携带KGF基因的减毒沙门氏菌（SPK）的构建及其治疗作用
发布时间：2018-10-10 07:41
【摘要】：角质细胞生长因子(keratinocyte growth factor, KGF)可调节多种上皮细胞的增殖、迁移和分化过程,目前已用于治疗多种疾病。但是,KGF蛋白在体内的半衰期较短,而大剂量多次给药又会引起严重的不良反应,加之KGF重组蛋白价格昂贵,限制了其临床应用。因此,本研究拟采用减毒沙门氏菌作为载体,观察KGF基因药物对于疾病的治疗效果。 通过基因工程方法,将KGF基因连接至真核表达载体,经电转化方法将其导入减毒沙门氏菌中,并鉴定出了携带KGF基因的阳性菌株,命名为Salmonella-plasmid-KGF菌株,简称为SPK菌株,然后观察了SPK对三种常见难治性疾病的治疗效果。 辐射复合皮肤损伤是一种难愈合的创面,伤口愈合缓慢,而且易出现感染、水肿等并发症,目前仍无有效的治疗方法。首先用SPK菌株转染人角质形成细胞系HaCaT细胞,证明KGF基因可在细胞内高效表达,并促进细胞增殖。在大鼠辐射复合皮肤损伤模型中,局部伤口周围注射SPK菌株可抑制炎症反应,促进血管新生和肉芽组织的形成,提高皮肤伤口愈合速率。 放射治疗可引起辐射性肺炎、肺纤维化等严重的副作用,极大地影响病人的生活质量,干扰肿瘤治疗效果。通过气管滴注SPK菌株,可显著降低X射线引起的大鼠肺组织损伤,抑制炎症反应,降低肺灌洗液中的总细胞数;使得肺组织中超氧化物歧化酶(superoxide dismutase, SOD)活性升高,同时丙二醛(malondialdehyde, MDA)含量下降,抵御了氧自由基(reactive oxygen species, ROS)积累造成的损伤;此外,给予SPK还可调节某些细胞因子的表达水平,维持肺脏正常的生理功能。 溃疡性结肠炎是一种病因不明、复发率高、难治愈的炎症性肠病,目前尚缺乏有效的治疗药物。在乙酸诱导的大鼠溃疡性结肠炎中,口服SPK菌株,可明显缓解结肠炎症状;抑制肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)的过量表达,降低炎症反应;降低ROS对机体造成的损害;同时SPK治疗还能提高KGF受体(keratinocyte growth factor receptor, KGFR)的表达水平,促进肠上皮细胞的增殖和受损组织的修复。 三种动物模型的实验结果表明,SPK可有效感染上皮细胞,促进细胞增殖和迁移、抗氧化和抗炎症反应等,调节病理过程,修复损伤组织。SPK可能成为一种治疗上皮组织损伤的新型有效方法。
[Abstract]:Keratinocyte growth factor (keratinocyte growth factor, KGF) can regulate the proliferation, migration and differentiation of many kinds of epithelial cells. However, the half-life of KGF protein in vivo is short, and the high dose of KGF protein can cause serious adverse reactions, and the high price of KGF recombinant protein limits its clinical application. Therefore, in this study, attenuated Salmonella was used as a carrier to observe the therapeutic effect of KGF gene drugs on disease. The KGF gene was ligated into eukaryotic expression vector by genetic engineering method, and it was introduced into attenuated Salmonella by electrotransformation. The positive strain carrying KGF gene was identified as Salmonella-plasmid-KGF strain, or SPK strain for short. Then the therapeutic effect of SPK on three common refractory diseases was observed. Radiation combined skin injury is a difficult wound to heal. The wound healing is slow, and it is prone to infection, edema and other complications. At present, there is still no effective treatment. Firstly, SPK strain was transfected into human keratinocytes HaCaT cell line. It was proved that KGF gene could be highly expressed in the cells and promote cell proliferation. In the model of combined skin injury induced by radiation injection of SPK strain around the wound could inhibit inflammatory reaction promote angiogenesis and granulation tissue formation and improve the healing rate of skin wound. Radiation therapy can cause serious side effects such as radiation pneumonia, pulmonary fibrosis and so on. The trachea drip of SPK strain could significantly reduce the lung tissue injury, inhibit inflammatory reaction, decrease the total number of cells in lung lavage fluid, and increase the activity of superoxide dismutase (superoxide dismutase, SOD) in lung tissue. At the same time, the content of malondialdehyde (malondialdehyde, MDA) decreased, which could resist the damage caused by the accumulation of oxygen free radical (reactive oxygen species, ROS), in addition, SPK could regulate the expression of some cytokines and maintain the normal physiological function of lung. Ulcerative colitis is an inflammatory bowel disease with unknown etiology, high recurrence rate and difficult to cure. In acetic acid-induced ulcerative colitis of rats, oral administration of SPK strain could significantly relieve the symptoms of colitis, inhibit the overexpression of tumor necrosis factor- 伪 (tumor necrosis factor- 伪, TNF- 伪, reduce inflammatory reaction, reduce the damage caused by ROS. At the same time, SPK can also increase the expression of KGF receptor (keratinocyte growth factor receptor, KGFR), promote the proliferation of intestinal epithelial cells and repair of damaged tissues. The experimental results of three animal models showed that SPK could effectively infect epithelial cells, promote cell proliferation and migration, antioxidation and anti-inflammatory reaction, and regulate the pathological process. Repair of injured tissue. SPK may be a novel and effective method for the treatment of epithelial tissue injury.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R818
本文编号：2261200
[Abstract]:Keratinocyte growth factor (keratinocyte growth factor, KGF) can regulate the proliferation, migration and differentiation of many kinds of epithelial cells. However, the half-life of KGF protein in vivo is short, and the high dose of KGF protein can cause serious adverse reactions, and the high price of KGF recombinant protein limits its clinical application. Therefore, in this study, attenuated Salmonella was used as a carrier to observe the therapeutic effect of KGF gene drugs on disease. The KGF gene was ligated into eukaryotic expression vector by genetic engineering method, and it was introduced into attenuated Salmonella by electrotransformation. The positive strain carrying KGF gene was identified as Salmonella-plasmid-KGF strain, or SPK strain for short. Then the therapeutic effect of SPK on three common refractory diseases was observed. Radiation combined skin injury is a difficult wound to heal. The wound healing is slow, and it is prone to infection, edema and other complications. At present, there is still no effective treatment. Firstly, SPK strain was transfected into human keratinocytes HaCaT cell line. It was proved that KGF gene could be highly expressed in the cells and promote cell proliferation. In the model of combined skin injury induced by radiation injection of SPK strain around the wound could inhibit inflammatory reaction promote angiogenesis and granulation tissue formation and improve the healing rate of skin wound. Radiation therapy can cause serious side effects such as radiation pneumonia, pulmonary fibrosis and so on. The trachea drip of SPK strain could significantly reduce the lung tissue injury, inhibit inflammatory reaction, decrease the total number of cells in lung lavage fluid, and increase the activity of superoxide dismutase (superoxide dismutase, SOD) in lung tissue. At the same time, the content of malondialdehyde (malondialdehyde, MDA) decreased, which could resist the damage caused by the accumulation of oxygen free radical (reactive oxygen species, ROS), in addition, SPK could regulate the expression of some cytokines and maintain the normal physiological function of lung. Ulcerative colitis is an inflammatory bowel disease with unknown etiology, high recurrence rate and difficult to cure. In acetic acid-induced ulcerative colitis of rats, oral administration of SPK strain could significantly relieve the symptoms of colitis, inhibit the overexpression of tumor necrosis factor- 伪 (tumor necrosis factor- 伪, TNF- 伪, reduce inflammatory reaction, reduce the damage caused by ROS. At the same time, SPK can also increase the expression of KGF receptor (keratinocyte growth factor receptor, KGFR), promote the proliferation of intestinal epithelial cells and repair of damaged tissues. The experimental results of three animal models showed that SPK could effectively infect epithelial cells, promote cell proliferation and migration, antioxidation and anti-inflammatory reaction, and regulate the pathological process. Repair of injured tissue. SPK may be a novel and effective method for the treatment of epithelial tissue injury.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R818
【参考文献】
相关期刊论文 前1条
1 马增翼;张军;李雪丽;席子明;胡延忠;;热休克转录因子4b的克隆表达及MAP激酶P38对其磷酸化调控[J];细胞与分子免疫学杂志;2010年04期
，本文编号：2261200
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