CIDEA基因在脂肪瘤间充质干细胞中的表达及其与脂肪肿瘤的关系

目的 检测CIDEA基因在脂肪瘤间充质干细胞中的表达,并探讨其与脂肪组织肿瘤的关系.方法 采用实时荧光定量逆转录-聚合酶链反应(RT-PCR)检测6例脂肪瘤间充质干细胞和6例正常脂肪组织来源干细胞中CIDEA的表达水平.结果 CIDEA在脂肪瘤间充质干细胞中的表达显著低于正常脂肪来源干细胞,提示其在脂肪瘤间充质干细胞中表达的下降或缺失可能与肿瘤干细胞引发脂肪组织肿瘤相关.结论 CIDEA与脂肪细胞的分化呈现相关性,其表达的下降或缺失可能与脂肪组织肿瘤的发生发展有关.     

CIDEA基因在脂肪瘤间充质干细胞中的表达及其与脂肪肿瘤的关系

目的 检测CIDEA基因在脂肪瘤间充质干细胞中的表达,并探讨其与脂肪组织肿瘤的关系.方法 采用实时荧光定量逆转录-聚合酶链反应(RT-PCR)检测6例脂肪瘤间充质干细胞和6例正常脂肪组织来源干细胞中CIDEA的表达水平.结果 CIDEA在脂肪瘤间充质干细胞中的表达显著低于正常脂肪来源干细胞,提示其在脂肪瘤间充质干细胞中表达的下降或缺失可能与肿瘤干细胞引发脂肪组织肿瘤相关.结论 CIDEA与脂肪细胞的分化呈现相关性,其表达的下降或缺失可能与脂肪组织肿瘤的发生发展有关.     

婴幼儿血管瘤间充质干细胞的分离鉴定及其在血管瘤组织中的定位观察

目的 探索周细胞是否是血管瘤间充质干细胞的来源,及间充质干细胞与婴幼儿血管瘤消退过程中的脂肪组织增生的关系.方法 采用贴壁筛选法从增生期血管瘤中分离间充质干细胞,应用流式细胞仪和免疫荧光染色检测细胞抗原表型.免疫组化染色观察CD133和PPAR-γ在增生期血管瘤组织中的表达,并以CD31和α-SMA行共染色.结果 血管瘤间充质干细胞为成纤维细胞样,表达CD133、PPAR-γ、CD105、CD90、CD29和Vimentin,不表达CD45、CD34、CD31和flt-1.部分细胞表达α-SMA.大部分细胞表达PDGFR-β.NG2、Desmin和RGS-5无表达.免疫组化染色证实CD133、PPAR-γ和α-SMA共表达于血管瘤组织的微血管周围.结论 细胞形态和抗原表型初步证实,本研究分离的细胞为间充质干细胞,免疫组化染色证实间充质干细胞存在于血管瘤组织的微血管周围.研究结果提示,周细胞是血管瘤间充质干细胞的来源.     

脂肪组织来源干细胞与脂肪瘤间充质干细胞特性的比较研究

目的 比较体外培养的脂肪来源干细胞(adipose-derived stem cells,ASCs)与脂肪瘤间充质干细胞(lipoma-derived mesenchymal stem cells,LMSCs)的生物特性,以探讨ASCs移植的安全性.方法 对正常脂肪组织和脂肪瘤组织进行切片染色,分离培养ASCs和LMSCs,观察细胞形态;MTS比色法检测细胞活性并绘制细胞生长曲线;流式细胞仪测定细胞周期及表面分子表达;QRT-PCR检测高迁移率族蛋白2(HMGA2)表达水平;免疫组织化学染色法鉴定端粒酶逆转录酶(hTERT)的表达.结果 正常脂肪组织和脂肪瘤组织切片差异明显;ASCs细胞形态一致性好,而LMSCs具有不均质性;MTS活性测定ASCs增殖活性要远低于LMSCs细胞(P=0.000);流式细胞仪检测结果显示ASCs与LMSCs在干细胞标志CD29、CD44、CD105上表达类似,而在肿瘤干细胞标志CD133表达上,ASCs(5.35%)要远低于LMSCs(26.87%);细胞周期显示ASCs的增殖能力低于LMSCs;定量PR-PCR显示ASCs中HMGA2平均aQ值为1,远低于在LMSCs中的表达(1.79),两者差异具有统计学意义(P<0.01);免疫细胞化学结果:hTERT在ASCs和LMSCs中的累计吸光度A值分别为1 379.597±498.617和3 328.108±902.856,面积分别为132 390.27±35 568.945和238 000.53±49 264.289,平均吸光度A值分别为0.009±0.003和0.014±0.003,ASCs中hTERT表达远低于LMSCs.结论 体外培养的ASCs生物学特性与LMSCs存在显著差异,未发现其恶性转、化为肿瘤干细胞的证据.     

核心结合因子α1过表达促进脂肪组织来源干细胞向成骨细胞分化

目的 检测核心结合因子α1(Cbfα1)的特异性成骨作用以及脂肪组织来源干细胞的成骨分化潜能.方法 采用Cbfα1重组的腺病毒载体感染脂肪来源干细胞,通过间接免疫荧光的方法检测脂肪来源干细胞中Cbfα1的表达.Cbfα1转染脂肪组织来源干细胞后1、3、7 d利用RT-PCR检测成骨特异性基因和成脂特异性基因的表达变化.同时通过检测Cbfα1转染后7、10 d脂肪来源干细胞碱性磷酸酶活性的变化,观察Cbfα1过表达后诱导脂肪组织来源干细胞向成骨细胞分化.结果 转染后脂肪组织来源干细胞细胞数目增长一倍.在特定诱导条件下,可向成脂细胞、成骨细胞分化.Cbfα1重组的腺病毒载体对脂肪组织来源干细胞的转染效率为82.4%.转染后第1、3、7天脂肪组织来源干细胞中骨钙素、骨桥素、Ⅰ型胶原的表达增强;脂蛋白脂酶的表达逐渐下降.转染Cbfα1后,脂肪组织来源干细胞中碱性磷酸酶活性显著增加.结论 核心结合因子过表达促进脂肪组织来源干细胞向成骨细胞分化、抑制成脂分化.     

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.     

DKK1在大鼠骨髓间充质干细胞成骨、成脂分化早期的差异性表达

目的：分析DKK1在大鼠骨髓间充质干细胞成脂、成骨分化早期的表达,探讨其调节机制。方法 Real－time PCR检测骨髓间充质干细胞在成脂、成骨诱导培养早期DKK1基因表达的水平;检测激素处理骨髓间充质干细胞DKK1基因的表达水平。结果成脂组与正常对照组,在成脂诱导6、12、24、48时DKK1基因表达水平较对照组增高,差异有统计学意义, P＜0．01。成骨组与正常对照组,0．5、6两个时间点DKK1基因表达量较对照组明显降低,差异有统计学意义,P＜0．05。骨髓间充质干细胞激素处理3、6、12、24时检测,结果显示激素作用下,骨髓间充质干细胞DKK1表达明显增加,差异有统计学意义,P＜0．05。结论 DKK1在骨髓间充质干细胞成脂、成骨分化早期可能起重要的调节作用,激素可能通过调节DKK1的表达而发挥调节成脂、成骨分化的作用。     

脂肪间充质干细胞的基本生物学特性及向成骨细胞诱导分化的实验研究

目的　研究脂肪间充质干细胞的基本生物学特性以及在特定培养条件下向成骨细胞分化 ,探讨其作为骨组织工程的种子细胞的可行性。方法　取 3周龄Lewis大鼠的腹股沟脂肪垫 ,消化法获得脂肪间充质干细胞 ,分别用脂肪诱导培养基和成骨诱导培养基诱导其向脂肪细胞与成骨细胞分化 ,组织化学染色、免疫细胞化学染色和Westernblot检测细胞分化的情况。结果　从成体大鼠脂肪组织中培养出脂肪间充质干细胞 ,原代脂肪间充质干细胞能自发分化为脂肪细胞 ,传代细胞在胰岛素和呋塞米的作用下生成脂滴 ,过氧化物酶体增殖物激活受体 (PPAR)γ表达增强 ,向脂肪细胞分化 ;在呋塞米、抗坏血酸、β 甘油磷酸钠的诱导下 ,脂肪间充质干细胞的碱性磷酸酶(ALP)活性检测显示诱导组与对照组差异有显著性 (P <0 .0 1) ,vonKossa染色出现钙结节 ,骨桥蛋白 (OPN)、骨形态发生蛋白 (BMP) 2免疫细胞化学染色阳性 ,Westernblot检测到诱导后细胞OPN、BMP2的表达。结论　从脂肪组织中可获得具有多分化潜能的间充质干细胞 ,经诱导后可分化为脂肪细胞和成骨细胞 ,有可能成为骨组织工程较理想的种子细胞之一     

不同年龄小鼠骨髓间充质干细胞中miR203的差异及 其对细胞增殖的调控

目的探索不同年龄骨髓间充质干细胞中miR203的表达变化,及其对骨髓间充质干细胞自我更新的作用机制。方法分别分离培养4周龄和18~24月龄的Balb/c小鼠BMSCs,对比不同年龄小鼠BMSCs增殖潜能的差异,并检测不同年龄小鼠BMSCs中miR-203的表达变化差异,从而探讨miR-203在骨髓间充质干细胞增殖调节中的作用机制。结果根据干细胞贴壁特性获得了稳定的骨髓间充质干细胞,其在分化诱导条件下可获得经茜素红染色呈红色结节及油红O染色显示有脂质沉淀,且成骨诱导后Ⅰ型胶原蛋白显著表达。在增殖条件下,与年轻BMSCs相比,老年BMSCs增殖(传代)能力明显下降。年轻小鼠(4周龄)BMSCs中miR-203远低于老年小鼠(18~24月龄)BMSCs中miR-203表达(P0.05)。结论年轻骨髓间充质干细胞增殖能力优于老年骨髓间充质干细胞,可能与miR-203表达较低有关。     

肿瘤相关间充质干细胞的研究进展

间充质干细胞普遍存在于组织间质中，对组织损伤修复、机体免疫调控等均发挥重要作用。近年研究发现，肿瘤组织中普遍存在间充质干细胞，称为肿瘤相关间充质干细胞，且与正常组织内的间充质干细胞在调控功能、基因表型等方面有所区别。可以说，肿瘤相关间充质干细胞是独立于正常间充质干细胞的特殊亚型。本文对肿瘤相关间充质干细胞的来源、转化、功能及其临床价值进行综述。     

Stem cells in burn eschar

This study compares mesenchymal cells isolated from excised burn wound eschar with adipose-derived stem cells (ASCs) and dermal fibroblasts in their ability to conform to the requirements for multipotent mesenchymal stem cells (MSCs). A population of multipotent stem cells in burn eschar could be an interesting resource for tissue engineering approaches to heal burn wounds. Cells from burn eschar, dermis, and adipose tissue were assessed for relevant CD marker profiles using flow cytometry and for their trilineage differentiation ability in adipogenic, osteogenic, and chondrogenic conditions. Although the different cell types did not differ significantly in their CD marker expression, the eschar-derived cells and ASCs readily differentiated into adipocytes, osteoblasts, and chondrocytes, while dermal fibroblasts only exhibited some chondrogenic potential. We conclude that the eschar-derived mesenchymal cells represent a population of multipotent stem cells. The origin of the cells from burn eschar remains unclear, but it is likely they represent a population of adult stem cells mobilized from other parts of the body in response to the burn injury. Their resemblance to ASCs could also be cause for speculation that in deep burns the subcutaneous adipose tissue might be an important stem cell source for the healing wound.     

Characterization and multilineage potential of cells derived from isolated microvascular fragments

Background

A number of therapies are being developed that use microvessels isolated from adipose tissue (microvascular fragments [MVFs]) to improve tissue perfusion and implant survival. Because it has been demonstrated that stem cells are associated with microvessels, the purpose of these studies was to gain further insight into the stem cells associated with MVFs to better understand their therapeutic potential.

Materials and methods

Cells derived from MVF explants were compared with adipose-derived stem cells (ASCs) based on the expression of cell surface proteins for mesenchymal stem cells and their capacity for angiogenic, neurogenic, adipogenic, and osteogenic differentiation.

Results

The expression of cell surface proteins for mesenchymal stem cell markers was similar between MVF-derived cells and ASCs; however, the increase in markers consistent with endothelial cells and pericytes was accompanied by an improved ability to form capillary-like networks when cultured on matrigel. MVF-derived cells had increased neuregulin, leptin, and osteopontin expression compared with ASCs when exposed to neurogenic, adipogenic, and osteogenic induction media, respectively.

Conclusions

The stem cell functionality of cells derived from MVFs is retained after their isolation. This helps to explain the ability of MVFs to improve tissue perfusion and has implications for the use of MVFs as a means to deliver stem cells within their niche.     

Characterization of two distinct lipomas: a comparative analysis from surgical perspective

Background: Lipomas are common benign soft tissue tumors that are well-circumscribed and encapsulated. However, adipose masses that are not demarcated from the surrounding fat are often encountered. Two distinct types of lipomas were analyzed from surgical perspective.

Methods: Thirty patients were enrolled after lipoma excision and diagnosed with either encapsulated (n?=?20) or non-encapsulated lipoma (n?=?10). Comparison of clinical variables, histologic analyses and characterization of the lipoma adipose-derived stem cells (ASCs) between the two lipomas were performed.

Results: Non-encapsulated lipomas were associated with older age at operation, larger tumor and increased seroma formation. The density of lymphatic vessels and gene expressions related to lymphatic vessel, inflammation and proliferation were increased in non-encapsulated lipoma. ASCs of non-encapsulated lipoma showed enhanced proliferation when cultured with serum.

Conclusions: Non-encapsulated lipomas and their ASCs showed distinct lymphatic histology and cellular response. These findings elucidated the pathogenesis and pathophysiology of lipomas.     

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.     

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.     

Autologous lung-derived mesenchymal stem cell transplantation in experimental emphysema

Autologous lung-derived mesenchymal stem cells (LMSCs) were transplanted endoscopically into sheep with experimental emphysema to assess their capacity to regenerate functional tissue. LMSC lines were derived from transbronchial biopsies, cloned at passage 2, expanded in culture, and labeled. A delivery scaffold containing 1% fibrinogen, 20 μg/ml of fibronectin, and 20 μg/ml of poly-L-lysine was used to promote cell attachment and spreading. Treatment animals received scaffold containing 5-10 × 10(6) cells/site; control animals received scaffold alone. Phenotypic markers, differentiation capacity, extracellular matrix protein expression, and paracrine function of LMSCs were characterized in vitro. Responses to LMSC transplantation in vivo were assessed in terms of clinical toxicity, lung physiology, change in tissue mass (measured by CT scanning) and perfusion (measured by scintigraphy scanning), and tissue histology. At 4-week follow-up, transplants were well tolerated and associated with increased tissue mass and lung perfusion compared to control treatment. Histology confirmed cell retention, increased cellularity, and increased extracellular matrix content following LMSC treatment. Labeled cells were distributed in the alveolar septum and peribronchiolar interstitium. Some label was also present within phagocytes, indicating that a fraction of autologous LMSCs do not survive transplantation. These results suggest that endobronchial delivery of autologous LMSCs has potential therapeutic utility for regenerating functional lung in emphysema.