KGF噬菌体活性肽构建及防治放射性口炎的实验研究
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摘要
口腔癌是严重危害人类健康的恶性疾患,对口腔癌治疗的长期探索提示放射治疗是口腔癌最有效的治疗方法之一。但是,口腔癌患者接受放射治疗后常发生放射性口炎,目前应用的多种治疗方法均难以达到满意的治疗效果,这严重影响了患者的生活质量,并干扰了患者的系统治疗。因此,促进口腔黏膜上皮细胞增殖迁移,修复缺损的口腔黏膜,是防治放射性口炎的关键,也是目前肿瘤支持治疗的研究热点。
角化细胞生长因子(Keratinocyte growth factor, KGF)是一种上皮细胞特异性的生长因子,是目前已知的与上皮细胞创伤愈合密切相关的细胞因子,能够与上皮细胞上的特异性受体相结合,在上皮损伤修复过程中大量表达,并具有促进上皮细胞增殖和迁移的作用,可促进上皮细胞从伤口边缘移行到基质,加速再上皮化。有研究表明KGF有助于修复放射性口炎。KGF能够抑制口腔黏膜上皮细胞凋亡和DNA损伤,并促进口腔黏膜上皮细胞增殖和迁移,从而保护口腔黏膜组织,促进其再生。口腔局部应用KGF喷雾剂或注射KGF,能够显著促进口腔黏膜细胞增殖和迁移,增强其活性,促进口腔黏膜炎明显好转。但是很多研究证实KGF与肿瘤的发生、发展及转移密切相关。KGF因为其促肿瘤生长的潜在风险,被禁止用于治疗实体瘤患者发生的口腔黏膜炎。而且目前应用的KGF产品生物半衰期短,稳定性差,易降解失活。这些因素限制了KGF的广泛应用。
噬菌体随机肽库涵盖性好,己广泛用于抗原表位的模拟和蛋白质工程的改造等方面,特别是小分子活性肽的展示,可用于研制新型创面药物和生长因子受体激动剂。利用该技术己成功筛选获得多种生长因子的模拟肽,例如：FGF, CTGF和VEGF等。利用噬菌体筛选技术筛选获得多种可编码生物活性KGF的关键基因片段,应用噬菌体展示技术,构建展示KGF噬菌体活性肽。与天然蛋白相比,合成肽具有分子量小、作用单一、稳定性好、免疫原性低、可溶性好和可大量生产等多种优点。因此,利用噬菌体随机肽库筛选技术筛选KGF的关键基因,继而采用噬菌体构建和展示技术,构建展示制备KGF噬菌体活性肽,有望增强其天然蛋白的促上皮细胞增殖、迁移的作用,同时增加稳定性,降低致瘤性等副作用,为局部治疗防治放射性口炎提供新型药物。
本研究为提高KGF的活性、应用效率和应用安全性,为人类口腔癌放射治疗后的放射性口炎的防治提供一种新型药物,有助于改善肿瘤患者的生活质量,有助于患者接受正规的治疗,为最终治愈肿瘤提供较好的辅助性治疗。
第一部分KGF噬菌体模拟肽筛选及离体实验
目的以KGF的单克隆抗体为靶,应用噬菌体随机7肽库生物淘选具有刺激人口腔黏膜上皮细胞增殖作用的KGF噬菌体模拟肽,分析KGF的序列结构。
方法以KGF的单克隆抗体为靶,进行4轮生物淘选噬菌体随机7肽库富集包含KGF特异性模拟肽的噬菌体,对随机挑选的噬菌体克隆进行扩增,并进行ELISA检测,选择ELISA结果较好的单克隆噬菌体,提取DNA进行测序和序列分析,并分析序列的相拟性；应用MTT方法检测KGF噬菌体模拟肽促进人口腔黏膜上皮细胞增殖的作用；用免疫荧光法鉴定KGF噬菌体活性肽与人口腔黏膜上皮细胞的细胞亲和力；用实时荧光定量PCR法检测KGFR,人防御素β3, c-Fos和c-Jun的表达情况。
结果经过4轮生物淘选,富集了特异性噬菌体。ELISA结果显示部分噬菌体包含KGF特异性的模拟肽。测序发现两种KGF噬菌体模拟肽,MTT结果显示这两种KGF噬菌体模拟肽能够促进人口腔黏膜上皮细胞增殖。免疫荧光鉴定结果显示这两种KGF噬菌体模拟肽具有较好的细胞亲和力。RT-PCR结果显示KGF和两种KGF噬菌体模拟肽能够促进KGFR的表达。人防御素β3在KGF对照组和两种KGF噬菌体模拟肽组中高表达,c-Fos和c-Jun在KGF对照组中高表达,而在两种KGF噬菌体模拟肽组中表达不增加,或表达较弱。
结论从噬菌体随机7肽库中生物淘选到了两种KGF噬菌体模拟肽,能够显著促进人口腔黏膜上皮细胞增殖；这两种KGF噬菌体模拟肽能够促进KGFR的表达,可能是一种正反馈反应；这两种KGF噬菌体模拟肽能够促进人防御性β3的表达,可能有助于控制溃疡创面的感染,有助于创面早日封闭；这两种KGF噬菌体模拟肽不会促进肿瘤基因c-Fos和c-Jun的表达,具有较好的应用安全性。通过序列分析了解了KGF的关键序列。
第二部分KGF噬菌体活性肽构建展示及离体实验
目的依据第一部分生物淘选和离体细胞实验的结果,构建pComb3-KGF噬菌粒,应用噬菌体展示技术获得KGF噬菌体活性肽,检测其对人口腔黏膜上皮细胞的生物学作用。
方法以噬菌体pComb3为载体,选取载体的重链作为基因插入位置,以Spel和Xhol作为限制性内切酶；依据生物淘选和离体实验的结果,选取4个序列,设计引物；培养和保存人口腔黏膜上皮细胞,提取人口腔黏膜上皮细胞总RNA,应用RT-PCR方法获得目的基因；Spel和Xhol双酶切目的基因及噬菌粒pComb3,将目的基因亚克隆至噬菌粒pComb3;应用噬菌体呈现技术,将插入的目的基因展示于噬菌体表面,获得KGF噬菌体活性肽；抽提纯化噬菌体DNA,进行酶切、PCR和测序鉴定；应用MTT方法检测KGF噬菌体活性肽促进人口腔黏膜上皮细胞增殖的作用；应用免疫荧光方法检测KGF噬菌体活性肽的细胞亲和力；应用RT-PCR方法检测KGF噬菌体活性肽对KGFR、人防御素β3、c-Fos和c-Jun的表达水平的影响。
结果应用RT-PCR方法获得目的的基因,并构建在噬菌粒pComb3中,目的基因成功表达于载体表面；酶切、PCR和测序结果证实目的基因己成功构建在载体中；MTT结果显示4种KGF噬菌体活性肽能够促进人口腔黏膜上皮细胞增殖；免疫荧光检测结果显示4种KGF噬菌体活性肽具有较好的细胞亲和力；RT-PCR结果显示KGF及两种KGF噬菌体活性肽能够促进KGFR的表达,KGF及3种KGF噬菌体活性肽能够促进人防御素β3的表达,4种KGF噬菌体活性肽促进c-Fos和c-Jun表达的作用均弱于野生型的KGF。
结论成功构建展示获得KGF噬菌体活性肽,能够显著促进人口腔黏膜上皮细胞增殖,可能有助于控制创面感染,并具有较好的应用安全性。
第三部分KGF噬菌体活性肽修复放射性口炎的实验研究
目的建立大鼠放射性口炎模型,将KGF噬菌体活性肽用于治疗大鼠放射性口炎,促进口腔溃疡的愈合。
方法采用瓦里安23-EX加速器6MVX线,源皮距100cm,照射野10×10cm2,机架角180。,单次20GY照射,吸收剂量率为40CGy/min,照射时间6分钟。清洁级雄性Wistar大鼠25只,随机抽出20只以上述方法进行投照,剩余5只作为对照组。每天观察大鼠的一般情况、体重变化、舌及颊部口腔黏膜的变化,对口腔黏膜的红斑(充血)、糜烂和溃疡等主要体征进行评估。照射3、6和9天后随机处死2只大鼠并做病理分析,以明确建立大鼠放射性口腔黏膜炎模型的最佳条件。然后,按照己确认的最佳条件取20只Wistar大鼠建立放射性口腔黏膜炎模型,随机抽出15只放射性口腔黏膜炎Wistar实验鼠,在照射第3天(A组)后开始出现充血水肿时,第6天(B组)后开始出现小面积溃疡时,第9天(C组)后出现大面积溃疡时,皮下注射KGF噬菌体活性肽1mg/kg/d,连续5天,每天观察大鼠一般情况、体重变化,颊部及舌部口腔黏膜炎的愈合情况,并做病理分析。剩余5只作为对照组。
结果共有20只大鼠接受照射,照射20Gy后第一天摄食、饮水量无明显变化,第二天开始出现每日摄食、饮水量减少。体重呈缓慢下降,照射结束后9-12天体重下降到最低点,以后缓慢回升。舌部和颊部口腔黏膜在单次照射20Gy后第3天开始出现充血水肿；镜下见舌部棘细胞空泡变性,固有层有少量的炎症细胞浸润。照射后第6天时开始出现小溃疡面；镜下见舌部丝状乳头脱落,棘细胞变性、脱落,固有层少量炎症细胞浸润：颊黏膜表面上皮细胞脱落。照射结束后9天时,舌部和颊部出现大的溃疡面,舌部上皮脱落；镜下见舌部丝状乳头脱落,基底细胞、棘细胞变性,固有层有大量的炎症细胞浸润；颊黏膜上皮表层细胞脱落。皮下注射KGF噬菌体活性肽后A组未出现溃疡；B组溃疡未见明显扩大,并在治疗结束后3天溃疡基本愈合。C组溃疡稍微扩大,并在治疗结束后6天溃疡基本愈合；镜下见治疗后舌部上皮结构完整,固有层仍可见少量炎症细胞浸润。对照组3周后溃疡基本愈合。
结论通过放疗所致大鼠放射性口炎模型的动物试验,研究KGF噬菌体活性肽防治放射性口炎的效果及最佳的应用途径、干预时机和安全有效剂量。结果显示KGF噬菌体活性肽能有效降低口腔黏膜炎的严重程度,促进创面上皮化,尽早封闭创面,缩短愈合时间,有效地预防放射性口炎发生。
Oral cancer is a detrimental and malignant disease. After a longtime exploration, it is widely accepted that radiotherapy (RT) is one of the most effective treatments for oral cancer. However, radiotherapy-induced oral mucositis (RTOM) often occurs after RT, and to date, effective treatment for RTOM is still unavailable. This situation significantly impairs the living quality of the patients and inevitably disturbs the systemic therapy for oral cancer. To prevent RTOM, it is of particular importance to stimulate the proliferation of the oral mucosal cells and repair the damaged oral mucosa, which has been the priority in the research field of tumor supportive care.
Keratinocyte growth factor (KGF) is a growth factor specifically expressed in epithelial cells and plays an important role in epithelial wound healing. KGF is highly expressed during the wound healing processes and induces a series of intracellular signaling cascades via interaction with its receptor on the epithelial cells. KGF stimulates the cell growth, promotes the epithelial cells to migrate from wound edge to the deeper matrix, and accelerates reepithelialization. Several studies have suggested that KGF can be used to treat RTOM. KGF could reduce apoptosis and DNA damage of the oral epithelial cells, and stimulate the proliferation and migration of these cells, which makes it a promising, novel therapy to protect oral mucosa and accelerate tissue regeneration. Indeed, it has been reported that local application of an RGF spray or an oral injection of RGF is capable of promoting the healing of oral mucositis through enhancing the proliferation and migration of the epithelial cells and increasing the biological activity of these cells.
However, many studies confirmed that KGF is closely correlated with the development, progression and metastasis of tumors. Therefore, KGF cannot be used to treat oral mucositis in cancer patients because of its potential capability of promoting tumor development. Moreover, the clinical application of FGF is significantly limited by other problems such as a short half-time, poor stability, easy degradation and inactivation.
Phage random peptide library (PRPL), encompassing a great range, has been widely used in imitating antigenic epitopes and protein engineering, especially in producing molecular polypeptide. It has been used to develop new medications of accelerating wound healing and receptor agonists of the growth factors. Using this technique, the researchers have successfully developed various mimic peptides for growth factors such as such as FGF, CTGF and VEGF. Compared with the natural protein, synthetic peptide has a smaller molecular weight, more specific biological effects, greater stability, lower immunogenicity, higher solubility, and easier to be largely manufactureed.
In our previous studies, we obtained multiple pivotal gene segments which encode biologically active KGF fragments (see attachment for the detailed results). In this study, we selected the pivotal gene segments of KGF using the solid phase peptide synthesis (SPPS) technology, and then synthetized and optimized the obtained biologically active peptide of KGF. In this way, we provide a new medicine and a novel biomaterial for preventing and treating complications of the oral cancer patients who are receiving RT. As an auxiliary therapy for oral cancer, our results considerably improve the living quality of the patients and make it easier for the patients to receive standard treatment for oral cancer.
Part I:The harvest of KGF phage model peptide and in vitro experiment
Objective The objective of the study was to isolate KGF phage model peptides from a phage display7-mer peptide library to evaluate their effect on stimulating the proliferation of oral mucosal epithelial cells.
Methods KGF monoclonal antibodies were used to select the biologically active KGF fragments from the phage library for four times. Phage titer was determined to evaluate the recovery of the phages. ELISA was performed to select monoclonal phages of the fourth round with good binding activity. DNA sequencing was used to evaluate the genetic similarities of model peptides. MTT assay was used to evaluate the effect of phage model peptides on the proliferation of oral mucosal epidermal cells. Immunofluorescence assay was employed to evaluate the cell affinity of phage model peptides. Quantitative RT-PCR analysis was employed to evaluate the expressions of KGFR, human β-defensin3, c-Fos and c-Jun in the epidermal cells.
Results After the four rounds of screening, specific phage model peptides were harvested. Twenty-five phage modeling peptides were isolated from the phage display7-mer peptide library. Some of the isolated phage modeling peptides exhibited high binding activity as shown by ELISA. MTT assay showed that four phage modeling peptides could promote the proliferation of oral mucosal epidermal cells. Immunofluorescence assay displayed that the phage modeling peptides had a high cell affinity. The expression levels of keratinocyte growth factor receptor (KGFR) and human defensin β3were increased in the KGF group and the two phage model peptide groups when compared with the negative control group. The expression levels of c-Fos and c-Jun were increased in the KGF group when compared with the negative control group, while showed no difference between the phage model peptide groups and the negative control groups.
Conclusions KGF phage model peptides can be isolated from the phage display7-mer peptide library, which can safely promote the proliferation of the oral mucosal epidermal cells. Two phage model peptides can promote the expression of human defensinP3in the epidermal cells to control the wound infection. Phage model peptides can not promote the expression of c-Fos and c-Jun.
Part II:The construction of KGF phage model active peptide and in vitro experiment
Objective Based on the sequence analysis and the in vitro experiments, the specific sequences encoding the biologically active KGF fragments were selected. pComb3-KGF active peptides were constructed and expressed to be used to evaluate the effect of these peptides on promoting the proliferation of oral mucosal epidermal cells.
Methods The heavy chain of pComb3was selected to be the location to insert the exogenous gene. SpeⅠ and Xhol were chosen as the restriction endonuclease. Based on the sequence analysis and the in vitro experiments, four sequences were chosen. Primers were designed. The normal oral mucosal epidermal cells were gathered using adherent tissue culture method. The total RNA was extracted from the cells. The selected genes were obtained using RT-PCR method. The genes were subcloned into the pComb3vector after double-digested with Spel and Xhol. The technology of phage display was used to display the inserted genes on the phage surface. Phage DNA was extracted, purified, digested with restriction endonucleases, and evaluated with PCR and gene sequencing. MTT assay was used to evaluate the effect of pComb3-KGF active peptides on promoting the proliferation of oral mucosal epidermal cells. Immunofluorescence assay was employed to evaluate the cell affinity of pComb3-KGF active peptides. Quantitative RT-PCR analysis was employed to evaluate the expression of KGFR, human β-defensin3, c-Fos and c-Jun in epidermal cells.
Results The genes were obtained by RT-PCR, and subcloned into pComb3vector. The results of restriction enzyme digestion, PCR and gene sequencing proved that the genes were inserted into the pComb3vector. The proteins of the genes were expressed on the surface of the vector. MTT assay showed that all of the four pComb3-KGF active peptides could promote the proliferation of oral mucosal epidermal cells. The results of immunofluorescence assay showed that these active peptides had high cell affinity. Moreover, the expression level of KGFR was significantly increased in the KGF group and two of the pComb3-KGF active peptide groups, and the expression level of human β-defensin3was elevated in the KGF group and three of the pComb3-KGF active peptide groups. The expression levels of c-Fos and c-Jun were significantly lower in the four pComb3-KGF active peptides groups when compared with those in the KGF group.
Conclusions pComb3-KGF active peptides are successfully constructed and expressed, which can promote the proliferation of oral mucosal epidermal cells. These active peptides can also promote the expression of human defensin β3in oral mucosal epidermal cells to control the wound infection. However, these active peptides displayed a weaker effect on enhancing the expression levels of c-Fos and c-Jun when compared with the wild-type KGF.
Part three:The effect of KGF phage model active peptide used in the treatment of radiotherapy-induced oral mucositis
Objective To establish a rat model of radiation-induced oral mucositis, and to investigate the in vivo effects of KGF phage model active peptides on treating radiation-induced oral mucositis and promoting the healing of oral ulcers.
Methods A Varian23EX linear accelerator was used for the irradiation process. The distance between the irradiation source and the skin was100cm, with a radiation area of10×10cm2and a rack angle of180d°. Twenty-fire male Wistar rats were used in this study,20of which received the irradiation and the other non-irradiated rats served as controls. Tongue and buccal mucosa of the20rats was irradiated for6minutes to receive a radiation dose of20Gy with a dose rate of40CGy/min. The general situation, body weight and the oral mucosa of the irradiated and the non-irradiated rats were examined on a daily basis. The animals were sacrificed on3,6and9days after the irradiation. Histological examination of the oral mucosa was performed to evaluate the optimal conditions to establish a rat RTOM model. Twenty Wistar rats were then used to establish the RTOM model using the aforementioned method. Fifteen of the RTOM rats were randomly divided into3groups to receive subcutaneous injections of the KGF phage active peptides at a dose of lmg/kg/d for5days on3(Group A).6(Group B), or9(Group C) days after the irradiation. The general situation, body weight and the oral mucosa of the rats were examined daily until the animals were sacrificed and subjected to histological analysis. The other five RTOM rats received only vehicle injections served as controls.
Results One day after the irradiation, the rats showed no significant changes in food or water intake, which was decreased on2days after the irradiation. The body weight of the irradiated rats was decreased slowly, reached its lowest point on9～12days after the irradiation, and then showed a slow recovery. Erythema was observed in the tongue and buccal mucosa on3days after the irradiation. Histological examination showed vacuolar degeneration in the tongue squamous cells, and a small amount of infiltrated inflammatory cells were observed in the lamina propria. Ulcers were observed on the6th day after irradiation. Histological examination showed that the filiform papillae of the tongue were missing, the spinous cells were degenerated and exfoliated, and a small amount of inflammatory cells infiltrated into the lamina propria. The epithelial cells of the buccal mucosa also exfoliated.
Larger ulcers were observed on the9th day after the irradiation. Histological examination showed that the filiform papillae of the tongue exfoliated, the basal cells and the squamous cells were degenerated, and a massive amount of inflammatory cells infiltrated into the lamina propria. The epithelium cells of the buccal mucosa exfoliated. The radiation-induced ulcer in the buccal mucosa could be self-repaired3weeks after a single20Gy irradiation.
After subcutaneous injections of KGF phage active peptides, Group A showed no ulcers. Moreover, the ulcers of Group B showed no progression after the application of KGF active peptides, and the ulcers were healed3days after the KGF peptide injections. The ulcers of Group C progressed slightly after the irradiation, and the ulcers were healed6days after the KGF peptide injections. Histological analysis showed a normal structure of the tongue epithelium and a few infiltrated inflammatory cells in the lamina propria. In contrast, the ulcers of the control group were not healed until3weeks after the irradiation.
Conclusion A rat model of radiation-induced oral mucositis (RTOM) was successfully established. Using this rat model, the in vivo effect of the KGF phage active peptides on the prevention and treatment of RTOM was investigated. The optimal conditions of applying the KGF phage active peptides were also evaluated. We found that the KGF active peptides can effectively reduce the severity of RTOM. promote epithelialization of the wound area, and accelerate the healing of the ulcers, indicating that KGF active peptides are a promising therapy for RTOM.
角化细胞生长因子(Keratinocyte growth factor, KGF)是一种上皮细胞特异性的生长因子,是目前已知的与上皮细胞创伤愈合密切相关的细胞因子,能够与上皮细胞上的特异性受体相结合,在上皮损伤修复过程中大量表达,并具有促进上皮细胞增殖和迁移的作用,可促进上皮细胞从伤口边缘移行到基质,加速再上皮化。有研究表明KGF有助于修复放射性口炎。KGF能够抑制口腔黏膜上皮细胞凋亡和DNA损伤,并促进口腔黏膜上皮细胞增殖和迁移,从而保护口腔黏膜组织,促进其再生。口腔局部应用KGF喷雾剂或注射KGF,能够显著促进口腔黏膜细胞增殖和迁移,增强其活性,促进口腔黏膜炎明显好转。但是很多研究证实KGF与肿瘤的发生、发展及转移密切相关。KGF因为其促肿瘤生长的潜在风险,被禁止用于治疗实体瘤患者发生的口腔黏膜炎。而且目前应用的KGF产品生物半衰期短,稳定性差,易降解失活。这些因素限制了KGF的广泛应用。
噬菌体随机肽库涵盖性好,己广泛用于抗原表位的模拟和蛋白质工程的改造等方面,特别是小分子活性肽的展示,可用于研制新型创面药物和生长因子受体激动剂。利用该技术己成功筛选获得多种生长因子的模拟肽,例如：FGF, CTGF和VEGF等。利用噬菌体筛选技术筛选获得多种可编码生物活性KGF的关键基因片段,应用噬菌体展示技术,构建展示KGF噬菌体活性肽。与天然蛋白相比,合成肽具有分子量小、作用单一、稳定性好、免疫原性低、可溶性好和可大量生产等多种优点。因此,利用噬菌体随机肽库筛选技术筛选KGF的关键基因,继而采用噬菌体构建和展示技术,构建展示制备KGF噬菌体活性肽,有望增强其天然蛋白的促上皮细胞增殖、迁移的作用,同时增加稳定性,降低致瘤性等副作用,为局部治疗防治放射性口炎提供新型药物。
本研究为提高KGF的活性、应用效率和应用安全性,为人类口腔癌放射治疗后的放射性口炎的防治提供一种新型药物,有助于改善肿瘤患者的生活质量,有助于患者接受正规的治疗,为最终治愈肿瘤提供较好的辅助性治疗。
第一部分KGF噬菌体模拟肽筛选及离体实验
目的以KGF的单克隆抗体为靶,应用噬菌体随机7肽库生物淘选具有刺激人口腔黏膜上皮细胞增殖作用的KGF噬菌体模拟肽,分析KGF的序列结构。
方法以KGF的单克隆抗体为靶,进行4轮生物淘选噬菌体随机7肽库富集包含KGF特异性模拟肽的噬菌体,对随机挑选的噬菌体克隆进行扩增,并进行ELISA检测,选择ELISA结果较好的单克隆噬菌体,提取DNA进行测序和序列分析,并分析序列的相拟性；应用MTT方法检测KGF噬菌体模拟肽促进人口腔黏膜上皮细胞增殖的作用；用免疫荧光法鉴定KGF噬菌体活性肽与人口腔黏膜上皮细胞的细胞亲和力；用实时荧光定量PCR法检测KGFR,人防御素β3, c-Fos和c-Jun的表达情况。
结果经过4轮生物淘选,富集了特异性噬菌体。ELISA结果显示部分噬菌体包含KGF特异性的模拟肽。测序发现两种KGF噬菌体模拟肽,MTT结果显示这两种KGF噬菌体模拟肽能够促进人口腔黏膜上皮细胞增殖。免疫荧光鉴定结果显示这两种KGF噬菌体模拟肽具有较好的细胞亲和力。RT-PCR结果显示KGF和两种KGF噬菌体模拟肽能够促进KGFR的表达。人防御素β3在KGF对照组和两种KGF噬菌体模拟肽组中高表达,c-Fos和c-Jun在KGF对照组中高表达,而在两种KGF噬菌体模拟肽组中表达不增加,或表达较弱。
结论从噬菌体随机7肽库中生物淘选到了两种KGF噬菌体模拟肽,能够显著促进人口腔黏膜上皮细胞增殖；这两种KGF噬菌体模拟肽能够促进KGFR的表达,可能是一种正反馈反应；这两种KGF噬菌体模拟肽能够促进人防御性β3的表达,可能有助于控制溃疡创面的感染,有助于创面早日封闭；这两种KGF噬菌体模拟肽不会促进肿瘤基因c-Fos和c-Jun的表达,具有较好的应用安全性。通过序列分析了解了KGF的关键序列。
第二部分KGF噬菌体活性肽构建展示及离体实验
目的依据第一部分生物淘选和离体细胞实验的结果,构建pComb3-KGF噬菌粒,应用噬菌体展示技术获得KGF噬菌体活性肽,检测其对人口腔黏膜上皮细胞的生物学作用。
方法以噬菌体pComb3为载体,选取载体的重链作为基因插入位置,以Spel和Xhol作为限制性内切酶；依据生物淘选和离体实验的结果,选取4个序列,设计引物；培养和保存人口腔黏膜上皮细胞,提取人口腔黏膜上皮细胞总RNA,应用RT-PCR方法获得目的基因；Spel和Xhol双酶切目的基因及噬菌粒pComb3,将目的基因亚克隆至噬菌粒pComb3;应用噬菌体呈现技术,将插入的目的基因展示于噬菌体表面,获得KGF噬菌体活性肽；抽提纯化噬菌体DNA,进行酶切、PCR和测序鉴定；应用MTT方法检测KGF噬菌体活性肽促进人口腔黏膜上皮细胞增殖的作用；应用免疫荧光方法检测KGF噬菌体活性肽的细胞亲和力；应用RT-PCR方法检测KGF噬菌体活性肽对KGFR、人防御素β3、c-Fos和c-Jun的表达水平的影响。
结果应用RT-PCR方法获得目的的基因,并构建在噬菌粒pComb3中,目的基因成功表达于载体表面；酶切、PCR和测序结果证实目的基因己成功构建在载体中；MTT结果显示4种KGF噬菌体活性肽能够促进人口腔黏膜上皮细胞增殖；免疫荧光检测结果显示4种KGF噬菌体活性肽具有较好的细胞亲和力；RT-PCR结果显示KGF及两种KGF噬菌体活性肽能够促进KGFR的表达,KGF及3种KGF噬菌体活性肽能够促进人防御素β3的表达,4种KGF噬菌体活性肽促进c-Fos和c-Jun表达的作用均弱于野生型的KGF。
结论成功构建展示获得KGF噬菌体活性肽,能够显著促进人口腔黏膜上皮细胞增殖,可能有助于控制创面感染,并具有较好的应用安全性。
第三部分KGF噬菌体活性肽修复放射性口炎的实验研究
目的建立大鼠放射性口炎模型,将KGF噬菌体活性肽用于治疗大鼠放射性口炎,促进口腔溃疡的愈合。
方法采用瓦里安23-EX加速器6MVX线,源皮距100cm,照射野10×10cm2,机架角180。,单次20GY照射,吸收剂量率为40CGy/min,照射时间6分钟。清洁级雄性Wistar大鼠25只,随机抽出20只以上述方法进行投照,剩余5只作为对照组。每天观察大鼠的一般情况、体重变化、舌及颊部口腔黏膜的变化,对口腔黏膜的红斑(充血)、糜烂和溃疡等主要体征进行评估。照射3、6和9天后随机处死2只大鼠并做病理分析,以明确建立大鼠放射性口腔黏膜炎模型的最佳条件。然后,按照己确认的最佳条件取20只Wistar大鼠建立放射性口腔黏膜炎模型,随机抽出15只放射性口腔黏膜炎Wistar实验鼠,在照射第3天(A组)后开始出现充血水肿时,第6天(B组)后开始出现小面积溃疡时,第9天(C组)后出现大面积溃疡时,皮下注射KGF噬菌体活性肽1mg/kg/d,连续5天,每天观察大鼠一般情况、体重变化,颊部及舌部口腔黏膜炎的愈合情况,并做病理分析。剩余5只作为对照组。
结果共有20只大鼠接受照射,照射20Gy后第一天摄食、饮水量无明显变化,第二天开始出现每日摄食、饮水量减少。体重呈缓慢下降,照射结束后9-12天体重下降到最低点,以后缓慢回升。舌部和颊部口腔黏膜在单次照射20Gy后第3天开始出现充血水肿；镜下见舌部棘细胞空泡变性,固有层有少量的炎症细胞浸润。照射后第6天时开始出现小溃疡面；镜下见舌部丝状乳头脱落,棘细胞变性、脱落,固有层少量炎症细胞浸润：颊黏膜表面上皮细胞脱落。照射结束后9天时,舌部和颊部出现大的溃疡面,舌部上皮脱落；镜下见舌部丝状乳头脱落,基底细胞、棘细胞变性,固有层有大量的炎症细胞浸润；颊黏膜上皮表层细胞脱落。皮下注射KGF噬菌体活性肽后A组未出现溃疡；B组溃疡未见明显扩大,并在治疗结束后3天溃疡基本愈合。C组溃疡稍微扩大,并在治疗结束后6天溃疡基本愈合；镜下见治疗后舌部上皮结构完整,固有层仍可见少量炎症细胞浸润。对照组3周后溃疡基本愈合。
结论通过放疗所致大鼠放射性口炎模型的动物试验,研究KGF噬菌体活性肽防治放射性口炎的效果及最佳的应用途径、干预时机和安全有效剂量。结果显示KGF噬菌体活性肽能有效降低口腔黏膜炎的严重程度,促进创面上皮化,尽早封闭创面,缩短愈合时间,有效地预防放射性口炎发生。
Oral cancer is a detrimental and malignant disease. After a longtime exploration, it is widely accepted that radiotherapy (RT) is one of the most effective treatments for oral cancer. However, radiotherapy-induced oral mucositis (RTOM) often occurs after RT, and to date, effective treatment for RTOM is still unavailable. This situation significantly impairs the living quality of the patients and inevitably disturbs the systemic therapy for oral cancer. To prevent RTOM, it is of particular importance to stimulate the proliferation of the oral mucosal cells and repair the damaged oral mucosa, which has been the priority in the research field of tumor supportive care.
Keratinocyte growth factor (KGF) is a growth factor specifically expressed in epithelial cells and plays an important role in epithelial wound healing. KGF is highly expressed during the wound healing processes and induces a series of intracellular signaling cascades via interaction with its receptor on the epithelial cells. KGF stimulates the cell growth, promotes the epithelial cells to migrate from wound edge to the deeper matrix, and accelerates reepithelialization. Several studies have suggested that KGF can be used to treat RTOM. KGF could reduce apoptosis and DNA damage of the oral epithelial cells, and stimulate the proliferation and migration of these cells, which makes it a promising, novel therapy to protect oral mucosa and accelerate tissue regeneration. Indeed, it has been reported that local application of an RGF spray or an oral injection of RGF is capable of promoting the healing of oral mucositis through enhancing the proliferation and migration of the epithelial cells and increasing the biological activity of these cells.
However, many studies confirmed that KGF is closely correlated with the development, progression and metastasis of tumors. Therefore, KGF cannot be used to treat oral mucositis in cancer patients because of its potential capability of promoting tumor development. Moreover, the clinical application of FGF is significantly limited by other problems such as a short half-time, poor stability, easy degradation and inactivation.
Phage random peptide library (PRPL), encompassing a great range, has been widely used in imitating antigenic epitopes and protein engineering, especially in producing molecular polypeptide. It has been used to develop new medications of accelerating wound healing and receptor agonists of the growth factors. Using this technique, the researchers have successfully developed various mimic peptides for growth factors such as such as FGF, CTGF and VEGF. Compared with the natural protein, synthetic peptide has a smaller molecular weight, more specific biological effects, greater stability, lower immunogenicity, higher solubility, and easier to be largely manufactureed.
In our previous studies, we obtained multiple pivotal gene segments which encode biologically active KGF fragments (see attachment for the detailed results). In this study, we selected the pivotal gene segments of KGF using the solid phase peptide synthesis (SPPS) technology, and then synthetized and optimized the obtained biologically active peptide of KGF. In this way, we provide a new medicine and a novel biomaterial for preventing and treating complications of the oral cancer patients who are receiving RT. As an auxiliary therapy for oral cancer, our results considerably improve the living quality of the patients and make it easier for the patients to receive standard treatment for oral cancer.
Part I:The harvest of KGF phage model peptide and in vitro experiment
Objective The objective of the study was to isolate KGF phage model peptides from a phage display7-mer peptide library to evaluate their effect on stimulating the proliferation of oral mucosal epithelial cells.
Methods KGF monoclonal antibodies were used to select the biologically active KGF fragments from the phage library for four times. Phage titer was determined to evaluate the recovery of the phages. ELISA was performed to select monoclonal phages of the fourth round with good binding activity. DNA sequencing was used to evaluate the genetic similarities of model peptides. MTT assay was used to evaluate the effect of phage model peptides on the proliferation of oral mucosal epidermal cells. Immunofluorescence assay was employed to evaluate the cell affinity of phage model peptides. Quantitative RT-PCR analysis was employed to evaluate the expressions of KGFR, human β-defensin3, c-Fos and c-Jun in the epidermal cells.
Results After the four rounds of screening, specific phage model peptides were harvested. Twenty-five phage modeling peptides were isolated from the phage display7-mer peptide library. Some of the isolated phage modeling peptides exhibited high binding activity as shown by ELISA. MTT assay showed that four phage modeling peptides could promote the proliferation of oral mucosal epidermal cells. Immunofluorescence assay displayed that the phage modeling peptides had a high cell affinity. The expression levels of keratinocyte growth factor receptor (KGFR) and human defensin β3were increased in the KGF group and the two phage model peptide groups when compared with the negative control group. The expression levels of c-Fos and c-Jun were increased in the KGF group when compared with the negative control group, while showed no difference between the phage model peptide groups and the negative control groups.
Conclusions KGF phage model peptides can be isolated from the phage display7-mer peptide library, which can safely promote the proliferation of the oral mucosal epidermal cells. Two phage model peptides can promote the expression of human defensinP3in the epidermal cells to control the wound infection. Phage model peptides can not promote the expression of c-Fos and c-Jun.
Part II:The construction of KGF phage model active peptide and in vitro experiment
Objective Based on the sequence analysis and the in vitro experiments, the specific sequences encoding the biologically active KGF fragments were selected. pComb3-KGF active peptides were constructed and expressed to be used to evaluate the effect of these peptides on promoting the proliferation of oral mucosal epidermal cells.
Methods The heavy chain of pComb3was selected to be the location to insert the exogenous gene. SpeⅠ and Xhol were chosen as the restriction endonuclease. Based on the sequence analysis and the in vitro experiments, four sequences were chosen. Primers were designed. The normal oral mucosal epidermal cells were gathered using adherent tissue culture method. The total RNA was extracted from the cells. The selected genes were obtained using RT-PCR method. The genes were subcloned into the pComb3vector after double-digested with Spel and Xhol. The technology of phage display was used to display the inserted genes on the phage surface. Phage DNA was extracted, purified, digested with restriction endonucleases, and evaluated with PCR and gene sequencing. MTT assay was used to evaluate the effect of pComb3-KGF active peptides on promoting the proliferation of oral mucosal epidermal cells. Immunofluorescence assay was employed to evaluate the cell affinity of pComb3-KGF active peptides. Quantitative RT-PCR analysis was employed to evaluate the expression of KGFR, human β-defensin3, c-Fos and c-Jun in epidermal cells.
Results The genes were obtained by RT-PCR, and subcloned into pComb3vector. The results of restriction enzyme digestion, PCR and gene sequencing proved that the genes were inserted into the pComb3vector. The proteins of the genes were expressed on the surface of the vector. MTT assay showed that all of the four pComb3-KGF active peptides could promote the proliferation of oral mucosal epidermal cells. The results of immunofluorescence assay showed that these active peptides had high cell affinity. Moreover, the expression level of KGFR was significantly increased in the KGF group and two of the pComb3-KGF active peptide groups, and the expression level of human β-defensin3was elevated in the KGF group and three of the pComb3-KGF active peptide groups. The expression levels of c-Fos and c-Jun were significantly lower in the four pComb3-KGF active peptides groups when compared with those in the KGF group.
Conclusions pComb3-KGF active peptides are successfully constructed and expressed, which can promote the proliferation of oral mucosal epidermal cells. These active peptides can also promote the expression of human defensin β3in oral mucosal epidermal cells to control the wound infection. However, these active peptides displayed a weaker effect on enhancing the expression levels of c-Fos and c-Jun when compared with the wild-type KGF.
Part three:The effect of KGF phage model active peptide used in the treatment of radiotherapy-induced oral mucositis
Objective To establish a rat model of radiation-induced oral mucositis, and to investigate the in vivo effects of KGF phage model active peptides on treating radiation-induced oral mucositis and promoting the healing of oral ulcers.
Methods A Varian23EX linear accelerator was used for the irradiation process. The distance between the irradiation source and the skin was100cm, with a radiation area of10×10cm2and a rack angle of180d°. Twenty-fire male Wistar rats were used in this study,20of which received the irradiation and the other non-irradiated rats served as controls. Tongue and buccal mucosa of the20rats was irradiated for6minutes to receive a radiation dose of20Gy with a dose rate of40CGy/min. The general situation, body weight and the oral mucosa of the irradiated and the non-irradiated rats were examined on a daily basis. The animals were sacrificed on3,6and9days after the irradiation. Histological examination of the oral mucosa was performed to evaluate the optimal conditions to establish a rat RTOM model. Twenty Wistar rats were then used to establish the RTOM model using the aforementioned method. Fifteen of the RTOM rats were randomly divided into3groups to receive subcutaneous injections of the KGF phage active peptides at a dose of lmg/kg/d for5days on3(Group A).6(Group B), or9(Group C) days after the irradiation. The general situation, body weight and the oral mucosa of the rats were examined daily until the animals were sacrificed and subjected to histological analysis. The other five RTOM rats received only vehicle injections served as controls.
Results One day after the irradiation, the rats showed no significant changes in food or water intake, which was decreased on2days after the irradiation. The body weight of the irradiated rats was decreased slowly, reached its lowest point on9～12days after the irradiation, and then showed a slow recovery. Erythema was observed in the tongue and buccal mucosa on3days after the irradiation. Histological examination showed vacuolar degeneration in the tongue squamous cells, and a small amount of infiltrated inflammatory cells were observed in the lamina propria. Ulcers were observed on the6th day after irradiation. Histological examination showed that the filiform papillae of the tongue were missing, the spinous cells were degenerated and exfoliated, and a small amount of inflammatory cells infiltrated into the lamina propria. The epithelial cells of the buccal mucosa also exfoliated.
Larger ulcers were observed on the9th day after the irradiation. Histological examination showed that the filiform papillae of the tongue exfoliated, the basal cells and the squamous cells were degenerated, and a massive amount of inflammatory cells infiltrated into the lamina propria. The epithelium cells of the buccal mucosa exfoliated. The radiation-induced ulcer in the buccal mucosa could be self-repaired3weeks after a single20Gy irradiation.
After subcutaneous injections of KGF phage active peptides, Group A showed no ulcers. Moreover, the ulcers of Group B showed no progression after the application of KGF active peptides, and the ulcers were healed3days after the KGF peptide injections. The ulcers of Group C progressed slightly after the irradiation, and the ulcers were healed6days after the KGF peptide injections. Histological analysis showed a normal structure of the tongue epithelium and a few infiltrated inflammatory cells in the lamina propria. In contrast, the ulcers of the control group were not healed until3weeks after the irradiation.
Conclusion A rat model of radiation-induced oral mucositis (RTOM) was successfully established. Using this rat model, the in vivo effect of the KGF phage active peptides on the prevention and treatment of RTOM was investigated. The optimal conditions of applying the KGF phage active peptides were also evaluated. We found that the KGF active peptides can effectively reduce the severity of RTOM. promote epithelialization of the wound area, and accelerate the healing of the ulcers, indicating that KGF active peptides are a promising therapy for RTOM.
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