人脂肪干细胞分离培养鉴定及多向分化潜能的实验研究

目的探讨在不同诱导条件下人脂肪干细胞（ADSC）的体外多向分化情况。方法取人脂肪组织,用酶消化法分离、培养ADSC,用免疫细胞化学法鉴定ADSC。体外培养并测定第二、五、七代细胞增殖速率,绘制细胞生长曲线,计算群体倍增时间。流式细胞仪检测抗原CD44、CD49d、CD34,分析酶消化法获得的细胞分别向表皮细胞、成骨细胞、脂肪细胞定向诱导的能力,明确其分化能力。结果第二、五、七代细胞生长稳定,群体倍增时间约60h。10代以内的脂肪干细胞随着传代次数的增加其增殖能力未现明显降低的趋势。免疫细胞化学显示,干细胞相关抗原CD44、CD49d为阳性。但与造血系统相关抗原CD34阴性。成表皮诱导组示：免疫组织化学鉴定结构显示有CK19的表达。成骨诱导组示：细胞碱性磷酸酶染色阳性。成脂肪细胞诱导组示：油红0染色胞质内脂滴均被染成红色,证实为脂性液体。结论脂肪干细胞体外增殖能力强,来源充足,细胞获得方便,又避免了伦理问题且具有向其他多种细胞分化的潜能,可能成为理想的组织工程种子细胞。     

人脂肪间充质干细胞体外向黑素细胞分化的研究

【摘要】 目的 建立体外诱导人脂肪间充质干细胞（hADSC）向黑素细胞分化的方法。 方法 由人皮下脂肪分离培养获得hADSC,流式细胞仪检测细胞表型,成骨成脂分化证明分化潜能。将干细胞生长因子（SCF）、内皮素3 （EDN-3）、碱性成纤维细胞生长因子（bFGF）等加到条件培养基中,促使第5代hADSC向黑素细胞方向诱导,诱导至第10周。未诱导组为正常对照组。使用实时荧光定量PCR检测黑素相关基因小眼畸形转录因子（MITF）,酪氨酸激酶受体c-Kit（KIT）,多巴色素异构酶（DCT）,酪氨酸酶（TYR）,酪氨酸酶相关蛋白1（TYRP1）,性别决定区域相关转录因子10（SOX10） mRNA的表达量,统计学分析采用单因素方差分析及LSD-t检验。诱导结束后,通过免疫细胞化学法、细胞免疫荧光法进行鉴定。 结果 流式细胞仪显示分离培养获得的hADSC高表达CD29、CD44、CD73、CD90、CD105、CD166,阳性率均在95%以上;低表达或不表达CD31、CD34、CD45,人白细胞DR抗原（HLA-DR）。成骨成脂分化结果显示hADSC具有分化潜能。实时荧光定量PCR结果显示,诱导10周后,MITF、KIT、DCT、TYR、TYRP1、SOX10 mRNA的表达水平分别为0.325 ± 0.012,0.042 ± 0.006,0.046 ± 0.013,0.036 ± 0.005,0.041 ± 0.003,0.225 ± 0.014,与诱导0周组相比,均有上调（P < 0.05）。诱导结束后,免疫细胞化学MITF、HMB45染色阳性,细胞免疫荧光TYRP1、S100阳性表达。 结论 由人脂肪间充质干细胞诱导所得细胞具有一定的黑素细胞生物学特性。     

Differentiation potential of human embryonic mesenchymal stem cells for skin-related tissue

BACKGROUND: Mesenchymal stem cells (MSC) have the capacity to differentiate into cells of connective tissue lineages, including bone, fat, cartilage and muscle, but the differentiation of embryonic MSC into epidermal cells by mesenchymal-epithelial transition has not been confirmed. OBJECTIVES: To determine the biological characteristics of human embryonic MSC (hMSC) and their potential for differentiation into epithelial cells. METHODS: hMSC were derived from 4-7-week-old embryos; they were localized and isolated, then primary culture was done. The biological characteristics of hMSC were detected by immunohistochemical methods and flow cytometry. Their differentiation potential was determined by coculture with conditioning medium and in vivo injection. RESULTS: hMSC express the relative specific antigens of MSC, such as SH2, alpha-smooth actin, CD29, CD44, CD90 and S100. After stimulation and in vivo transplantation, hMSC possess the potential to differentiate into epidermal cells with the production of keratin 19 and E-cadherin. CONCLUSIONS: hMSC derived from the early human embryo have the ability to transform into epidermal cells in vivo and in vitro.     

Beneficial effects of a skin tolerance-tested moisturizing cream on the barrier function in experimentally-elicited irritant and allergic contact dermatitis

In experimentally-induced irritant (ICD) and allergic (ACD) contact dermatitis, an oil-in-water (o/w) cream was applied to investigate its effects on a disturbed barrier function compared to untreated physiological barrier repair. Transepidermal water loss (TEWL) measurements were performed. Before the start of the experiments, the skin tolerance of the cream was examined, revealing the non-irritating characteristics of the ingredients and the absence of any contact allergic patch test reaction. In the ICD study, sodium lauryl sulfate (SLS) patches were applied to the forearms of young female volunteers. Consequently, it was observed that repeated cream application (14 days, 2x/day) significantly improved the TEWL of SLS-damaged skin, leading to a complete recovery on day 15. In the ACD study, disruption of skin barrier function was obtained by a nickel-mediated contact allergy patch (CAP) test. The cream was then applied 2x/day for 4 consecutive days. Assessment of TEWL clearly showed that recovery of the disrupted skin significantly improved after cream application in comparison to untreated barrier repair.     

Immunophenotyping of the human bulge region: the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche

Abstract:  Since the discovery of epithelial hair follicle stem cells (eHFSCs) in the bulge of human hair follicles (HFs) an important quest has started: to define useful markers. In the current study, we contribute to this by critically evaluating corresponding published immunoreactivity (IR) patterns, and by attempting to identify markers for the in situ identification of human eHFSCs and their niche. For this, human scalp skin cryosections of at least five different individuals were examined, employing standard immunohistology as well as increased sensitivity methods. Defined reference areas were compared by quantitative immunohistochemistry for the relative intensity of their specific IR.
According to our experience, the most useful positive markers for human bulge cells turned out to be cytokeratin 15, cytokeratin 19 and CD200, but were not exclusive, while β1 integrin and Lhx2 IR were not upregulated by human bulge keratinocytes. Absent IR for CD34, connexin43 and nestin on human bulge cells may be exploited as negative markers. α6 integrin, fibronectin, nidogen, fibrillin-1 and latent transforming growth factor (TGF)-beta-binding protein-1 were expressed throughout the connective tissue sheath of human HFs. On the other hand, tenascin-C was upregulated in the bulge and may thus constitute a component of the bulge stem cell niche of human HFs.
These immunophenotyping results shed further light on the in situ expression patterns of claimed follicular 'stem cell markers' and suggest that not a single marker alone but only the use of a limited corresponding panel of positive and negative markers may offer a reasonable and pragmatic compromise for identifying human bulge stem cells in situ .     

Improved method of differentiation,selection and amplification of human melanocytes from the hair follicle cell pool

Hair root harbours a complex cell pool with an immense developmental potential. Several lineages, including skin, can be differentiated from the multipotent to pluripotent cells of outer root sheath (ORS) of hair follicle. Outer root sheath presents the most opulent non‐invasively gained adult stem cell source known. For the purposes of cultivating melanocytes designated for graft‐based treatments of depigmentation disorders, we have established an ex vivo/in vitro cultivation method by introducing several methodological improvements to the ORS explant method of Dieckmann. As a result, we gained a higher, purer yield of differentiated melanocytes in half the time (at least 10⁶ of 95% pure cells in 4 weeks). This reliable cultivation procedure begins with the epilation of 60 hairs and yields high numbers of ORS melanocytes that could be used for grafting applications. The procedure not only utilises the developmental potential of hair root cell pool and favors differentiation into melanocytes, but also contributes to the general trend of minimal‐to‐non‐invasive strategies for regenerative medicine.     

人真皮间充质干细胞对白癜风患者皮损周围CD8+ T淋巴细胞表达和分泌白介素13的影响

【摘要】 目的 探讨人真皮间充质干细胞（DMSC）对白癜风患者皮损周围CD8+ T淋巴细胞分泌表达白介素（IL）13的影响。 方法 细胞增殖检测法（MTS）检测重组IL-13（rIL-13）对黑素细胞增殖的影响。逆转录聚合酶链反应（RT-PCR）和Western印迹法分别检测6例进展期寻常型白癜风患者皮损周围及外周血中CD8+ T淋巴细胞中IL-13基因和蛋白表达。实时荧光定量反转录聚合酶链反应（qRT-PCR）技术和酶联免疫吸附试验（ELISA）分别检测DMSC与白癜风患者皮损周围CD8+ T淋巴细胞共培养前后细胞IL-13 mRNA水平和上清蛋白水平。 结果 不同浓度（10、50、100、250、500 μg/L）的rIL-13作用黑素细胞24、48、72、96 h后黑素细胞的增殖与对照组比较均未见明显变化（均P > 0.05）。白癜风患者皮损周围及外周血中CD8+ T淋巴细胞均可表达IL-13,但皮损周围CD8+ T淋巴细胞表达IL-13更显著。将皮损周围CD8+ T淋巴细胞和DMSC共培养,CD8+ T淋巴细胞IL-13 mRNA（0.100 0 ± 0.002 4）和蛋白［（1 509.62 ± 48.44） ng/L］的表达水平均显著低于单独培养的CD8+ T淋巴细胞［mRNA：0.383 2 ± 0.018 7,蛋白：（5 507.98 ± 34.11） ng/L,均P < 0.05］。 结论 白癜风患者皮损周围CD8+ T淋巴细胞高表达IL-13,DMSC能够有效地抑制其表达IL-13,或许可作为白癜风的治疗靶点之一。