SFN基因治疗促进创面愈合的在体研究

发布时间：2018-05-15 06:08

本文选题：SFN + 创面愈合　；参考：《西北大学》2016年硕士论文

【摘要】：研究背景创面愈合是外科学的主要问题之一。近期有研究表明通过对Hippo通路中关键组成元件Yap的调控可以促进创面愈合。SFN作为14-3-3家族成员之一,调节信号转导通路及细胞周期,调节细胞增殖,分化,存活,凋亡。已有报道证明SFN蛋白通过与家族其他成员发生同源或是异源二聚化影响Yap的核浆穿梭。因此,我们推测截短型SFN可以通过控制Hippo通路中Yap的核浆穿梭调节表皮细胞增殖及肉芽组织形成。基于这些研究,我们设计了缺少C端的40个氨基酸的截短型SFN,它与14-3-3家族成员形成异源二聚体影响了该家族与Yap的结合,导致Yap发生核转位,Yap与PP2A结合进入到细胞核,与TEAD发生作用,共同起始转录,因而,表皮细胞持续增殖,促进创面愈合。实验目的探索野生型和截短型SFN基因治疗对创面愈合的影响,分析截短型SFN体内影响Yap胞浆分布来调节Hippo信号通路促进表皮细胞增殖的分子机制,为基因治疗创面愈合的高效性提供科学依据。实验方法1)利用RT-PCR扩增目的基因,分子克隆技术对野生型及截短型真核表达载体进行构建；2)使用Si02法大提质粒并使用TritonX-114等温相分离法去除内毒素；3)将40只8w的健康雌性C57小鼠随机分为5组,每只小鼠麻醉后背部脱毛,在脊柱中线两侧构建小鼠全层皮肤缺损模型,PEI体内介导F-127,空载体pSecX,野生型pSecX-SFN1和截短型pSecX-SFN2的基因治疗,在各组小鼠治疗第3d,第5d,第7d伤口组织取材,测量伤口愈合面积,分析血管化,对组织包埋切片,进行HE染色和Masson染色,进一步通过再上皮化及再生胶原纤维探讨该基因对创面愈合的治疗效果。实验结果1)成功构建截短型和野生型真核表达载体,通过双酶切及测序鉴定了其正确性；2)使用Si02等温相内毒素去除法提取到高纯度质粒；3)基因治疗小鼠创面愈合。第3d时,截短型SFN2治疗组残余伤口面积64.43±1.18mm2,伤口边缘有少量较厚再上皮化和胶原纤维生成,各组均有肉芽组织形成。治疗第5d时,治疗组与对照组统计学均有显著性差异P0.05,野生型SFN1治疗组残留伤口面积为70.94±3.09mm2,再上皮化和胶原纤维最少,伤口附近的血管化较为丰富,截短型SFN2治疗组的愈合速率远远超过其他组,残留伤口面积仅剩16.02±2.59mm2,增殖的表皮细胞数量及沉积的胶原纤维最多,伤口及其周围血管化较少。治疗第7d时,各组表皮细胞由边缘向中心位置增殖迁移,截短型SFN2治疗组与野生型SFN1治疗组统计学差异显著P0.05,截短型SFN2治疗组完全再上皮化,大量胶原纤维排列在表皮细胞下,血管化较少,结果显示截短型pSecX-SFN2促进伤口愈。
[Abstract]:Background wound healing is one of the major problems in surgery. Recent studies have shown that regulation of Yap, a key component of Hippo pathway, can promote wound healing as a member of 14-3-3 family, regulate signal transduction pathway and cell cycle, regulate cell proliferation, differentiation, survival and apoptosis. It has been reported that SFN protein affects the nuclear and cytoplasmic shuttle of Yap by homology or heterodimerization with other family members. Therefore, we speculate that truncated SFN can regulate epidermal cell proliferation and granulation tissue formation by controlling the nuclear and cytoplasmic shuttle of Yap in Hippo pathway. Based on these studies, we designed 40 truncated SFNs lacking C-terminal amino acids, which formed heterodimers with 14-3-3 family members to affect the binding of the family to Yap, resulting in the nuclear translocation of Yap, Yap and PP2A binding into the nucleus. It acts with TEAD and begins transcription together. Therefore, epidermal cells proliferate continuously and promote wound healing. Objective to investigate the effects of wild-type and truncated SFN gene therapy on wound healing, and to analyze the molecular mechanism of Hippo signaling pathway in regulating the cytoplasmic distribution of Yap in truncated SFN. To provide scientific basis for the high efficiency of gene therapy wound healing. Method 1) the target gene was amplified by RT-PCR. The wild and truncated eukaryotic expression vectors were constructed by molecular cloning technique. The plasmid was extracted by Si02 method and the endotoxin was removed by TritonX-114 isothermal phase separation method. 40 healthy female C57 mice were randomly divided into 5 groups. After anesthesia, each mouse was treated with hair loss on the back, and the full-thickness skin defect model was constructed on both sides of the midline of the spine. The gene therapy of F-127, pSecX, wild type pSecX-SFN1 and truncated pSecX-SFN2 was mediated in vivo. The wound tissue was taken from each group on the 3rd, 5th and 7th day of treatment. The wound healing area was measured, vascularization was analyzed, tissue embedded sections were stained with HE and Masson staining, and the therapeutic effect of the gene on wound healing was further studied by re-epithelialization and regeneration of collagen fibers. Results 1) truncated eukaryotic expression vectors and wild type eukaryotic expression vectors were successfully constructed, and their correctness was confirmed by double enzyme digestion and sequencing. The high purity plasmids were extracted by Si02 isothermal phase endotoxin removal method. On the 3rd day, the area of residual wound in truncated SFN2 group was 64.43 卤1.18mm-2, there was a little thicker reepithelialization and collagen fiber formation at the edge of the wound, and granulation tissue was formed in each group. At the 5th day of treatment, there was significant difference between the treatment group and the control group (P 0.05). The area of residual wound in the wild-type SFN1 treatment group was 70.94 卤3.09m2, and the reepithelialization and collagen fiber were the least, and the vascularization near the wound was abundant. The healing rate of truncated SFN2 group was much higher than that of other groups. The area of residual wound was only 16.02 卤2.59mm2.The number of proliferative epidermis cells and collagen fibers deposited were the most, and the vascularization of wound and its surroundings was less. At the 7th day of treatment, the epidermal cells proliferated and migrated from the edge to the center in each group. There was a significant difference between the truncated SFN2 treatment group and the wild-type SFN1 treatment group (P0.05). The truncated SFN2 treatment group was completely re-epithelialized and a large number of collagen fibers were arranged under the epidermal cells. The results showed that truncated pSecX-SFN2 promoted wound healing.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R450;R64

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