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.     

脂肪来源细胞体外构建组织工程软骨的初步探索

目的 探讨脂肪来源细胞(adipose-derived cells,ADCs)在体外构建特定形态软骨的可行性.方法 脂肪组织由整形外科吸脂术获得.酶消化法分离抽吸物中细胞,体外扩增.以第3代细胞接种PLGA生物支架,在成软骨培养基中体外诱导4周,大体观察、组织学检测构建组织的成软骨能力.结果 大体观察见诱导组能维持圆柱形态.非诱导组失去原有形态,单纯支架组完全塌陷.组织学上诱导组局部检测到软骨陷窝包埋于嗜碱性基质中,Massens's染色和Safranin'O染色示胶原、蛋白多糖呈阳性,免疫组织化学染色示Ⅱ型胶原轻度阳性;非诱导组呈典型的疏松结缔组织结构,组织学特殊染色均呈阴性.结论 脂肪来源细胞虽为多种细胞混合群体,但作为构建特定形态软骨的种子细胞,具有可行性.     

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.     

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.     

复合水凝胶负载人脂肪间充质干细胞体内分化的研究

目的 寻求一种为人脂肪间充质干细胞生长提供优越环境的生物材料.方法 将人脂肪间充质干细胞分别悬浮于多肽水凝胶、泊洛沙姆水凝胶和复合水凝胶中,接种于裸鼠颈部皮下,通过组织病理、免疫组化观察接种部位脂肪再生、血管再生及细胞分化等情况.结果 在复合水凝胶组,注射部位发现再生的脂肪组织,且毛细血管增生明显;而在多肽水凝胶和泊洛沙姆水凝胶组,注射部位无脂肪再生.再生的脂肪组织中,免疫组化鼠抗人细胞核单克隆抗体阳性表达,注射部位的皮肤组织中亦发现了人源性细胞且鼠抗人角蛋白抗体阳性表达.结论 复合水凝胶同时具有泊洛沙姆良好的机械性能和多肽水凝胶优越的生物活性,为人脂肪干细胞提供良好的生长分化环境,为软组织缺损修复提供新的组织工程支架材料.     

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。     

人细胞外基质支架联合脂肪干细胞构建脂肪组织

目的 探讨人脂肪组织细胞外基质(ECM)支架联合人脂肪来源干细胞(ADSCs)构建工程化脂肪组织的可行性.方法 以酶消化法从人抽脂术抽吸物脂质部分获取人ADSCs,体外进行多向分化诱导鉴定,并行DiI荧光标记.从抽脂术的脂质部分分离提取人脂肪组织细胞外基质,经过低温冻干、粉碎、灭菌等处理,制备成粉末状,电镜扫描观察表面特征并将其与ADSCs进行黏附实验,探讨其作为支架材料的可行性.收集人ADSCs,以2×109/L的细胞密度与提取的细胞外基质支架复合后移植于裸鼠背部皮下,同鼠对侧背部皮下移植ECM支架和细胞培养液作为对照,每侧移植0.5 ml,共6只实验鼠.8周后取材,称量标本湿重.取出的标本行苏木素-伊红(HE)染色和油红O染色进行定性判断,分析人脂肪组织ECM支架联合人ADSCs构建工程化脂肪组织的能力.结果 从脂肪组织中分离得到人ADSCs和ECM支架.ADSCs在相应的诱导环境下能够分化成为脂肪细胞、骨细胞和软骨细胞.ECM支架电镜扫描和大体观察具有疏松、多孔的结构特征,适合ADSCs的黏附生长.ADSCs与支架相容性良好,黏附率达(89.87±2.59)％,细胞在支架表面可充分伸展生长.体内移植8周后,实验组和对照组都能够形成新生物,湿重比较实验组较对照组重(P＜0.05).经HE切片及油红O染色均证实实验组形成成熟的脂肪组织,对照组不能形成脂肪组织.结论 人脂肪组织ECM支架联合人ADSCs在体内能够成功构建成熟的脂肪组织,8周后支架并无明显吸收.     

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。     

不同层次脂肪干细胞在裸鼠脂肪移植的比较研究

目的探讨静置后脂肪颗粒提取脂肪干细胞(PLA-ADSC)和静置后下层滤液提取的脂肪干细胞(LAF-ADSC)诱导裸鼠成脂的机制, 为细胞辅助的脂肪颗粒移植提供思路。方法 2019年6月至2021年3月, 于成都八大处医疗美容医院整形外科和成都市第三人民医院医学美容部手术室行正常人体的脂肪抽吸手术, 将通过手术获得的脂肪组织抽吸物静置, 得到上层脂肪颗粒组织进行消化、分离培养PLA-ADSC并鉴定;得到下层液体组织离心后取沉淀物, 沉淀物进行消化、分离培养LAF-ADSC并鉴定;比较PLA-ADSC和LAF-ADSC分化特性及其生长能力。动物实验分实验1组和实验2组, 将基质胶Matrigel移植至裸鼠体内设为对照组, 比较3组裸鼠体内的成脂能力。结果细胞实验结果显示, 静置后脂肪颗粒及下层滤液沉淀物提取细胞均能贴壁生长并顺利传代, 上述两种不同来源细胞均表达CD44、CD73、CD105, 阳性率分别为99.5%、99.99%、99.7%, CD19、CD31、CD45则为阴性表达。成脂诱导分化结果显示, 胞质内有脂滴形成, 细胞油红O染色后可见脂滴呈橘红色。动物实验结果显示, 移植后...     

基因转染脂肪间充质干细胞作为软骨组织工程种子细胞的研究

目的 观察人转化生长因子(hTGF)β2基因转染诱导脂肪间充质干细胞向软骨细胞的定向分化能力,探讨脂肪间充质干细胞作为种子细胞和在基因增强的软骨组织工程中应用的可行性.方法 取3周龄Lewis大鼠的脂肪组织,消化法获得脂肪间充质干细胞,pcDNA 3.1(+)/hTGFD2通过脂质体介导转染脂肪间充质干细胞,用免疫化学染色、逆转录-聚合酶链反应(RT-PCR)和Western blot检测筛选的阳性克隆细胞中hTGFB2基因与软骨特异性蛋白-Ⅱ型胶原和蛋白多糖表达的情况;然后将基因转染的脂肪间充质干细胞与PLGA支架体外构建细胞-载体复合物,再将其植入裸鼠体内,12周后观察基因增强的组织工程软骨的形成情况.结果 从成体大鼠脂肪组织中培养出脂肪问充质干细胞,能大量稳定增殖传代.hTGFB2基因在脂肪间充质干细胞内能瞬时及稳定表达,并促使Ⅱ型胶原和蛋白多糖合成;细胞.载体复合物在裸鼠体内经12周的培养,可以形成形态、结构接近正常软骨的组织工程软骨.结论 PODNA 3.1(+)/hTGFl32成功转染脂肪间充质干细胞,诱导其向软骨细胞分化,脂肪间充质干细胞可作为基因增强的软骨组织工程较理想的种子细胞.     

Adipose tissues differentiated by adipose-derived stem cells harvested from transgenic mice

Objective: To induce adipocyte differentiation in vitro by adipose-derived stromal cells (ASCs) harvested from transgenic mice with green fluorescent protein (GFP) and assess the possibility of constructing adipose tissues via attachment of ASCs to typeⅡcollagen scaffolds. Methods: Inguinal fat pads from GFP transgenic mice were digested by enzymes for isolation of ASCs (primary culture). After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks, and the adipocyte differentiation by ASCs in vitro was assessed by morphological observation and Oil Red O staining. Then they were attached to collagen scaffolds and co-cultured for 12 hours, followed by hypodermic implantation to the dorsal skin of nude mice for 2 months. The newly-formed tissues were detected by HE staining. Results: The cultured primary stem cells were fibroblast-like and showed active proliferation. After being incubated in an adipocyte differentiation medium, the lipid droplets in the cytoplasm accumulated gradually and finally developed into mature adipocytes, which showed positive in Oil Red O staining. A 0. 5-cm3 new tissue clot was found under the dorsal skin of the nude mice and it was confirmed as mature adipose tissues by fluorescent observation and HE staining. Conclusions: ASCs can successfully differentiate adipose tissues into mature adipocytes, which exhibit an adipocyte-like morphology and express as intracytoplasmic lipid droplets. It is an efficient model of adipose tissues engineered with ASCs and type I collagen scaffolds.     

裸鼠原位种植大肠癌肝转移三种建模方法的比较

目的 比较3种不同的人大肠癌裸鼠原位种植肝转移模型,建立稳定有效的人大肠癌裸鼠原位种植肝转移模型.方法 逐步传代法皮下成瘤传5代,采用84只BALB/C-nu/nu裸鼠,随机分为4组,结肠原位荷包缝合法和结肠原位纤维蛋白胶黏合法和结肠微注射1×106和1×105SW1116细胞法建立大肠癌裸鼠原位种植肝转移模型.各组裸鼠死亡后立即解剖观察原位以及肝脏、肺、脾脏、胰腺、肠系膜等脏器组织的转移并且进行苏木素-伊红(HE)染色进行镜下观察有无转移.结果 荷包缝合组和纤维蛋白胶组结肠原位种植肝转移成功率均为100%比微注射法50.0%和14.3%高,纤维蛋白胶法比其他两种方法所形成的结肠原位肿瘤和肝转移瘤均大而多.HE染色表明肝转移肿瘤性质与结肠原位种植瘤相同.结论 结肠原位纤维蛋白胶黏合法比较其他二种原位种植肝转移模型的手术成功率高,肝转移率高、操作简单.     

A comparative histologic analysis of tissue-engineered bone using platelet-rich plasma and platelet-enriched fibrin glue.

OBJECTIVE: The aim of this study was to compare the effects of platelet-rich plasma (PRP) and platelet-enriched fibrin glue on bone formation in bone tissue engineering. STUDY DESIGN: PRP was mixed with bone marrow mesenchymal stem cells and bone morphogenetic protein-2 (BMP-2), and the composites were injected into the subcutaneous space on the dorsum of nude mice. On the contralateral side of the dorsum, platelet-enriched fibrin glue/bone marrow mesenchymal stem cells/BMP-2 composites were injected. Bone formation was evaluated after 12 weeks. RESULTS: The volumes of subcutaneous nodules formed in nude mice were 55 +/- 18 microL at the PRP/bone marrow mesenchymal stem cells/BMP-2 sites and 135 +/- 27 microL at the platelet-enriched fibrin glue/bone marrow mesenchymal stem cells/BMP-2 sites. Histomorphometric analysis demonstrated that the nodules contained 14.9 +/- 4.1% newly formed bone when using PRP and 19.8 +/- 3.6% newly formed bone when using platelet-enriched fibrin glue. CONCLUSION: The results indicated that the osteogenic characteristics of platelet-enriched fibrin glue are superior to PRP in bone tissue engineering.     

角质细胞与成纤维细胞混合移植修复裸鼠全层皮肤缺损的初步报告

目的：利用组织工程原理探讨修复全层皮肤缺损的理想方式。方法：以裸鼠为动物模型,在皮肤全层缺损区域分别移植纤维蛋白胶(n=10),纤维蛋白胶角质细胞悬液（n=10）,纤维蛋白胶成纤维细胞悬液（n=10）以及纤维蛋白胶角质细胞成纤维细胞悬液（n=10）,术后每天对伤口进行大体观察,第5,7,10,14,21,35d,分别取材活检行组织学及免疫组织化学检查。结果：移植有角质细胞组（2和4组）的创面愈合快,术后10d组织学提示创面完全上皮化,抗人特异性HLA－1型抗原、抗involucrin染色和抗Ⅶ型胶原染色阳性证明新生上皮由移植的人角质细胞形成,抗involucrin染色阳性又证明角质细胞分化成熟有角质层形成,抗Laminin染色、抗Ⅶ型胶原染色阳性提示早期基底膜形成。组织学检查提示第4组新生上皮有许多类似皮钉样结构。结论：培养的角质细胞,成纤维细胞结合纤维蛋白胶移植到创面上后,可以形成复层分化良好、接近正常结构和功能的新生成肤组织。     

Engineering of Adipose Tissue by Injection of Human Preadipocytes in Fibrin

Background Despite efforts of plastic surgeons in recent years to discover new alternatives, the techniques currently used for restoration of soft tissue defects still have disadvantages. The gold standard for soft tissue reconstruction remains autologous pedicled/free tissue transfer. This technique often results in high rates of operative morbidity and donor site deformity. Results obtained by autologous fat tissue transfer usually are disappointing because of a high graft resorption rate with unpredictable outcomes. Different tissue engineering approaches have been used in the past to generate adipose tissue. However, long-term results in terms of volume persistence have been disappointing. Methods In this study, different concentrations of undifferentiated human preadipocytes in fibrin were injected into athymic nude mice (n = 8). Mice that had fibrin injection without cells served as control subjects (n = 8). The specimens were explanted after 1, 2, 6, and 9 months, with subsequent qualitative and quantitative analysis of adipose tissue formation by histologic and image analysis. Results Within the first 4 weeks after initial volume reduction of the implants, the volume and shape of the implants with preadipocytes remained stable. The implants without cells were completely resorbed within 3 weeks. Histologic analysis demonstrated generation of stable adipose tissue with no signs of an inflammatory response or evidence of tissue necrosis in the implants containing preadipocytes. The best results were obtained after implantation of 30 million preadipocytes. Adipose tissue formation was not observed in the control group. Conclusions The findings demonstrate that long-term stable adipose tissue can be engineered in vivo by simple injection of human preadipocytes using fibrin as a carrier material. After further investigation, this approach may represent an alternative to the techniques currently used for soft tissue restoration. Presented at the 36th Conference of the Association of German Plastic Surgeons (DGPRA) in Munich, Germany, September 2005, and at the 8th Conference of the Association of French Aesthetic and Plastic Surgeons (SOFCEP) in Paris, France, June 2005     

前脂肪细胞凝胶植入预防腰椎术后硬膜外腔瘢痕粘连的实验研究

目的：探讨自体前脂肪细胞凝胶植入法预防腰椎术后硬膜外腔瘢痕粘连的效果。方法：将60只SD大鼠随机分为A组(前脂肪细胞凝胶植入组)、B组(自体脂肪颗粒植入组)、C组(空白对照组)，将A组动物先进行前脂肪细胞培养，待其脂肪细胞培养传代至适量后，制备各组大鼠的椎板缺损模型，A组取培养的单层脂肪细胞与生物蛋白胶制成混合物，B组取自体脂肪颗粒分别植入椎板缺损区，C组不植入任何物质，于术后8周行大体、光镜、电镜及MRI检查。结果：A组预防硬膜外腔瘢痕粘连效果良好，优于B组。结论：自体前脂肪细胞与生物蛋白胶制成的混合物植入预防硬膜外腔瘢痕的形成与粘连有良好的效果，是一种预防椎板切除术后硬膜外腔瘢痕形成与粘连的新方法。     

The effect of fibrin glue on fat graft survival.

Autologous fat transplantation for filling defects or augmenting tissue is a common procedure but may have unreliable results. While fibrin glues lead to increased proliferation of fibroblasts and local accumulation of vascular endothelial growth factor, which enhances the neovascularisation, in this study the efficacy of fibrin glues on fat graft survival was investigated. Inguinal fat pads from Sprague-Dawley rats were harvested and same volumes of autogenous fat grafts were implanted into the separate pockets with the aid of fibrin glue (Group 1) and saline solution (Group 2). All the fat grafts were harvested, washed, blotted dry, and volumetrically measured with the same method used peroperatively at 6 months after implantation. Mean graft survival values for Group 1 were compared with Group 2 and histopathological evaluation of the grafts was also made. There was a significantly higher survival rate of the grafts in Group 1 than control group (79+/-4% and 55+/-6%, respectively). Histopathological examination of the grafts demonstrated evident increase in neovascularisation of the fat grafts in the experimental group. The authors conclude that the fibrin glue significantly diminishes the fat graft resorption and further well-controlled studies are required before using fibrin glues for clinical purposes.