【摘要】：目的：1.體外傳代培養(yǎng)，觀察人臍帶間充質(zhì)干細(xì)胞(Human umbilical cordmesenchymal stem cells,hUC-MSCs)多向分化能力的改變，探索其體外誘導(dǎo)分化的最佳時(shí)間窗。2.尋求一種更佳的，體外無支架培養(yǎng)條件下誘導(dǎo)hUC-MSCs向組織工程軟骨細(xì)胞分化的方法。 方法： 1.體外傳代培養(yǎng)，觀察hUC-MSCs多向分化能力的改變——采用膠原酶消化法分離hUC-MSCs，胰酶消化傳代并培養(yǎng)。流式細(xì)胞術(shù)檢測不同代細(xì)胞表面標(biāo)志物與細(xì)胞周期；MTT法檢測不同代細(xì)胞增殖能力；對(duì)不同代細(xì)胞行成骨細(xì)胞、成軟骨細(xì)胞和成脂肪細(xì)胞誘導(dǎo)并鑒定；實(shí)時(shí)定量PCR檢測不同代細(xì)胞Oct-4、Sox-2、Nanog的mRNA表達(dá)水平。 2.hUC-MSCs向組織工程軟骨細(xì)胞分化的誘導(dǎo)——收集第3代hUC-MSCs，通過兩種無支架培養(yǎng)體系，體外誘導(dǎo)其向組織工程軟骨細(xì)胞分化。HE染色觀察離心管聚集成團(tuán)培養(yǎng)誘導(dǎo)后組織的形態(tài)；免疫組織化學(xué)法檢測離心管聚集成團(tuán)培養(yǎng)體系誘導(dǎo)后組織細(xì)胞II型膠原表達(dá)；免疫熒光法檢測平面誘導(dǎo)培養(yǎng)體系誘導(dǎo)后組織細(xì)胞II型膠原表達(dá)；阿利新藍(lán)及甲苯胺藍(lán)染色檢測兩種培養(yǎng)體系誘導(dǎo)后組織細(xì)胞糖胺聚糖(Glycosaminoglycans,GAG)的表達(dá)；羥脯氨酸法和阿新藍(lán)法分析兩種培養(yǎng)體系誘導(dǎo)前后Ⅱ型膠原與GAG的含量變化；實(shí)時(shí)定量PCR分析兩種培養(yǎng)體系誘導(dǎo)前后Sox-9、Ⅱ型膠原及GAG的mRNA表達(dá)水平。 結(jié)果： 1.體外傳代培養(yǎng)至23代，hUC-MSCs細(xì)胞表面標(biāo)志物表達(dá)率及細(xì)胞周期無顯著差異。細(xì)胞生長曲線相似，細(xì)胞增殖率無明顯下降。細(xì)胞均可被誘導(dǎo)成成骨細(xì)胞、軟骨細(xì)胞和脂肪細(xì)胞。不同代細(xì)胞Oct-4、Sox-2、Nanog的mRNA表達(dá)量無顯著差異。 2.HE染色結(jié)果顯示，，離心管聚集成團(tuán)培養(yǎng)誘導(dǎo)后組織具有軟骨組織特有形態(tài)。免疫組化、免疫熒光及阿利新藍(lán)染色、甲苯胺藍(lán)染色結(jié)果顯示，兩種培養(yǎng)體系誘導(dǎo)后組織細(xì)胞均分別表達(dá)Ⅱ型膠原與GAG；Ⅱ型膠原與GAG定量檢測(羥脯氨酸法及阿利新藍(lán)法)結(jié)果顯示，兩種培養(yǎng)體系的實(shí)驗(yàn)組較各自對(duì)照組Ⅱ型膠原與GAG表達(dá)量高，離心管聚集成團(tuán)培養(yǎng)實(shí)驗(yàn)組的表達(dá)量顯著高于平面誘導(dǎo)培養(yǎng)實(shí)驗(yàn)組；實(shí)時(shí)定量PCR結(jié)果顯示，誘導(dǎo)后細(xì)胞較誘導(dǎo)前均高表達(dá)GAG、Ⅱ型膠原和Sox-9的mRNA；離心管聚集成團(tuán)誘導(dǎo)培養(yǎng)與平面誘導(dǎo)培養(yǎng)比較，其GAG和Ⅱ型膠原的mRNA表達(dá)量更高，有顯著性差異。 結(jié)論： 1.體外培養(yǎng)至23代，隨著傳代次數(shù)的增加，hUC-MSCs多向分化能力未受影響。 2.離心管聚集成團(tuán)培養(yǎng)可顯著提高誘導(dǎo)后組織Ⅱ型膠原及GAG的表達(dá)水平，其更有利于hUC-MSCs向軟骨細(xì)胞分化。
[Abstract]:Objective: 1. The multidirectional differentiation of human umbilical cord mesenchymal stem cells (Human umbilical cordmesenchymal stem cells,hUC-MSCs) was observed in vitro, and the optimal time window of differentiation was explored. 2. To find a better way to induce hUC-MSCs to differentiate into tissue engineering chondrocytes in vitro without scaffold culture. Methods: 1. The ability of multidirectional differentiation of hUC-MSCs was observed by in vitro subculture. HUC-MSCs, trypsin was digested and cultured by collagenase digestion. Flow cytometry was used to detect cell surface markers and cell cycle; MTT assay was used to detect the proliferation of different generation cells; osteoblasts, chondroblasts and adipocytes were induced and identified by MTT assay. The mRNA expression of Oct-4,Sox-2,Nanog in different generations of cells was detected by real time quantitative PCR. Induction of 2.hUC-MSCs differentiation into tissue engineered chondrocytes: the third generation of hUC-MSCs, was collected through two scaffolding free culture systems. The cells were induced to differentiate into tissue engineering chondrocytes in vitro. The morphology of the cells was observed by HE staining. Immunohistochemical method was used to detect the expression of II type collagen in tissue cells induced by colony culture system of centrifuge tube, and immunofluorescence method was used to detect the expression of II type collagen in tissue cells induced by plane-induced culture system. The expression of glycosaminoglycan (Glycosaminoglycans,GAG) was detected by alimine blue and toluidine blue staining, and the changes of type 鈪

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