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
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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。
Part1The Establishment Of The Diabetic Mice Full-thickness Skin Defect Model
Objective: 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 Mice
Objective: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.
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。
Part1The Establishment Of The Diabetic Mice Full-thickness Skin Defect Model
Objective: 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 Mice
Objective: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.
引文
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[20]Jensen LJ,Denner L,Schrijvers BF,et al.Renal effects of a neutralising RAGE-antibody in long-term streptozo-tocin-diabetic mice[J].J Endocrinol,2006,183(3):493-501
[21]Yang W,Lu J,Weng J,et al. Prevalence of Diabetes among Men and Women in China[J].N Engl J Med.2010,362(12):1090-1101
[22]陆树良,谢挺,牛轶雯。创面难愈机制研究-糖尿病皮肤的“微环境污染”[J]。中华烧伤杂志,2008,24(1)：3-5
[23]Weckroth, M,Vaheri A,Myohanen,et al. Differential effects of acute and chronic wound fluids on urokinase-type plasminogen activator,urokinase-type plasminogen activator receptor,and tissue-type plasminogen activator in cultured human keratinocytes and fibroblasts[J]..Wound Repair Regen,2001,9(4):314-322
[24]Theoret CL,Barber SM, Moyana TN,et al.Expression of transforming growth factor beta(1),beta(3),and basic fibroblast growth factor in full-thickness skin wound of equine limbs and thorax[J]..Vet Surg,2001,30(3):269-277
[25]Cook KM,Figg WD.Angiogenesis inhibitiors;Current strategies and future prospects[J].CA Cancer J Clin,2010.60(4):222-243
[26]Gereon Lauer,Stephan Sollberg,Melanie Cole,et al.Expression and Proteolysis of Vascular Endothelial Growth Factor is increased in chronic wound[J].J invest Dermatol,2000,15:12-18
[27]余传林,朱正光,雷林生等,链脲佐菌素糖尿病模型动物血糖及体征动态变化的研究。南方医科大学学报,2008,28(1)：132-133
[28]陆树良.青春,谢挺,等。糖尿病皮肤“隐性损害”的机制研究。中华创伤杂志,2004,20(8)： 468-4
[1]Singer AJ.Clark.RA.Cutaneous wound healing .N Engl J Med,1999,341(10):738-746
[2]Kao HK, Chen B, Murphy GF, et al. Peripheral blood fibrocytes:enhancement of wound healing by cell proliferation, re-epithelialization, contraction, and angiogenesis. Ann Surg. 2011:254(6):1066-1074
[3]曹大勇,陈斌,付晋凤。基因治疗在创伤愈合中的应用[J].中华损伤与修复杂志：电子版,2011,6(6)：1012-1018.
[4]Mustoe TA, O'Shaughnessy K, Kloeters O. Chronic wound pathogenesis and current treatment strategies:a unifying hypothesis. J Plast Reconstr Surg. 2006:117:35-1
[5]Brem H,Balledux J,Bloom T,et al. Healing of diabetic foot ulcers and pressure ulcers with human skin equivalent:a new paradigm in wound healing.Arch Surg,2000,135(6):627-634
[6]Hartlapp I,Abe R,Saeed RW,et al.Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo[J].Faseb J.2001.15(12):2215-2224.
[7]Yang W,Lu J,Weng J,et al. Prevalence of Diabetes among Men and Women in China.N Engl J Med.2010,362(12):1090-1101.
[8]Robson MC.Exogenous growth factor application effect on human wound healing.Prog Dermatol,1996,30:1-7
[9]Wieman TJ.Smilell JM,Su Y.Efficacy and safety of a topical gel formulation of recombinant human platelet-derived growth factor-BB(Becaplemin) in patients with chromic neuropathic diabetic ulcers.Diabetes Care,1998,21:822-827
[10]Spraul CW,Kaven C,Lang GK.et al.Effect of growth factors on bovine retinal pigment epithelial cell migration and proliferation.Ophthalmic Res,2004,36(3):166-171
[11]Ferrara N.Vascular endothelial growth factor[J]Arterioscler Thromb Vasc Biol,2009,29(6) :789-791.
[12]Kolostova K,Taltynov O,Pinterova D,et al. Wound healing gene therapy:cartilage regeneration induced by vascular endothelial growth factor plasmid[J].Am J Otolaryngol. 2012V33Nl:68-74.
[13]Atiyeh BS, Hayek SN, Gunn SW. New technologies for burn wound closure and healing-review of the literature. Burns,2005,31 (8):944-956
[14]Liu W. Cao Y, Longaker MT. Gene therapy of scarring:a lesson learned from fetal scarless wound healing. Yonsei Med J, 2001,42 (6):634-645
[15]Southwood LL, Frisbie DD. Kawcak CE, et al. Delivery of growth factors using gene therapy to enhance bone healing. Vet Surg, 2004, 33 (6):565-578
[16]Hengge UR, Chan EF, Foster RA, et al. Cytokine gene expression in epidermis with biological effects following injection of naked DNA[J]. Nat Genet, 1995,10(2):161-166.
[17]李勤勤,耿欣。不同处理的精制玉米麸皮对链脲佐菌素(STZ)诱导的糖尿病小鼠的降糖作用。食品科学,2011,,32(19)：236-239.
[18]Isner JM,Pieczek A,Schainfeld R,et,al.Clinical evidence of angiogenesis after arterial gene transfer of phVEGF 145 in patient with ischemic limb.Lancet,1996,348:370
[19]Baumgartner I,Pieczek A,Manor O,et al.Constitutive expression of phVEGF165 after intramuscular gene transfer promote collateral vessel development in patient with critical limb ischemia.Circulation,1998.97:1114-1123
[20]陆树良,谢挺,牛轶雯。创面难愈机制研究-糖尿病皮肤的“微环境污染”。中华烧伤杂志,2008,24(1)：3-5
[21]Hon KM,Belperio JA,Keane MP,et al.Differentiation of human circulating fibrocytes as mediated by transforming growth factor-beta and peroxisome proliferator-activated receptor gamma.J Biol Chem,2007,282(31):22910-22920
[22]Weisberg SP,McCann D,Desai M,et al.Obesity is associated with macrophage accumulation in adipose tissue.J Clin Invest.2003,112(12):1796-1808
[23]Klagsgrun M,D'Amore PA.Regulators of angiogenesis. Annu Rev Physiol 1991,53:217-239
[24]Ferrara N,Gerber HP,Le Couter J.The biology of VEGF and its receptorsNat Med,2003,9: 669-676
[25]Compernolle V,Brusselmans K,Acker T,et al.Loss of HIF-2alpha and inhibition of VEGF impair fetal lung maturation,whereas treatment with VEGF prevents fatal respiratory distress in premature mice.Nat Med 2002,8:702-710
[26]Neufield G,Cohen T,Gengrinovith S,et al.Vascular endothelial growth factor and its receptors. FASEB J,1999,13:9-22
[27]Branski LK, Gauglitz GG, Herndon DN, et al. A review of gene and stem cell therapy in cutaneous wound healing. Bums,2009,35(2):171-180.
[28]张向荣,郭荣光,刘德伍。血管内皮细胞生长因子165基因转染人骨髓间充质干细胞构建组织工程皮肤的实验研究[J].南昌大学博士论文：2009
[29]杜丽,张庆林,莘旭妮等。携带人肝细胞生长因子的重组裸质粒对放射复合皮肤创伤促愈作用的实验研究。创伤外科杂志,2010,12(4)：314-318
[30]刘亮明,罗杰,张吉翔等。裸质粒流体力学注射法-基因治疗研究的利器。世界华人消化杂志,2006,14(28)：2780-2784
[31]Dicken S,Vermeulen P.Hendrickx B,et al.Regulable vascular endothelial growth factorl65 overexpression by vivo expand kerationcyte cultures promotes matrix formation,angiogenesis, and healing in porcine full-thickness wound.Tissue Eng Part A,2008,14(1):19-27
1 Jeschke MG, Hemdon DN, Baer W, et al. Possibilities of non-viral gene transfer to improve cutaneous wound healing[J]. Curr Gene Ther, 2001.1(3):267-278.
2 Branski LK, Gauglitz GG, Herndon DN. et al. A review of gene and stem cell therapy in cutaneous wound healing[J]. Burns, 2009, 35(2): 171-180.
3 Wu Y, Chen L, Scott PG, et al. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis[J]. Stem Cells, 2007,25(10):2648-2659.
4 Branski LK, Pereira CT, Herndon DN, et al. Gene therapy in wound healing: present status and future directions[J]. Gene Ther,2007,14(1):1-10.
5 曹明媚,戚中田.基因治疗载体的研究进展[J].国外医学：肿瘤学分册.2004,31(1)：22-26.
6 Weber E, Anderson WF, Kasahara N. Recent advances in retrovirus vector-mediated gene therapy: teaching an old vector new tricks[J]. Curr Opin Mol Ther, 2001,3(5):439-453.
7 Blaese RM, Culver KW, Miller AD, et al. T lymphocyte-directed gene therapy for ADA-SCID:initial trial results after 4 years[J]. Science, 1995,270 (5235):475-480.
8 Olsen JC. EIAV, CAEV and other lentivirus vector systems[J]. Somat Cell Mol Genet, 2001, 26(1/6):131-145.
9 Badillo AT, Chung S, Zhang L, et al. Lentiviral gene transfer of SDF-1 alpha to wounds improves diabetic wound healing[J]. J Surg Res, 2007,143(1): 35-42.
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32 Maeda T, Fujimoto K. A reduction-triggered delivery by a liposomal carrier possessing membrane-permeable ligands and a detachable coating[J]. Colloids Surf B Bionterfaces, 2006, 49(1):15-21.
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[6]曹大勇,陈斌,付晋凤。基因治疗在创伤愈合中的应用[J].中华损伤与修复杂志：电子版,2011.6(6)：1012-1018
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[18].Kolostova K,Taltynov O.Pinterova D,et al. Wound healing gene therapy: cartilage regeneration induced by vascular endothelial growth factor plasmid[J].Am J Otolaryngol. 2012V33N 1:68-74
[19]Sharma A, Singh A, Warren S, et al. Differential regulation of angiogenic genes in diabetic wound healing. J Invest Dermatol,2006, 126(7):2323-2331
[20]Jensen LJ,Denner L,Schrijvers BF,et al.Renal effects of a neutralising RAGE-antibody in long-term streptozo-tocin-diabetic mice[J].J Endocrinol,2006,183(3):493-501
[21]Yang W,Lu J,Weng J,et al. Prevalence of Diabetes among Men and Women in China[J].N Engl J Med.2010,362(12):1090-1101
[22]陆树良,谢挺,牛轶雯。创面难愈机制研究-糖尿病皮肤的“微环境污染”[J]。中华烧伤杂志,2008,24(1)：3-5
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[24]Theoret CL,Barber SM, Moyana TN,et al.Expression of transforming growth factor beta(1),beta(3),and basic fibroblast growth factor in full-thickness skin wound of equine limbs and thorax[J]..Vet Surg,2001,30(3):269-277
[25]Cook KM,Figg WD.Angiogenesis inhibitiors;Current strategies and future prospects[J].CA Cancer J Clin,2010.60(4):222-243
[26]Gereon Lauer,Stephan Sollberg,Melanie Cole,et al.Expression and Proteolysis of Vascular Endothelial Growth Factor is increased in chronic wound[J].J invest Dermatol,2000,15:12-18
[27]余传林,朱正光,雷林生等,链脲佐菌素糖尿病模型动物血糖及体征动态变化的研究。南方医科大学学报,2008,28(1)：132-133
[28]陆树良.青春,谢挺,等。糖尿病皮肤“隐性损害”的机制研究。中华创伤杂志,2004,20(8)： 468-4
[1]Singer AJ.Clark.RA.Cutaneous wound healing .N Engl J Med,1999,341(10):738-746
[2]Kao HK, Chen B, Murphy GF, et al. Peripheral blood fibrocytes:enhancement of wound healing by cell proliferation, re-epithelialization, contraction, and angiogenesis. Ann Surg. 2011:254(6):1066-1074
[3]曹大勇,陈斌,付晋凤。基因治疗在创伤愈合中的应用[J].中华损伤与修复杂志：电子版,2011,6(6)：1012-1018.
[4]Mustoe TA, O'Shaughnessy K, Kloeters O. Chronic wound pathogenesis and current treatment strategies:a unifying hypothesis. J Plast Reconstr Surg. 2006:117:35-1
[5]Brem H,Balledux J,Bloom T,et al. Healing of diabetic foot ulcers and pressure ulcers with human skin equivalent:a new paradigm in wound healing.Arch Surg,2000,135(6):627-634
[6]Hartlapp I,Abe R,Saeed RW,et al.Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo[J].Faseb J.2001.15(12):2215-2224.
[7]Yang W,Lu J,Weng J,et al. Prevalence of Diabetes among Men and Women in China.N Engl J Med.2010,362(12):1090-1101.
[8]Robson MC.Exogenous growth factor application effect on human wound healing.Prog Dermatol,1996,30:1-7
[9]Wieman TJ.Smilell JM,Su Y.Efficacy and safety of a topical gel formulation of recombinant human platelet-derived growth factor-BB(Becaplemin) in patients with chromic neuropathic diabetic ulcers.Diabetes Care,1998,21:822-827
[10]Spraul CW,Kaven C,Lang GK.et al.Effect of growth factors on bovine retinal pigment epithelial cell migration and proliferation.Ophthalmic Res,2004,36(3):166-171
[11]Ferrara N.Vascular endothelial growth factor[J]Arterioscler Thromb Vasc Biol,2009,29(6) :789-791.
[12]Kolostova K,Taltynov O,Pinterova D,et al. Wound healing gene therapy:cartilage regeneration induced by vascular endothelial growth factor plasmid[J].Am J Otolaryngol. 2012V33Nl:68-74.
[13]Atiyeh BS, Hayek SN, Gunn SW. New technologies for burn wound closure and healing-review of the literature. Burns,2005,31 (8):944-956
[14]Liu W. Cao Y, Longaker MT. Gene therapy of scarring:a lesson learned from fetal scarless wound healing. Yonsei Med J, 2001,42 (6):634-645
[15]Southwood LL, Frisbie DD. Kawcak CE, et al. Delivery of growth factors using gene therapy to enhance bone healing. Vet Surg, 2004, 33 (6):565-578
[16]Hengge UR, Chan EF, Foster RA, et al. Cytokine gene expression in epidermis with biological effects following injection of naked DNA[J]. Nat Genet, 1995,10(2):161-166.
[17]李勤勤,耿欣。不同处理的精制玉米麸皮对链脲佐菌素(STZ)诱导的糖尿病小鼠的降糖作用。食品科学,2011,,32(19)：236-239.
[18]Isner JM,Pieczek A,Schainfeld R,et,al.Clinical evidence of angiogenesis after arterial gene transfer of phVEGF 145 in patient with ischemic limb.Lancet,1996,348:370
[19]Baumgartner I,Pieczek A,Manor O,et al.Constitutive expression of phVEGF165 after intramuscular gene transfer promote collateral vessel development in patient with critical limb ischemia.Circulation,1998.97:1114-1123
[20]陆树良,谢挺,牛轶雯。创面难愈机制研究-糖尿病皮肤的“微环境污染”。中华烧伤杂志,2008,24(1)：3-5
[21]Hon KM,Belperio JA,Keane MP,et al.Differentiation of human circulating fibrocytes as mediated by transforming growth factor-beta and peroxisome proliferator-activated receptor gamma.J Biol Chem,2007,282(31):22910-22920
[22]Weisberg SP,McCann D,Desai M,et al.Obesity is associated with macrophage accumulation in adipose tissue.J Clin Invest.2003,112(12):1796-1808
[23]Klagsgrun M,D'Amore PA.Regulators of angiogenesis. Annu Rev Physiol 1991,53:217-239
[24]Ferrara N,Gerber HP,Le Couter J.The biology of VEGF and its receptorsNat Med,2003,9: 669-676
[25]Compernolle V,Brusselmans K,Acker T,et al.Loss of HIF-2alpha and inhibition of VEGF impair fetal lung maturation,whereas treatment with VEGF prevents fatal respiratory distress in premature mice.Nat Med 2002,8:702-710
[26]Neufield G,Cohen T,Gengrinovith S,et al.Vascular endothelial growth factor and its receptors. FASEB J,1999,13:9-22
[27]Branski LK, Gauglitz GG, Herndon DN, et al. A review of gene and stem cell therapy in cutaneous wound healing. Bums,2009,35(2):171-180.
[28]张向荣,郭荣光,刘德伍。血管内皮细胞生长因子165基因转染人骨髓间充质干细胞构建组织工程皮肤的实验研究[J].南昌大学博士论文：2009
[29]杜丽,张庆林,莘旭妮等。携带人肝细胞生长因子的重组裸质粒对放射复合皮肤创伤促愈作用的实验研究。创伤外科杂志,2010,12(4)：314-318
[30]刘亮明,罗杰,张吉翔等。裸质粒流体力学注射法-基因治疗研究的利器。世界华人消化杂志,2006,14(28)：2780-2784
[31]Dicken S,Vermeulen P.Hendrickx B,et al.Regulable vascular endothelial growth factorl65 overexpression by vivo expand kerationcyte cultures promotes matrix formation,angiogenesis, and healing in porcine full-thickness wound.Tissue Eng Part A,2008,14(1):19-27
1 Jeschke MG, Hemdon DN, Baer W, et al. Possibilities of non-viral gene transfer to improve cutaneous wound healing[J]. Curr Gene Ther, 2001.1(3):267-278.
2 Branski LK, Gauglitz GG, Herndon DN. et al. A review of gene and stem cell therapy in cutaneous wound healing[J]. Burns, 2009, 35(2): 171-180.
3 Wu Y, Chen L, Scott PG, et al. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis[J]. Stem Cells, 2007,25(10):2648-2659.
4 Branski LK, Pereira CT, Herndon DN, et al. Gene therapy in wound healing: present status and future directions[J]. Gene Ther,2007,14(1):1-10.
5 曹明媚,戚中田.基因治疗载体的研究进展[J].国外医学：肿瘤学分册.2004,31(1)：22-26.
6 Weber E, Anderson WF, Kasahara N. Recent advances in retrovirus vector-mediated gene therapy: teaching an old vector new tricks[J]. Curr Opin Mol Ther, 2001,3(5):439-453.
7 Blaese RM, Culver KW, Miller AD, et al. T lymphocyte-directed gene therapy for ADA-SCID:initial trial results after 4 years[J]. Science, 1995,270 (5235):475-480.
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