【作者】 杨磊；

【导师】 高建华；

【作者基本信息】 第一军医大学 ， 整形外科学， 2004， 博士

【摘要】 研究目的：创面愈合是烧伤治疗的难题，多种因素影响往往导致创面愈合延迟，愈合后瘢痕增生。本研究对如何优化创面愈合，即加速上皮生长促进创面愈合、又可防止瘢痕过度增生进行了基因治疗的尝试。通过脂质体介导外源基因转导烧伤创面的手段，将Ⅰ型胰岛素样生长因子(IGF-Ⅰ)和单纯疱疹病毒胸苷激酶(HSV-tk)基因联合转导烧伤创面，检测IGF-Ⅰ、HSV-tk的表达，观察IGF-Ⅰ对创面愈合的促进作用及HSV-tk／GCV对瘢痕增生的抑制作用，从而为基因治疗用于烧伤外科领域打下基础。研究方法：本研究共分两个部分内容。 第一部分IGF-Ⅰ和HSV-tk重组真核表达载体的构建。在已有的原核表达质粒pUChIGF-Ⅰ和pUChHyTk的研究基础上，利用PCR技术分别扩增IGF-Ⅰ和tk基因，将其克隆至真核表达载体pcDNA3.1中，构建重组真核表达质粒pcDNA3.1／IGF-Ⅰ和pcDNA3.1／tk。 本部分研究分为5步骤： 1．原核表达质粒pUChIGF-Ⅰ和pUChHyTk的分别PCR扩增，获取IGF-Ⅰ和HSV-tk基因DNA片段 2．IGF-Ⅰ和HSV-tk基因片段与载体pcDNA3.1体外重组 3．重组体pcDNA3.1／IGF-Ⅰ和pcDNA3.1／tk转化大肠杆菌BL21(DE3)感受态细胞 4．转化子的筛选和鉴定第一军医大学博士研究生学位论文嘟 5.重组质粒peDNA3 .1/IGF一I和peDNA3 .1/tk中插入基因IGF一I和tk的序列测定和分析 第二部分脂质体介导IGF一I和tk联合基因转移对烫伤大鼠创面愈合作用的研究。通过脂质体介导PcDNA3 .1/I GF一I、PcDNA3.1/tk重组质粒联合转染烫伤大鼠皮肤组织，观察烫伤大鼠体重、创面愈合情况等指标，并分别检测IGF一I和tk基因的表达情况。 可分为5项内容: 1.烫伤动物模型的制作和动物实验的实施、标本的获取 2.免疫组化染色检测IGF一I基因在创面局部及肝脏组织的表 达情况 3.放射免疫方法检测血清中IGF一I的水平 4.RT一PCR方法检测tk基因在创面局部的表达 5.透射电镜观察注射GCV后tk基因表达阳性的成纤维细胞 的凋亡结果:第一部分结果显示:重组质粒经酶切鉴定与预期结果一致，DNA测序结果表明IGF一I和HsV一tk基因已分别正确插入到peDNA3 .1质粒，构建成功了各自的重组真核表达载体。 第二部分结果显示:免疫组化染色显示脂质体介导的IGF一I基因转移可在创面成纤维细胞中出现阳性表达，而肝脏组织中未出现阳性表达;RT一PCR检测脂质体介导tk基因也可在创面组织出现阳性表达;大鼠创面注射IGF一I重组质粒后，创面愈合较对照组显著加快(p<0.05)，大鼠体重同未注射IGF一I的大鼠比较未出现减轻现象(p<0.05);创面局部注射IGF一I重组质粒不会影响血清中IGF一I的水平;创面局部注射GCV后，可促进tk基因表达阳性的成纤维第4页第一军医大学博士研究生学位论文嘟细胞出现凋亡。本部分研究结果说明，脂质体介导的IGF一I和tk联合基因转移可以促进创面愈合，避免伤后体重下降，并可适当地控制瘫痕增生;研究结果同时也提示，创面局部实施基因联合转移所产生的生理效应，是局部基因表达的结果，不会对全身产生影响。结论:本研究采用基因工程技术，成功地将原核表达载体中的IGF一I和tk基因亚克隆至真核表达载体peDNA3 .1上，构建成功重组真核表达质粒peDNA3 .1/l GF一I和pcDNA3.1/tk;脂质体介导PcDNA3 .1/I GF一I和pcDNA3.1/tk到烫伤大鼠创面周围，可以观察到IGF一I和tk基因在注射点附近皮肤均可出现阳性表达;伤后适当时间注射GCV可以促进tk基因表达阳性的成纤维细胞出现凋亡;脂质体介导IGF一I和tk联合基因转移，可促进创面愈合，并对瘫痕增生有一定的抑制作用;本研究通过脂质体介导IGF一I和tk联合基因转移，为创面愈合提供了一条基因治疗的新思路。更多还原

【Abstract】 OBJECTIVE: Wound healing is of major important to the survival and clinical outcome of burn patients, but it meets with many problems during treatment. In order to probe into the effect of gene therapy for treatment of wound healing, combined exogenous gene of IGF-I and HSV-tk transported by liposome were conducted to the wound of scald rats. The gene expression of IGF-I and HSV-tk were detected respectively. In the meantime, the benefit of IGF-I gene transduction on wound healing, and inhibition of tk gene transfer on fibroblast were observed. The gene therapy of this study has made a positive trial in burn and plastic surgery. METHODS: The study is composed of two parts.The first part tends to actualize construction of recombinant eukaryotic expression vector of IGF-1 and HSV-tk. In this period, IGF-I and tk gene from prokaryotic plasmids were amplified with PCR techniques, and were cloned to eukaryotic plasmid of pcDNA3.l respectively, then eukaryotic recombinant plasmid of pcDNA3.1/IGF-I and pcDNA3.1/tk were constructed successfully.There were 5 steps in the first part. First, Harvest for DNA fragments of IGF-I and HSV-tk from prokaryotic vectors of pUChIGF-I and pUChHyTk by PCR methods respectively. Second, Recombination of DNA fragments of IGF-I and HSV-tk with plasmid pcDNA3.1 in vitro. Third, Transformation of permissive cell of Bacillus coli conducted byrecombinant pcDNA3.1/IGF-I and pcDNA3.1/tk. Fourth, screening and evaluation of transformant. Finally, Assay and analysis of gene sequence of IGF-I and tk in recombinant plasmid pcDNA3.1/IGF-I and pcDNA3.1/tk.The second part aims to probe into the effect of combined gene transfer of IGF-I and tk conducted by liposome on wound healing of scald rat.In this part, pcDNA3.1/IGF-I and pcDNA3.1/tk construct transported by liposome were injected subdermally in wound region of scald rats. Changes of body weight, tendency of wound healing were observed carefully within 5 weeks postburn. The expression of IGF-1 gene transfer was observed with immunohistochemical staining, and expression of tk gene transfer was detected by RT-PCR technique; on the other hand, after GCV was injected, apoptosis of fibroblast positive to tk gene transfer was observed under transmission electron microscope.Results: The results of agarose gel electrophoresise evidenced that IGF-I and tk gene fragment was correctly inserted into the cloning site of the eukaryotic expression plasmid vector pcDNA3.1 respectively. The gene fragments from pcDNA3.1/IGF-I and pcDNA3.1/tk, digested with Hind III and EcoR I restriction enzymes, were as same as those digested with the same restriction enzymes from pUChIGF-I and pUChHyTk. Furthermore, DNA sequence analysis of IGF-I and tk gene indicated that target gene was corrected reconstructed separately.The results of animal experiment showed that IGF-I gene transported by liposome expressed positively in fibroblast in the burn wound withimmunohistochemical staining, and expression of tk gene transfer can also be detected by RT-PCR technique; meanwhile, after GCV was injected, apoptosis of fibroblast positive to tk gene transfer was observed under transmission electron microscope.There were no significant differences in the serum IGF-I concentrations among treatment groups and control group (p>0.05). The speed of wound healing for animals injected with IGF-I reconstruct was faster than those which didn’t injected IGF-I (p<0.05); The body weight of the animals in groups injected with IGF-I (group A,C1,C2) hadn’t a loss in 5 weeks postburn, but those which didn’t injected IGF-I (group B,D) had a loss in body during post burn days (p<0.05); After GCV injection, apoptosis of fibroblast positive to tk gene transfer could be detected under transmission electron microscope. CONCLUSION: Eukaryotic recombinant plasmid of pcDNA3.1/ IGF-I and pcDNA3.1/tk were constructed successfully by subcloning method. The recombinant DNA plasmids were introduced into burn wound of rat respectively or collectively by liposome-mediated transfection. Positive expression更多还原