【作者】 赵春生；

【导师】 李俊峡；

【作者基本信息】 河北医科大学 ， 内科学， 2010， 硕士

【摘要】 第一部分人脐血间充质干细胞体外分离培养与鉴定目的:探索建立一种稳定的人脐血间充质干细胞的体外分离培养方法。方法:无菌条件下采集正常足月剖宫产胎儿脐血,采用密度梯度离心法结合直接贴壁法分离脐血间充质干细胞(human umbilical cord blood mesenchymal stem cells ,UCB-MSCs) ,用DMEM/F12培养基加10%胎牛血清并添加生长因子(GM-CSF和IL-3)后,接种于塑料培养瓶培养并传代,对其增殖情况进行观察并利用流式细胞仪检测细胞表面抗原表达情况。结果:24h后可见有少量圆形细胞贴壁,1周左右可见贴壁细胞增多,出现少量单极梭形细胞,2周后可见细胞形成集落,经培养3-4周左右,形成平行排列、辐射状或旋涡状细胞界限不清楚的似成纤维样细胞,且80～90 %呈现融合。第二代细胞接种后12小时开始贴壁,10d左右即可达80～90 %融合。流式细胞分析显示此类细胞稳定地表达相关的抗原标记CD29、CD44、CD105 ,但不表达造血细胞系的表面标记CD34、CD45、CD14。结论:此种培养方法可成功体外培养UCB-MSCs,细胞纯度较高,贴壁细胞表面抗原表达与骨髓间充质干细胞有较强的一致性。第二部分超顺磁性氧化铁纳米粒子(SPIO)标记人脐血间充质干细胞的可行性研究目的:探讨SPIO标记人脐血间充质干细胞干细胞(UCB-MSCs)的方法及安全性、有效性。方法:采用密度梯度离心法结合直接贴壁法分离培养UCB-MSCs,应用不同浓度的SPIO标记UCB-MSCs,分别通过普鲁士蓝染色测标记率;台盼蓝染色检测标记后细胞活性;MTT比色实验评价SPIO对细胞生长能力的影响。结果:普鲁士蓝染色证实SPIO标记阳性率均在95%以上,低浓度的SPIO对细胞活性无明显影响,当SPIO浓度达140ug/ml以上时对UCB-MSCs活性有一定程度的影响。结论:适宜浓度的SPIO标记UCB-MSCs方法简单、安全、有效。第三部分两种方法制备犬急性心肌梗死模型目的:探索建立稳定的急性心肌梗死模型的造模方法。方法:将16只健康杂种犬随机分为两组,每组8只,一组采用开胸冠脉结扎的方法造模,另一组采用闭胸冠脉栓塞的方法造模。术后均通过心电图、心肌酶学、病理组织切片对急性心肌梗死模型建立情况作出评价。结果:两种方法均可成功建立犬急性心肌梗死模型,并经心电图、心肌酶学和病理检测证实。其中开胸结扎法8只动物中,4只存活,4只死亡;闭胸介入法8只动物中,7只存活,1只死亡。结论:开胸结扎法效果确实可靠,但操作难度高,创伤大,动物不容易存活,而介入法简便易行,创伤小,死亡率低,是一种值得推广的犬急性心肌梗死造模方法。第四部分标记干细胞移植于梗死心肌的核磁动态观察目的:探讨利用磁共振成像活体示踪SPIO标记的人脐血间充质干细胞(UCB-MSCs)心肌移植的可行性及准确性。方法:采用密度梯度离心法结合直接贴壁法分离培养UCB-MSCs,体外进行SPIO标记,采用开胸及闭胸两种方法制备犬急性心肌梗死模型,将标记细胞或纯SPIO以开胸直接注射和闭胸经导管冠脉注入两种方式进行细胞移植,分别于移植前、植入后即刻、1-4周行核磁扫描,每次核磁扫描后处死动物1只,取心脏组织切片行HE染色和普鲁士蓝染色。结果:通过开胸心肌直接注射方式植入的标记细胞可通过磁共振成像检测到,并为病理所证实。4周后植入细胞仍可被核磁检测到,病理结果显示植入细胞在心梗区域存活,并向同一方向排列,而通过冠脉途径植入的标记细胞未能被磁共振成像及病理检测到。结论:以开胸心肌直接注射方式植入的SPIO标记细胞可以通过磁共振成像技术准确实现活体示踪,而通过冠脉途径植入的标记细胞未被检测到。更多还原

【Abstract】 Part 1 Optimization and identification of in vitro isolation and culture condition of human umbilical cord blood mesenchymal stem cellsOjective: To explore establish an optimized methord to isolate,culture and expend mesenchymal stem cells (MSCs) from human umibilical cord blood (UCB).Method: Under sterile condition, Human UCB sample was harvested from full term delivery by cesarean section. MSCs were isolated from UCB by combination of gradient centrifugation and different adherent time method. And the isolated cells(MSCs) were cultivated in plastic culture flask containing DMEM/F12, 10% fetal calf serum (GM-CSF , IL-3). The cells were observed and their immunophenotypes were determined by flowcytometry(FCM).Results: After 24 hours , there gave rise to a small quantity adherent round cells,after 1 week the adherent cells increased and part of them exhibited single -point -shuttled,after 2 weeks formed cells group, after 3-4 weeks cultured to show parallelly arranged, radiat or whirlpool-shaped fibroblast-like cells and 80%-90% cells were fused. Serial subcultivation cells were found within 12 hours adhered and after about 10 days 80%-90% cells were fused. FCM showed that these cells expressed CD29,CD44,CD105 ,but no hematopoietic lineage markers , such as CD34,CD45,CD14.Conclusion: MSCs can be successfully isolated from human cord blood by using this method,and the cells purification is higher, and the adherent cells have the same immunophenotype with bone marrow derived. Part 2 The Study of the Feasibility of Human Umbilical Cord Blood Mesenchymal Stem CellsObjective:To evaluate the safety and efficiency of a new labeling agent,superparamagnetic iron oxide(SPIO).Methods:MSCs were isolated from UCB by combination of gradient centrifugation and different adherent time method. UCB-MSCs were labeled with different concentration of SPIO agent.The labeling efficiency was tested through Prussian blue staining ,cells viability were tested through Trypan blue rejection method,and the ability of growth was evaluated through MTT.Results:There were intracyto plasmic blue part- icles in nearly every cell in Prussian blue staining.The positive rate was more than 95%.With the concentration of SPIO increasing (14ug/ml~140ug/ml),there was no obvious diffenence in cell viability of UCB-MSCs(p>0.05).When the concentration of SPIO exceeded 280ug/ml,there was a significient difference(p<0.05).Conclusion:The UCB-MSCs could be safety and effi- ciently labledwith suitable concentration of SPIO.Part 3 Development and evaluation of the canine models of acute myocardial infarction using two waysObjective:To explore establish an optimized methord to develop and evaluate the canine model of acute myocardial infarction.Method: The 16 healthy canines were divided into two groups randomly,and each group contained 8 canines . One group was established the myocardial infarction model by using open-chest coronary artery ligation method,and another was received closed-chest coronary artery embolization method of modeling. Then electrocardiogram, CK-MB and histopathologic slide were investigated to confirm AMI. Results: The two methods both can successfully establish canine model of acute myocardial infarction, and be confirmed by the electrocardiogram, CK-MB and pathology.In the group of coronary artery ligation, 4 canines survived and 4 canines died ,while another group of coronary artery embolization,7 canines survived and 1 canines died.Conclusion:The method of coronary artery ligation is reliable,but the operation is difficult and the animal is not easy to survive.While the coronary artery embolization method of modeling is simple, convenient and safe, it is worth to spread.Part 4 Imaging Stem Cells Implanted in Infarcted MyocardiumObjective:To investigate the feasibility of in vivo tracking for umbilical cord blood mesenchymal stem cells delivered to infracted myocardium by MRI.Methods:MSCs were isolated from UCB by combination of gradient centrifugation and different adherent time method and were labeled with different concentration of SPIO agent. The acute myocardial infarction models were established by two methods of coronary artery ligation and coronary artery embolization.Then the labeled cells or SPIO were transplanted into the myocardium in two ways-direct injection and through coronary catheter. The MRI was respectively carried out immediately and 1–4 weeks. After MRI the hearts were excised, the segment in which injections were performed were thin cut and stained with hematoxylin-eosin and Prussian blue staining.Results:The labeled cells transplanted by direct injection could be detected through MRI and were confirmed on pathology. After 4 weeks the injected labeled cells could still be detected through MRI, and the pathology showed the injected cells could survive in the MI area, and parallel in the same direction.While the labeled cells transplanted through coronary catheter could not be detected by MRI and pathology.Conclusion:The labeled cells could be reliably detected by MRI in vivo.更多还原