基于损伤范围控制的大鼠胰腺创伤模型建立及其胰腺干细胞增殖规律的研究

发布时间：2019-03-02 20:14

【摘要】：目的:1、研制一种多功能小动物撞击系统并评估其初步使用效果,为进一步动物实验建立创伤模型及为满足创伤基础医学研究提供仪器需要。2、建立一种新型的大鼠单纯胰腺创伤模型,并基于损伤范围评估其伤情变化,为后续研究提供模型条件。3、研究胰腺干细胞在本研究的大鼠胰腺大范围创伤模型中增殖变化规律。方法:1、撞击仪系统基于储能换能装置、简便式多功能撞击装置、撞击参数测量装置等三大部件的相关原理设计,并进一步检验其有效性和稳定性。2、将大鼠随机分为2组,采用自主研发多功能撞击仪的3cm2和6cm2撞击头,分别撞击各组处于开腹状态的胰腺,使之在400kpa的压强下形成对应不等范围的损伤,并设置对照;建模24h后观察各组大鼠存活率、一般情况、大体病理和组织病理学改变,并检测血钾、血钙的浓度和血清、腹水中淀粉酶、脂肪酶的水平。3、将大鼠随机分为创伤组和对照组,前者接受6cm2/400Kpa的撞击强度,后者为假手术组;建模后1、2、3、7天处死大鼠,处死前12h按100mg/kg体重经腹腔注射5-溴-2'脱氧脲嘧啶核苷(Brd U);取胰腺组织,荧光TUNEL检测细胞凋亡,Brd U的免疫组化染色检测细胞增殖,RT-PCR、免疫组化(荧光)和Western blot等方法检测PDX-1的表达并探讨其变化规律。结果:1、多功能小动物撞击系统研制完成,其撞击应力峰值连续可调节且量程在0-200kg,压缩和挤压应力连续可调节且量程在0-100kg;经验证,该仪器具有良好的有效性(P(27)0.05)和可重复性(P(29)0.05)。2、成功构建了大鼠胰腺创伤模型;3cm2和6cm2致伤面积组均能形成明显的损伤,其中致伤组死亡率、腹水量、胰腺湿重、病理评分、血清酶学指标均高于对照组(P(27)0.05),血钾、血钙较对照组低(P(27)0.05),而6cm2致伤面积组的损伤比3cm2组更显著。3、胰腺创伤模型中,伤后24h胰腺即可发生凋亡与代偿性增殖;PDX-1的表达增强主要在建模后的第2-3天,随后下降,至第7天恢复至正常水平附近,伤后第2,3天与同时间点对照组比较,有显著性差异(P(27)0.05)。结论:1、研制的撞击仪操作简单,撞击方式多样,撞击参数可被实时记录;经过调试和动物实验,效果显著且性能稳定,可对不同实验动物形成不同强度和方式的创伤;适用于撞击伤的建模和形态、机能学的研究。2、该胰腺创伤建模简单、模型有效、操作的可重复性好,伤情单一且稳定。适合用于伤情演化机制和创伤施救等研究。3、胰腺创伤后,胰腺干细胞将发生活化,在创伤后修复期增殖并分化为各类功能细胞,一定程度补偿了胰腺受损害的功能。
[Abstract]:Objective: 1, to develop a multi-functional impact system for small animals and evaluate its preliminary effect, to establish trauma models for further animal experiments and to provide instruments for the basic medical research of trauma. A new rat model of simple pancreatic trauma was established, and the changes of injury were evaluated based on the range of injury, which provided the model condition for further study. 3, To investigate the proliferation of pancreatic stem cells in the rat pancreatic trauma model. Methods: 1. The impactor system was designed based on the principle of energy storage and energy exchanger, simple multi-function impact device and impact parameter measurement device, and further tested its effectiveness and stability. 2, the impact instrument system was designed based on the energy storage device, the convenient multi-function impact device and the impact parameter measurement device. The rats were randomly divided into two groups. The 3cm2 and 6cm2 impingement heads of the self-developed multi-function impingement instrument were used to impact the pancreas in the open state of each group respectively, so that the corresponding range of injury was formed under the pressure of 400kpa, and the control group was set up. The survival rate, general condition, gross pathological and histopathological changes of rats in each group were observed 24 hours after modeling, and the levels of serum potassium, serum calcium and amylase and lipase in serum and ascitic fluid were measured, and the levels of amylase and lipase in serum and ascitic fluid were measured. The rats were randomly divided into trauma group and control group, the former received the impact intensity of 6cm2/400Kpa, and the latter was sham-operated group. The rats were killed at 1, 2, 3, 7 days after modeling. The rats were injected intraperitoneally with 5-bromo-2'- deoxyuracil nucleoside (Brd U); 12 hours before death according to the body weight of 100mg/kg. The expression of PDX-1 in pancreatic tissue was detected by immunohistochemical staining of apoptotic, Brd U, and the expression of PDX-1 was detected by RT-PCR, immunohistochemistry (fluorescence) and Western blot, respectively. The expression of PDX-1 was detected by fluorescence TUNEL. Results: 1, the multi-function small animal impact system has been developed, the peak value of impact stress is continuously adjustable and the measuring range is 0 ~ 200 kg, the compression and extrusion stress is continuously adjustable and the measurement range is 0 ~ 100 kg; It was proved that the instrument was effective and reproducible (P (27) and repeatable (P (29 (0.05). (2) the rat pancreatic trauma model was successfully established. Both 3cm2 and 6cm2 injured area group could form obvious injury, in which death rate, ascites volume, wet weight of pancreas, pathological score, serum enzyme index were higher than those of control group (P (27), and serum potassium level was significantly higher than that of control group (P < 0.05). The level of serum calcium was lower than that of control group (P (27), but the injury of 6cm2 injury area group was more significant than that of 3cm2 group. 3. In pancreatic trauma model, 24 hours after injury, apoptosis and compensatory proliferation of pancreas could occur. The expression of PDX-1 increased mainly at the 2nd-3rd day after the model, then decreased, and returned to the normal level on the 7th day. There was a significant difference between the control group on the 2nd and 3rd day after injury and the control group at the same time (P (27). Conclusion: 1, the developed impactor is simple in operation, diverse in impact modes and can be recorded in real time, after debugging and animal experiments, the effect is remarkable and the performance is stable, which can cause trauma of different intensity and mode to different experimental animals; It is suitable for modeling and morphological, functional study of impact injury. 2, this pancreatic trauma model is simple, effective, repeatable, simple and stable. (3) Pancreatic stem cells will be activated after pancreatic trauma, proliferate and differentiate into various functional cells in post-traumatic period, which can compensate the damaged function of pancreas to a certain extent. 3) it can be used to study the mechanism of wound evolution and wound rescue. 3) after pancreatic trauma, pancreatic stem cells will be activated and differentiated into various functional cells.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R657.5;R-332

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