CD29和CD31流式鉴定;脂肪移植裸鼠实验分为3组:SVF、ASCs、和空白对照组(DMEM/F12),每组4只裸鼠,沿背部脊柱两侧对称部位4个移植位点,每组共16个注射移植位点,每点0.3ml脂肪颗粒(adi pose granul e,AG)+0.2ml细胞成分;术后4m时取材称重、固定行HE染色观察移植脂肪组织结构,行CD31免疫组化染色观察新生血管及其密度。结果:家兔ASCs体外分离培养成功,为贴壁生长,第3代细胞形态均呈长梭形。成脂诱导实验油红O染色显示形成脂滴,成骨诱导实验茜素红染色显示形成钙化结节。流式鉴定显示SVF:CD29:17.0%,CD31:1.3%;ASCs:CD29:96.2%,CD31:3.8%。SVF、ASCs和空白对照组各组移植脂肪成活量分别为0.2096±0.0024g,0.1798±0.0033g,0.1350±0.0020g,两两比较差异均有统计学意义,P<0.05。HE染色显示SVF组移植脂肪成活较好,组织结构完整,脂滴大小均一,可见脂肪组织中间有丰富的血管存在;ASCs组移植脂肪成活尚可,组织结构尚完整,脂滴大小一般均一,可见脂肪组织中有结缔组织纤维间隔和新生血管形成;空白对照组结缔组织纤维间隔明显增多,脂滴大小不一,有少量较大空泡形成。各组移植脂肪新生血管密度分别32.6±2.1条/mm2,29.3±1.6条/mm2,23.3±1.9条/mm2,两两比较均有统计学意义,P<0.05。结论:新鲜的干细胞成分SVF比体外扩增的ASCs能更好的促进移植脂肪成活。     

人脂肪干细胞的体外培养鉴定及分化特性

目的：在体外从脂肪块中分离出脂肪干细胞（adipose derived stem/stromal cells,ASCs）,对其进行形态观察、干细胞鉴定、增殖和分化能力检测。方法：将腹部取皮术的皮下脂肪利用胶原酶消化法,进行体外分离培养,取第3代的细胞进行细胞爬片HE染色、流式鉴定、MTT、细胞周期检测等,利用成脂和成骨培养液诱导,油红O和茜素红染色鉴定诱导结果。结果：原代培养第1次换液时细胞多呈多角形和短梭形,第3代ASCs细胞爬片HE染色显示形态多为长梭形,呈漩涡状生长;流式鉴定显示：CD29＋,CD31-,CD34-,CD44＋,CD45-,CD49＋,CD106-,CD133-;MTT显示ASCs生长增殖活性强;细胞周期检测结果显示：G1=86.8%,G2=8.77%;成脂诱导后油红O染色阳性,对照组为阴性;成骨诱导后茜素红染色阳性,对照组为阴性。结论：人ASCs具有贴壁生长、多向分化以及干细胞表型等特征,且生长增殖活性强,是一种很有应用前景的间充质干细胞。     

高成脂脂肪干细胞系的分子克隆筛选

目的 探讨并筛选具备高成脂能力的脂肪干细胞系表面标志、可应用于脂肪组织工程的种子细胞,以提高组织工程化脂肪的构建效率.方法 胶原酶消化人脂肪组织,获得脂肪干细胞,培养扩增后成脂诱导,收集诱导成熟的脂肪细胞,天花板贴壁培养得到去分化脂肪细胞.比较去分化脂肪细胞与人脂肪干细胞的增殖能力,成脂分化能力及表面抗原表达的变化.结果 去分化脂肪细胞与脂肪干细胞的形态和增殖能力相似;去分化脂肪细胞的成脂分化能力高于脂肪干细胞;两种细胞表面抗原的表达大致相同,但去分化脂肪细胞的CD54的阳性表达高于脂肪干细胞.结论 CD54的表达可能与去分化脂肪细胞的高成脂分化能力密切相关,可能是高成脂系干细胞的特异性表面抗原标志.     

去分化脂肪细胞与脂肪来源干细胞体内成脂能力比较的初步研究

目的 通过比较去分化脂肪细胞(dedifferentiated adipocytes,DA)与脂肪来源干细胞(adipose-derived stem cells,ASCs)在体内的成脂分化能力,为脂肪组织工程筛选出成脂效率高的种子细胞.方法 取健康女性脂肪抽吸术的抽吸物,使用酶消化法获取成熟脂肪细胞及脂肪来源干细胞,采用天花板贴壁法培养成熟脂肪细胞使其去分化,获取去分化脂肪细胞,各取第3代细胞进行实验.将两种细胞分别与纤维蛋白胶(fibrin glue,FG)支架体外混合培养,光镜及扫描电镜检测细胞与支架的相容性;DiI荧光染料标记细胞,将DiI标记过的细胞支架复合物(DA-FG组,去分化脂肪细胞-支架,n=8;ASCs-FG组,脂肪来源干细胞-支架,n=8)以及空白支架(空白FG组,n=8)注射于裸鼠皮下.术后8周将新生组织取出,进行大体观察和湿重测定,HE染色、H染色和油红"O"染色后进行组织学观察、纤维化比率测定以鉴定新生物的性质、来源.结果 成熟脂肪细胞经天花板贴壁法培养后变为长梭形成纤维细胞状,即去分化脂肪细胞,脂肪来源干细胞呈长梭形.细胞-支架复合物体外培养3 d后光镜及扫描电镜检测发现细胞在支架上生长良好.术后8周DA-FG、ASCs-FG组裸鼠背部皮下均有新生组织块形成,经检测后证实其为成熟脂肪块并来源于植入的种子细胞,DA-FG组新生物平均湿重大于ASCs-FG组,平均纤维化比率则低于ASCs-FG组;空白FG组无新生组织形成,支架被降解吸收.结论 去分化脂肪细胞与脂肪来源干细胞均可作为种子细胞在裸鼠皮下构建出脂肪组织,较之脂肪来源干细胞,去分化脂肪细胞构建出的组织块具有湿重大,纤维化比率低的优点.     

大鼠脂肪干细胞体外诱导成平滑肌样细胞的研究

目的:研究大鼠脂肪干细胞(ADSCs)体外单层培养条件下诱导分化为平滑肌样细胞的可行性。方法:从SD大鼠的腹股沟脂肪垫分离获取脂肪干细胞,测定其生长曲线。取第4代细胞用成脂诱导液诱导分化,并用油红O染色鉴定。取第4代细胞用成骨诱导液诱导分化,并用Von Kossa染色鉴定。取第4代细胞用含有β-巯基乙醇的成平滑肌诱导液诱导,并用免疫组化的方法检测α平滑肌肌动蛋白(α-SMA)的表达。结果:脂肪干细胞成梭形和多角形,体外生长迅速,生长曲线表明传代2 d后细胞进入对数生长期。第4代细胞成脂诱导后,油红O染色证实细胞内存在脂滴;成骨诱导后,Von Kossa染色证实有矿化结节。脂肪干细胞诱导平滑肌样细胞免疫组化结果,β-巯基乙醇诱导组和未诱导组细胞α-SMA的表达阳性率分别为(29.80±6.89)%、(2.89±1.24)%。诱导组细胞α-SMA的表达阳性率高于未诱导组,差异有统计学意义(P<0.01)。结论:脂肪干细胞经诱导后出现明显的平滑肌细胞特征,可成为平滑肌相关疾病在组织工程研究上新的种子细胞来源。     

脂肪组织来源干细胞提高游离脂肪移植存活率的研究

目的 探讨脂肪组织来源干细胞移植在体内促进游离移植脂肪组织的再血管化,提高移植脂肪组织存活率的可行性.方法 自人体吸脂术中脂质部分分离、培养AScs,行成脂、骨和软骨分化.将ASCs以DiI标记后与人体吸脂术获得的脂肪组织混合移植于18只裸鼠背部,裸鼠背部随机注入3组移植物:ASCs组(A组)、胰岛素组(B组)及培养基对照组(C组).术后6个月观察移植物情况,通过组织观察、HE染色和免疫组化进行分析.结果 抽脂术脂质部分能获取大量的ASCs,能分化成脂肪、成骨和软骨细胞.术后6个月3组移植脂肪的湿重分别为A组(165.97±5.51)mg,B组(93.42±5.12)mg,C组(67.64±5.09)lIIg.A组移植脂肪的存活率高于B、C组(P=0.000).纤维化程度的测定,"网格点计数"3组移植物的点个数分别为A组(152.2±9.8)个/10HF,B组(743.9±20.4)个/10HF,C组(892.2±16.5)个/10HF.A组移植脂肪的纤维化及坏死程度低于B、C组(P=0.000).免疫组化证实:ASCs散在分布于部分脂肪细胞及小叶间隔中,ASCs在体内能够部分转化为血管内皮细胞.结论 脂肪组织来源干细胞移植在体内可促进游离移植脂肪组织的再血管化,提高移植脂肪组织的存活率并改善了移植物的质地.AsCs辅助移植可能是一种较为理想的细胞疗法.     

人脂肪组织来源干细胞植入裸鼠的成脂效应

目的 探讨脂肪抽吸术液态部分获取的人脂肪组织来源干细胞(adipose tissue-derivedstem cells,ADSC)植入裸鼠的成脂效应.方法 自人体脂肪抽吸术中液体部分分离、培养和鉴定ADSC,行成脂、骨和软骨分化.植入物分三组:空支架(第1组),人ADSC接种胶原海绵(第2组),经成脂诱导的ADSC接种胶原海绵架(第3组),植入裸鼠皮下.在2和8周时,通过组织观察、HE染色和油红O染色来分析新生组织.结果 抽脂术液态部分能获取大量的ADSC,能分化成脂肪、成骨和软骨细胞.8周时,第1组植入物已降解,第2组和第3组均有脂肪组织形成,但第2组比第3组在形成体积上小、成脂效应差.结论 从脂肪抽吸术液态部分分离和培养的ADSC和胶原支架混合培养能在体内形成脂肪组织,此方法 获得的ADSC多能干细胞可作为脂肪组织工程的种子细胞.     

A human chondrogenic cell line retains multi-potency that differentiates into osteoblasts and adipocytes

We investigated the differentiation potential into various mesenchymal cell lineages of the clonal cell line (USAC) that was isolated from a human chondrogenic osteosarcoma. USAC cells produced types II and X collagens and proteoglycan, indicating chondrocyte differentiation. Production of type I collagen and osteocalcin by USAC cells demonstrated osteoblastic properties. They also differentiated into adipocytes generating numerous lipid droplets in their cytoplasm. Recombinant human bone morphogenetic protein-2 (rhBMP-2) increased production of proteoglycan, types II and X collagens and osteocalcin. This indicates that rhBMP-2 promotes both chondroblastic and osteoblastic differentiation in USAC cells. rhBMP-2 inhibited adipocyte differentiation. USAC cells transplanted with rhBMP-2 into the peritoneal cavities of athymic mice using diffusion chambers generated cartilage and bone more effectively in the diffusion chambers than those produced without rhBMP-2. Although USAC cells also produced adipose tissue in the diffusion chambers following transplantation with or without rhBMP-2, rhBMP-2 treatment reduced the amounts of adipose tissue. These results demonstrate that USAC is a suitable model to explore regulatory mechanisms involved in human mesenchymal cell differentiation.     

Metabolic Rescue of Obese Adipose-Derived Stem Cells by Lin28/Let7 Pathway

Adipose-derived stem cells (ASCs) are promising candidates for autologous cell-based regeneration therapies by virtue of their multilineage differentiation potential and immunogenicity; however, relatively little is known about their role in adipose tissue physiology and dysfunction. Here we evaluated whether ASCs isolated from nonobese and obese tissue differed in their metabolic characteristics and differentiation potential. During differentiation to mature adipocytes, mouse and human ASCs derived from nonobese tissues both increased their insulin sensitivity and inhibition of lipolysis, whereas obese-derived ASCs were insulin-resistant, showing impaired insulin-stimulated glucose uptake and resistance to the antilipolytic effect of insulin. Furthermore, obese-derived ASCs showed enhanced release of proinflammatory cytokines and impaired production of adiponectin. Interestingly, the delivery of cytosol from control ASCs into obese-derived ASCs using a lipid-based, protein-capture methodology restored insulin sensitivity on glucose and lipid metabolism and reversed the proinflammatory cytokine profile, in part due to the restoration of Lin28 protein levels. In conclusion, glucose and lipid metabolism as well as maturation of ASCs is truncated in an obese environment. The reversal of the altered pathways in obese cells by delivery of normal subcellular fractions offers a potential new tool for cell therapy.Adipose tissue is now recognized as an important endocrine and metabolic organ that, when accumulated in excess, increases the risk of chronic diseases such as diabetes, stroke, and arterial hypertension (1). Recently, new mechanisms that control the obesity phenotype have been identified such as the equilibrium between white and brown adipose tissue, the localization of adipose mass (visceral vs. ventral), and the presence of adipose stem cells (ASCs) and mesenchymal stem cells (MSCs) (1–4). Although the relative importance of fat tissue type and localization are being actively unraveled, the role of stem cells in adipose tissue physiology and dysfunction is still poorly understood.Adult stem cells are multipotent cells that contribute to the homeostasis of various organs, including adipose tissue. ASCs are a class of MSCs localized in adipose tissue that have attracted increasing interest because of their potential to differentiate into adipogenic, osteogenic, chondrogenic, and other mesenchymal lineages (5–8). Other clinically attractive properties attributed to ASCs include proangiogenic and anti-inflammatory actions (9–11). Moreover, depending on the environmental conditions, ASCs can be beneficial or detrimental to health. ASCs thus represent a possible target for therapies aimed at modulating the response of the body to obesity and diabetes as well as a potential tool for regenerative medicine.Adipocytes are central to the control of energy balance and lipid homeostasis (12). In response to prolonged obesity, adipocytes become hypertrophic, and new adipocytes are required to counter the metabolic dysfunction of the hypertrophic cells (13,14). It has been postulated that the adipose tissue depots of obese individuals have already committed all of their stem cell reserves to the adipocyte lineage and, therefore, have no capacity to generate new adipocytes (15–17).In this study, we demonstrate that the differentiation of mouse and human adipose MSCs into mature well-functioning adipocytes is truncated in an obese environment, resulting in impaired metabolic function. We also validate a novel approach to restore normal adipocyte metabolic responsiveness in obese-derived stem cells by cytosolic transfer.     

Novel genes regulated by the insulin sensitizer rosiglitazone during adipocyte differentiation

Thiazolidinediones (TZDs) are a new class of compounds that improve insulin sensitivity in type 2 diabetic patients as well as in rodent models of this disease. These compounds act as ligands for a member of the nuclear hormone receptor superfamily, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), which is highly expressed in adipose tissue and, moreover, has been shown to play an important role in adipocyte differentiation. The strong correlation between the antidiabetic activity of TZDs and their ability to activate PPAR-gamma suggests that PPAR-gamma, through downstream-regulated genes, mediates the effects of TZDs. In this report, we present the isolation and characterization of 81 genes, encoding proteins of known function, differentially expressed during TZD-stimulated differentiation of 3T3-L1 cells. By the use of different reverse- Northern blot techniques, the differential expression of 50 of these genes could be verified, and 21 genes were specifically regulated by a potent TZD during the course of adipocyte differentiation, whereas no effect of a PPAR-gamma antagonist could be observed in mature adipocytes. The differential expression of a large fraction of the isolated genes was also shown to occur in white adipose tissue of ob/ob mice treated with rosiglitazone; combined, our results suggest that an important effect of rosiglitazone in adipose tissue is based on activation of PPAR-gamma in preexisting preadipocytes found among the mature adipocytes, resulting in subsequent adipocyte differentiation.     

大鼠脂肪组织来源干细胞的分离、培养和生物学特性的研究

目的:建立一种分离和培养大鼠脂肪干细胞的方法,并探讨获得的脂肪干细胞的部分生物学特性。方法:取SD大鼠腹股沟处的皮下脂肪,I型胶原酶消化法获取原代脂肪干细胞,接种至含10%胎牛血清的DMEM培养液,培养并适时传代,每日在倒置显微镜下观察记录细胞形态和增殖特征,测定其生长曲线,进行冻存复苏实验。第3代细胞使用成骨诱导液诱导其向成骨细胞分化,进行VonKossa染色;使用成脂诱导液诱导其向成脂细胞分化,进行油红O染色。结果:从大鼠脂肪组织中分离出脂肪干细胞,其在体外生长增殖迅速,呈成纤维样细胞生长。生长曲线表明第3代脂肪干细胞增殖能力最强,可以在地塞米松、维生素C、β-甘油磷酸钠的诱导下,表现出成骨细胞特性,VonKossa染色出现矿化结节;在IBMX(3-异丁基-1-甲基黄嘌呤)、吲哚美辛、胰岛素的诱导下,表现出脂肪细胞特性,油红O染色后发现胞浆内有脂肪滴。脂肪干细胞在液氮中冻存1个月后,其生长增殖活性和多向分化能力没有明显降低。结论:成功建立了一种简单有效的分离和培养大鼠脂肪干细胞的方法,获得的脂肪干细胞生长增殖旺盛,具有多向分化能力,可以方便地保存,有望作为细胞治疗和组织工程的种子细胞。在分离大鼠的脂肪干细胞时,NH4Cl裂解红细胞这一步可以省略,地塞米松在成脂诱导过程中不是必需的。     

Role of gender and anatomical region on induction of osteogenic differentiation of human adipose-derived stem cells

Adipose-derived stem cells (ASCs) display multilineage plasticity and, under appropriate conditions, can mineralize their extracellular matrix and undergo osteogenesis. The aims of this study are to examine in vitro osteogenic differentiation properties of ASCs to assess the role of gender, fat depot, and optimal duration as variables for differentiation.Human ASCs were isolated from superficial and deep adipose layers of the abdominoplasty specimens obtained from patients undergoing elective surgeries. ASCs were cultured in osteogenic media (OM). After 1, 2, and 4 weeks of differentiation, cultures were assessed for markers of osteogenesis. Alkaline phosphatase (AP), alizarin red (AR) and Masson trichrome (MT) stainings for osteoblastic transformation, matrix mineralization, and collagen production; enzyme-linked immunosorbent assay (ELISA) for Gla-osteocalcin; and Western blot analysis for osteonectin protein expression were performed.Osteogenic differentiation began as early as 1 week. Cells exhibited a vertical growth pattern, lacunae formed in the cultures, matrix volume increased, and mineralization was observed. Differences in AP staining were most evident during the first week. AR activity progressively increased over 4 weeks, and collagen was secreted only by differentiated ASCs. There was no significant difference in the degree of osteogenic differentiation between the ASCs from both depots in the female. In the male, the superficial depot ASCs differentiated faster and more efficiently than those of the deep depot. Male ASCs from both depots differentiated more effectively than female ASCs from both depots.We describe a hierarchy of osteogenic differentiation potential based on gender and anatomic harvest site by layering adipose tissues of the abdominal wall. ASCs derived from male superficial layer were most efficient in achieving osteogenesis. In future clinical applications using stem cells for osseous healing, these gender and depot differences will guide our clinical methods.