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目的:探索局部注射siRNA对小鼠牙胚生长发育产生影响的实验方法。方法:选择健康新生昆明小鼠作为实验对象,将阴性对照siRNA进行壳聚糖(chitosan,CS)包裹,分别将其注入小鼠的两个不同部位:①.腹腔注射组,将Cy5.5标记的CS包裹的siRNA纳米粒注射于新生昆明小鼠腹腔,于不同时间点在动物活体成像仪下观察荧光分布情况。②.颌骨局部注射组,将Cy5.5标记的siRNA/CS纳米粒局部直接注射于新生小鼠左上颌第一磨牙牙胚,通过动物活体成像结合冰冻切片观察荧光分布情况,验证局部注射方法的可行性。结果:在腹腔注射组,随着时间延长,荧光开始在腹腔内扩散,随后在腹腔内淬灭,荧光始终局限于腹腔,腹腔外没有观察到荧光;颌骨局部注射组通过活体动物成像显示注射的牙胚处有荧光,进一步的冰冻切片验证在牙胚处有荧光,且牙胚结构无明显损伤。结论:本实验将CS包裹siRNA纳米粒技术应用于牙胚发育的相关研究,首次成功构建了新生昆明小鼠牙胚局部注射siRNA的方法,为研究牙胚发育相关基因的作用开辟了新的途径。
Objective: To explore the experimental method of local injection of siRNA on mouse tooth germ growth and development. Methods: Healthy neonatal Kunming mice were selected as the experimental subjects. The negative control siRNAs were encapsulated by chitosan (CS) and injected into two different parts of mice respectively. ①. The intraperitoneal injection of Cy5.5 The CS-encapsulated siRNA nanoparticles were injected into the peritoneal cavity of neonatal Kunming mice to observe the fluorescence distribution at different time points under the living animal imager. Local injection of Cy5.5-labeled siRNA / CS nanoparticles directly into the first maxillary first molar tooth germ of neonatal mice by local injection of xanthine and observation of fluorescence distribution by animal biopsy and frozen sections Feasibility. Results: In the intraperitoneal injection group, with the passage of time, the fluorescence began to diffuse into the peritoneal cavity and then quenched in the peritoneal cavity. The fluorescence was always confined to the peritoneal cavity, and no fluorescence was observed outside the peritoneal cavity. The local injection group of the jaws was injected with live animal imaging Of the tooth germ at the fluorescence, further frozen section verified that there is fluorescence in the tooth germ, and no significant damage to the tooth germ structure. Conclusion: The CS wrapped siRNA nanoparticles technology applied to the development of tooth germ-related research, the first successful construction of a newborn Kunming mice tooth germ siRNA injection method for the study of the role of tooth germ development-related genes opened up a new way .