發(fā)布時(shí)間：2018-10-10 07:41

【摘要】：角質(zhì)細(xì)胞生長(zhǎng)因子(keratinocyte growth factor, KGF)可調(diào)節(jié)多種上皮細(xì)胞的增殖、遷移和分化過(guò)程,目前已用于治療多種疾病。但是,KGF蛋白在體內(nèi)的半衰期較短,而大劑量多次給藥又會(huì)引起嚴(yán)重的不良反應(yīng),加之KGF重組蛋白價(jià)格昂貴,限制了其臨床應(yīng)用。因此,本研究擬采用減毒沙門(mén)氏菌作為載體,觀察KGF基因藥物對(duì)于疾病的治療效果。 通過(guò)基因工程方法,將KGF基因連接至真核表達(dá)載體,經(jīng)電轉(zhuǎn)化方法將其導(dǎo)入減毒沙門(mén)氏菌中,并鑒定出了攜帶KGF基因的陽(yáng)性菌株,命名為Salmonella-plasmid-KGF菌株,簡(jiǎn)稱(chēng)為SPK菌株,然后觀察了SPK對(duì)三種常見(jiàn)難治性疾病的治療效果。 輻射復(fù)合皮膚損傷是一種難愈合的創(chuàng)面,傷口愈合緩慢,而且易出現(xiàn)感染、水腫等并發(fā)癥,目前仍無(wú)有效的治療方法。首先用SPK菌株轉(zhuǎn)染人角質(zhì)形成細(xì)胞系HaCaT細(xì)胞,證明KGF基因可在細(xì)胞內(nèi)高效表達(dá),并促進(jìn)細(xì)胞增殖。在大鼠輻射復(fù)合皮膚損傷模型中,局部傷口周?chē)⑸銼PK菌株可抑制炎癥反應(yīng),促進(jìn)血管新生和肉芽組織的形成,提高皮膚傷口愈合速率。 放射治療可引起輻射性肺炎、肺纖維化等嚴(yán)重的副作用,極大地影響病人的生活質(zhì)量,干擾腫瘤治療效果。通過(guò)氣管滴注SPK菌株,可顯著降低X射線引起的大鼠肺組織損傷,抑制炎癥反應(yīng),降低肺灌洗液中的總細(xì)胞數(shù);使得肺組織中超氧化物歧化酶(superoxide dismutase, SOD)活性升高,同時(shí)丙二醛(malondialdehyde, MDA)含量下降,抵御了氧自由基(reactive oxygen species, ROS)積累造成的損傷;此外,給予SPK還可調(diào)節(jié)某些細(xì)胞因子的表達(dá)水平,維持肺臟正常的生理功能。 潰瘍性結(jié)腸炎是一種病因不明、復(fù)發(fā)率高、難治愈的炎癥性腸病,目前尚缺乏有效的治療藥物。在乙酸誘導(dǎo)的大鼠潰瘍性結(jié)腸炎中,口服SPK菌株,可明顯緩解結(jié)腸炎癥狀;抑制腫瘤壞死因子-α(tumor necrosis factor-α, TNF-α)的過(guò)量表達(dá),降低炎癥反應(yīng);降低ROS對(duì)機(jī)體造成的損害;同時(shí)SPK治療還能提高KGF受體(keratinocyte growth factor receptor, KGFR)的表達(dá)水平,促進(jìn)腸上皮細(xì)胞的增殖和受損組織的修復(fù)。 三種動(dòng)物模型的實(shí)驗(yàn)結(jié)果表明,SPK可有效感染上皮細(xì)胞,促進(jìn)細(xì)胞增殖和遷移、抗氧化和抗炎癥反應(yīng)等,調(diào)節(jié)病理過(guò)程,修復(fù)損傷組織。SPK可能成為一種治療上皮組織損傷的新型有效方法。
[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- 偽 (tumor necrosis factor- 偽, TNF- 偽, 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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R818

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 馬增翼;張軍;李雪麗;席子明;胡延忠;;熱休克轉(zhuǎn)錄因子4b的克隆表達(dá)及MAP激酶P38對(duì)其磷酸化調(diào)控[J];細(xì)胞與分子免疫學(xué)雜志;2010年04期

，

本文編號(hào)：2261200