【摘要】：目的培養(yǎng)人臍帶間充質(zhì)干細(xì)胞并分析細(xì)胞的生物安全性，從而為臨床應(yīng)用提供依據(jù)。方法剝除新鮮臍帶血管，取華通式膠，采用組織貼壁法培養(yǎng)細(xì)胞，將不同代數(shù)的細(xì)胞（P1～P9）采用形態(tài)學(xué)、表面標(biāo)志物測(cè)定、分化能力檢測(cè)及增殖能力等手段進(jìn)行鑒定，對(duì)P3～P6細(xì)胞進(jìn)行病原學(xué)、核型、急慢性毒性、過敏及致瘤性的研究，以明確臍帶間充質(zhì)干細(xì)胞的安全性。結(jié)果人臍帶間充質(zhì)干細(xì)胞呈梭型流水狀生長(zhǎng)，并可在體外穩(wěn)定擴(kuò)增，細(xì)胞增殖呈‘S’型曲線；流式檢測(cè)陽性標(biāo)志CD90、CD44、CD73表達(dá)量均在90%以上，陰性標(biāo)志表達(dá)量在P3～P6表達(dá)量較低；所培養(yǎng)細(xì)胞可以分化為成脂細(xì)胞、成骨細(xì)胞以及成軟骨細(xì)胞。通過對(duì)細(xì)胞病原學(xué)檢測(cè)，未發(fā)現(xiàn)細(xì)菌、真菌及其他病原微生物；采用染色體G顯帶法對(duì)P3～P6臍帶間充質(zhì)干細(xì)胞進(jìn)行染色核型分析，細(xì)胞在傳代過程中未發(fā)現(xiàn)染色體畸變；通過動(dòng)物實(shí)驗(yàn)觀察臍帶間充質(zhì)干細(xì)胞的安全性，注射人臍帶間充質(zhì)干細(xì)胞后未出現(xiàn)急性中毒，慢性毒性實(shí)驗(yàn)顯示各組織器官未出現(xiàn)病理改變，致瘤實(shí)驗(yàn)在注射部位未發(fā)現(xiàn)腫瘤細(xì)胞，過敏實(shí)驗(yàn)未發(fā)現(xiàn)實(shí)驗(yàn)動(dòng)物出現(xiàn)皮膚局部及全身的過敏反應(yīng)。結(jié)論人臍帶可以分離培養(yǎng)間充質(zhì)干細(xì)胞，間充質(zhì)干細(xì)胞可在體外穩(wěn)定擴(kuò)增培養(yǎng)，臍帶來源的間充質(zhì)干細(xì)胞具有生物安全性，可為今后臨床治療提供選擇。 目的探索經(jīng)鼻粘膜移植不同類型干細(xì)胞治療大鼠腦白質(zhì)損傷方法的可行性。方法培養(yǎng)鼠源少突膠質(zhì)前體細(xì)胞、鼠源骨髓間充質(zhì)干細(xì)胞、人臍帶間充質(zhì)干細(xì)胞。7日齡的SD大鼠制作腦白質(zhì)損傷模型，共40只，隨機(jī)分為四組，每組10只：（Ⅰ）鼠源少突膠質(zhì)前體細(xì)胞移植組、（Ⅱ）鼠源骨髓間充質(zhì)干細(xì)胞移植組、（Ⅲ）人臍帶間充質(zhì)干細(xì)胞移植組、（Ⅳ）對(duì)照組。10日齡模型大鼠經(jīng)鼻粘膜進(jìn)行細(xì)胞移植，移植前30min經(jīng)鼻黏膜滴入透明質(zhì)酸酶200U，細(xì)胞移植量5×105/20μl，于移植后24h、48h、72h以及7d取腦組織固定脫水，進(jìn)行冰凍切片，觀察細(xì)胞移植后的遷移情況。結(jié)果所培養(yǎng)的三種細(xì)胞生長(zhǎng)良好，大鼠腦白質(zhì)損傷模型患側(cè)MBP表達(dá)量明顯減少，，經(jīng)鼻粘膜細(xì)胞移植后，模型鼠損傷部位可以發(fā)現(xiàn)標(biāo)記的鼠源少突膠質(zhì)前體細(xì)胞和鼠源骨髓間充質(zhì)干細(xì)胞，并隨著時(shí)間的延長(zhǎng)遷移至損傷部位的細(xì)胞數(shù)量逐漸增多，損傷側(cè)未發(fā)現(xiàn)移植的人臍帶間充質(zhì)干細(xì)胞。結(jié)論鼠源少突膠質(zhì)前體細(xì)胞和鼠源骨髓間充質(zhì)干細(xì)胞具有向損傷部位遷移的能力。經(jīng)鼻粘膜細(xì)胞移植治療腦白質(zhì)損傷是一種簡(jiǎn)便、無創(chuàng)、可行的治療方法。
[Abstract]:Objective to culture human umbilical cord mesenchymal stem cells and analyze their biological safety. Methods the fresh umbilical cord blood tube was stripped, the Huatong glue was taken, and the cells were cultured by tissue adherent method. The cells of different generations (P1~P9) were identified by means of morphology, surface marker measurement, differentiation ability test and proliferation ability. The etiology, karyotype, acute and chronic toxicity, allergy and tumorigenicity of P3~P6 cells were studied to determine the safety of umbilical cord mesenchymal stem cells. Results the mesenchymal stem cells of human umbilical cord were spindle-like and could be expanded stably in vitro. The proliferation of human umbilical cord mesenchymal stem cells was'S' curve. The CD90,CD44,CD73 expression of positive markers was above 90%, and the expression of negative markers was lower in P3~P6. The cultured cells could differentiate into adipoblasts, osteoblasts and chondroblasts. No bacteria, fungi and other pathogenic microorganisms were found in the cell pathogeny, and chromosome aberration was not found in the passage of P3~P6 umbilical cord mesenchymal stem cells by chromosome G banding method. The safety of umbilical cord mesenchymal stem cells was observed by animal experiments. There was no acute poisoning after injection of human umbilical cord mesenchymal stem cells. Chronic toxicity test showed no pathological changes in tissues and organs. Tumor cells were not found in the injection site in tumorigenic test, and allergic reaction in skin and whole body was not found in allergic test. Conclusion Mesenchymal stem cells can be isolated from human umbilical cord and cultured stably in vitro. Mesenchymal stem cells derived from umbilical cord have biological safety and can provide a choice for clinical treatment in the future. Objective to explore the feasibility of different types of stem cells transplanted through nasal mucosa in the treatment of brain white matter injury in rats. Methods Rat oligodendrocyte precursor cells, mouse bone marrow mesenchymal stem cells and human umbilical cord mesenchymal stem cells were cultured. The white matter injury model was made in 7-day-old SD rats. 40 rats were randomly divided into four groups. There were 10 rats in each group: (I) rodent oligodendrocyte precursor cell transplantation group, (鈪

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