發(fā)布時(shí)間：2018-05-02 00:43

  本文選題：乙型肝炎 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《廣西醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的：通過體外培養(yǎng)人的骨髓問充質(zhì)干細(xì)胞(MSCs),評價(jià)其生物學(xué)特性及長期體外培養(yǎng)的安全性,為行MSCs移植治療終末期肝病的臨床應(yīng)用的質(zhì)量控制提供深入基礎(chǔ) 研究對象及方法：選擇中山大學(xué)附屬第三醫(yī)院感染科2009年10月——2010年4月行自體干細(xì)胞移植治療的乙肝肝硬化和肝衰竭患者的骨髓液作為培養(yǎng)對象,采用全骨髓貼壁培養(yǎng)的方法,用無血清培養(yǎng)基聯(lián)合成纖維生長因子(FGF)行患者的MSCs的體外培養(yǎng),觀察細(xì)胞形態(tài),采用流式細(xì)胞檢測儀行細(xì)胞表面分子鑒定,對細(xì)胞懸液行細(xì)菌、病毒、內(nèi)毒素及血清殘留檢測, T淋巴細(xì)胞抑制試驗(yàn)觀察其對T淋巴細(xì)胞增殖抑制率,軟瓊脂克隆實(shí)驗(yàn)觀察其致瘤性,染色體核型分析其遺傳穩(wěn)定性,并用培養(yǎng)過MSCs的條件培養(yǎng)基培養(yǎng)腫瘤細(xì)胞,觀察其對腫瘤細(xì)胞增殖的影響。 結(jié)果：患者M(jìn)SCs經(jīng)過體外培養(yǎng)傳代至第10代,染色體核型及表面分子表達(dá)穩(wěn)定,未見細(xì)菌、病毒污染,計(jì)算T淋巴細(xì)胞抑制率約為45%(P=0.00),無致瘤性,但在腫瘤細(xì)胞存在情況下,體外培養(yǎng)時(shí)可能通過直接作用或其分泌可溶性因子的間接作用促進(jìn)肝癌細(xì)胞的生長。 結(jié)論：患者的MSCs在無血清培養(yǎng)基中亦具有較高的增殖潛能,經(jīng)過體外傳代后可以大量擴(kuò)增,滿足移植細(xì)胞數(shù)量需求,長期傳代核型及細(xì)胞表型穩(wěn)定,無致瘤性,可以抑制T淋巴細(xì)胞,具有降低移植物抗宿主反應(yīng)的可能性,是作為細(xì)胞治療和基因治療的理想種子。但在體外與肝癌細(xì)胞共同培養(yǎng)時(shí)可能通過其分泌細(xì)胞因子的的營養(yǎng)作用促進(jìn)肝癌細(xì)胞的增殖,是否能將其作為移植物治療肝臟腫瘤引起的肝衰竭有待于進(jìn)一步深入的探討。
[Abstract]:Objective: to evaluate the biological characteristics and long-term safety of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro so as to provide a foundation for quality control of clinical application of MSCs transplantation in the treatment of end-stage liver diseases. Subjects and methods: bone marrow fluid of patients with hepatitis B cirrhosis and liver failure treated by autologous stem cell transplantation from October 2009 to April 2010 were selected as culturing objects in the third Hospital infection Department affiliated to Sun Yat-sen University. Whole bone marrow adherent culture was used to culture patients with MSCs in vitro using serum-free medium combined with synthetic fiber growth factor (FGFs). Cell morphology was observed, cell surface molecular identification was performed by flow cytometry, and bacteria were isolated from cell suspension. Virus, endotoxin, serum residues, T lymphocyte inhibition test, soft Agar clone assay were used to detect the tumorigenicity of T lymphocytes, and chromosome karyotype was used to analyze its genetic stability. Tumor cells were cultured on MSCs conditioned medium and their effects on tumor cell proliferation were observed. Results: the chromosome karyotype and surface molecular expression of MSCs were stable, no bacteria and virus contamination were observed. The inhibition rate of T lymphocytes was calculated to be 45P0. 00G, no tumorigenicity, but in the presence of tumor cells. In vitro culture may promote the growth of hepatoma cells by direct action or indirect effect of soluble factor secretion. Conclusion: the MSCs of the patients also has high proliferative potential in serum-free medium. After passage in vitro, it can be expanded in large quantities to meet the needs of the number of transplanted cells. The long-term subculture karyotype and cell phenotype are stable and non-tumorigenic. It can inhibit T lymphocytes, which has the possibility of reducing graft versus host reaction and is an ideal seed for cell therapy and gene therapy. However, in vitro co-culture with hepatoma cells may promote the proliferation of hepatoma cells through the nutrient effect of cytokines. Whether it can be used as graft in the treatment of liver failure caused by liver tumors needs further study.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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本文編號：1831690