超声微泡靶向基质金属蛋白酶抑制剂1下调的腺病毒治疗大鼠肝纤维化效果
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摘要
目的分析超声微泡联合超声辐照介导基质金属蛋白酶抑制剂1(TIMP-1)下调的腺病毒治疗肝纤维化的效果及机制。方法构建下调TIMP-1基因的重组腺病毒,制备重组腺病毒超声造影剂。建立大鼠肝纤维化模型,分为:模型组、腺病毒组(重组腺病毒)、腺病毒微泡组(重组腺病毒超声造影剂)、实验组(超声辐照+重组腺病毒超声造影剂)、超声腺病毒组(超声辐照+重组腺病毒)。24 h后处死大鼠,制备肝脏切片,观察各组EGFP的表达情况,分析转染率;Masson染色判断肝纤维化分期;运用单因素方差分析对比各组大鼠丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)、羟脯氨酸(Hyp)、透明质酸(HA)、Ⅳ型胶原(CIV)、层粘连蛋白(LN)水平差异。Western blot检测各组大鼠TIMP-1、基质金属蛋白酶13(MMP-13)蛋白的相对表达含量。结果实验组转染率高于腺病毒组(q=3.418)、腺病毒微泡组(q=3.756)、超声腺病毒组(q=5.502),差异有统计学意义(P <0.05);病理检查可见实验组纤维化程度较轻且肝纤维化分级整体较低(P <0.01);对比各组血清学指标检测结果可知实验组大鼠ALT、AST、HA、LN、CIV、Hyp含量活性低于其余4组。Western blot显示,与其他各组相比,实验组大鼠TIMP-1蛋白表达更高,MMP-13蛋白表达更低。结论超声微泡联合超声辐照靶向TIMP-1下调的腺病毒治疗能抑制TIMP-1活性,改善肝纤维化程度,基因治疗具有潜在应用价值。
Objective Analysis of the effect and the mechanism of adenovirus with down regulation of matrix metalloproteinase inhibitor 1(TIMP-1) achieved targeting by ultrasound microbubbles combined with ultrasound irradiation for liver fibrosis in rats. Methods Recombinant adenovirus-mediated with down regulation of TIMP-1 gene was constructed and a mixture of recombinant adenovirus and ultrasound contrast agent was prepared.The rat liver fibrosis model was established and divided into 5 groups : model group; adenovirus group(recombinant adenovirus); adenovirus microbubble group(mixture of recombinant adenovirus and ultrasound contrast agent); experimental group(ultrasound irradiation + mixture of recombinant adenovirus and ultrasound contrast agent); ultrasound adenovirus group(ultrasound irradiation + recombinant adenovirus). The rats were sacrificed after 24 hours and liver sections were prepared. The expression of EGFP in each group was observed and the transfection rate was analyzed. The liver slices were stained by Masson to judge the stage of liver fibrosis. ANOVA analysis was used to compare the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),bydroxyproline(Hyp), hyaluronic acid(HA), type IV collagen(CIV) and laminin(LN) in each group. The relative expression levels of TIMP-1 and matrix metalloproteinase 13(MMP-13) in each group were detected by Western blot. Results The transfection rate of the experimental group was higher than that of the adenovirus group(q = 3.418), the adenovirus microbubble group(q = 3.756) and the ultrasonic adenovirus group(q = 5.502),and the difference was statistically significant(P < 0.05); Pathological examination showed that the degree of fibrosis in the experimental group and the grade of liver fibrosis were lower than the other groups(P < 0.01). The activities of ALT, AST, HA, LN, CIV and Hyp in the experimental group were lower than those in the other 4 groups.Western blot showed that the level of TIMP-1 protein expression was highest while the level of MMP-13 protein expression was lowest in the experimental group than those in the other groups. Conclusion Adenovirus with down regulation of TIMP-1 achieved targeting by ultrasound microbubbles combined with ultrasound irradiation can inhibit the activity of TIMP-1 and improve the degree of liver fibrosis. Gene therapy is an potential therapeutic method in the application of treating liver fibrosis.
Objective Analysis of the effect and the mechanism of adenovirus with down regulation of matrix metalloproteinase inhibitor 1(TIMP-1) achieved targeting by ultrasound microbubbles combined with ultrasound irradiation for liver fibrosis in rats. Methods Recombinant adenovirus-mediated with down regulation of TIMP-1 gene was constructed and a mixture of recombinant adenovirus and ultrasound contrast agent was prepared.The rat liver fibrosis model was established and divided into 5 groups : model group; adenovirus group(recombinant adenovirus); adenovirus microbubble group(mixture of recombinant adenovirus and ultrasound contrast agent); experimental group(ultrasound irradiation + mixture of recombinant adenovirus and ultrasound contrast agent); ultrasound adenovirus group(ultrasound irradiation + recombinant adenovirus). The rats were sacrificed after 24 hours and liver sections were prepared. The expression of EGFP in each group was observed and the transfection rate was analyzed. The liver slices were stained by Masson to judge the stage of liver fibrosis. ANOVA analysis was used to compare the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),bydroxyproline(Hyp), hyaluronic acid(HA), type IV collagen(CIV) and laminin(LN) in each group. The relative expression levels of TIMP-1 and matrix metalloproteinase 13(MMP-13) in each group were detected by Western blot. Results The transfection rate of the experimental group was higher than that of the adenovirus group(q = 3.418), the adenovirus microbubble group(q = 3.756) and the ultrasonic adenovirus group(q = 5.502),and the difference was statistically significant(P < 0.05); Pathological examination showed that the degree of fibrosis in the experimental group and the grade of liver fibrosis were lower than the other groups(P < 0.01). The activities of ALT, AST, HA, LN, CIV and Hyp in the experimental group were lower than those in the other 4 groups.Western blot showed that the level of TIMP-1 protein expression was highest while the level of MMP-13 protein expression was lowest in the experimental group than those in the other groups. Conclusion Adenovirus with down regulation of TIMP-1 achieved targeting by ultrasound microbubbles combined with ultrasound irradiation can inhibit the activity of TIMP-1 and improve the degree of liver fibrosis. Gene therapy is an potential therapeutic method in the application of treating liver fibrosis.
引文
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[11]ARFFA M L,ZAPF M A,KOTHARI A N,et al.Epigallocatechin-3-Gallate Upregulates miR-221 to Inhibit Osteopontin-Dependent Hepatic Fibrosis[J].PLos One,2016,11(12):e0167435.
[12]WU K,YE C,LIN L,et al.Inhibiting miR-21 attenuates experimental hepatic fibrosis by suppressing both ERK1 pathway in HSC and hepatocyte EMT[J].Clin Sci,2016,130(16):1469.
[13]夏冬,吴斌,梁建群,等.包载TIMP-1重组腺病毒微球的制备及其对肝癌细胞增殖的抑制[J].中国肿瘤生物治疗杂志,2010,17(1):57-61.
[14]沈宇宙,刘俐.超声微泡造影剂介导基因转染的研究进展[J].中国介入影像与治疗学,2016,13(6):382-386.
[15]于姣姣,赵云.高分子材料超声微泡的应用研究[J].中国医学影像学杂志,2016,24(7):557-560.
[16]郑智唯,赵云,刘朝奇.靶向超声微泡介导miRNA在疾病治疗中的研究[J].实用医学杂志,2017,33(4):654-656.
[17]安祯祥,何远利,王敏.金钗石斛多糖对肝纤维化大鼠转化生长因子及基质金属蛋白酶的影响[J].中华中医药学刊,2017(3):530-533.
[2]郑荣琴,金洁玚.超声剪切波弹性成像在肝脏疾病中的应用进展[J].器官移植,2017,8(4):260-266.
[3]马振增,陆伦根.肝纤维化药物治疗的新进展[J].临床肝胆病杂志,2016,32(6):1183-1187.
[4]LI X,PENG J,SUN Z,et al.Chinese medicine CGA formula ameliorates DMN-induced liver fibrosis in rats via inhibiting MMP2/9,TIMP1/2 and the TGF-β/Smad signaling pathways[J].Acta Pharmacologica Sinica,2016,37(6):783-793.
[5]张燕,郎清,石小枫,等.TIMP-1siRNA对肝纤维化大鼠TIMP-1及胶原表达的影响[J].重庆医科大学学报,2016(4):364-368.
[6]杨灵洁,刘丽云,穆玉明.携pAd-EGFP/SDF-1α和pAd-RFP/BMP2双基因微泡的制备及其特性检测[J].中华超声影像学杂志,2016,25(11):1002-1007.
[7]杨恒丽,蔡文斌,张莉,等.纳米级分子靶向超声造影剂的制备及其体外肿瘤靶向性实验研究[J].中国超声医学杂志,2016,32(4):367-370.
[8]李汇,毛用敏,赵莉莉,等.携带人金属蛋白酶组织抑制剂1重组腺病毒的构建及在人脐静脉内皮细胞中的表达[J].重庆医学,2013,42(8):841-844.
[9]WAHSH E,ABU-ELSAAD N,EL-KAREF A,et al.The vitamin D receptor agonist,calcipotriol,modulates fibrogenic pathways mitigating liver fibrosis in-vivo:An experimental study[J].Eur J Pharmacol,2016,789:362-369.
[10]KIM I H,XU J,LIU X,et al.Aging increases the susceptibility of hepatic inflammation,liver fibrosis and aging in response to high-fat diet in mice[J].AGE,2016,38(4):1-12.
[11]ARFFA M L,ZAPF M A,KOTHARI A N,et al.Epigallocatechin-3-Gallate Upregulates miR-221 to Inhibit Osteopontin-Dependent Hepatic Fibrosis[J].PLos One,2016,11(12):e0167435.
[12]WU K,YE C,LIN L,et al.Inhibiting miR-21 attenuates experimental hepatic fibrosis by suppressing both ERK1 pathway in HSC and hepatocyte EMT[J].Clin Sci,2016,130(16):1469.
[13]夏冬,吴斌,梁建群,等.包载TIMP-1重组腺病毒微球的制备及其对肝癌细胞增殖的抑制[J].中国肿瘤生物治疗杂志,2010,17(1):57-61.
[14]沈宇宙,刘俐.超声微泡造影剂介导基因转染的研究进展[J].中国介入影像与治疗学,2016,13(6):382-386.
[15]于姣姣,赵云.高分子材料超声微泡的应用研究[J].中国医学影像学杂志,2016,24(7):557-560.
[16]郑智唯,赵云,刘朝奇.靶向超声微泡介导miRNA在疾病治疗中的研究[J].实用医学杂志,2017,33(4):654-656.
[17]安祯祥,何远利,王敏.金钗石斛多糖对肝纤维化大鼠转化生长因子及基质金属蛋白酶的影响[J].中华中医药学刊,2017(3):530-533.
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