hITF基因治疗肠源性感染的体内外实验研究
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摘要
背景:
肠三叶因子(intestinal trefoil factor,ITF)是由肠杯状细胞特异分泌的小分子多肽,它的59个氨基酸序列中有一段特殊的核心结构域,其间的6个半胱氨酸按特定(1-5,2-4和3-6)的顺序,依次以二硫键两两相接,形成3个环状结构,整个肽链由此发生弯曲和折叠,形如“三叶草”,故名肠三叶因子(ITF)。ITF一经发现便受到广大学者的广泛关注,大量的研究表明,ITF在肠黏膜的自我保护及修复机制中占据重要地位。作为一个具有广泛应用前景的药物前体,却因为复杂的制备过程、较低的产量限制了它的应用。基因治疗是近年来出现的崭新的治疗手段,是将人正常基因或有治疗作用的基因通过一定方式导入人体靶细胞以纠正基因的缺陷或者发挥治疗作用,从而达到治疗疾病目的的生物医学高技术。若通过基因治疗的途径将ITF基因导入体内,可以使ITF在体内大量表达,有望简化繁琐的操作程序、降低昂贵的生产成本。
目的:
筛选并构建hITF理想的基因传递系统;探索hITF基因治疗对烧伤后肠源性感染的作用。
方法:
1.首先从人小肠内膜提取总RNA,再利用RT-PCR技术获得hITF信号肽及成熟肽cDNA,然后克隆至质粒pEGFP-N1获得重组载体,最后双酶切及测序鉴定。
2.利用复凝法制备壳聚糖纳米粒,用透射电镜观察纳米粒的形态及大小,以Zeta电位仪检测不同N/P比及pH对Zeta电位的影响,琼脂糖电泳分析纳米粒对基因的包裹和保护能力,并计算纳米粒的包封率和载药量,最后分析其释放曲线。3.利用壳聚糖纳米粒转染HEK293细胞,荧光显微镜、流式细胞仪检测转染效率,RT-PCR检测转录情况,Western Blot检测hITF表达情况。4.将穿梭质粒(pAdTrack-CMV)、目的基因(pEGFP-N1-hITF)双酶切,以T4DNA连接酶连接构建重组穿梭质粒pAdTrack-CMV-hITF。重组质粒PmeⅠ线性化后与骨架质粒pAdEasy-1在大肠杆菌BJ5183中同源重组,PacⅠ酶切鉴定。重组腺病毒载体在HEK293细胞中包装,根据Karbers公式计算病毒颗粒滴度。
5.将重组腺病毒感染人结肠癌细胞株(HT-29、Caco-2),然后行RT-PCR及WB检测,从转录及蛋白水平检测hITF表达情况;感染病毒细胞做爬片后行细胞免疫荧光检查;最后通过划痕实验,检测重组腺病毒对结肠癌细胞迁移功能的影响。
6.首先制备30%TBSAⅢ°烧伤小鼠模型,并给予重组腺病毒灌胃,在相应时相点取标本检测肠黏膜损伤指数、绒毛高度、隐窝深度、病理切片、冰冻切片、肠道菌群移位率以及血清LPS,以观察Ad-hITF对小鼠烧伤后肠源性感染是否具有治疗组用。
结果:
1.获得hITF信号肽+成熟肽全长基因序列,并成功插入真核表达载体pEGFP-N1构建出重组载体,双酶切鉴定后送基因测序,结果证明目的基因与Genbank数据库所收录的基因序列完全一致,遂命名为pEGFP-N1-hITF。
2.利用复凝法制备了不同N/P比的壳聚糖纳米粒,透射电镜观察到粒度集中在300-500nm,粒径呈窄分布;Zeta电位与N/P比呈正相关,而与pH值呈负相关;凝胶阻滞实验说明壳聚糖在N/P比大于1时可以完全包裹质粒;DNA保护实验说明纳米粒可以保护内部质粒免受DNAse I的降解;包封率随着N/P比的增加而增加,载药量则随着N/P比的增加而减小;体外释放试验显示,纳米粒释放速度先快后满,而且随着N/P比的增加而逐渐减慢。
3.荧光显微镜及流式细胞仪检测显示,壳聚糖纳米粒对HEK293细胞的转染率约为80%,和脂质体的转染率基本一致;RT-PCR和WB检测说明,hITF被成功转录并分泌表达。
4.成功构建出重组穿梭质粒pAdTrack-CMV-hITF,PmeⅠ线性化后与骨架质粒pAdEasy-1同源重组,PacⅠ酶切鉴定后命名为Ad-hITF;重组腺病毒载体在HEK293细胞中包装,获得重组腺病毒颗粒;根据Karbers公式计算病毒颗粒滴度为2×109pfu/ml。
5.重组腺病毒在MOI=50的滴度下可以感染人结肠癌细胞株(HT-29、Caco-2),感染效率接近100%;RT-PCR和WB检测说明,hITF被成功转录并分泌表达;细胞免疫荧光显示,hITF可表达于细胞核和细胞浆,但以细胞浆为主;划痕实验证明感染重组腺病毒对结肠癌细胞具有很强的促迁移能力。
6.成功制备了30%TBSAⅢ°烧伤小鼠模型,给予重组腺病毒灌胃后,烧伤小鼠肠黏膜损伤指数明显低于B组,绒毛高度、隐窝深度显著高于B组;病理切片提示Ad-hITF组损伤情况明显较轻,冰冻切片显示hITF已表达于肠黏膜;肠道菌群移位率以及血清LPS检测提示,Ad-hITF组显著低于B组。
结论:
1.基因测序证明重组真核表达质粒pEGFP-N1-hITF构建成功。
2.复凝法制备出壳聚糖纳米粒,其直径小于500nm,Zeta电位阳性,能有效保护内部基因。
3.壳聚糖纳米粒能高效转染HEK293细胞,并分泌表达出成熟hITF。
4.成功构建出含hITF基因的重组腺病毒载体Ad-hITF。
5.Ad-hITF能高效感染结肠癌细胞,并分泌表达出成熟hITF,且对细胞迁移具有显著的促进作用。
6.Ad-hITF可显明显减轻烧伤小鼠肠黏膜损伤、促进修复,对烧伤后肠源性感染具有良好的治疗作用。
Background: Intestinal trefoil factor (ITF, also known as TFF3), a member of the trefoil factor family (TFF), is a small peptide secreted by intestinal goblet cells. ITF is composed of one structurally characteristic trefoil domain. A trefoil domain is defined as a sequence of 38 or 39 amino acid residues in which 6 cysteine residues are linked in the configuration 1-5, 2-4, 3-6, thus forming a characteristic three-leaved structure. ITF is essential for protecting the epithelial layer of the gastrointestinal tract from damage and repairing epithelium after injury. ITF is attracting the researchers’attention for its potential pharmacological value. Though ITF is a novel polypeptide with potential pharmacological value, its production is too limited for to be clinical useful. Gene delivery is the process in which plasmid DNA is introduced into target cells, transcribed and the genetic information ultimately translated into the corresponding protein. If hITF gene therapy was used, the major drawbacks limiting its clinical use would be overcomed. Objective: To select an optimal hITF gene delivery systems, and to explore the effect of ITF gene therapy on the treatment of gut derived infection.
Methods:
1. hITF gene (73 aa), encoding both a signal peptide and a mature secretory peptide, was generated by RT-PCR technology. After digestion with XhoI and EcoRI, the gene was inserted into the multiple cloning sites of the E. coli vector, pEGFP-N1, to construct a GFP N-terminal tagged recombinant plasmid. Following confirmation by restriction analysis plasmid DNA was sent to Shanghai Invitrogen Corp for DNA sequencing.
2. Chitosan nanoparticles were prepared by a complex coacervation technique, and its size and morphology was assessed using TEM. Zeta potentials were determined using a Zetasizer Nano. Gel retarding analysis and DNA integrity analysis were assayed by gel electrophoresis. In addition, DNA loading efficiency and loading capacity were evaluated and in vitro release of DNA from the chitosan nanoparticles was analysed.
3. Gene transfer capability was assessed in HEK293 cells, and transfection efficiency was evaluated by fluorescence microscopy and flow cytometry. The transcription and expression of hITF was assessed by RT-PCR and Western Blot.
4. After digestion with BglⅡand NotⅠ, the gene was inserted into the multiple cloning sites of pAdTrack-CMV, to construct recombinant vector.Then the recombinant vector was co-transfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying hITF was generated with homologous recombinantion in bacteria. The adenoviral plasmid was transfected in 293 cells and the recombinant adenovirus Ad-hITF was packed in 293 cells.
5. Ad-hITF was transfected to human colon adenocarcinoma cell line (HT-29, Caco-2), and the expression of hITF mRNA and protein was detected by RT-PCR ,Western blot and immunofluorescence. Then, wounds were established in confluent monolayers of HT-29 cells infected with pAd-hITF, and wounded monolayers were cultured for 24 h after addition of DMEM. The width of the wound border was measured in a blind manner. 6. Kunming mice were inflicted with 30 % total body surface area full thickness burns and administrated with Ad-hITF orally. Injury index, villous height, crypt depth, LPS, intestinal bacterial translocation rate and so on were determined on 1,3 ,5and 7 PBDs.
Results:
1. As a result of RT-PCR, a 240bp DNA fragment was obtained and identified by gel electrophoresis. Then, the DNA fragment was inserted into plasmid pEGFP-N1 to construct recombinant vector. The recombinant vector was confirmed by restriction analysis and gene sequencing to be the same as the original sequences of hITF gene from GenBank(Accession No. NM003226), and the recombinant vector was named pEGFP-N1-hITF.
2. The chitosan nanoparticles were successfully prepared by a complex coacervation technique with sizes 300-500 nm and positive zeta potentials. The nanoparticles could protect DNA from nuclease degradation, and release profiles of DNA were dependent on N/P ratios.The loading efficiency of chitosan nanoparticles increased depending on the N/P ratio, while the loading capacity of nanoparticles was lower with decreasing N/P ratios.
3. The transfection efficiency was studied by fluorescence microscopy and flow cytometry. The results showed that the transfection efficiencies of chitosan nanoparticles were about 80%, similar to the transfection efficiency of Lipofectamine. RT-PCR and Western Blot assay demonstrated that hITF was expressed successfully and the proteins had much good antigenicity and specificity.
4. The recombinant vector pAdTrack-CMV-hITF was constructed.After digestion with restriction enzyme PmeⅠ, linearized recombinant vector was transformed into BJ5183 bacteria and the adenoviral plasmid Ad-hITF carrying hITF was generated with homologous recombination. The mature viruses would be packaged in 293 cells. The titer of virus was 2×109pfu/ml by TCID50 method.
5. Transfection efficiency was about 100% as the MOI of the recombinant adenoviruses was 50.RT-PCR and Western Blot assay demonstrated that hITF was transcribed and expressed successfully. hITF was proved to distribute in cytoplasm by immunofluorescence assay.In addition, Ad-hITF enhanced migration activity of HT-29 cells obviously.
6. Ad-hITF treatment significantly decreased the injury index, increased villous height and crypt depth, alleviated pathological damage, lowered LPS and intestinal bacterial translocation rate in comparison with the control group.
Conclusion:
1. Recombinant eukaryotic expression vector pEGFP-N1-hITF was constructed successfully.
2. The chitosan nanoparticles were successfully prepared by a complex coacervation technique with sizes 300-500 nm and positive zeta potentials and could protect DNA from nuclease degradation.
3. The chitosan nanoparticles could be transfected to HEK293 cells with high transfection efficiency, and hITF was expressed in the cultures.
4. The recombinant adenovirus Ad-hITF carrying hITF was constructed successfully.
5. Ad-hITF could infect human colon adenocarcinoma cell line(HT-29, Caco-2) with transfection efficiency close to 100%, and hITF was expressed in the cultures. Ad-hITF has the ability to enhance cell migration activity.
6. Ad-hITF treatment significantly alleviated intestinal mucosal damage, promoted the healing of mucosa and had efficient therapeutic effect on gut derived infection.
肠三叶因子(intestinal trefoil factor,ITF)是由肠杯状细胞特异分泌的小分子多肽,它的59个氨基酸序列中有一段特殊的核心结构域,其间的6个半胱氨酸按特定(1-5,2-4和3-6)的顺序,依次以二硫键两两相接,形成3个环状结构,整个肽链由此发生弯曲和折叠,形如“三叶草”,故名肠三叶因子(ITF)。ITF一经发现便受到广大学者的广泛关注,大量的研究表明,ITF在肠黏膜的自我保护及修复机制中占据重要地位。作为一个具有广泛应用前景的药物前体,却因为复杂的制备过程、较低的产量限制了它的应用。基因治疗是近年来出现的崭新的治疗手段,是将人正常基因或有治疗作用的基因通过一定方式导入人体靶细胞以纠正基因的缺陷或者发挥治疗作用,从而达到治疗疾病目的的生物医学高技术。若通过基因治疗的途径将ITF基因导入体内,可以使ITF在体内大量表达,有望简化繁琐的操作程序、降低昂贵的生产成本。
目的:
筛选并构建hITF理想的基因传递系统;探索hITF基因治疗对烧伤后肠源性感染的作用。
方法:
1.首先从人小肠内膜提取总RNA,再利用RT-PCR技术获得hITF信号肽及成熟肽cDNA,然后克隆至质粒pEGFP-N1获得重组载体,最后双酶切及测序鉴定。
2.利用复凝法制备壳聚糖纳米粒,用透射电镜观察纳米粒的形态及大小,以Zeta电位仪检测不同N/P比及pH对Zeta电位的影响,琼脂糖电泳分析纳米粒对基因的包裹和保护能力,并计算纳米粒的包封率和载药量,最后分析其释放曲线。3.利用壳聚糖纳米粒转染HEK293细胞,荧光显微镜、流式细胞仪检测转染效率,RT-PCR检测转录情况,Western Blot检测hITF表达情况。4.将穿梭质粒(pAdTrack-CMV)、目的基因(pEGFP-N1-hITF)双酶切,以T4DNA连接酶连接构建重组穿梭质粒pAdTrack-CMV-hITF。重组质粒PmeⅠ线性化后与骨架质粒pAdEasy-1在大肠杆菌BJ5183中同源重组,PacⅠ酶切鉴定。重组腺病毒载体在HEK293细胞中包装,根据Karbers公式计算病毒颗粒滴度。
5.将重组腺病毒感染人结肠癌细胞株(HT-29、Caco-2),然后行RT-PCR及WB检测,从转录及蛋白水平检测hITF表达情况;感染病毒细胞做爬片后行细胞免疫荧光检查;最后通过划痕实验,检测重组腺病毒对结肠癌细胞迁移功能的影响。
6.首先制备30%TBSAⅢ°烧伤小鼠模型,并给予重组腺病毒灌胃,在相应时相点取标本检测肠黏膜损伤指数、绒毛高度、隐窝深度、病理切片、冰冻切片、肠道菌群移位率以及血清LPS,以观察Ad-hITF对小鼠烧伤后肠源性感染是否具有治疗组用。
结果:
1.获得hITF信号肽+成熟肽全长基因序列,并成功插入真核表达载体pEGFP-N1构建出重组载体,双酶切鉴定后送基因测序,结果证明目的基因与Genbank数据库所收录的基因序列完全一致,遂命名为pEGFP-N1-hITF。
2.利用复凝法制备了不同N/P比的壳聚糖纳米粒,透射电镜观察到粒度集中在300-500nm,粒径呈窄分布;Zeta电位与N/P比呈正相关,而与pH值呈负相关;凝胶阻滞实验说明壳聚糖在N/P比大于1时可以完全包裹质粒;DNA保护实验说明纳米粒可以保护内部质粒免受DNAse I的降解;包封率随着N/P比的增加而增加,载药量则随着N/P比的增加而减小;体外释放试验显示,纳米粒释放速度先快后满,而且随着N/P比的增加而逐渐减慢。
3.荧光显微镜及流式细胞仪检测显示,壳聚糖纳米粒对HEK293细胞的转染率约为80%,和脂质体的转染率基本一致;RT-PCR和WB检测说明,hITF被成功转录并分泌表达。
4.成功构建出重组穿梭质粒pAdTrack-CMV-hITF,PmeⅠ线性化后与骨架质粒pAdEasy-1同源重组,PacⅠ酶切鉴定后命名为Ad-hITF;重组腺病毒载体在HEK293细胞中包装,获得重组腺病毒颗粒;根据Karbers公式计算病毒颗粒滴度为2×109pfu/ml。
5.重组腺病毒在MOI=50的滴度下可以感染人结肠癌细胞株(HT-29、Caco-2),感染效率接近100%;RT-PCR和WB检测说明,hITF被成功转录并分泌表达;细胞免疫荧光显示,hITF可表达于细胞核和细胞浆,但以细胞浆为主;划痕实验证明感染重组腺病毒对结肠癌细胞具有很强的促迁移能力。
6.成功制备了30%TBSAⅢ°烧伤小鼠模型,给予重组腺病毒灌胃后,烧伤小鼠肠黏膜损伤指数明显低于B组,绒毛高度、隐窝深度显著高于B组;病理切片提示Ad-hITF组损伤情况明显较轻,冰冻切片显示hITF已表达于肠黏膜;肠道菌群移位率以及血清LPS检测提示,Ad-hITF组显著低于B组。
结论:
1.基因测序证明重组真核表达质粒pEGFP-N1-hITF构建成功。
2.复凝法制备出壳聚糖纳米粒,其直径小于500nm,Zeta电位阳性,能有效保护内部基因。
3.壳聚糖纳米粒能高效转染HEK293细胞,并分泌表达出成熟hITF。
4.成功构建出含hITF基因的重组腺病毒载体Ad-hITF。
5.Ad-hITF能高效感染结肠癌细胞,并分泌表达出成熟hITF,且对细胞迁移具有显著的促进作用。
6.Ad-hITF可显明显减轻烧伤小鼠肠黏膜损伤、促进修复,对烧伤后肠源性感染具有良好的治疗作用。
Background: Intestinal trefoil factor (ITF, also known as TFF3), a member of the trefoil factor family (TFF), is a small peptide secreted by intestinal goblet cells. ITF is composed of one structurally characteristic trefoil domain. A trefoil domain is defined as a sequence of 38 or 39 amino acid residues in which 6 cysteine residues are linked in the configuration 1-5, 2-4, 3-6, thus forming a characteristic three-leaved structure. ITF is essential for protecting the epithelial layer of the gastrointestinal tract from damage and repairing epithelium after injury. ITF is attracting the researchers’attention for its potential pharmacological value. Though ITF is a novel polypeptide with potential pharmacological value, its production is too limited for to be clinical useful. Gene delivery is the process in which plasmid DNA is introduced into target cells, transcribed and the genetic information ultimately translated into the corresponding protein. If hITF gene therapy was used, the major drawbacks limiting its clinical use would be overcomed. Objective: To select an optimal hITF gene delivery systems, and to explore the effect of ITF gene therapy on the treatment of gut derived infection.
Methods:
1. hITF gene (73 aa), encoding both a signal peptide and a mature secretory peptide, was generated by RT-PCR technology. After digestion with XhoI and EcoRI, the gene was inserted into the multiple cloning sites of the E. coli vector, pEGFP-N1, to construct a GFP N-terminal tagged recombinant plasmid. Following confirmation by restriction analysis plasmid DNA was sent to Shanghai Invitrogen Corp for DNA sequencing.
2. Chitosan nanoparticles were prepared by a complex coacervation technique, and its size and morphology was assessed using TEM. Zeta potentials were determined using a Zetasizer Nano. Gel retarding analysis and DNA integrity analysis were assayed by gel electrophoresis. In addition, DNA loading efficiency and loading capacity were evaluated and in vitro release of DNA from the chitosan nanoparticles was analysed.
3. Gene transfer capability was assessed in HEK293 cells, and transfection efficiency was evaluated by fluorescence microscopy and flow cytometry. The transcription and expression of hITF was assessed by RT-PCR and Western Blot.
4. After digestion with BglⅡand NotⅠ, the gene was inserted into the multiple cloning sites of pAdTrack-CMV, to construct recombinant vector.Then the recombinant vector was co-transfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying hITF was generated with homologous recombinantion in bacteria. The adenoviral plasmid was transfected in 293 cells and the recombinant adenovirus Ad-hITF was packed in 293 cells.
5. Ad-hITF was transfected to human colon adenocarcinoma cell line (HT-29, Caco-2), and the expression of hITF mRNA and protein was detected by RT-PCR ,Western blot and immunofluorescence. Then, wounds were established in confluent monolayers of HT-29 cells infected with pAd-hITF, and wounded monolayers were cultured for 24 h after addition of DMEM. The width of the wound border was measured in a blind manner. 6. Kunming mice were inflicted with 30 % total body surface area full thickness burns and administrated with Ad-hITF orally. Injury index, villous height, crypt depth, LPS, intestinal bacterial translocation rate and so on were determined on 1,3 ,5and 7 PBDs.
Results:
1. As a result of RT-PCR, a 240bp DNA fragment was obtained and identified by gel electrophoresis. Then, the DNA fragment was inserted into plasmid pEGFP-N1 to construct recombinant vector. The recombinant vector was confirmed by restriction analysis and gene sequencing to be the same as the original sequences of hITF gene from GenBank(Accession No. NM003226), and the recombinant vector was named pEGFP-N1-hITF.
2. The chitosan nanoparticles were successfully prepared by a complex coacervation technique with sizes 300-500 nm and positive zeta potentials. The nanoparticles could protect DNA from nuclease degradation, and release profiles of DNA were dependent on N/P ratios.The loading efficiency of chitosan nanoparticles increased depending on the N/P ratio, while the loading capacity of nanoparticles was lower with decreasing N/P ratios.
3. The transfection efficiency was studied by fluorescence microscopy and flow cytometry. The results showed that the transfection efficiencies of chitosan nanoparticles were about 80%, similar to the transfection efficiency of Lipofectamine. RT-PCR and Western Blot assay demonstrated that hITF was expressed successfully and the proteins had much good antigenicity and specificity.
4. The recombinant vector pAdTrack-CMV-hITF was constructed.After digestion with restriction enzyme PmeⅠ, linearized recombinant vector was transformed into BJ5183 bacteria and the adenoviral plasmid Ad-hITF carrying hITF was generated with homologous recombination. The mature viruses would be packaged in 293 cells. The titer of virus was 2×109pfu/ml by TCID50 method.
5. Transfection efficiency was about 100% as the MOI of the recombinant adenoviruses was 50.RT-PCR and Western Blot assay demonstrated that hITF was transcribed and expressed successfully. hITF was proved to distribute in cytoplasm by immunofluorescence assay.In addition, Ad-hITF enhanced migration activity of HT-29 cells obviously.
6. Ad-hITF treatment significantly decreased the injury index, increased villous height and crypt depth, alleviated pathological damage, lowered LPS and intestinal bacterial translocation rate in comparison with the control group.
Conclusion:
1. Recombinant eukaryotic expression vector pEGFP-N1-hITF was constructed successfully.
2. The chitosan nanoparticles were successfully prepared by a complex coacervation technique with sizes 300-500 nm and positive zeta potentials and could protect DNA from nuclease degradation.
3. The chitosan nanoparticles could be transfected to HEK293 cells with high transfection efficiency, and hITF was expressed in the cultures.
4. The recombinant adenovirus Ad-hITF carrying hITF was constructed successfully.
5. Ad-hITF could infect human colon adenocarcinoma cell line(HT-29, Caco-2) with transfection efficiency close to 100%, and hITF was expressed in the cultures. Ad-hITF has the ability to enhance cell migration activity.
6. Ad-hITF treatment significantly alleviated intestinal mucosal damage, promoted the healing of mucosa and had efficient therapeutic effect on gut derived infection.
引文
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