摘要
目的:1.改良兔2型糖尿病模型的构建方法并建立兔2型糖尿病早期皮肤缺损模型,观察阻碍兔2型糖尿病早期皮肤缺损愈合的相关因素;2.将碱性成纤维细胞生长因子基因以基因转染的方法转染至骨髓间充质干细胞,并复合猪小肠黏膜下层构建组织工程皮肤;3.将组织工程皮肤移植修复兔2型糖尿病早期皮肤缺损,观察组织工程皮肤对2型糖尿病早期皮肤缺损的疗效。
方法:本研究分为三个部分:
第一部分,兔2型糖尿病模型早期皮肤缺损愈合相关因素的研究:1、全身状况:通过高糖高脂饲料2月+链脲菌素耳缘静脉注射改良诱导兔2型糖尿病模型,并定期监测血糖、血脂、胰岛素水平的变化情况;2、局部观察:在兔2型糖尿病模型的基础上制作糖尿病早期皮肤缺损模型,与正常皮肤缺损对比其在造模后3天、1周、2周、4周缺损皮肤修复的情况,包括周围有无炎症、瘢痕面积的大小及形状、修复后皮肤的物理特性、颜色,并比较修复面积;取缺损皮肤行HE染色,观察空白组及糖尿病皮肤缺损组各时相点表皮及真皮组织组织修复情况、炎症反应、肉芽组织生长情况;在HE染色的基础上,进行免疫组化检测,对比2型糖尿病早期皮肤缺损与正常皮肤缺损创面PCNA、MMP9、CD8及胶原纤维的表达。
第二部分:转染碱性成纤维细胞生长因子骨髓间充质干细胞复合小肠黏膜下层构建的组织工程皮肤的实验研究:日本大耳兔骨髓间充质干细胞进行体外培养扩增后,通过pCDNA3.1质粒将碱性成纤维细胞生长因子基因转染至生长状态良好的骨髓间充质干细胞,并将转染后的细胞接种子制备好的猪小肠黏膜下层上,进行体外联合培养,构建组织工程皮肤。观察细胞生长情况,流式细胞术检测骨髓间充质干细胞表型,WesternBlot和免疫荧光检测pCDNA-bFGF真核表达载体转染兔骨髓间充质干细胞的结果,并通过光镜和电镜观察组织工程皮肤的结构。
第三部分:组织工程皮肤治疗兔2型糖尿病早期皮肤缺损的实验研究:构建好的组织工程皮肤移植修复2型糖尿病早期皮肤缺损,并设空白支架对照组,于术后3天、1周、2周、4周取材,进行大体观察和HE染色观察各时相点组织修复情况、炎症反应、肉芽组织生长情况,并行免疫组化染色观察创面PCNA、MMP9、CD8及胶原纤维的阳性表达。
采用SPSS13.5统计软件进行数据录入和统计处理,采用方差分析、卡方检验(X2检验)进行统计学分析。
结果:
第一部分高糖高脂饲料2月+链脲菌素改良诱导兔2型糖尿病成模率为70%,不成模的原因主要为因腹膜炎及反应性低血糖导致的死亡(约占不成模率20%)和因胰腺自愈能力较强而致的早期复原(约占不成模率10%)。皮肤缺损愈合模型大体观察结果:各时间点位中,空白对照修复皮肤缺损的时间明显快于糖尿病早期皮肤缺损,创面面积小于糖尿病早期皮肤缺损,两组比较有统计学意义(P＜0.05)。HE染色提示:2型糖尿病早期皮肤缺损愈合过程中肉芽组织出现时间较晚,肉芽组织形成较正常组少,成熟晚,肉芽组织内血管新生不活跃,炎症反应持续时间长,在愈合的晚期仍可出现较为严重的炎性反应,有微坏死灶形成。免疫组化染色提示:糖尿病情况下PCNA在创伤后2周到4周其表达水平与正常组相近,差异不明显,在造模后3天及1周,2型糖尿病皮肤缺损的PCNA表达明显低于空白对照组;CD8表达在伤后3天、1周均高于空白对照组,在2周时较正常2周为低,4周时检测发现高表达;MMP9表达在糖尿病组中各时间点位表达均高于空白对照组;Van Geisen染色空白组颜色在各时相点均较糖尿病组深。
第二部分传代后骨髓间充质干细胞生长迅速,呈长梭形,形态良好。细胞的表面抗原CD90、CD44有阳性表达,而CD45为阴性。构建了碱性成纤维细胞生长因子基因和pCDNA-bFGF真核表达载体,将载体转染入兔骨髓基质干细胞通过检测证明能稳定表达,转染后的骨髓间充质干细胞在猪小肠黏膜下层中生长良好,可体外构建组织工程皮肤。
第三部分大体观察和HE染色提示:术后3天组织工程皮肤移植组和空白支架对照组修复面积未见明显统计学差异;术后1周,组织工程皮肤移植组修复面积较空白支架对照组高;2周:组织工程皮肤组修复快于空白支架组,组织工程皮肤移植组修复面积较空白支架对照组高;在术后4周,组织工程皮肤移植组修复面积仍较空白支架对照组略高。免疫组化染色提示:组织工程皮肤移植组和空白支架对照组CD8分子表达水平均不高,两组间无统计学差异;PCNA表达在组织工程皮肤移植组术后3天、1周、2周均高于空白对照组,在术后4周时,组织工程批租移植组的PCNA水平要低于空白支架对照组:MMP9的表达水平在创伤后3天和1周时空白支架组与组织工程皮肤移植组之间相比未见明显差异,但在2周及4周时差异明显;Van Geisen染色组织工程皮肤移植组颜色在各时相点均较空白支架对照组深,HCK染色在组织工程皮肤移植组4周为阳性。
结论:
第一部分:1、高糖高脂饮食加STZ小剂量注射可成功构建2型糖尿病动物模型,成模率高,稳定。单纯高糖高脂饮食是无法诱导出2型糖尿病的。2、2型糖尿病兔早期即可皮肤缺损愈合能力差,出现愈合慢甚至不愈合。3、2型糖尿病早期皮肤缺损愈合过程中,其肉芽组织出现时间较晚,成熟晚,肉芽组织明显少于正常情况;组织增殖和修复能力是滞后、紊乱的;局部免疫功能是异常的,过度的,紊乱的;细胞外基质过度分解,其中炎性反应重,且炎性反应紊乱,持续时间长可能造成是糖尿病皮肤缺损难愈的主要原因。
第二部分:1、本实验体外扩增培养骨髓间充质干细胞,并经检测加以证实,可作为组织工程的种子细胞;2、本实验于体外克隆了碱性成纤维细胞生长因子基因,构建了pCDNA-bFGF真核表达载体,并将pCDNA-bFGF真核表达载体转染至骨髓间充质干细胞,经检测可持续的表达;3、将转染了bFGF的骨髓间充质干细胞与猪小肠粘膜下层支架复合培养构建了组织工程皮肤。
第三部分:1、BMSCs在体内的微环境下可分化为表皮样细胞;2、构建的组织工程皮肤可明显的改善2型糖尿病早期皮肤缺损创面的愈合情况,是治疗糖尿病皮肤缺损的有效方法;3、组织工程皮肤由于bFGF有效存在而具有更好修复作用
Objective:
1. To establish the rabbit type 2 diabetes mellitus models and the skin defect model with type 2 diabetes mellitus, then some data of histology and immunohistochemistry was detected to approach the factors related to the skin healing with type 2 diabetes mellitus.
2. Basic fibroblast growth factor (bFGF) gene was transfected into bone marrow mesenchymal stem cells (BMSCs), and combined the cells with SIS to construct tissue-engineered skin.
3. The tissue-engineered skin was transplanted to heal the skin defect with type 2 diabetes mellitus, then the effect of the tissue-engineered skin was observed.
Methods:
The hole experiment was devided into 3 parts:
Part one: The study of healing factors of skin defect with type 2 diabetes. The rabbit type 2 diabetes mellitus models was induced by 2 month feeding with high glucose and grease and streptozocin injection. The blood sugar, blood fat and insulin was detected at the times of 2 month feeding, 2 weeks , 4 weeks, and 8 weeks after the injection. Then the skin defect was estabolished based on the type 2 diabetes model, and the defect skin healing was compared at the 3 days, 1 week, 2 weeks, 4 weeks with control group, included the inflammation, area of healing and crust. Also the HE stain and immunohistochemistry stain was taken to compared the expression of PCNA, CD8, MMP9 and collagen fibers.
Part two: Construction of tissue-engineered skin using basic fibroblast growth factor gene transfected bone marrow mesenchymal stem cells combined with small intestinal submucosa. BMSCs were obtained from Japanese big-eared rabbits, and in vitro cultured. Then the subcultured BMSCs were transfected by pCDNA3.1 plasmid, followed by incubation on swine SIS to construct the tissue-engineered skin. The growth of cells and phenotype of BMSCs were detected by flow cytometry. In addition, the result of transfecting BMSCs with pCDNA-bFGF vector was measured by Western blot, and the structure of tissue-engineered skin was observed.
Part three: The tissue-engineered skin constructed in part 2 was transplanted to the skin defect with type 2 diabetes, and the control group was also set up. Then the defect skin healing was compared at the 3 days, 1 week, 2 weeks, 4 weeks with control group, included the inflammation, area of healing and crust. Also the HE stain and immunohistochemistry stain was taken to compared the expression of PCNA, CD8, MMP9 and collagen fibers.
Results:
Part one: The rate of models of type 2 diabetes mellitus induced by 2 month feeding with high glucose and grease and streptozocin injection was 70%. In the models of skin healing, the healing of control group was faster than the DM group, as same as the area of healing(P<0.05). HE stain suggested: the emergence time of granulation tissue was later and less. The neogenesis of vessels in granulation tissue was not active. The during of Inflammatory reaction was longer, and severe Inflammatory reaction and necrosis could be deteced in advanced stage. The immunohistochemistry stain showed: the expression of PCNA in DM group was higher than control group at 3 days and 1 week after surgery(P<0.05), but at 2 and 4 weeks there were no Significant difference between the 2 groups; the expression of CD8 in DM group was higher at 3days, 1 week and 4 weeks, but lower at 2 weeks(P<0.05); the expression of MMP9 in DM group was higher at all time, as some as the Van Geisen stain.
Part two: After passaged, BMSCs were grown quickly with long-fusiform shape. The cells were positive expressed CD90 and CD44, but negative expressed CD45. bFGF had been transfected into BMSCs, and stable expressed. The transfected BMSCs grew well in SIS.
Part three: The macroscopy and HE stain showed: there were no significant difference between the tissue-engineered skin group and control group at the 3 days, but showed the significant difference between two groups at 1,2 and 4 weeks(P<0.05). The immunohistochemistry stain suggested: the expression of PCNA in tissue-engineered skin group was higher than control group at 3 days, 1 and 2 weeks, but lower at 4 weeks(P<0.05); there were no significant difference between the two groups in the expression of CD8; there were no significant difference between the two groups in the expression of MMP9 at 3 days and 1 weeks, but significant difference at 2 and 4 weeks(P<0.05); the color were more obvious in tissue-engineered skin group by Van Geisen stain at all time; the HCK stain werepositive at 4 weeks.
Conclusion:
Part one: 1. The rabbit type 2 diabetes mellitus model could be induced by 2 monthfeeding with high glucose and grease and streptozocin injection. 2. The capability ofskin healing in diabetes mellitus was poor, the slow union and disunion occurrence. 3. Theemergence time of granulation tissue was later and less; the neogenesis of vessels in granulationtissue was not active; the capability of hyperblastosis and repairing was hysteresis and disorder;the ECM disintegration was excess and local immunity was abnormal, excess and disorder; andthe main reason of poor capability in skin healing with DM might be the severe and long duringinflammatory disorder.
Part two: 1. BMSCs were grown quickly with long-fusiform shape. 2. bFGF had been transfectedinto BMSCs, and stable expressed. 3. The transfected BMSCs grew well in SIS, andtissue-engineered skin was constructed successfully.
Part three: 1. The BMSCs could differentiate into epidermis cells.2. The tissue-engineeredskin could improve the skin healing in DM. 3. The better repairing could be obtained as bFGF.
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