西帕依溃结安作用机理及基因枪联合RNAi技术治疗溃疡性结肠炎的实验研究
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摘要
溃疡性结肠炎(Ulcerative colitis,UC)病程长、病变范围广,易出现癌变,它的治疗是临床上一大难题,而维医治疗溃疡性结肠炎有其独特的方法。长期以来,维医广泛运用维药西帕依溃结安治疗UC,疗效确切。本研究通过建立大鼠UC模型并进行维药西帕依溃结安干预,在分子水平上检测诱导型一氧化氮合酶(induced -nitricoxide synthase,iNOS)、环氧合酶-2(cyclooxygenase-2,COX-2)、核转录因子-κB(nuclear factor-κB,NF-кB)、白细胞介素-6(interleukin-6,IL-6)、即刻早期原癌基因c-jun、c-fos等候选基因mRNA及蛋白质表达水平;应用高效二维液相色谱分离技术探讨大鼠溃疡性结肠炎模型组及药物干预组大鼠结肠组织中差异表达蛋白;同时建立RNA干扰(RNA interference,RNAi)技术平台,并借助其高特异性联合基因枪技术对大鼠溃疡性结肠炎进行基因治疗。
1.方法与结果
1.1大鼠溃疡性结肠炎动物模型的建立及维药西帕依溃结安的干预本研究采用2,4-二硝基氯苯(2,4-dinitrochlorobenzene,DNCB)复合乙酸法成功复制大鼠溃疡性结肠炎动物模型。模型大鼠的症状、体征及组织病理学改变与UC的相关指标相接近。半定量RT-PCR及Western blot法检测结果显示大鼠溃疡性结肠炎模型组中iNOS、COX-2、NF-кB、IL-6基因mRNA及蛋白质表达水平与正常组相比均上调,差异有统计学意义(P<0.05),c-jun、c-fos mRNA表达水平两组间差异无统计学意义,c-Jun蛋白表达水平与正常组相比上调,差异有统计学意义(P<0.05),c-Fos蛋白表达水平两组间差异无统计学意义。
将实验动物分为模型组、生理盐水阴性对照组、5-氨基水杨酸(5-ASA)阳性对照组、维药西帕依溃结安干预组(大、中、小剂量三组),研究维药西帕依溃结安的治疗作用及其机理。组织病理学研究结果显示,维药西帕依溃结安对大鼠溃疡性结肠炎治疗有效,大、中剂量治疗组间的疗效差异无统计学意义(P>0.05),而大中剂量治疗组与小剂量治疗组间疗效差异有统计学意义(P<0.05)。通过半定量RT-PCR及Western blot方法分别检测各组大鼠结肠组织中相关基因mRNA及蛋白质表达水平,发现维药西帕依溃结安大剂量治疗组中iNOS、COX-2、c-Jun的蛋白质表达水平与生理盐水阴性对照组相比均明显下调,差异有统计学意义(P<0.05);中剂量治疗组中COX-2的mRNA表达水平及iNOS蛋白质表达水平与生理盐水阴性对照组相比也明显下调,差异有统计学意义(P<0.05);5-氨基水杨酸治疗组中COX-2的mRNA表达水平与生理盐水阴性对照组相比明显下调,差异有统计学意义(P<0.05),但iNOS、c-jun的mRNA及蛋白质表达水平与生理盐水组相比差异无统计学意义(P>0.05)。而NF-кB、c-fos的mRNA及蛋白质表达水平虽然在各实验组中有所变化,但与生理盐水阴性对照组相比差异均无统计学意义。
1.2应用高效二维液相色谱分离技术探讨溃疡性结肠炎动物及药物干预组结肠组织中差异表达蛋白
本研究应用蛋白质组学技术对溃疡性结肠炎的发病及维药西帕依溃结安的作用机理进行研究。通过对各实验组大鼠的结肠组织裂解物进行一维色谱聚焦和二维液相色谱分离,利用ProteoVue软件将二维色谱数据转换成模拟胶图,再应用DeltaVue软件对各组蛋白表达谱进行比较和分析。研究发现,在pH8.5～4.0的范围内差异表达的蛋白条带在大鼠溃疡性结肠炎模型组和正常组间有296条、生理盐水阴性对照组和维药西帕依溃结安治疗组间有132条、生理盐水阴性对照组和正常组间有186条、正常组和维药西帕依溃结安治疗组间有162条、生理盐水阴性对照组和大鼠溃疡性结肠炎模型组间有286条、大鼠溃疡性结肠炎模型组和维药西帕依溃结安治疗组间有312条,并且差异表达蛋白主要集中在等电点为7.0～8.5区域的蛋白质中。
1.3基因枪联合RNAi技术治疗大鼠溃疡性结肠炎
1.3.1应用RNAi技术调节溃疡性结肠炎发病相关基因的表达本研究成功构建大鼠iNOS、COX-2、NF-κB、c-jun、c-fos等UC相关基因的pcDNA3.1/CT-GFP-TOPO重组真核表达载体和相应pSilencerTM1.0-U6-siRNA干扰载体,通过脂质体转染方法进行转染,在COS-7细胞中表达目的基因-GFP(Green fluorescence protein,绿色荧光蛋白)融合蛋白,并将pcDNA3.1/CT-GFP-TOPO重组真核表达载体和相应pSilencerTM1.0-U6-siRNA干扰载体进行共转染,经倒置荧光显微镜及Western blot法检测目的基因- GFP融合蛋白表达,筛选有效siRNA。
1.3.2 RNAi联合基因枪技术治疗大鼠溃疡性结肠炎本研究运用基因枪技术将特异性的有效pSilencerTM1.0-U6-c-jun干扰载体导入活体大鼠结肠组织中,成功调节大鼠结肠组织中目的基因c-jun的表达,并确定其有效浓度为5μg,作用持续时间为9天。基因枪联合RNAi技术治疗大鼠溃疡性结肠炎,进行组织病理学鉴定,发现有效pSilencerTM1.0-U6-c-jun干扰载体促进溃疡性结肠炎大鼠结肠组织中肉芽组织的生成,且有效干扰载体与空白载体组间溃疡性结肠炎的愈合率的差异有统计学意义(P<0.05)。
2.结论
经DNCB和乙酸复合法成功建立了溃疡性结肠炎动物模型,即刻早期原癌基因c-jun在UC动物模型中迅速而短暂地表达;组织病理学转归表明维药西帕依溃结安对治疗UC有效;药物干预实验表明其治疗作用与iNOS、COX-2、c-jun的表达下调有关,维药西帕依溃结安调节COX-2的表达可能发生在转录水平上,而调节iNOS和c-jun的表达可能发生在转录后水平上;蛋白质组学研究发现大鼠溃疡性结肠炎的发生及维药西帕依溃结安的治疗作用与多种蛋白质有关,并且差异表达蛋白多集中在等电点为7.0～8.5区域的蛋白质中;成功构建真核表达载体并在COS-7细胞中表达且筛选出有效pSilencer干扰载体,并应用基因枪介导的RNAi技术对大鼠溃疡性结肠炎进行基因治疗,发现UC大鼠结肠组织中c-jun的表达下调,对大鼠UC的愈合有促进作用。
Ulcerative colitis (UC) is a long term and wide range of disease that easily develops to cancer. The treatment of UC is a major clinical problem. Uyghur traditional Medicine has unique method in treating UC. Xipayi Kui Jie’an has been used for the treatment of UC effectively. However, the treatment mechanism of Xipayi Kui Jie’an is still unclear. In this study, UC animal model was established and which has been used for the treatment with Xipayi Kui Jie’an;the mRNA and the protein level of induced -nitricoxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-κB (NF-кB), interleukin-6 (IL-6) early oncogene c-jun, c-fos candidate gene were measured. Differentially expressed proteins in the tissue of the UC model group and the treatment group rats were tested by using the technology of high performance two-dimensional liquid chromatography. At the same time, the RNAi technology platform has been established, the strategy of RNAi gene therapy was used for the treatment of rat ulcerative colitis.
1.Methods and Results
1.1 Development of UC animal model and the intervention of Xipayi Kui Jie’an In this research, rat UC model was successfully developed by using 2, 4-dinitrochlorobenzene (DNCB) and acetic acid. The symptoms, physical signs and histopathology changes of UC rats were closely correspondent to the UC indexes. The methods of semi-quantity RT-PCR and Western blot were used to detect the different expression of mRNAs and proteins in normal control and model groups. The results showed that the expression levels of mRNA and protein of iNOS, COX-2, NF-кB, IL-6 gene were up-regulated, there was a statistical significance between the normal control group and the model group (P<0.05). Meanwhile, there was no statistical significance in the mRNA expression level of c-jun and c-fos between these two groups. The expression level of c-Jun protein was up-regulated in model group compared to the normal control group (P<0.05) ; no statistical significance has been found in the protein expression level for c-fos between these two groups.
In order to study the therapeutic effect of Uyghur traditional medicine Xipayi Kui Jie’an, animals were divided into four groups, which include the model group, NS negative control group, the 5-ASA positive control group and the Xipayi Kui Jie’an treatment group (large-dosage group, middle-dosage group and small-dosage group). The results showed that, Xipayi Kui Jie’an, Uyghur traditional medicine has a therapeutic effect on UC rats, but there was no statistical significance in the therapeutic effect between the large-dosage group and the middle-dosage group (P>0.05). The statistical significance between the large-middle dosage groups and the small-dosage group (P<0.05). The methods of semi-quantity RT-PCR and Western blot were used to detect the different expression of mRNA and protein in each group. The result showed that expression levels of iNOS?COX-2 and c-Jun protein in the large-dosage group were down-regulated compared to the normal saline treated negative control group (NS) (P<0.05). The expressed mRNA of COX-2 and the protein of iNOS were significantly down-regulated in the middle-dosage group compared to the normal saline treated negative control group (NS) (P<0.05). There was no statistical significance between the Xipayi Kui Jie’an group and the ulcerative colitis (UC) model group in the expression level of mRNA and protein for each candidate gene (P>0.05). The COX-2 mRNA was significantly down-regulated in the amino salicylic acid positive control group (5-ASA) compared to the normal saline treated negative control group (NS) (P<0.05). There was no statistical significance between the amino salicylic acid positive control group (5-ASA) and the normal saline treated negative control group in the expression level of iNOS?c-jun mRNA and protein (P>0.05). Although the expression level of NF-кB?c-fos mRNA and protein were changed in each experimental groups, there was no statistical significance between the UC group and the normal saline treated negative control group (P>0.05).
1.2 To analyse the differentially expressed proteins in UC model group and the Xipayi Kui Jie’an treated group by using the technology of high performance two-dimensional liquid chromatography.
In this study, Proteomics technology was used to analysis the pathogenesis of Ulcerative colitis and the mechanism of Xipayi Kuijie’an. First of all, the lysis product of ulcerative colitis tissues were separated by using the technology of one-dimensional focusing chromatography and two-dimensional reverse liquid chromatography. Two-dimensional data was transformed into simulating pattern by ProteoVue software and the comparison and analysis of the expressed protein in each group were conducted by DeltaVue software. The data show 296 different expressed bands between the model group and the normal group, 132 bands between the model groups and NS negative control group, 286 bands between the model group and Uyghur traditional medicine Xipayi Kui Jie’an group, 186 bands between the normal group and NS negative control group, 162 bands between normal group and Uyghur traditional medicine Xipayi Kui Jie’an groups, 132 bands between normal saline treated negative control group, and Uyghur traditional medicine Xipayi Kui Jie’an group, which in the range of pH8.5～4.0 region. The expressed proteins in different groups was concentrated in the range of pH 7.0～8.5 of isoelectric point.
1.3 The treatment of UC rat by using the gene gun jointed with RNAi technology
1.3.1 Regulation of UC related gene by using RNAi technology In this study, pcDNA3. 1/CT-GFP-TOPO recombinant eukaryotic expression vectors of iNOS, COX-2, NF-κB, c-jun, c-fos genes and corresponding pSilencerTM 1.0-U6-siRNA relevant interference vectors were successfully constructed. The GFP fused target protein was expressed in the COS-7 cell. The pcDNA3. 1/CT-GFP-TOPO recombinant eukaryotic expression vectors and the pSilencerTM 1.0-U6-siRNA relevant interference vectors were co-transfected in the same cell line. The expression of GFP fused target protein was detected by inverted fluorescent microscope and the Western blot.
1.3.2 Treatment of UC rat by using gene gun jointed with RNAi technology Interference vector of pSilencerTM1.0-U6-c-jun was successfully transformed into the colon tissue in vivo by gene gun technology and the expression level of c-jun in the colon tissue of the rats was regulated by this construct. The effective concentration of the target gene was confirmed to be 5μg and the time of activity was lasted for 9 days. The ulcerative colitis tissue of the rats was interfered by pSilencerTM1. 0-U6-c-jun interference vector, and the histopathological evaluation was determined. The result showed that the granulation tissue was facilitated effectively by the interference vector, and there was a statistical significance (P<0.05) in the healing ratio of ulcerative colitis between the groups, which has been used interference vector and blank vector.
2.Conclusion
UC model was developed by using DNCB and acetic acid, we found that early oncogene c-jun was expressed shortly in UC animal model. Histopothology results showed that Uyghur traditional medicine Xipayi Kui Jie’an is effective for treating UC;Medicine intervention experiment results showed that, the treatment effect of Xipayi Kui Jie’an might be related to the action of down regulation of iNOS, COX-2, c-jun expression. Uyghur traditional medicine Xipayi Kui Jie’an may regulate COX-2 in the transcription level, whereas the regulation to the gene of iNOS and c-jun may occur at the post-transcription level. Proteomics analysis results showed that the protein expression of different groups were concentrated in the range of pH7.0～8.5 region of isoelectric point. That result imply that the occurrence of ulcerative colitis in the rats and the therapeutic effect of Uyghur traditional medicine Xipayi Kui Jie’an are highly related to many different kinds of proteins. We also successfully constructed eukaryotic expression vector and the pSilencer interference carrier in COS-7 cells. After applied the gene gun-mediated RNAi technique on rat UC model, we found that the expression of c-jun is down regulated, and the technique of RNAi gene therapy is effective in treating UC.
1.方法与结果
1.1大鼠溃疡性结肠炎动物模型的建立及维药西帕依溃结安的干预本研究采用2,4-二硝基氯苯(2,4-dinitrochlorobenzene,DNCB)复合乙酸法成功复制大鼠溃疡性结肠炎动物模型。模型大鼠的症状、体征及组织病理学改变与UC的相关指标相接近。半定量RT-PCR及Western blot法检测结果显示大鼠溃疡性结肠炎模型组中iNOS、COX-2、NF-кB、IL-6基因mRNA及蛋白质表达水平与正常组相比均上调,差异有统计学意义(P<0.05),c-jun、c-fos mRNA表达水平两组间差异无统计学意义,c-Jun蛋白表达水平与正常组相比上调,差异有统计学意义(P<0.05),c-Fos蛋白表达水平两组间差异无统计学意义。
将实验动物分为模型组、生理盐水阴性对照组、5-氨基水杨酸(5-ASA)阳性对照组、维药西帕依溃结安干预组(大、中、小剂量三组),研究维药西帕依溃结安的治疗作用及其机理。组织病理学研究结果显示,维药西帕依溃结安对大鼠溃疡性结肠炎治疗有效,大、中剂量治疗组间的疗效差异无统计学意义(P>0.05),而大中剂量治疗组与小剂量治疗组间疗效差异有统计学意义(P<0.05)。通过半定量RT-PCR及Western blot方法分别检测各组大鼠结肠组织中相关基因mRNA及蛋白质表达水平,发现维药西帕依溃结安大剂量治疗组中iNOS、COX-2、c-Jun的蛋白质表达水平与生理盐水阴性对照组相比均明显下调,差异有统计学意义(P<0.05);中剂量治疗组中COX-2的mRNA表达水平及iNOS蛋白质表达水平与生理盐水阴性对照组相比也明显下调,差异有统计学意义(P<0.05);5-氨基水杨酸治疗组中COX-2的mRNA表达水平与生理盐水阴性对照组相比明显下调,差异有统计学意义(P<0.05),但iNOS、c-jun的mRNA及蛋白质表达水平与生理盐水组相比差异无统计学意义(P>0.05)。而NF-кB、c-fos的mRNA及蛋白质表达水平虽然在各实验组中有所变化,但与生理盐水阴性对照组相比差异均无统计学意义。
1.2应用高效二维液相色谱分离技术探讨溃疡性结肠炎动物及药物干预组结肠组织中差异表达蛋白
本研究应用蛋白质组学技术对溃疡性结肠炎的发病及维药西帕依溃结安的作用机理进行研究。通过对各实验组大鼠的结肠组织裂解物进行一维色谱聚焦和二维液相色谱分离,利用ProteoVue软件将二维色谱数据转换成模拟胶图,再应用DeltaVue软件对各组蛋白表达谱进行比较和分析。研究发现,在pH8.5～4.0的范围内差异表达的蛋白条带在大鼠溃疡性结肠炎模型组和正常组间有296条、生理盐水阴性对照组和维药西帕依溃结安治疗组间有132条、生理盐水阴性对照组和正常组间有186条、正常组和维药西帕依溃结安治疗组间有162条、生理盐水阴性对照组和大鼠溃疡性结肠炎模型组间有286条、大鼠溃疡性结肠炎模型组和维药西帕依溃结安治疗组间有312条,并且差异表达蛋白主要集中在等电点为7.0～8.5区域的蛋白质中。
1.3基因枪联合RNAi技术治疗大鼠溃疡性结肠炎
1.3.1应用RNAi技术调节溃疡性结肠炎发病相关基因的表达本研究成功构建大鼠iNOS、COX-2、NF-κB、c-jun、c-fos等UC相关基因的pcDNA3.1/CT-GFP-TOPO重组真核表达载体和相应pSilencerTM1.0-U6-siRNA干扰载体,通过脂质体转染方法进行转染,在COS-7细胞中表达目的基因-GFP(Green fluorescence protein,绿色荧光蛋白)融合蛋白,并将pcDNA3.1/CT-GFP-TOPO重组真核表达载体和相应pSilencerTM1.0-U6-siRNA干扰载体进行共转染,经倒置荧光显微镜及Western blot法检测目的基因- GFP融合蛋白表达,筛选有效siRNA。
1.3.2 RNAi联合基因枪技术治疗大鼠溃疡性结肠炎本研究运用基因枪技术将特异性的有效pSilencerTM1.0-U6-c-jun干扰载体导入活体大鼠结肠组织中,成功调节大鼠结肠组织中目的基因c-jun的表达,并确定其有效浓度为5μg,作用持续时间为9天。基因枪联合RNAi技术治疗大鼠溃疡性结肠炎,进行组织病理学鉴定,发现有效pSilencerTM1.0-U6-c-jun干扰载体促进溃疡性结肠炎大鼠结肠组织中肉芽组织的生成,且有效干扰载体与空白载体组间溃疡性结肠炎的愈合率的差异有统计学意义(P<0.05)。
2.结论
经DNCB和乙酸复合法成功建立了溃疡性结肠炎动物模型,即刻早期原癌基因c-jun在UC动物模型中迅速而短暂地表达;组织病理学转归表明维药西帕依溃结安对治疗UC有效;药物干预实验表明其治疗作用与iNOS、COX-2、c-jun的表达下调有关,维药西帕依溃结安调节COX-2的表达可能发生在转录水平上,而调节iNOS和c-jun的表达可能发生在转录后水平上;蛋白质组学研究发现大鼠溃疡性结肠炎的发生及维药西帕依溃结安的治疗作用与多种蛋白质有关,并且差异表达蛋白多集中在等电点为7.0～8.5区域的蛋白质中;成功构建真核表达载体并在COS-7细胞中表达且筛选出有效pSilencer干扰载体,并应用基因枪介导的RNAi技术对大鼠溃疡性结肠炎进行基因治疗,发现UC大鼠结肠组织中c-jun的表达下调,对大鼠UC的愈合有促进作用。
Ulcerative colitis (UC) is a long term and wide range of disease that easily develops to cancer. The treatment of UC is a major clinical problem. Uyghur traditional Medicine has unique method in treating UC. Xipayi Kui Jie’an has been used for the treatment of UC effectively. However, the treatment mechanism of Xipayi Kui Jie’an is still unclear. In this study, UC animal model was established and which has been used for the treatment with Xipayi Kui Jie’an;the mRNA and the protein level of induced -nitricoxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-κB (NF-кB), interleukin-6 (IL-6) early oncogene c-jun, c-fos candidate gene were measured. Differentially expressed proteins in the tissue of the UC model group and the treatment group rats were tested by using the technology of high performance two-dimensional liquid chromatography. At the same time, the RNAi technology platform has been established, the strategy of RNAi gene therapy was used for the treatment of rat ulcerative colitis.
1.Methods and Results
1.1 Development of UC animal model and the intervention of Xipayi Kui Jie’an In this research, rat UC model was successfully developed by using 2, 4-dinitrochlorobenzene (DNCB) and acetic acid. The symptoms, physical signs and histopathology changes of UC rats were closely correspondent to the UC indexes. The methods of semi-quantity RT-PCR and Western blot were used to detect the different expression of mRNAs and proteins in normal control and model groups. The results showed that the expression levels of mRNA and protein of iNOS, COX-2, NF-кB, IL-6 gene were up-regulated, there was a statistical significance between the normal control group and the model group (P<0.05). Meanwhile, there was no statistical significance in the mRNA expression level of c-jun and c-fos between these two groups. The expression level of c-Jun protein was up-regulated in model group compared to the normal control group (P<0.05) ; no statistical significance has been found in the protein expression level for c-fos between these two groups.
In order to study the therapeutic effect of Uyghur traditional medicine Xipayi Kui Jie’an, animals were divided into four groups, which include the model group, NS negative control group, the 5-ASA positive control group and the Xipayi Kui Jie’an treatment group (large-dosage group, middle-dosage group and small-dosage group). The results showed that, Xipayi Kui Jie’an, Uyghur traditional medicine has a therapeutic effect on UC rats, but there was no statistical significance in the therapeutic effect between the large-dosage group and the middle-dosage group (P>0.05). The statistical significance between the large-middle dosage groups and the small-dosage group (P<0.05). The methods of semi-quantity RT-PCR and Western blot were used to detect the different expression of mRNA and protein in each group. The result showed that expression levels of iNOS?COX-2 and c-Jun protein in the large-dosage group were down-regulated compared to the normal saline treated negative control group (NS) (P<0.05). The expressed mRNA of COX-2 and the protein of iNOS were significantly down-regulated in the middle-dosage group compared to the normal saline treated negative control group (NS) (P<0.05). There was no statistical significance between the Xipayi Kui Jie’an group and the ulcerative colitis (UC) model group in the expression level of mRNA and protein for each candidate gene (P>0.05). The COX-2 mRNA was significantly down-regulated in the amino salicylic acid positive control group (5-ASA) compared to the normal saline treated negative control group (NS) (P<0.05). There was no statistical significance between the amino salicylic acid positive control group (5-ASA) and the normal saline treated negative control group in the expression level of iNOS?c-jun mRNA and protein (P>0.05). Although the expression level of NF-кB?c-fos mRNA and protein were changed in each experimental groups, there was no statistical significance between the UC group and the normal saline treated negative control group (P>0.05).
1.2 To analyse the differentially expressed proteins in UC model group and the Xipayi Kui Jie’an treated group by using the technology of high performance two-dimensional liquid chromatography.
In this study, Proteomics technology was used to analysis the pathogenesis of Ulcerative colitis and the mechanism of Xipayi Kuijie’an. First of all, the lysis product of ulcerative colitis tissues were separated by using the technology of one-dimensional focusing chromatography and two-dimensional reverse liquid chromatography. Two-dimensional data was transformed into simulating pattern by ProteoVue software and the comparison and analysis of the expressed protein in each group were conducted by DeltaVue software. The data show 296 different expressed bands between the model group and the normal group, 132 bands between the model groups and NS negative control group, 286 bands between the model group and Uyghur traditional medicine Xipayi Kui Jie’an group, 186 bands between the normal group and NS negative control group, 162 bands between normal group and Uyghur traditional medicine Xipayi Kui Jie’an groups, 132 bands between normal saline treated negative control group, and Uyghur traditional medicine Xipayi Kui Jie’an group, which in the range of pH8.5～4.0 region. The expressed proteins in different groups was concentrated in the range of pH 7.0～8.5 of isoelectric point.
1.3 The treatment of UC rat by using the gene gun jointed with RNAi technology
1.3.1 Regulation of UC related gene by using RNAi technology In this study, pcDNA3. 1/CT-GFP-TOPO recombinant eukaryotic expression vectors of iNOS, COX-2, NF-κB, c-jun, c-fos genes and corresponding pSilencerTM 1.0-U6-siRNA relevant interference vectors were successfully constructed. The GFP fused target protein was expressed in the COS-7 cell. The pcDNA3. 1/CT-GFP-TOPO recombinant eukaryotic expression vectors and the pSilencerTM 1.0-U6-siRNA relevant interference vectors were co-transfected in the same cell line. The expression of GFP fused target protein was detected by inverted fluorescent microscope and the Western blot.
1.3.2 Treatment of UC rat by using gene gun jointed with RNAi technology Interference vector of pSilencerTM1.0-U6-c-jun was successfully transformed into the colon tissue in vivo by gene gun technology and the expression level of c-jun in the colon tissue of the rats was regulated by this construct. The effective concentration of the target gene was confirmed to be 5μg and the time of activity was lasted for 9 days. The ulcerative colitis tissue of the rats was interfered by pSilencerTM1. 0-U6-c-jun interference vector, and the histopathological evaluation was determined. The result showed that the granulation tissue was facilitated effectively by the interference vector, and there was a statistical significance (P<0.05) in the healing ratio of ulcerative colitis between the groups, which has been used interference vector and blank vector.
2.Conclusion
UC model was developed by using DNCB and acetic acid, we found that early oncogene c-jun was expressed shortly in UC animal model. Histopothology results showed that Uyghur traditional medicine Xipayi Kui Jie’an is effective for treating UC;Medicine intervention experiment results showed that, the treatment effect of Xipayi Kui Jie’an might be related to the action of down regulation of iNOS, COX-2, c-jun expression. Uyghur traditional medicine Xipayi Kui Jie’an may regulate COX-2 in the transcription level, whereas the regulation to the gene of iNOS and c-jun may occur at the post-transcription level. Proteomics analysis results showed that the protein expression of different groups were concentrated in the range of pH7.0～8.5 region of isoelectric point. That result imply that the occurrence of ulcerative colitis in the rats and the therapeutic effect of Uyghur traditional medicine Xipayi Kui Jie’an are highly related to many different kinds of proteins. We also successfully constructed eukaryotic expression vector and the pSilencer interference carrier in COS-7 cells. After applied the gene gun-mediated RNAi technique on rat UC model, we found that the expression of c-jun is down regulated, and the technique of RNAi gene therapy is effective in treating UC.
引文
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