本文選題：間充質(zhì)干細(xì)胞 + 人臍帶　； 參考：《中國(guó)輸血雜志》2017年08期

【摘要】：目的建立從人臍帶Wharton's jelly組織中直接培養(yǎng)間充質(zhì)干細(xì)胞(MSCs)的方法。方法將人臍帶切成5 cm左右的片段,縱向剖開,剔除血管,把富含Wharton's jelly組織的臍帶面置于10 cm2塑料培養(yǎng)皿的培養(yǎng)面上,加入含20%FBS的LG-DMEM培養(yǎng)基連續(xù)培養(yǎng)10 d;移去臍帶組織后,培養(yǎng)皿中的細(xì)胞繼續(xù)培養(yǎng)至80%-90%融合。傳代和擴(kuò)增3代(次)后,以倒置光學(xué)顯微鏡觀察細(xì)胞形態(tài),流式細(xì)胞儀檢測(cè)細(xì)胞免疫表型及細(xì)胞周期,用含有成骨細(xì)胞誘導(dǎo)劑、脂肪細(xì)胞誘導(dǎo)劑對(duì)傳代培養(yǎng)至第3代的細(xì)胞分別定向誘導(dǎo)分化為成骨細(xì)胞、脂肪細(xì)胞,并對(duì)誘導(dǎo)后細(xì)胞用堿性磷酸酶檢測(cè)試劑、Von-Kossa染色檢測(cè)試劑和茜蘇紅染色試劑作細(xì)胞生物學(xué)檢測(cè)。結(jié)果臍帶組織在貼壁培養(yǎng)5 d,組織周圍可見有少量細(xì)胞貼壁生長(zhǎng),主要呈"成纖維樣",形狀不規(guī)則,移去臍帶組織后繼續(xù)培養(yǎng)至14-15 d時(shí),細(xì)胞達(dá)到80%-90%匯合;細(xì)胞表面表達(dá)CD73、CD105、CD90、CD44、CD29、CD71及CD13,不表達(dá)CD34、CD14、CD133、CD45、HLA-DR;培養(yǎng)至第4代時(shí)約72.724%的細(xì)胞處在G1期,S期細(xì)胞占18.069%,第6代時(shí),G1期細(xì)胞約為83.875%、S期細(xì)胞僅為9.606%左右。細(xì)胞經(jīng)成骨細(xì)胞誘導(dǎo)分化后,堿性磷酸酶染色顯示呈強(qiáng)陽(yáng)性,茜蘇紅染色和Von-Kossa染色顯示細(xì)胞有鈣鹽沉積并形成鈣結(jié)節(jié);細(xì)胞經(jīng)成脂細(xì)胞誘導(dǎo)分化后,油紅O染色呈紅色,顯示胞漿內(nèi)有大量甘油三酯的聚集。結(jié)論采用非酶解法從人臍帶Wharton's jelly中分離及培養(yǎng)擴(kuò)增的細(xì)胞具備MSCs的基本特性,具有分化為成骨細(xì)胞和脂肪細(xì)胞的能力。
[Abstract]:Objective to establish a direct culture method of mesenchymal stem cells (MSCs) from human umbilical cord Wharton's jelly tissue. Methods the human umbilical cord was cut into about 5 cm segment, cut out longitudinally, removed the blood vessels, placed the cord surface rich in Wharton's jelly tissue on the culture surface of 10 cm2 plastic petri dish, added 20s LG-DMEM medium for 10 days, and removed the umbilical cord tissue for 10 days. The cells in the dish continued to grow to 80-90% fusion. Cell morphology was observed by inverted optical microscope, immunophenotype and cell cycle were detected by flow cytometry and osteoblast inducer was used. Adipocytes were induced to differentiate into osteoblasts and adipocytes respectively. After induction, the cells were detected by Von-Kossa staining reagent and hematoxylin staining reagent by alkaline phosphatase assay. Results after 5 days of culture, there were a few cells around the cord tissue, mainly "fibroblast" and irregular shape. When the umbilical cord tissue was removed and cultured for 14-15 days, the cells converged to 80-90%. The cell surface expressed CD73T, CD105CD90, CD44, CD29, CD71 and CD13, and did not express CD34, CD14, CD133, CD45 and HLA-DR.At the fourth passage, about 72.724% of the cells were in the G 1 phase of S phase, and the number of cells in the G 1 phase was about 9.606% in the sixth generation. After differentiation induced by osteoblasts, alkaline phosphatase staining showed strong positive, hematoxylin staining and Von-Kossa staining showed that the cells had calcium salt deposition and formed calcium nodules, oil red O staining was red after differentiation induced by adipogenic cells. There is a large accumulation of triglycerides in the cytoplasm. Conclusion the cells isolated and cultured from human umbilical cord Wharton's jelly by non-enzymatic hydrolysis have the basic characteristics of MSCs and the ability to differentiate into osteoblasts and adipocytes.
【作者單位】： 安徽省血液中心;解放軍第105醫(yī)院;Global
【基金】：合肥市衛(wèi)生計(jì)生委2016年應(yīng)用醫(yī)學(xué)研究項(xiàng)目(hwk2016zc020)
【分類號(hào)】：R329.2

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李茜,韓俊領(lǐng),句鳳華,孫樂靜,董舒文,閆鳳英,邱錄貴;天津市臍帶血庫(kù)質(zhì)量管理體系的建立和實(shí)施[J];生物醫(yī)學(xué)工程與臨床;2004年02期

2 馬旭東;臍帶血干細(xì)胞:嬰兒帶給人類的厚禮[J];國(guó)外科技動(dòng)態(tài);2001年06期

3 馬麗萍,李偉,李玉林,張麗紅,邵明玉;臍帶血中血管內(nèi)皮前期細(xì)胞的分離[J];吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2004年01期

4 孫成文，，韓穎;臍帶血和成人外周血淋巴細(xì)胞免疫活性的區(qū)別[J];上海免疫學(xué)雜志;1995年02期

5 王有國(guó);可代替骨髓移植的臍帶血──臍帶血自述[J];醫(yī)藥與保健;1998年03期

6 李玉明;臍帶血的基礎(chǔ)研究及臨床應(yīng)用[J];中國(guó)實(shí)用內(nèi)科雜志;1998年06期

7 黃友章,羅暉,楊平地,沈建良;臍帶血清在骨髓造血祖細(xì)胞培養(yǎng)中的效應(yīng)[J];中國(guó)應(yīng)用生理學(xué)雜志;2000年01期

8 張秀榮,張京秀,劉復(fù)強(qiáng);臍帶血免疫球蛋白、補(bǔ)體及補(bǔ)體復(fù)合物的研究[J];實(shí)用婦產(chǎn)科雜志;1997年04期

9 江淑芳,史春夢(mèng);臍帶血免疫學(xué)特性的研究進(jìn)展[J];免疫學(xué)雜志;2002年S1期

10 陳光明;王海鷹;張紹志;方立英;韓曉紅;;臍帶血干細(xì)胞玻璃化保存研究[J];浙江大學(xué)學(xué)報(bào)(工學(xué)版);2007年05期

相關(guān)會(huì)議論文 前4條

1 韓俊領(lǐng);趙英新;武文杰;伍盛舉;楊俊曄;邱錄貴;;臍帶血造血干細(xì)胞的分離和冷凍保存[A];第九屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要匯編[C];2003年

2 劉[?;范華驊;高躒;聶曉絢;楊懿銘;陸華中;高峰;;臍帶血與外周血NKT細(xì)胞的體外擴(kuò)增及其表型比較[A];第10屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要匯編[C];2005年

3 張彥;武大林;邢獻(xiàn)志;;臍帶血造血干細(xì)胞在4℃、-80℃、-196℃保存技術(shù)比較分析[A];第九屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要匯編[C];2003年

4 孫漢英;劉文勵(lì);周劍鋒;譚細(xì)友;孫嵐;孟凡凱;吳寧;劉丹;;控制臍帶血造血干/祖細(xì)胞增殖的關(guān)鍵細(xì)胞周期蛋白D亞型的確定[A];第九屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要匯編[C];2003年

相關(guān)重要報(bào)紙文章 前2條

1 趙焱邋陳威華;科學(xué)家在人類臍帶上發(fā)現(xiàn)新干細(xì)胞源[N];人民日?qǐng)?bào);2007年

2 新華;干細(xì)胞與造血干細(xì)胞[N];光明日?qǐng)?bào);2001年

相關(guān)博士學(xué)位論文 前2條

1 李云濤;臍帶血多能成體祖細(xì)胞生物學(xué)特性及分離相關(guān)影響因素的研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2006年

2 旭日;異基因臍帶血移植后急性GVHD的預(yù)測(cè)研究[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2004年

本文編號(hào)：1935081