【作者】 曹大勇；

【导师】 付晋凤；

【作者基本信息】 昆明医科大学 ， 外科学， 2012， 硕士

【摘要】 第一部分小鼠糖尿病全层皮肤缺损模型的建立目的：本实验在链脲佐菌素(STZ)诱导ICR小鼠糖尿病模型的基础上复制全层皮肤缺损模型,观察创面愈合时间的变化及VEGF的表达水平,旨在探索构建小鼠糖尿病创面修复模型的新方法,为后续研究提供可靠的动物模型。方法：36只四周龄ICR雄性小鼠随机分为两组,其中普通对照组(ND组)12只,高脂饲料+STZ组(HS组)24只。两组小鼠均于4-15周龄各周测量体重,于第4、8、9及第10-15周龄时每周测量空腹血糖值。喂养4周后,HS组小鼠腹腔注射STZ80mg/Kg诱导糖尿病,将空腹微量血糖值≥11.lmmol/L的小鼠纳入糖尿病组(DM组)。糖尿病建模成功4周后(13周龄)两组小鼠进行全层皮肤缺损模型的复制。术后分别进行创面换药、照相、取材。NIH ImageJ图像分析软件进行创面愈合率的测定,Real time-PCR检测创面组织mVEGF-A的表达。结果：经STZ腹腔注射后,DM组小鼠血糖显著升高,达18.76±1.75mmol/L,与ND组的7.30±0.62mmol/L相比有统计学差异(P<0.05),同时观察到建模后小鼠尿量增加、体重下降,糖尿病成模率为100%：且高血糖水平至少可维持6周。糖尿病小鼠建模4周复制全层皮肤缺损创面后,其创面愈合较ND组延迟,术后第7、10、14天均有统计学差异(P<0.05),而术后第7天,DM组创面组织mVEGF-A mRNA表达水平也低于ND组(P<0.05)。结论：高脂饮食+STZ诱导小鼠成糖尿病模型方法切实可行,建模成功率达100%,且高血糖状态持续稳定,最少可保持6周。构建的糖尿病小鼠全层皮肤缺损模型创面愈合率较正常小鼠明显延迟、mVEGF-A的表达亦减低,可应用创面愈合的相关的动物研究。第二部分pcDNA3/hVEGF165裸质粒对糖尿病小鼠皮肤全层缺损创面愈合的促进作用目的：前期实验构建的糖尿病小鼠全层皮肤缺损创面模型具有糖尿病创面愈合延迟的特征。因此在本部分实验中,我们将采用该模型观察重组质粒pcDNA3/hVEGF165促进创面愈合的作用进行研究。方法：36只四周龄ICR雄性小鼠随机分为2组,分别给予普通鼠粮(C组,n=12)或15%猪油强化的高脂鼠粮饲养(DM组,n=24)。经高脂鼠粮喂养4周后(即8周龄),高脂鼠粮饲养的24只小鼠腹腔注射STZ80mg/Kg诱导糖尿病模型。以空腹血糖≥11lmmol/L作为糖尿病小鼠的入组标准,随机分为pcDNA3空质粒十全层皮肤缺损组(P组)、pcDNA3/hVEGF165+全层皮肤缺损组(V组)。糖尿病建模成功4周后(13周龄)两组小鼠进行全层皮肤缺损模型的复制。C、P、V组背部全层皮肤缺损创面建模后即刻分别于创缘皮内注射200u1PBS溶液、60ug/100ul的pcDNA3空质粒、60ug/100ul的pcDNA3/hVEGF165质粒。术后当天、第3、7、10、14天分别进行创面定焦照相,第3、7、10、14天创面换药,术后第7、14天创面取材。NIH ImageJ图像分析软件进行创面愈合率的测定,Realtime-PCR检测创面组织hVEGF-A、 mCOL-Ia、 mVCAM的表达,并进行组织病理检查。结果：与C组相比,P、V两组血糖值明显升高(P<0.01)。P、V两组血糖值差异不明显,且高血糖状态至少维持至实验结束(即术后14天)。C组与P组比较,P组创面愈合率第7天时降低(P<0.05),第10、14天时创面愈合率明显降低(P<0.01,表3)。C组与V组比较,各时相点创面愈合率无明显统计学差异(P>0.05)。V组与P组比较,V组创面愈合较快,在术后第10天具有统计学差异(P<0.05)。V组小鼠hVEGF-A相对表达量较P、C组明显增高,在术后第7天、第14天均具有明显的统计学差异(P<0.05)。V、P组小鼠创面组织mCOL-Ia、 mVCAM mRNA的表达均低于C组,但无统计学差异(P>0.05)。在术后第7天,V组小鼠创面组织mCOL-Ia、 mVCAM mRNA的表达均高于P组,但未发现统计学差异(P>0.05)。结论：pcDNA3/hVEGF165裸质粒注射可使hVEGF165成功转染,并可促进糖尿病小鼠创面的愈合。其作用机制与直接增加小鼠创面组织hVEGFl65的表达有关。同时,转染hVEGF165后,创面组织中mVEGF-A mRNA的表达同步上升。更多还原

【Abstract】 Part1The Establishment Of The Diabetic Mice Full-thickness Skin Defect ModelObjective: In this study,replication of full-thickness skin defect model on the basis of ICR mice which induced by streptozotocin(STZ).We observed changes in the wound healing time and the level of expression of VEGF,to explore a new method of building diabetic mice wound healing model for the follow-up study to provide a reliable animal model.Methods:36four weeks old ICR mice were randomly divided into two groups. including normal control(ND group)12, high fat diet+STZ group(HS group)24.Two groups of mice were taken weekly measurement of body weight in4to15weeks of age and weekly fasting blood glucose levels on4、8、9and10to15weeks of age.4weeks after the feeding, the HS mice by intraperitoneal injection of STZ80mg/Kg induction of diabetes, fasting trace glucose value≥II.lmmol/L mice were included in the diabetic group(DM group).A copy of the full-thickness skin defect model after the diabetes modeling successfully after4weeks(13weeks).After surgery were wound dressing,photography,drawing.The NIH Image analysia software for the determination of the wound healing rate,Real time-PCR detection wound organization mVEGF-A expression.Results:After STZ induced by intraperitoneal injection, the DM group, blood glucose was significantly increased up to18.76+1.75mmol/L, compared with the ND group7.30±0.62mmol/L.There were significant differences (P<0.05), at the same time observed modeling the increase in mouse urine,weight loss, diabetes was100%into a mold,and high blood sugar levels at least for6weeks. The wound healing of DM group delayed compared with the ND group,7、10、14days after surgery were statistically significant (P<0.05),while7th postoperative day.the expression levels of mVEGF-A mRNA of DM group were also lower than the ND group(P<0.05).Conclusions: High fat diet+STZ induced mice diabetes model approach practicable, modeling a success rate of100%,and high blood sugar steady state can be maintained for at least6weeks. The diabetic mice full-thickness skin defect model wound healing rate than normal mice significantly reduced, expression of mVEGF-A is also reduced.in line with the characteristics of wound healing can be applied to animal studies.PartⅡThe Role Of pcDNA3/hVEGF165Naked Plasmid In Promoting The Healing Of Full-thickness Skin Wounds Of Diabetic MiceObjective:The full-thickness skin defect model of diabetic mice constructed by the previous experimental has the characteristics of the diabetic wound healing delay.Therefore, in this part of the experiment.we will be using the model to observe the role of recombinant plasmid pcDNA3/hVEGF165promote wound healing.Methods:36four weeks old ICR mice were randomly divided into2groups were given normal rat food (group C,n=12) or15%lard-enhanced high fat rat grain feeding(DM group,n=24). After high-fat rat food fed for4weeks(8weeks old).the high-fat rat food kept in24mice by intraperitoneal injection of STZ80mg/Kg induced diabetes model. Fasting plasma glucose≥ll.lmmol/L as the inclusion criteria of the diabetic mice.Then were randomly divided into pcDNA3empty plasmid+full-thickness skin defects(Group P), pcDNA3/hVEGF165+full-thickness skin defect group(Group V).Diabetes modeling success after4weeks(13weeks), two groups of mice.a copy of the full-thickness skin defect model.C、 P、 V groups had a margin of intradermal injection of200ul PBS、60ug/100ul pcDNA3empty plasmid、60ug/100ul pcDNA3/hVEGF165plasmid on the back of each mice immediately after full-thickness skin wounds modeling. Postoperative day、3、7、10、14days were wound fixed focus camera,3、7、10、14days wound dressing.7and14days drawing.The NIH ImageJ image analysis software for the determination of wound healing rate.Real-time PCR detect the expression of hVEGF-A、 mVEGF-A、 mCOL-Ia、 mVCAM of wound tissue, and histopathologic examination.Results:Compared with group C, the levels of blood glucose of group P and V are significantly high (P＜.01) but the blood glucose between group P and V have no significant difference, and the hyperglycemic state maintain at least the end of the experiment (ie,14days after surgey). Compared with group C, the wound healing rate of group P decreased at day7(P<0.05) and the wound healing rate at day10、14was significantly lower((P<0.01, Table3).Group C and group V.the wound healing rate was no significant differences at each time point (P>0.05).Compare with group P, the wound healing of group V was more faster, and have a statistically difference after10days of surgery. The relative expression of hVEGF-A of group V was significantly higher than group C and P. and has a statistically significant difference after7、14days of surgery (P<0.05).The expression of mCOL-Ia and mVCAM mRNA of group V and P were lower than group C in wound tissue of each mice,but no significant difference (P>0.05). The expression of mCOL-Ia and mVCAM mRNA of group V was higher than group P,but no significant difference (P>0.05).Conclusions:The injection of pcDNA3/hVEGF165naked plasmid allows hVEGF165successfully transfected, and contribute to diabetic mice wound healing. Its mechanism wound improve the expression of hVEGF165directly in the wound tissue of mice. And we know that the expression of mVEGF-A also improvement follow the transfer of hVEGF165.更多还原