【作者】 张强；

【导师】 傅跃先；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 硕士

【摘要】 目的:1.通过对兔脂肪干细胞的分离、培养及鉴定,获得纯化的兔脂肪干细胞(ADSCs);建立一种稳定、高效的兔ADSCs的培养方法。2.寻求一种BMP-9基因转染靶细胞ADSCs的稳定的实验方法;证实BMP-9基因转染后的ADSCs可向成骨细胞分化,以获得可用于骨组织工程的基因修饰的种子细胞。方法:1.ADSCs的分离、培养及鉴定:采用I型胶原酶消化法获取原代ADSCs。扩增和纯化ADSCs后,显微镜下观察兔ADSCs形态,MTT、细胞计数分析ADSCs的体外增殖情况,免疫组化检测ADSCs表面抗原CD29、CD44的表达。2.BMP-9转染ADSCs的实验:载BMP-9的腺病毒直接感染P3代的ADSCs。荧光显微镜观察转染效果,流式细胞仪测定转染率,RT-PCR检测转染后ADSCs内BMP-9 mRNA的表达。碱性磷酸酶活性、碱性磷酸酶和茜素红钙结节染色检测转染后ADSCs的成骨活性。结果:1.ADSCs的分离、培养及鉴定:(1)分离、培养的原代ADSCs细胞形态呈梭形或多角状,类似成纤维细胞,团簇状生长,在10 d左右达到90%融合。(2)经过传代、纯化的ADSCs呈成纤维细胞样,分布均匀、大小一致、细胞紧密排列,漩涡状生长,5 d左右达90%融合,显示出活跃的增殖能力。(3)生长曲线显示:1~3 d为传代培养的滞留期,4~6 d为对数增殖期,7~9 d为生长抑制期,此时细胞生长速度变慢。(4)累计倍增曲线显示:群体倍增时间大约为55 h,P3代细胞增殖速度最快,P8代以后的细胞增殖速度减慢,细胞出现衰老征象。(5)免疫组化染色显示:ADSCs细胞内表面抗原CD29、CD44均阳性表达。2.BMP-9转染ADSCs的实验:(1)BMP-9基因腺病毒载体可成功转染ADSCs。(2)腺病毒介导的BMP-9基因转染ADSCs后24 h即有荧光表达,并随时间延长荧光强度逐渐增强,转染3d转染效率达到80%以上。(3)转染后ADSCs停滞期略延长,数量轻度下降,倍增时间稍延长,但不影响细胞增殖。(4) RT-PCR检测结果显示,各转染组细胞内hBMP-9的mRNA持续阳性表达,未转染组未见阳性条带。(5)ADSCs转染后,ALP染色及茜素红钙结节染色为阳性,细胞内ALP活性呈增长趋势,转染组显著高于未转染组。结论:1.从兔脂肪组织中分离、培养、纯化的ADSCs具有明显的干细胞特征,体外生长旺盛,扩增迅速,长期传代仍能保持稳定的增殖能力,为BMP-9转染ADSCs诱导成骨的实验奠定了基础。2.腺病毒介导的BMP-9基因可以安全、有效地转染ADSCs,其转染的BMP-9基因可获得较高水平的表达,且具有明显的诱导ADSCs向成骨细胞转化的作用;而携带人全长BMP-9基因的腺病毒转染ADSCs可作为研究BMP-9诱导ADSCs成骨作用实验的可靠方法。更多还原

【Abstract】 Objective:1.To obtain purification ADSCs and set up a ADSCs cultural method stably and high efficiently.2.To explore an empirical method about ADSCs transfected with BMP-9, confirm ADSCs with BMP-9 can differentiate to the osteoblast and gain gene-modified seed cells which can be used for bone tissue engineering.Methods:1.Separation、cultivation and identification of ADSCs: Obtained primary ADSCs was with the I-collagenase digestion. Following amplification and purification of ADSCs, morphology was observed by microscope, proliferation in vitro was detected by MTT and cell count, and expression of surface antigen CD29 and CD44 was detected by immunohistochemistry.2.Transfection into ADSCs with BMP-9: adenovirus containing BMP-9 infected ADSCs in P3 period, effect of infection was observed by fluorescence microscope and infection efficiency was determined by flow cytometry. Expression of BMP-9 mRNA in ADSCs infected was detected by RT-PCR. Osteoblastic activity of ADSCs infected was detected by semi-quantity kit of alkaline phosphatase, alkaline phosphatase and alizarin red staining of calcium nodules.Results:1.Separation、cultivation and identification of ADSCs(:1)Morphology of primary ADSCs were spindle-shaped or angular, similar to fibroblasts, cluster-like growth. At the time of 10 days 90% cells were fusions.(2)ADSCs with passage and purification were fibroblast-like, distribution、size, consistent with cells close and swirl-like growth. At the time of 5 days 90% cells were fusions, which showing activity proliferation.(3)Growth curve showed: day 1 to 3 was lag phase of serial subcultivation, day 4 to 6 was logarithmic growth phase, and day 7 to 9 was growth inhibiting phase, in which cells growth slowed down.(4)Cumulative multiplication curve showed: population doubling time was about 55 hours, proliferation rate of cells in P3 period was fastest of all, and after P8 period, proliferation rate of cells slowed down and cells showed aging sign.(5)Immunohistochemistry showed: surface antigen CD29 and CD44 of ADSCs were both positive.2.Transfection into ADSCs with BMP-9:(1)Adenovirus containing BMP-9 could infect ADSCs successfully.(2)Fluorescent was observed at the time of 24h and the fluorescence intensity over time gradually after ADSCs infected by adenovirus containing BMP-9. After 3 days, infect rate could reach to more than 80%(.3)Lag phase of ADSCs with adenovirus was slightly extended, quantity reduced slightly and doubling time was slightly extended, which didn’t affect cell proliferation.(4)BMP-9 mRNA in cells with adenovirus was positive and the expression was sustained. There was no expression in cells without adenovirus.(5)Alkaline phosphatase and alizarin red staining of calcium nodules were positive in ADSCs infected. The activity of alkaline phosphatase was growing trend and infected group was significantly higher than non-infected.Conclusions:1.ADSCs, which were separated from adipose tissue of rabbit, cultivated and purified, had obvious characteristics of stem cells. The characteristics were high growth in vitro, amplified rapidly and maintained stable reproductive activity after passage of long-term. It will offer basis for ADSCs infected with BMP-9 osteoinduction further.2.Adenovirus containing BMP-9 could be infected ADSCs safely and efficiently. BMP-9 gene infected had higher expression and a significant role in osteogenesis. Therefore, ADSCs infected with adenovirus containing the length of human BMP-9 could be a reliable method to research on BMP-9 inducting ADSCs osteogenesis.更多还原