【摘要】：目的：利用紅色熒光蛋白(RFP)和熒光染料CyI (Cy5的衍生物)標(biāo)記F344大鼠骨髓間充質(zhì)干細(xì)胞(BMSCs),研究光學(xué)標(biāo)記干細(xì)胞的安全及有效性、在大鼠體表對標(biāo)記干細(xì)胞成像的可行性,并結(jié)合超小超順磁性氧化鐵顆粒(USPIO)同時標(biāo)記BMSCs,探索在體雙模態(tài)標(biāo)記干細(xì)胞示蹤成像的可能。 方法：使用成功轉(zhuǎn)染、穩(wěn)定表達(dá)RFP的F344大鼠BMSCs (BMSCs/RFP);同時,使用含不同濃度的CyI培養(yǎng)基與BMSCs/RFP共孵育培養(yǎng)24h后。在體外,用熒光檢測儀分別檢測熒光強度,用光學(xué)成像系統(tǒng)分別檢測體表注射光學(xué)標(biāo)記細(xì)胞的F344大鼠成像效果。使用含USPIO (40μg/ml)和多聚賴氨酸(PLL,1.5μg/ml)的培養(yǎng)基與BMSCs/RFP共孵育24h,標(biāo)記后行普魯士藍(lán)染色觀察鐵顆粒在細(xì)胞內(nèi)的分布。通過皮下或肌肉注射RFP或CyI標(biāo)記的BMSCs,進(jìn)行大鼠體表的光學(xué)成像�；铙w條件下,開胸結(jié)扎冠狀動脈左前降支(LAD)建立F344大鼠急性心梗模型,通過心肌局部注射移植三種標(biāo)記細(xì)胞(BMSCs/RFP、CyI或USPIO標(biāo)記的BMSCs/RFP,其中以USPIO/RFP雙標(biāo)的為主),利用光學(xué)成像及MRI進(jìn)行標(biāo)記細(xì)胞的在體示蹤。取病理行普魯士藍(lán)染色及熒光成像觀察心臟中USPIO顆粒及RFP的分布情況。 結(jié)果：體外熒光強度檢測顯示RFP或CyI標(biāo)記(CyI孵育細(xì)胞濃度為5.0×10-5mol/L)的BMSCs在細(xì)胞濃度達(dá)到5.0×105/ml以上時有較強的信號強度；當(dāng)CyI與BMSCs/RFP共孵育時,其孵育濃度分別在5.0×10-6、1.0×10-5及5.0×104mol/L時細(xì)胞形態(tài)及死細(xì)胞數(shù)未見明顯變化。大鼠體表成像時在注射細(xì)胞數(shù)為1.0×106條件下,CyI的熒光成像效果優(yōu)于RFP。呼吸機輔助通氣條件下通過開胸結(jié)扎冠狀動脈左前降支(LAD)可成功建立F344大鼠急性心梗模型。通過心肌局部注射的標(biāo)記細(xì)胞在光學(xué)條件下,在體成像沒有檢測到熒光信號,離體成像能檢測到心臟表面有較弱的熒光：MRI在標(biāo)記后2周內(nèi)均能觀察到注射區(qū)域信號強度明顯降低。病理普魯士藍(lán)染色及熒光成像提示心肌內(nèi)鐵顆粒及RFP分布與移植干細(xì)胞分布一致。 結(jié)論： 1、RFP或CyI(CyI孵育細(xì)胞濃度為5.0×10-5mol/L)可以安全有效地標(biāo)記BMSCs; 2、RFP或CyI標(biāo)記的BMSCs在F344大鼠體表的光學(xué)成像是可行的； 3、離體成像可示蹤心臟局部注射光學(xué)標(biāo)記的BMSCs; 4、MRI在標(biāo)記后2周仍可在體示蹤USPIO標(biāo)記的BMSCs.
[Abstract]:Objective: to study the safety and efficacy of F344 rat bone marrow mesenchymal stem cells (BMSCs) labeled with red fluorescent protein (RFP) and fluorescent dye CyI (derivatives of Cy5). The feasibility of imaging the labeled stem cells on the surface of the rat was studied, and the possibility of bimodal labeled stem cell tracer imaging in vivo was explored by combining ultra-small superparamagnetic iron oxide particles with (USPIO) and labeling BMSCs, simultaneously. Methods: F344 rat BMSCs (BMSCs/RFP) expressing RFP stably was successfully transfected and co-cultured with BMSCs/RFP with different concentration of CyI for 24 h. In vitro, the fluorescence intensity was measured by fluorescence detector, and the imaging effect of F344 rats injected with optically labeled cells on the body surface was detected by optical imaging system. The medium containing USPIO (40 渭 g / ml) and polylysine (PLL, 1.5 渭 g / ml) was incubated with BMSCs/RFP for 24 h. The distribution of iron particles in the cells was observed by Prussian blue staining. Optical imaging of rat body surface was performed by subcutaneous or intramuscular injection of RFP or CyI labeled BMSCs,. In vivo, a F344 rat model of acute myocardial infarction was established by ligating the left anterior descending branch of the coronary artery (LAD) by thoracotomy. Three kinds of labeled cells (BMSCs/RFP,CyI or USPIO labeled BMSCs/RFP, in which USPIO/RFP was double labeled) were injected into the myocardium of F344 rats. Optical imaging and MRI were used to label the cells in vivo. Prussian blue staining and fluorescence imaging were used to observe the distribution of USPIO particles and RFP in heart. Results: in vitro fluorescence intensity analysis showed that BMSCs labeled with RFP or CyI (5.0 脳 10-5mol/L in CyI) had a strong signal intensity when the cell concentration was higher than 5.0 脳 105/ml. When the concentration of CyI and BMSCs/RFP were 5.0 脳 10 ~ (- 6), 1.0 脳 10 ~ (- 5) and 5.0 脳 104mol/L respectively, the cell morphology and the number of dead cells did not change significantly at the concentration of 5.0 脳 10 ~ (- 6), 1.0 脳 10 ~ (- 5) and 5.0 脳 104mol/L. When the number of injected cells was 1.0 脳 10 6, the fluorescence imaging effect of CyI was better than that of RFP. in rat body surface imaging. The acute myocardial infarction model of F344 rats was successfully established by ligating the left anterior descending coronary artery (LAD) under ventilator-assisted ventilation. Labeled cells injected locally into the myocardium did not detect fluorescence signals in vivo imaging under optical conditions. In vitro imaging was able to detect weak fluorescence on the heart surface: the signal intensity of the injection region was significantly decreased by MRI within 2 weeks after labeling. Pathological Prussian blue staining and fluorescence imaging suggested that the distribution of iron particles and RFP in myocardium was consistent with that of stem cells transplantation. Conclusion: 1. The concentration of RFP or CyI (CyI is 5.0 脳 10-5mol/L to label BMSCs;-2 safely and effectively. The optical imaging of BMSCs labeled by RFP or CyI is feasible in F344 rats. 3. In vitro imaging can be used to trace the local injection of BMSCs;-4 into the heart. The BMSCs. labeled by USPIO can still be traced in vivo 2 weeks after labeling.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329.2

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