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。     

人脂肪来源细胞体内构建组织工程化脂肪的实验研究

目的探讨以组织工程技术，应用脂肪来源细胞（Adipose—derived cells，ADCs）体内构建脂肪组织的可行性。方法吸脂术获得人脂肪组织，一部分直接植入裸鼠体内；另一部分用酶消化法分离、培养ADCs，将第三代细胞接种于纤维凝胶（Fibrin glue）支架中，经成脂肪培养液诱导1周后，植入裸鼠体内，4周后取材，用称重法及油红、HE染色检测结果。实验分为直接注射脂肪组、单纯支架组、细胞-支架复合体脂肪诱导组、非诱导组。结果直接注射组在裸鼠皮下形成脂肪组织，但吸收量大；细胞-支架复合体诱导组有大量脂肪类组织形成，油红染色显示组织有脂滴形成；细胞-支架复合体非诱导组和单纯支架组均未发现脂肪组织形成。结论采用组织下程技术将吸脂术获得脂肪来源细胞接种纤维凝胶支架，体内构建脂肪组织，具有可行性。     

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.     

不同支架及接种培养方法对前脂肪细胞生长情况的影响

目的 通过对体外培养前脂肪细胞并分别与聚乳酸-羟基乙酸共聚物（PLGA）支架、胶原支架以及透明质酸支架复合,研究3种不同支架材料的细胞相容性.方法 切取成年女性腹部皮下脂肪组织,采用胶原酶消化的方法分离培养人前脂肪细胞,采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑臭盐（MTT）法观察3种不同支架材料的细胞相容性,并以透明质酸作为支架,考察静态和水平摇床两种接种培养方式对前脂肪细胞在支架上接种率的影响.结果 前脂肪细胞可以在体外成功地分离、培养,能定向分化为脂肪细胞,并可以进行传代扩增.前脂肪细胞在PLGA、胶原以及透明质酸3种材料上生长良好,其中透明质酸支架以及水平摇床接种培养效果更佳.结论 透明质酸更加适宜作为组织工程化脂肪的支架材料,水平摇床的接种培养可提高细胞与支架材料之间的接种率,优于静态培养.     

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.     

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.     

经RNAi诱导的前脂肪细胞构建组织工程化脂肪组织

目的：应用基因工程和组织工程的原理和方法,通过改造人前体脂肪细胞,提高其活性后,于裸鼠背部皮下构建组织工程化脂肪组织。方法：采用原代消化细胞培养法培养出前脂肪细胞,利用RNAi技术,诱导前脂肪细胞内的一个重要凋亡调控基因Bax基因表达沉默,然后接种到聚乳酸-乙醇酸共聚物（PLGA）支架上,种植在20只雌性裸鼠皮下,对获得的脂肪组织进行组织学评价。结果：经过改造后的前脂肪细胞活性良好,术后裸鼠全部成活,前脂肪细胞能够在支架上良好的存活,生长和增殖。结论：经过改造后的前脂肪细胞具有良好的活性,并且PLGA-前脂肪细胞复合体在裸鼠体内可形成脂肪细胞,值得进一步研究,以探索治疗软组织缺损的新途径。     

经RNM诱导的前脂肪细胞构建组织工程化脂肪组织

目的:应用基因工程和组织工程的原理和方法,通过改造人前体脂肪细胞,提高其活性后,于裸鼠背部皮下构建组织工程化脂肪组织.方法:采用原代消化细胞培养法培养出前脂肪细胞,利用RNAi技术,诱导前脂肪细胞内的一个重要凋亡调控基因Bax基因表达沉默,然后接种到聚乳酸一乙醇酸共聚物(PLGA)支架上,种植在20只雌性裸鼠皮下,对获得的脂肪组织进行组织学评价.结果:经过改造后的前脂肪细胞活性良好,术后裸鼠全部成活,前脂肪细胞能够在支架上良好的存活,生长和增殖.结论:经过改造后的前脂肪细胞具有良好的活性,并且PLGA-前脂肪细胞复合体在裸鼠体内可形成脂肪细胞,值得进一步研究,以探索治疗软组织缺损的新途径.     

人脐带间充质干细胞与蚕丝蛋白支架构建组织工程脂肪的研究

目的：将体外以人脐带间充质干细胞（hUCMSCs）与蚕丝蛋白支架初步构建的组织工程脂肪移植到大鼠体内,观察其演变过程。方法：hUCMSCs与蚕丝蛋白支架复合培养10天后,进行成脂诱导;6周后将其移植到Wi st ar大鼠后肢肌肉内,同时,以同体积支架材料作为对照;分别于移植后4周和8周取材,行油红O染色、HE染色以及扫描电镜观察。结果：hUCMSCs与蚕丝蛋白支架复合培养及成脂诱导6周后,见大量成脂样细胞生成,并与支架牢固粘附。移植4周,移植物体积略小,质稍硬,表面有透明薄膜形成,膜中分布新生血管网;油红O染色见支架内新生脂肪组织及细胞呈橙红色;HE染色显示支架网眼内有新生脂肪组织,并可见少量炎性细胞浸润;扫描电镜见支架网眼内有球形、表面光滑的脂肪细胞。移植8周,移植物体积进一步缩小,质变软,表面薄膜内血管网丰富;油红O染色见支架中着橙红色组织较前明显增多,部分呈片状融合;HE染色显示新生脂肪明显增多,仍有少量炎性细胞浸润;扫描电镜显示脂肪细胞较前增生明显。对照组同样可见炎性细胞浸润,未见新生脂肪组织生成,支架材料8周时较4周时降解更加明显。结论：随着时间推移,蚕丝蛋白支架网眼内脂肪细胞逐渐增多,支架材料在体内呈现逐步降解趋势,说明体内环境有利于组织工程化脂肪的进一步形成。同时,也提示支架材料在组织相容性方面尚存不足。     

聚β-羟基丁酸脂用于前脂肪细胞移植的大体及组织学观察

目的探讨聚β-羟基丁酸脂（PHB）支架材料用于前脂肪细胞移植的可行性。方法将SD（n=20）大鼠的前脂肪细胞体外纯化后转种于PHB支架上增殖，再将细胞支架移植于同一大鼠背部皮下组织内，术后3周取出部分（n=4）移植物行组织学观察，术后8周时（n=8）取出移植物，行大体及组织学观察。对照组（n=8）以无细胞的支架材料种植于对侧皮下。结果前脂肪细胞体外培养时在PHB支架材料上生长良好，植入体内后支架内细胞增多，体积、重量增加，组织学证实移植细胞成活，并有脂滴形成。结论前脂肪细胞可在PHB支架上黏附，增殖并分化为成熟的脂肪细胞。PHB材料作为前脂肪细胞的移植载体是可行的，值得进一步研究。     

Human bone marrow mesenchymal stem cells seeded on modified collagen improved dermal regeneration in vivo

In the correction of functional and aesthetic impairments, loss of soft connective tissue creates the need for adequate implant material. The reconstruction of defects resulting from radical excisions, trauma, or hereditary diseases has seen the use of combined grafts and flaps. With the aim of minimizing donor site morbidity, new methods have been evaluated. Because of a low rate of vascularization, with artificial dermal templates the take has only been poor. As shown in previous studies, improved angiogenetic potency and epidermal formation has been obtained in modified, cell-seeded collagen matrices. We have now investigated the suitability of adult bone marrow mesenchymal stem cells (hMSC) for soft tissue engineering. In this study, hMSC were isolated and expanded. Cells (10(6)) were seeded onto EDC cross-linked collagen sponges and implanted in 30 immunodeficient mice. Collagen sponges without cells were used as controls. The grafts were evaluated after 2 and 6 weeks. After explantation, macroscopic appearance, weights, and histology (scaffold degradation, cellularity, and invasion depth of the seeded cells) were all assessed. After 2 and 6 weeks in vivo, new vessels were found macroscopically on all cell-seeded collagen grafts. The control grafts appeared to be degraded with a lower rate of vessel ingrowth. In the experimental group, weight gain was significant after 2 and 6 weeks in vivo compared to the same grafts after 72 h in vitro, while weight increased only slightly in the control group. Histologically, populated scaffolds showed a high density of vascularization under a capsule. The control sponges showed single capillaries and a thicker capsule. Compared to the controls, cellularity (cells/field) was greater in cell-containing collagen grafts after 2 and 6 weeks. The results obtained demonstrate that in vitro cultured human mesenchymal stem cells seeded on modified collagen sponges may be able to act as a replacement for soft tissue.     

人脂肪来源干细胞与胶原支架共培养的实验研究

目的观察评价人脂肪来源干细胞(Human adipose derived stem cells,hADSCs)在胶原支架中的生长情况,为进一步体内组织修复研究提供依据。方法取人抽脂术后脂肪,经胶原酶解、过滤、离心获得hADSCs,代传代扩增后,接种到胶原支架上。细胞-材料复合物分别体外培养1周、2周,裸鼠体内培养2周、4周后,HE染色观察细胞在支架上的生长情况,免疫组化HLA-Ⅰ检测经裸鼠体内培养后的复合物上的细胞的种属来源。结果原代培养的hADSCs呈"梭形"或"成纤维细胞"样,并以克隆团形式生长。免疫荧光Vimentin染色阳性。第3代hADSCs经流式细胞鉴定,CD29、CD44、CD105表达阳性,CD45、CD34表达阴性。胶原支架复合hADSCs经过体外、体内培养,hADSCs均能长入胶原支架的空隙内,且体内培养比体外培养有更多的细胞长入支架。体外培养1周,已有细胞粘附生长在胶原支架的边缘,体外培养2周后,更多的细胞渗透到材料内部。体内培养2周后,大量细胞占据材料的边缘,有部分细胞能渗透到材料内部甚至材料全层。体内培养4周后,大量细胞渗透支架全层。HLA-Ⅰ抗体检测发现,支架材料内部细胞阳性表达,说明胶原支架内部的细胞来源于hADSCs。结论胶原支架与hADSCs具有较好的相容性,可作为hADSCs的载体材料,用于组织工程缺损修复的研究。     

Construction of a multi-layer skin substitute: Simultaneous cultivation of keratinocytes and preadipocytes on a dermal template

Background

After deep excision of burn eschar down to the muscle fascia patients have a non-reversible loss of the skin and underlying subcutaneous tissue. These patients would benefit from the development of a sufficient epidermal, dermal, and hypodermal tissue-engineered replacement provided by new technologies of tissue engineering.The aim of the present study was to determine whether keratinocytes and preadipocytes grow simultaneously on a bovine-derived collagen-elastin matrix under in vitro conditions in order to obtain a multi-layer skin substitute.

Methods

Human keratinocytes as well as human preadipocytes were seeded onto a collagen-elastin matrix (Matriderm^®). Human preadipocytes were isolated from human subcutaneous adipose tissue and seeded onto the scaffold directly after isolation. Keratinocytes were isolated from fresh human split-thickness skin harvests and seeded onto the surface of the scaffold after 4 days of proliferation.Twenty one days after seeding all scaffolds were histologically evaluated, using hematoxylin eosin, immunohistochemical staining with collagen IV as well as immunofluorescence labeling with anti-Ki67 antibody and DAPI (4′,6-diamidino-2-phenylindole).

Results

Simultaneous growth of keratinocytes and preadipocytes could be observed on the collagen-elastin matrix. Keratinocytes adhered well to the surface of the matrix and formed a confluent epidermis-like layer. Preadipocytes adhered well and also penetrated into the deeper layers of the matrix.

Conclusion

In this study, a collagen-elastin matrix served as a suitable scaffold for simultaneous culturing of preadipocytes and keratinocytes. Preadipocytes showed good penetration into deeper layers of the scaffold, whereas keratinocytes attached only to the uppermost surface of the matrix.This approach towards a multi-layered skin substitute might be a useful asset for future reconstructive surgery.     

经胶原包埋、修饰的聚羟基乙酸与软骨细胞体外培养的实验研究

目的：探讨经胶原包埋、修饰后的聚羟基乙酸（poloyglycolic acid简称PGA）作为组织技术中细胞培养支架的可行性；方法：将用胶原包埋的PGA和没有包埋的PGA分别与软骨细胞共同置于二氧化碳培养箱中培养，观察二者的亲水性，对细胞的吸附能力和细胞分泌基质的能力进行比较；结果：以胶原包埋的PGA和没有包埋的PGA亲水性没有明显的差异，二者亲水性均不强，而前者对细胞的吸附能力和细胞在其上面生长、分泌基质的能力明显增强；结论：以胶原包埋、修饰的PGA作为组织工程技术中细胞生长的支架，具有很大的应用前景。     

In vitro adipose tissue engineering using an electrospun nanofibrous scaffold

Electrospun 3-dimensional nanofibrous scaffolds share morphologic similarities to collagen fibrils, and promote favorable biologic responses of seeded cells. In this study, we have fabricated a 3-dimensional nanofibrous scaffold made of poly L-lactic acid, and examined its ability to support and maintain the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells in vitro. After a 21-day incubation, oil red O staining of constructs treated with adipogenic supplements revealed positive adipose-like staining, compared with lack of staining in untreated cultures. Semi-quantitative RT-PCR analysis of human bone marrow-derived mesenchymal stem cells cultured in adipogenic medium revealed highly elevated levels of adipogenesis-associated genes (1797-fold for lipoprotein lipase, and 5.6-fold for peroxisome proliferator-activated receptor gamma). Immunofluorescence staining of cellular constructs in adipogenic culture media showed the presence of lipoprotein lipase vesicles, a characteristic feature of adipose tissue. These results suggest that the poly L-lactic acid-based nanofibrous scaffold is a promising candidate for adult stem cell-based engineering of adipose tissue.     

人脐带间充质干细胞的分离培养及其与胶原支架材料的相容性

目的探讨人脐带间充质干细胞(Mesenchymal stem cells,MSCs)体外分离、培养的方法,观察其与胶原蛋白支架的相容性,为进一步研究提供依据。方法利用组织体外培养法分离人脐带MSCs,流式细胞学分析细胞表型,组织化学观察细胞分化能力;将细胞接种于胶原蛋白支块架上,观察细胞在材料中的形态、粘附情况和增殖特点。结果直接贴壁法分离的细胞表型均一,体外具有成骨和成脂肪能力;细胞在支架上有较好的粘附铺展,培养2周后,MSCs占据支架总体积的80%,细胞分布较均匀。结论组织块培养法可获得高纯度的人脐带MSCs,细胞在体外与胶原蛋白支架有较好的组织相容性。     

组织工程肌腱种子细胞的比较研究

目的 探讨猪肌腱细胞、真皮成纤维细胞、骨髓问充质干细胞中何种细胞最适宜作为体外组织工程化肌腱构建的种子细胞.方法 收集猪肌腱细胞、真皮成纤维细胞及骨髓间充质干细胞,以50×106个细胞密度均匀接种于圆柱状聚羟基乙酸(Polyglycolic acids,PGA)上,按细胞种类分为三组,每组n=3,体外培养,并于1、2、6周取材,进行组织学检测、免疫组化检测、胶原定量测定和大体观察.结果 细胞-PGA复合物体外培养时有细胞外基质产生,六周时肌腱细胞组产生胶原量最多,明显优于真皮成纤维细胞和骨髓间充质干细胞(p＜0.01).免疫组化显示形成的主要为Ⅰ型胶原.结论 体外构建组织工程肌腱时肌腱细胞合成胶原能力最强,在现有条件下是体外构建组织工程肌腱的最佳种子细胞.     

Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds

Tissue engineering approaches have been clinically tried to repair damaged articular cartilages. It is an essential step to uniformly seed chondrocytes into 3D scaffolds in order to reconstruct tissue-engineered cartilages in vitro, but the tissue engineering could not have been provided with efficient cell seeding methods. Type I collagen is clinically used and known as a cytocompatible material, having recognition sites for integrins. Collagen gel encapsulating chondrocytes has been tried for making regenerated cartilages, but it is found difficult to have the gel keep its original shape after long-term culture, because of shrinking. On the other hand, 3D scaffolds, either of a nonwoven structure or a sponge-like structure, involve difficulty in that chondrocytes could not be uniformly seeded, although they have adequate initial mechanical properties. In this study, by combining collagen gelation with a nonwoven PLLA scaffold, we achieved uniform cell seeding into the 3D scaffold. Bovine articular chondrocytes were mixed with type I collagen solution, and the solution was poured into the nonwoven PLLA scaffold (1.5 mm thick, diameter 15 mm). The collagen-chondrocyte mixture was made into gel at 37 degrees C for 1 h. The 0.39% collagen mixture was viscous enough to prevent cells from precipitating during gelation. Almost all chondrocytes were able to be incorporated into the PLLA scaffolds by mixing with collagen solution and subsequently making into gel, while 30-40% of the chondrocytes seeded as a cell suspension were not trapped into the PLLA scaffolds. The method presented, where chondrocytes were mixed with collagen solution, and the mixture was incorporated into a 3D scaffold, then made into gel in the scaffold, could serve as an alternative for in vitro cartilage regeneration, also simultaneously having the advantages of both materials.