人脂肪源性间充质干细胞向内耳毛细胞定向诱导分化的实验研究

目的 验证在体外将人脂肪源性间充质干细胞(human adipose-derived mesenchymalstem cells,hAD-MSC)定向分化为内耳毛细胞的可行性.方法 用特定的培养体系配合多种细胞因子定向诱导hAD-MSC向神经干/祖细胞样细胞分化,而后进一步将诱导后细胞与发育期鸡胚听泡细胞在体外进行共培养,以促使其向内耳毛细胞分化,通过免疫组化等方法对分化不同阶段的细胞特异性指标进行鉴定.结果 hAD-MSC诱导后呈现神经干/祖细胞样的形态并表达其特异性标志,与发育期鸡胚听泡细胞共培养后表达内耳毛细胞特异性标志.结论 hAD-MSC在体外可定向诱导分化为具有内耳毛细胞特异性标志的毛细胞样细胞.     

人骨髓基质干细胞体外诱导和鉴定

目的建立人自体骨髓基质干细胞(human menchymal stem cells,hMSCs)体外分离、鉴定体系,建立经诱导表达成骨、软骨、脂肪细胞表型的体外培养体系,探讨作为骨组织工程种子细胞的可能性,为组织工程技术应用打下基础。方法抽取患者骨髓5ml,以密度梯度法分离hMSCs,使用流式细胞仪鉴定细胞表型;应用免疫组化和分子生物学技术,对hMSCs进行成骨、软骨、脂肪细胞的诱导培养和表型鉴定。结果第2代hMSCs阳性细胞表达率CD105(78±6)%,CD166(43±7)%,CD29(69±12)%;hMSCs成骨诱导培养第3代平均扩增(163.4±13.4)倍,形成钙结节,骨细胞转录因子、骨钙素、骨桥蛋白和I型胶原免疫荧光阳性,RT-PCR证实有I型胶原、骨钙素、骨桥蛋白和骨结合素mRNA表达,对照组阴性;成软骨细胞诱导培养后II型胶原、SOX9(SRY-type HMGbox9,是在哺乳动物性别决定和软骨生成中起着关键调控作用的一个基因)。免疫荧光阳性,RT-PCR证实II型胶原、软骨聚集蛋白聚糖mRNA表达,对照组阴性;成脂肪细胞诱导培养后,油红-O染色阳性,RT-PCR证实PPAR2 mRNA...     

Regeneration of the vocal fold using autologous mesenchymal stem cells

The aim of this study was to regenerate the injured vocal fold by means of selective cultured autologous mesenchymal stem cells (MSCs). Eight adult beagle dogs were used for this experiment. Selective incubation of MSCs from bone marrow was done. These MSCs were submitted to 3-dimensional incubation in 1% hydrochloric acid atelocollagen. Three-dimensional incubated MSCs were injected into the left vocal fold, and atelocollagen only was injected into the right vocal fold of the same dog as a control. Four days after injection, the posterior parts of the vocal folds were incised. The regeneration of the vocal fold was estimated by morphological and histologic evaluations. Our results showed that 3-dimensional incubated MSCs were useful in the regeneration of the injured vocal fold. This study shows that damaged tissues such as an injured vocal fold would be able to be regenerated by tissue engineering.     

自体脂肪间充质干细胞移植促进声带修复再生的实验研究

目的 探讨自体脂肪间充质干细胞移植入损伤声带后的生长分布及对声带固有层及其主要细胞外基质变化的影响特点.方法 对34只实验用兔(68侧声带)的53侧声带进行全麻支撑喉镜下声带锐性损伤.分离培养、鉴定及定向诱导20只实验用兔脂肪间充质干细胞.将自体脂肪间充质干细胞植入20侧损伤声带后,示踪观察脂肪间充质干细胞在声带固有层内的生长分布;同时以单纯支架(胶原或透明质酸凝胶)植入18侧受损声带(胶原10侧,透明质酸8侧)及单纯损伤15侧声带作为对照组.术后15 d～12个月时采用HE染色、Masson染色、Alcian Blue染色及免疫组化染色观察声带形态学变化及声带固有层胶原、透明质酸、纤维连接蛋白的含量及分布变化.结果 脂肪间充质干细胞呈梭形贴壁生长,具有多向分化潜能,植入损伤兔声带后持续分布于声带固有层.损伤声带在脂肪间充质于细胞植入6个月时形态接近正常,12个月时部分组织学结构类似正常;声带细胞外基质中的胶原于植入3个月内含量有增高趋势,无序分布于固有层,后逐渐减少至正常水平附近,12个月时部分分布略显不规则;透明质酸于植入40 d含量亦有增高趋势,分布于固有层各层,6个月含量减至正常水平附近,局限于固有层浅中层;纤维连接蛋白于植入后始终在固有层内散在分布,含量在40 d内有增高趋势,后逐渐减少,12个月时含量接近正常.单纯声带损伤3个月后局部开始出现瘢痕挛缩,以胶原纤维为主的大量纤维组织增生,12个月时仍紊乱分布于声带固有层各层.单纯支架植入组变化介于二者之间.结论 脂肪间充质干细胞植入兔损伤声带后具有促进声带细胞外基质分泌、合理分布及部分有序化排列的功能,具有促进声带修复再生的作用.     

脂肪来源的间充质干细胞治疗变应性鼻炎的研究进展

变应性鼻炎(AR)是机体经变应原诱发产生的常见良性变态反应性疾病。除了与过敏相关的鼻部和眼部症状外,还会导致睡眠质量下降,白天生活质量降低等问题,影响患者的工作和生活。目前AR的治疗方法是以远离变应原及药物控制为主,且在疗效上有其局限性和弊端。干细胞疗法的应用在各疾病的治疗中正在得到极大地发展。脂肪来源的间充质干细胞(MSCs)因其独特的免疫调节及抗炎等作用以及获取方便等优势,在AR的治疗上具有相当的潜力。本文就脂肪来源MSCs治疗AR的实验研究进展作一综述。     

Nasal augmentation using injectable alginate and mesenchymal stem cells in the rabbit

BACKGROUND: The goal of this study was to evaluate the use of the autogenous mesenchymal stem cells (MSCs) impregnated in an injectable alginate gel containing platelet-rich plasma (PRP) for nasal augmentation in rabbit model. METHODS: Bone marrow-derived MSCs were isolated and expanded from New Zealand white rabbits. At confluence, the cells were mixed with sodium alginate solution. PRP was prepared from the rabbits and it was immediately mixed into the alginate-cell mixture. The cell-PRP-alginate mix was injected into a subcutaneous nasal area. Eight weeks after injection changes in facial contour, newly formed nasal hump was analyzed and the amount of chondroitin sulfate in tissue was measured. RESULTS: Augmented nasal dorsa maintained their original shape until harvest. Immunohistochemical staining revealed that the deposited matrix was composed of type II collagen and that it was distributed abundantly and widely in the connective tissue of the tissue generated. The amount of chondroitin sulfate (main component of the proteoglycan in cartilage) produced was significantly higher when MSCs and PRP-alginate were used. CONCLUSION: Injectable PRP-alginate gel containing autologous mesenchymal stem cells may offer a useful means of facial soft tissue augmentation.