超小超顺磁氧化铁标记大鼠脂肪源间充质干细胞生物学特性的初步实验研究

发布时间：2018-01-30 16:45

  本文关键词： 大鼠 脂肪源间充质干细胞 超小超顺磁氧化铁(USPIO) 多聚赖氨酸(PLL) 细胞培养　出处：《南方医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究背景 帕金森病(Parkinson's disease,PD)是一种常见的神经退行性疾病,特征性的病理改变是多巴胺神经元凋亡和黑质纹状体通路损害,主要临床表现为静止性震颤、肌强直、运动迟缓和姿势平衡障碍。随着人口老龄化的加速,其发病率呈逐年上升的趋势,是威胁老年人健康的一类重大疾病,65岁以上人群发病率超过1%,给家庭和社会都造成了沉重的负担。美国目前约有50万PD患者,且每年以5万例的速度递增,而我国现已逐步进入老龄化社会,PD患者已达到200万人,每年新增帕金森病患者近20万人。若不及时进行有效的治疗,患者病情呈慢性进行性加重,晚期往往全身僵硬、活动受限,约30%中晚期患者生活不能自理,最后常死于各种并发症。目前无论是药物治疗还是手术治疗只能暂时改善症状而不能阻止病情进行性发展。随着再生与组织工程医学的兴起,利用干细胞移植替代凋亡的多巴胺能神经元成为治疗PD一种新策略。 干细胞移植作为治疗中枢系统疾病的新治疗策略而被广泛研究。脂肪源间充质干细胞(adipose-derived stromal cells,ADSCs)是一种极具潜力的种子细胞,自Zuk于2001年发现以来,其生物学特性及分化潜能等方面与骨髓源间充质干细胞非常相似,并且由于ADSCs具有来源丰富、易获取、创伤小、增殖快等优势,使其成为一种更为理想的种子细胞。如何对移植的干细胞标记及活体示踪是近年的研究热点和难点,近年来,得益于分子影像学的快速发展为此提供了可能。磁共振成像(MRI)是目前临床普遍应用成熟的影像学技术,但是常规MR成像的空间、时间分辨率无法显示移植细胞,研究表明,借助新型磁共振造影增强剂可以反复无创地追踪移植的干细胞。其中超小超顺磁氧化铁(ultrasmallsuperparamagnetic particles of iron oxide,USPIO)标记是一种较为理想的MR示踪方法。目前已有不少学者相继报道利用超顺磁氧化铁颗粒(super-paramagnetic iron oxide,SPIO)可成功标记细胞并对其进行示踪,但关于ADSCs的标记及USPIO示踪还鲜有报道,如何提高标记效率同时又减少标记物对细胞的毒性是移植治疗过程中的前提。本课题旨在探讨USPIO对ADSCs的标记的适宜浓度及示踪。 本研究拟采用超小超顺磁氧化铁(USPIO)对大鼠脂肪源间充质干细胞(ADSCs)(?)进行标记,对比分析不同浓度的超小超顺磁氧化铁(USPIO)对ADSCs标记的效率,并分别用CCK-8及Alamar blue方法对已标记细胞的活力进行检测,探寻USPIO对ADSCs适宜的标记浓度。为观测已标记ADSCs在PD模型大鼠体内的存活、迁移提供了相关的实验基础。 目的：建立大鼠脂肪源间充质干细胞的分离和培养方法,并对其形态学、细胞表面标志物进行检测,为USPIO标记ADSCs提供细胞来源。 方法： 1.大鼠脂肪源间充质干细胞的原代培养、纯化、传代 大鼠脂肪源间充质干细胞的原代培养：SD大鼠,体重120+20g,采用36g/L水合氯醛,按1ml/100g体重的剂量进行腹腔注射麻醉,麻醉满意后将大鼠摆俯卧位,固定四肢于平板上,剃除背部及腹部的鼠毛。置于超净工作台上,依次用碘酊、酒精消毒,将解剖器械盒、三个玻璃培养皿(加入冷PBS液)等依次排放在超净台上。严格无菌条件下操作,逐层分离组织,尽量减少出血及红细胞污染,取出肾周脂肪组织,选取含血管较少的部分置于培养皿中,包裹好迅速转移至细胞房。用无菌的0.01mmol/L磷酸缓冲液(PBS)反复冲洗脂肪组织,尽量剔除软组织和血管,然后置于青霉素瓶中用眼科剪剪碎；再用吸管将细碎的脂肪组织转移至一次性离心管(15m1),加入0.075%的Ⅰ型胶原酶37℃振荡消化30min～40min;含10%FBS的DMEM/F12等体积中和,100gm nylonmesh过滤后离心(1200g×10min),弃上清,以含10%FBS的DMEM/F12重悬细胞,轻柔吹打制成单细胞悬液,混匀,细胞计数仪下计数后以1×106/ml密度接种于25cm2塑料培养瓶；置于37℃、5%CO2饱和温度培养箱中培养。48h后全量换液,去除悬浮细胞。以后每2-3天半量换液一次,待细胞生长至70%-80%融合后传代培养。 2.脂肪源间充质干细胞表面标志物的流式细胞仪检测 取第3代ADSCs,将培养好的脂肪源间充质干细胞吹打分离下来,收集于50m1离心管中,并以吸管轻轻吹打、将脂肪源间充质干细胞吹散,250xg4℃离心5分钟,弃上清；用0.01mol/L的PBS10m1重悬细胞,清洗1-2次；用适量0.01mol/L PBS调整细胞浓度1×106个/ml,分装于EP管中,共5管,每管1ml,分别做好标记。分别加入抗鼠CD29-PE、抗鼠CD90-FITC、抗鼠CD44-FITC和抗鼠CD45-FITC流式抗体5u1,室温避光孵育10min,0.01mol/LPBS清洗2遍,1000rmp离心5min,适量的0.01mol/L PBS重悬后用流式细胞仪检测。 结果 1.大鼠脂肪源间充质干细胞呈贴壁生长,细胞形态均一,呈鹅卵石样,一周以后大多数脂肪源间充质干细胞有胞浆突起,以梭形细胞为主,胞浆丰富、核大、核染色质细、核仁明显,可见细胞呈克隆样生长。传代后,于倒置显微镜下可见成纤维样细胞形态,细胞呈平行排列生长或旋涡状生长,在形态上很难与骨髓来源的MSC区别开来。 2.流式细胞仪检测结果显示,体外培养的第3代ADSCs的表型标志CD44、CD90和CD29呈阳性表达,CD90表达阳性率达92.76%,CD29表达阳性率达96.56%,CD44表达阳性率达91.05%；CD45呈阴性表达,表明ADSCs是比较均一的未分化干细胞。 结论本实验获得的大鼠脂肪源间充质干细胞经纯化和传代及流式细胞仪鉴定,达到理想的纯度,能够满足实验设计的需要,可以用于后续实验。 目的：采用不同浓度的USPIO-PLL复合物标记ADSCs,并分别用CCK-8及Alamar blue两种方法检测细胞活力,用普鲁士蓝染色检测标记效率,寻求较为理想的标记浓度,旨在为后续研究工作提供实验数据参考。 方法：将实验分为八个组,即(200μg/ml组,150μg/ml组,100μg/ml组,50μg/ml组,25μg/ml组,12.5μg/ml组,阴性对照组和空白对照组)。采用USPIO与正电荷转染剂PLL共培养方法制备USPIO-PLL复合物,将不同浓度的USPIO-PLL复合物标记ADSCs,置于37℃、5%CO2细胞培养箱中孵育。每天定时在被检组4个复孔中加入CCK-8和Alamar blue试剂各10μ1,孵育1h后使用酶标仪检测OD值,连续检测7天,整理数据,统计分析。用普鲁士蓝染色检查USPIO-PLL标记ADSCs的效率。 用SPSS13.0软件进行统计学分析,计量资料用均数±标准差(x±SD)表示方差齐时采用单因素方差分析和LSD的多重比较,不齐时采用非参数检验和Tunnett's T3多重比较。以P≤0.05为差异有统计学意义。 结果：ADSCs经不同浓度的USPIO-PLL复合物标记后经酶标仪测得OD值,首先进行方差齐性检验,方差齐时采用单因素方差分析和LSD的多重比较,不齐时采用非参数检验和Tunnett's T3多重比较。在标记的第1天,经Levene方差齐性检验,结果显示方差齐性(F=1.211,P=0.335),然后进行单因素方差分析显示浓度组之间的差异有统计学意义(F=3.049,P=0.019),进行LSD多重比较后发现空白对照组与其他组之间的差异有统计学意义(P0.01),其余各组之间两两比较差异没有统计学意义(P0.05),说明除空白对照组,各组之间是均衡一致的。各组OD值在第3天逐渐出现变化,经Levene方差齐性检验,结果显示方差齐性(F=2.233,P=0.067),然后进行单因素方差分析显示浓度组之间的差异有统计学意义(F=49.283,,P0.01),进行LSD多重比较后发现200μg/ml组各组比较差异有统计学意义,说明其细胞增殖已受到抑制。第5天,经Levene方差齐性检验,结果显示方差齐性(F=0.911,P=0.515),然后进行单因素方差分析显示浓度组之间的差异有统计学意义(F=110.356,,P0.01),进行LSD多重比较后发现200μg/ml组各组比较仍然存在显著差异。至第7天,经Levene方差齐性检验,结果显示方差齐性(F=1.111,P=0.388),然后进行单因素方差分析显示浓度组之间的差异有统计学意义(F=149.746,,P0.01),进行LSD多重比较后发现150μg/ml组的细胞增殖也出现抑制现象,200μg/ml组的细胞增殖明显受到抑制,与各组比较差异有统计学意义。而CCK-8和Alamar blue两种方法检测方法的结果一致表明：12.5gg/ml-100μg/ml不影响ADSCs的增殖能力和活力,可以安全有效地标记脂肪源间充质干细胞。USPIO浓度为50μg/ml时,ADSCs胞浆内可见蓝色颗粒,铁染率约95%；USPIO浓度为100gg/ml以上时,ADSCs胞浆内可见大量蓝色颗粒,铁染率约100%,呈剂量依赖关系。 结论：USPIO的浓度为2001μg/ml以上则明显影响ADSCs的活力,抑制细胞增殖；而12.5μg/ml～25μg/ml的USPIO标记效率较低,故可以选取50gg/ml-100μg/ml作为标记ADSCs的适宜浓度。
[Abstract]:Research background
Parkinson's disease (Parkinson's disease PD) is a common neurodegenerative disease, characterized by pathologic change and apoptosis of dopaminergic neurons nigrostriatal damage, the main clinical manifestations of resting tremor, rigidity, bradykinesia and postural. With the acceleration of population aging, the incidence rate is rising year by year the trend is a kind of major diseases threatening the health of the elderly people over the age of 65, the incidence rate of more than 1%, to the family and society have caused a heavy burden. The United States currently has about 500 thousand PD patients, and the annual increase of 50 thousand cases of speed, while China has gradually entered the aging society, PD patients have up to 2 million people, the new Parkinson's disease nearly 200 thousand people every year. If the effective treatment is not timely, the patient had chronic progressive, often late stiff and restricted activity, about 30% of patients with advanced life Can not take care of themselves, finally died of various complications. At present often either medication or surgery can temporarily improve symptoms but can not prevent progressive disease development. With the development of tissue engineering and regenerative medicine, using stem cell transplantation to replace apoptosis of dopaminergic neurons in the treatment of PD is a new strategy.
Stem cell transplantation as a new therapeutic strategy for the treatment of central nervous system diseases and has been studied. Adipose derived mesenchymal stem cells (adipose-derived stromal cells, ADSCs) is a kind of potential seed cells, since Zuk discovered in 2001, its biological characteristics and differentiation potential with bone marrow derived mesenchymal stem cells similar, and because ADSCs has rich source, easy to obtain, small trauma, rapid proliferation and other advantages, make it become a kind of more ideal seed cells. How to stem cell labeling and tracing in vivo transplantation is a hot and difficult topic in recent years, in recent years, thanks to the rapid development of molecular imaging offers possible. Magnetic resonance imaging (MRI) is a universal application of imaging technology, but the conventional MR imaging space and time resolution can not display the transplanted cells, research shows that, with the help of a new type of magnetic resonance Contrast agent can be repeated non-invasive tracking of transplanted stem cells. The ultrasmall superparamagnetic iron oxide (ultrasmallsuperparamagnetic particles of iron oxide, USPIO) marker is an ideal MR tracer method. At present many scholars have reported the use of superparamagnetic iron oxide particles (super-paramagnetic iron oxide, SPIO) can be successful the labeled cells and tracer, but mark and USPIO tracer on ADSCs has not been reported, how to improve the labeling efficiency and reduce the toxicity of cell markers is a prerequisite for transplantation in the treatment process. This study aims to investigate the suitable concentration of ADSCs and USPIO tracing marker.
This study intends to use ultra small superparamagnetic iron oxide (USPIO) on rat adipose derived mesenchymal stem cells (ADSCs) (?) mark, the comparative analysis of different concentrations of ultrasmall superparamagnetic iron oxide (USPIO) on the efficiency of ADSCs markers, and were used to detect cell viability using CCK-8 labeled Alamar and blue methods, explore the USPIO of marker concentration ADSCs appropriate. For the observation of labeled ADSCs in survival in PD model rats, to provide experimental basis for related migration.
Objective: to establish a method for isolation and culture of rat adipose derived mesenchymal stem cells, and to detect morphologic and cell surface markers, and to provide cell source for USPIO labeled ADSCs.
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