叶酸对糖尿病小鼠皮肤创面愈合延迟的保护作用

发布时间：2018-07-23 13:30

【摘要】：目的采用链脲佐菌素（STZ）建立糖尿病小鼠模型，并在此基础上构建糖尿病小鼠皮肤创面愈合延迟模型，探讨补充叶酸对糖尿病小鼠皮肤创面愈合延迟的效应并初步探讨其机制。方法ICR小鼠分为正常对照小鼠及建糖尿病模型小鼠，建糖尿病模型小鼠连续5日腹腔注射STZ50mg/kg，正常对照小鼠连续5日腹腔注射柠檬酸缓冲液。首次注射前及末次注射后72h采集小鼠尾静脉血液检测血糖浓度，以血糖浓度高于250mg/dl判定为糖尿病模型构建成功。在此基础上，正常对照小鼠纳入正常对照组（Control组），建糖尿病模型小鼠分为2组：糖尿病对照组（DM组）、糖尿病叶酸处理组（DM+FA组），，所有小鼠均制作皮肤创面，以创面制作成功之日定为第0天，实验过程中每日观察小鼠皮肤创面愈合情况，包括创面湿润度、颜色、分泌物、形状、创缘；于第0、3、6、9、12天以透明薄膜准确描绘创面，计算各时间点创面愈合率；于第0、3、6、9、12天，每组各6只小鼠麻醉后处死，取相同部位创面组织用于组织学检查，第3、6、9、12天部分创面组织用于测定羟脯氨酸含量；于第6天每组各6只小鼠麻醉后处死，留取创面组织测定谷胱甘肽（GSH）、丙二醛（MDA）、一氧化氮（NO）含量及采用Western blotting测定3-硝基酪氨酸（3-NT）蛋白表达水平，留取肝脏组织测定GSH含量；每组剩余16只小鼠持续观察至创面完全愈合，记录创面完全愈合时间，并于实验结束前再次采集小鼠尾静脉血液，检测血糖浓度。结果建糖尿病模型小鼠连续5日腹腔注射STZ后小鼠精神状态变差，毛发失去光泽，枯黄松散，体重减轻，末次注射后72h血糖浓度高于250mg/dl，实验结束前再次检测小鼠血糖浓度仍高于250mg/dl。在糖尿病小鼠模型建立成功基础上制作皮肤创面，Control组小鼠皮肤创面完全愈合时间为（12.08±1.08）天，DM组为（14.00±2.00）天与Control组比较明显延迟，DM+FA组创面完全愈合时间为（12.73±0.79）天与DM组比较明显缩短；第3、6、9、12天各时间点创面愈合率比较显示Control组与DM+FA组两组之间无差异，Control组与DM组、DM+FA组与DM组之间比较各时间点差异均有统计学意义；形态学及组织学观察可见DM组小鼠皮肤创面愈合延迟，补充叶酸改善创面愈合；第3、6、9、12天三组小鼠创面组织羟脯氨酸含量均呈递增趋势，DM组与Control组比较各时间点羟脯氨酸含量均显著降低，DM+FA组与DM组比较第6、9、12天羟脯氨酸含量升高。测定第6天三组小鼠创面皮肤组织GSH、MDA、NO含量和3-NT蛋白表达水平结果显示，与Control组相比较，DM组MDA含量升高、GSH含量降低、NO含量降低、3-NT蛋白表达水平升高，补充叶酸后可降低小鼠创面皮肤组织MDA、3-NT水平，NO水平升高，降低机体GSH耗竭，减轻机体氧化损伤。结论本研究得出以下结论：（1）补充叶酸对糖尿病小鼠皮肤创面愈合延迟具有保护作用；（2）叶酸对糖尿病小鼠愈合延迟的保护作用机制可能与叶酸减轻创面局部氧化应激，提高创面组织NO水平有关。
[Abstract]:Objective to establish a diabetic mouse model with streptozotocin (STZ), and to establish a delayed skin wound healing model in diabetic mice, and to explore the effect of folic acid supplementation on the delayed healing of diabetic skin wound and its mechanism. Methods ICR mice were divided into normal control mice and diabetic model mice. STZ 50 mg / kg was injected intraperitoneally for 5 days and citric acid buffer solution was injected intraperitoneally for 5 days in normal control mice. The blood glucose concentration of tail vein of mice was detected before the first injection and 72 hours after the last injection. The blood glucose concentration was higher than that of 250mg/dl and the diabetic model was successfully constructed. On this basis, the normal control mice were included in the normal control group (Control group), and the diabetic model mice were divided into two groups: diabetic control group (DM group) and diabetic folic acid treated group (DM FA group). The wound healing was observed daily in mice, including wound wetness, color, secretion, shape and wound margin. The wound healing rate was calculated at each time point, at the 12th day after anesthesia, 6 mice in each group were killed, the wound tissue of the same site was taken for histological examination, and the content of hydroxyproline was measured in some wound tissues on the 12th day of the 3rd day. Six mice in each group were killed after anesthesia on the 6th day. The content of glutathione (GSH), malondialdehyde (MDA),) nitric oxide (MDA),) (NO) and the expression of 3-nitrotyrosine (3-NT) protein were measured by Western blotting and GSH content in liver tissue was determined by Western blotting. The remaining 16 mice in each group were observed to heal completely and the time of complete wound healing was recorded. The blood samples of caudal vein of mice were collected again before the end of the experiment to detect the concentration of blood glucose. Results after intraperitoneal injection of STZ for 5 consecutive days, the diabetic model mice showed poor mental state, lost luster of hair, loose yellow and weight loss. The concentration of blood glucose was higher than 250 mg / dl at 72 h after the last injection, and the concentration of blood sugar in mice was still higher than 250 mg / dl before the end of the experiment. The complete healing time of skin wound in control group was (12.08 卤1.08) days and that in DM group was (14.00 卤2.00) days, which was significantly shorter than that in Control group (12.73 卤0.79) days. The comparison of wound healing rate between Control group and DM FA group showed that there was no difference between Control group and DM FA group. Morphological and histological observation showed that skin wound healing was delayed and folic acid supplementation improved wound healing in DM group. The content of hydroxyproline in wound tissue of DM group and Control group was significantly lower than that of DM FA group and DM group on the 12th day, and the hydroxyproline content in DM FA group was significantly higher than that in DM group. On the 6th day, the levels of GSH MDAN no and 3-NT protein expression in the skin tissue of the three groups were measured. The results showed that compared with the Control group, the MDA content in DM group was higher than that in the DM group, and the no content in DM group was lower than that in the DM group and the protein expression level of 3-NT was decreased. After folic acid supplementation, the level of MDA-3-NT in skin tissue of mice was decreased, the level of no increased, the depletion of GSH was decreased, and the oxidative damage of the body was alleviated. Conclusion the conclusions are as follows: (1) folic acid supplementation has protective effect on delayed wound healing in diabetic mice; (2) the protective mechanism of folic acid on delayed healing of diabetic mice may be associated with folic acid in relieving local oxidative stress. It is related to the increase of no level in wound tissue.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R622;R587.1

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