【摘要】：[目的]建立制備臍帶Wharton膠干細(xì)胞細(xì)胞外基質(zhì)(extracellular matrix,ECM)的方法并對(duì)其生物相容性進(jìn)行觀(guān)察。[方法]無(wú)菌條件下收集人臍帶組織,采用酶消化法分離培養(yǎng)臍帶干細(xì)胞,使用普通培養(yǎng)基培養(yǎng)7 d后,在原培養(yǎng)基中添加抗壞血酸,再連續(xù)培養(yǎng)8 d;加入含有0.5%Triton X-100和20 mM NH 4OH的PBS溶液進(jìn)行脫細(xì)胞處理后,用Hoechst 33258染色觀(guān)察其DNA殘留,采用掃描電鏡對(duì)其微結(jié)構(gòu)進(jìn)行觀(guān)察,通過(guò)天狼星紅、甲苯胺藍(lán)、番紅花O組織學(xué)染色和I型膠原、II型膠原、纖維粘連蛋白及層粘連蛋白免疫熒光染色觀(guān)察微載體的主要成分;將異體臍帶干細(xì)胞種植在無(wú)細(xì)胞核DNA殘留的ECM上,通過(guò)MTT法觀(guān)察臍帶干細(xì)胞的增殖生長(zhǎng)情況。[結(jié)果]通過(guò)本實(shí)驗(yàn)方法得到的天然脫細(xì)胞臍帶干細(xì)胞ECM,表面無(wú)細(xì)胞及DNA殘留,整體表面有細(xì)胞陷窩形成,基質(zhì)緊密連接。Hoechst 33258熒光染色陰性;天狼星紅、甲苯胺藍(lán)和番紅花O組織學(xué)染色陽(yáng)性;I型膠原、II型膠原、纖維粘連蛋白和層粘連蛋白免疫熒光染色陽(yáng)性;在MTT檢測(cè)細(xì)胞增殖中,種植于該ECM上的異體臍帶干細(xì)胞組的OD值高于單純平面培養(yǎng)組(P0.05)。[結(jié)論]此次實(shí)驗(yàn)所得的脫細(xì)胞臍帶干細(xì)胞外基質(zhì)具有良好的生物相容性,能夠?yàn)榻M織工程提供高質(zhì)量的種子細(xì)胞,有望成為組織工程種子細(xì)胞的新型培養(yǎng)載體。
[Abstract]:[objective] to establish a method for preparation of umbilical cord Wharton adhesive stem cell extracellular matrix (extracellular matrix,ECM) and to observe its biocompatibility. [methods] Human umbilical cord tissues were collected under aseptic condition. Umbilical cord stem cells were isolated and cultured by enzyme digestion. After 7 days of culture in normal culture medium, ascorbic acid was added to the original medium and cultured for 8 days. After acellular treatment with PBS solution containing 0.5%Triton X-100 and 20 mM NH 4OH, the DNA residue was observed by Hoechst 33258 staining, and its microstructure was observed by scanning electron microscope. The main components of microcarriers were observed by immunohistochemical staining of saffron O, type I collagen, II collagen, fibronectin and laminin. The allogeneic umbilical cord stem cells were implanted on ECM without nuclear DNA residue. The proliferation and growth of cord stem cells were observed by MTT method. [results] the ECM, surface of natural acellular umbilical cord stem cells obtained by this method was free of cells and DNA residues, cell lacunae were formed on the whole surface, matrix was tightly connected. Hoechst 33258 fluorescent staining was negative. Sirius red, toluidine blue and saffron O were positive for histological staining, type I collagen, II collagen, fibronectin and laminin were positive for immunofluorescence staining. In the MTT detection of cell proliferation, the OD value of cord allogeneic stem cells implanted on the ECM group was higher than that of the flat culture group (P0.05). [conclusion] the extracellular matrix of acellular umbilical cord stem cells obtained in this experiment has good biocompatibility and can provide high quality seed cells for tissue engineering, which is expected to be a new culture carrier for tissue engineering seed cells.
【作者單位】： 解放軍總醫(yī)院骨科;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(編號(hào):31240048) 全軍十二五重點(diǎn)項(xiàng)目(編號(hào):BWS11J025)
【分類(lèi)號(hào)】：R318.08

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本文編號(hào)：2325128