本文關(guān)鍵詞： 大鼠 脂肪源間充質(zhì)干細胞 超小超順磁氧化鐵(USPIO) 多聚賴氨酸(PLL) 細胞培養(yǎng)　出處：《南方醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景 帕金森病(Parkinson's disease,PD)是一種常見的神經(jīng)退行性疾病,特征性的病理改變是多巴胺神經(jīng)元凋亡和黑質(zhì)紋狀體通路損害,主要臨床表現(xiàn)為靜止性震顫、肌強直、運動遲緩和姿勢平衡障礙。隨著人口老齡化的加速,其發(fā)病率呈逐年上升的趨勢,是威脅老年人健康的一類重大疾病,65歲以上人群發(fā)病率超過1%,給家庭和社會都造成了沉重的負擔。美國目前約有50萬PD患者,且每年以5萬例的速度遞增,而我國現(xiàn)已逐步進入老齡化社會,PD患者已達到200萬人,每年新增帕金森病患者近20萬人。若不及時進行有效的治療,患者病情呈慢性進行性加重,晚期往往全身僵硬、活動受限,約30%中晚期患者生活不能自理,最后常死于各種并發(fā)癥。目前無論是藥物治療還是手術(shù)治療只能暫時改善癥狀而不能阻止病情進行性發(fā)展。隨著再生與組織工程醫(yī)學(xué)的興起,利用干細胞移植替代凋亡的多巴胺能神經(jīng)元成為治療PD一種新策略。 干細胞移植作為治療中樞系統(tǒng)疾病的新治療策略而被廣泛研究。脂肪源間充質(zhì)干細胞(adipose-derived stromal cells,ADSCs)是一種極具潛力的種子細胞,自Zuk于2001年發(fā)現(xiàn)以來,其生物學(xué)特性及分化潛能等方面與骨髓源間充質(zhì)干細胞非常相似,并且由于ADSCs具有來源豐富、易獲取、創(chuàng)傷小、增殖快等優(yōu)勢,使其成為一種更為理想的種子細胞。如何對移植的干細胞標記及活體示蹤是近年的研究熱點和難點,近年來,得益于分子影像學(xué)的快速發(fā)展為此提供了可能。磁共振成像(MRI)是目前臨床普遍應(yīng)用成熟的影像學(xué)技術(shù),但是常規(guī)MR成像的空間、時間分辨率無法顯示移植細胞,研究表明,借助新型磁共振造影增強劑可以反復(fù)無創(chuàng)地追蹤移植的干細胞。其中超小超順磁氧化鐵(ultrasmallsuperparamagnetic particles of iron oxide,USPIO)標記是一種較為理想的MR示蹤方法。目前已有不少學(xué)者相繼報道利用超順磁氧化鐵顆粒(super-paramagnetic iron oxide,SPIO)可成功標記細胞并對其進行示蹤,但關(guān)于ADSCs的標記及USPIO示蹤還鮮有報道,如何提高標記效率同時又減少標記物對細胞的毒性是移植治療過程中的前提。本課題旨在探討USPIO對ADSCs的標記的適宜濃度及示蹤。 本研究擬采用超小超順磁氧化鐵(USPIO)對大鼠脂肪源間充質(zhì)干細胞(ADSCs)(?)進行標記,對比分析不同濃度的超小超順磁氧化鐵(USPIO)對ADSCs標記的效率,并分別用CCK-8及Alamar blue方法對已標記細胞的活力進行檢測,探尋USPIO對ADSCs適宜的標記濃度。為觀測已標記ADSCs在PD模型大鼠體內(nèi)的存活、遷移提供了相關(guān)的實驗基礎(chǔ)。 目的：建立大鼠脂肪源間充質(zhì)干細胞的分離和培養(yǎng)方法,并對其形態(tài)學(xué)、細胞表面標志物進行檢測,為USPIO標記ADSCs提供細胞來源。 方法： 1.大鼠脂肪源間充質(zhì)干細胞的原代培養(yǎng)、純化、傳代 大鼠脂肪源間充質(zhì)干細胞的原代培養(yǎng)：SD大鼠,體重120+20g,采用36g/L水合氯醛,按1ml/100g體重的劑量進行腹腔注射麻醉,麻醉滿意后將大鼠擺俯臥位,固定四肢于平板上,剃除背部及腹部的鼠毛。置于超凈工作臺上,依次用碘酊、酒精消毒,將解剖器械盒、三個玻璃培養(yǎng)皿(加入冷PBS液)等依次排放在超凈臺上。嚴格無菌條件下操作,逐層分離組織,盡量減少出血及紅細胞污染,取出腎周脂肪組織,選取含血管較少的部分置于培養(yǎng)皿中,包裹好迅速轉(zhuǎn)移至細胞房。用無菌的0.01mmol/L磷酸緩沖液(PBS)反復(fù)沖洗脂肪組織,盡量剔除軟組織和血管,然后置于青霉素瓶中用眼科剪剪碎；再用吸管將細碎的脂肪組織轉(zhuǎn)移至一次性離心管(15m1),加入0.075%的Ⅰ型膠原酶37℃振蕩消化30min～40min;含10%FBS的DMEM/F12等體積中和,100gm nylonmesh過濾后離心(1200g×10min),棄上清,以含10%FBS的DMEM/F12重懸細胞,輕柔吹打制成單細胞懸液,混勻,細胞計數(shù)儀下計數(shù)后以1×106/ml密度接種于25cm2塑料培養(yǎng)瓶；置于37℃、5%CO2飽和溫度培養(yǎng)箱中培養(yǎng)。48h后全量換液,去除懸浮細胞。以后每2-3天半量換液一次,待細胞生長至70%-80%融合后傳代培養(yǎng)。 2.脂肪源間充質(zhì)干細胞表面標志物的流式細胞儀檢測 取第3代ADSCs,將培養(yǎng)好的脂肪源間充質(zhì)干細胞吹打分離下來,收集于50m1離心管中,并以吸管輕輕吹打、將脂肪源間充質(zhì)干細胞吹散,250xg4℃離心5分鐘,棄上清；用0.01mol/L的PBS10m1重懸細胞,清洗1-2次；用適量0.01mol/L PBS調(diào)整細胞濃度1×106個/ml,分裝于EP管中,共5管,每管1ml,分別做好標記。分別加入抗鼠CD29-PE、抗鼠CD90-FITC、抗鼠CD44-FITC和抗鼠CD45-FITC流式抗體5u1,室溫避光孵育10min,0.01mol/LPBS清洗2遍,1000rmp離心5min,適量的0.01mol/L PBS重懸后用流式細胞儀檢測。 結(jié)果 1.大鼠脂肪源間充質(zhì)干細胞呈貼壁生長,細胞形態(tài)均一,呈鵝卵石樣,一周以后大多數(shù)脂肪源間充質(zhì)干細胞有胞漿突起,以梭形細胞為主,胞漿豐富、核大、核染色質(zhì)細、核仁明顯,可見細胞呈克隆樣生長。傳代后,于倒置顯微鏡下可見成纖維樣細胞形態(tài),細胞呈平行排列生長或旋渦狀生長,在形態(tài)上很難與骨髓來源的MSC區(qū)別開來。 2.流式細胞儀檢測結(jié)果顯示,體外培養(yǎng)的第3代ADSCs的表型標志CD44、CD90和CD29呈陽性表達,CD90表達陽性率達92.76%,CD29表達陽性率達96.56%,CD44表達陽性率達91.05%；CD45呈陰性表達,表明ADSCs是比較均一的未分化干細胞。 結(jié)論本實驗獲得的大鼠脂肪源間充質(zhì)干細胞經(jīng)純化和傳代及流式細胞儀鑒定,達到理想的純度,能夠滿足實驗設(shè)計的需要,可以用于后續(xù)實驗。 目的：采用不同濃度的USPIO-PLL復(fù)合物標記ADSCs,并分別用CCK-8及Alamar blue兩種方法檢測細胞活力,用普魯士藍染色檢測標記效率,尋求較為理想的標記濃度,旨在為后續(xù)研究工作提供實驗數(shù)據(jù)參考。 方法：將實驗分為八個組,即(200μg/ml組,150μg/ml組,100μg/ml組,50μg/ml組,25μg/ml組,12.5μg/ml組,陰性對照組和空白對照組)。采用USPIO與正電荷轉(zhuǎn)染劑PLL共培養(yǎng)方法制備USPIO-PLL復(fù)合物,將不同濃度的USPIO-PLL復(fù)合物標記ADSCs,置于37℃、5%CO2細胞培養(yǎng)箱中孵育。每天定時在被檢組4個復(fù)孔中加入CCK-8和Alamar blue試劑各10μ1,孵育1h后使用酶標儀檢測OD值,連續(xù)檢測7天,整理數(shù)據(jù),統(tǒng)計分析。用普魯士藍染色檢查USPIO-PLL標記ADSCs的效率。 用SPSS13.0軟件進行統(tǒng)計學(xué)分析,計量資料用均數(shù)±標準差(x±SD)表示方差齊時采用單因素方差分析和LSD的多重比較,不齊時采用非參數(shù)檢驗和Tunnett's T3多重比較。以P≤0.05為差異有統(tǒng)計學(xué)意義。 結(jié)果：ADSCs經(jīng)不同濃度的USPIO-PLL復(fù)合物標記后經(jīng)酶標儀測得OD值,首先進行方差齊性檢驗,方差齊時采用單因素方差分析和LSD的多重比較,不齊時采用非參數(shù)檢驗和Tunnett's T3多重比較。在標記的第1天,經(jīng)Levene方差齊性檢驗,結(jié)果顯示方差齊性(F=1.211,P=0.335),然后進行單因素方差分析顯示濃度組之間的差異有統(tǒng)計學(xué)意義(F=3.049,P=0.019),進行LSD多重比較后發(fā)現(xiàn)空白對照組與其他組之間的差異有統(tǒng)計學(xué)意義(P0.01),其余各組之間兩兩比較差異沒有統(tǒng)計學(xué)意義(P0.05),說明除空白對照組,各組之間是均衡一致的。各組OD值在第3天逐漸出現(xiàn)變化,經(jīng)Levene方差齊性檢驗,結(jié)果顯示方差齊性(F=2.233,P=0.067),然后進行單因素方差分析顯示濃度組之間的差異有統(tǒng)計學(xué)意義(F=49.283,,P0.01),進行LSD多重比較后發(fā)現(xiàn)200μg/ml組各組比較差異有統(tǒng)計學(xué)意義,說明其細胞增殖已受到抑制。第5天,經(jīng)Levene方差齊性檢驗,結(jié)果顯示方差齊性(F=0.911,P=0.515),然后進行單因素方差分析顯示濃度組之間的差異有統(tǒng)計學(xué)意義(F=110.356,,P0.01),進行LSD多重比較后發(fā)現(xiàn)200μg/ml組各組比較仍然存在顯著差異。至第7天,經(jīng)Levene方差齊性檢驗,結(jié)果顯示方差齊性(F=1.111,P=0.388),然后進行單因素方差分析顯示濃度組之間的差異有統(tǒng)計學(xué)意義(F=149.746,,P0.01),進行LSD多重比較后發(fā)現(xiàn)150μg/ml組的細胞增殖也出現(xiàn)抑制現(xiàn)象,200μg/ml組的細胞增殖明顯受到抑制,與各組比較差異有統(tǒng)計學(xué)意義。而CCK-8和Alamar blue兩種方法檢測方法的結(jié)果一致表明：12.5gg/ml-100μg/ml不影響ADSCs的增殖能力和活力,可以安全有效地標記脂肪源間充質(zhì)干細胞。USPIO濃度為50μg/ml時,ADSCs胞漿內(nèi)可見藍色顆粒,鐵染率約95%；USPIO濃度為100gg/ml以上時,ADSCs胞漿內(nèi)可見大量藍色顆粒,鐵染率約100%,呈劑量依賴關(guān)系。 結(jié)論：USPIO的濃度為2001μg/ml以上則明顯影響ADSCs的活力,抑制細胞增殖；而12.5μg/ml～25μg/ml的USPIO標記效率較低,故可以選取50gg/ml-100μg/ml作為標記ADSCs的適宜濃度。
[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.
Method錛，

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