大鼠IL-10质粒DNA尾静脉注射对肝纤维化大鼠的治疗作用及其机制
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摘要
目的探讨大鼠IL-10质粒DNA尾静脉快速大容量注射对猪血清诱导大鼠肝纤维化的治疗作用及其机制。
方法采用RT-巢式PCR和分子克隆技术构建大鼠IL-10真核表达质粒PcDNA3-rIL-10,通过受体介导的脂质体转染试剂转染大鼠正常肝细胞BRL,RT-PCR及ELISA法检测转染后BRL细胞表达分泌型rIL-10基因的情况,MTT法及流式细胞术观察rIL-10重组质粒的转染对BRL细胞的影响;尾静脉快速大容量注射法将PcDNA3-rIL-10转移至大鼠体内,RT-PCR、ELISA和免疫组织化学法检测rIL-10在大鼠体内的分布及表达情况;30只清洁级SD大鼠被随机分为正常对照组(N组),纤维化模型组(M组), rIL-10质粒治疗组(I组),空质粒对照组(P组)。N组大鼠腹腔注射生理盐水,每周2次,每次0.5m L/只,共8周;其余各组大鼠腹腔注射猪血清,每周2次,每次0.5m L/只,共8周;从第5周开始, N组和M组大鼠尾静脉注射林格氏液作为试剂对照,I组大鼠尾静脉注射pcDNA3-rIL-10质粒, P组大鼠尾静脉注射pcDNA3.0空质粒,每周1次。所有大鼠于实验第8周末处死,收集肝组织和血清备用。HE染色检测肝组织病理形态学改变,生化检测血清丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)的含量、苦味酸-天狼星红染色检测各组肝组织中I、III胶原的改变,SP免疫组织化学检测各组肝组织中α-SMA、MMP-13、TIMP-1的表达。结果重组质粒DNA酶切和DNA测序证实真核表达载体PcDNA3-rIL-10构建成功;RT-PCR法及ELISA法证实转染rIL-10重组质粒的BRL细胞高表达分泌型rIL-10基因及rL-10的表达对质粒转染后肝细胞具有一定的保护作用; RT-PCR和免疫组织化学法证实PcDNA3-rIL-10尾静脉注射后主要在肝组织中转录及表达,ELISA法检证实血浆中rIL-10浓度可通过质粒的重复注射使其保持在一个稳态;肝病理组织学证实猪血清诱导大鼠肝纤维化模型构建成功,与P组和M组大鼠比较,I组大鼠肝细胞变性坏死程度减轻、汇管区及其周围炎细胞的浸润减少,胶原纤维沉积明显下降,显著降低肝组织HAI积分(P<0.01)。I组血清ALT和AST水平显著降低(P<0.01),天狼猩红染色显示肝组织中I、III型胶原的沉积明显减少(P<0.01),免疫组化结果显示肝组织中α-SMA和TIMP1的表达显著降低(P<0.01)。
结论rIL-10质粒DNA静脉注射能有效地阻止猪血清诱导的大鼠肝纤维化的进展,具有一定的治疗作用;其机制可能是rIL-10质粒DNA静脉注射可使rIL-10基因在肝组织中高表达及其能有效抑制肝组织中α-SMA和TIMP1的表达和促进肝组织中I、III型胶原的降解。
Aims: to investigate the therapeutic effects of rIL-10 plasmid DNA in large volume in short time tail vein injection on hepatic fibrosis in rat and its mechanisms.
Methods: RT-nest-PCR and gene recombination technique were used to construct rat interleukin-10 eukaryotic expression vector (pcDNA3-rIL-10). The reconstituted plasmid pcDNA3-rIL-10 and the empty vector pcDNA3 were transfected respectively into BRL cells by asialoglycoprotein receptor mediated liposome PEIjet-gal. The expression of rIL-10 gene was detected by RT-PCR and ELISA analysis. The influence of the transfection of reconstituted plasmid pcDNA3-rIL-10 on BRL cells was detected by MMT and flow cytometric analysis. Reconstituted plasmid pcDNA3-rIL-10 was rapidly injected into rat in large volume in a short time through tail vein. RT-PCR, ELISA and immunohistochemistry analysis were used to detect the expression and distribution of rIL-10 gene in rat. Thirty SD rats were divided randomLy into control group (group N), fibrosis model group (group M), rIL-10 gene therapy group (group I) and empty vector control group (group P). Rats in group N were treated with 0.5mL normal sodium twice a week for 8 wk via intraperitoneal injection, and rats in other groups were treated with 0.5mL pig serum twice a week for 8 wk. At the beginning of the 5th wk, rats in group N and M were injected weekly with Ringer,s solution, rats in group I were injected weekly with the rIL-10 recombinant plasmid pcDNA3-rIL-10 and rats in group P were injected weekly with empty vector pcDNA3 via tail vein. At the end of the 8th wk, all rats were put to death and then collected sample of liver tissue and serum. The grading and staging of hepatic fibrosis were measured by HE staining. The change of ALT and AST in serum was measured by biochemistry analysis. The change of collagen type I、III was measured by Picrosirius staining and the expression ofα-SMA、MMP-13 and TIMP-1 in liver tissue were measured by S-P immunohistochemistry.
Results: DNA sequencing and restriction endonuclease digestion confirmed that eukaryotic expression vector pcDNA3-rIL-10 was constructed successfully. RT-PCR and ELISA assay confirmed that the BRL cells transfected with plasmid pcDNA3-rIL-10 expressed highly secretary type rIL-10 protein and the expression of rIL-10 show a few protection effect on RBL cells transfected. RT-PCR and immunohistochemistry confirmed the presence of the rIL-10 transcription and expression mainly in liver and ELISA assay confirmed maintaining a stable expression of rIL-10 in serum can be assessed by repeated administration. The liver pathohistology analysis confirmed experimental rat hepatic fibrosis model induced by pig serum was established successfully. Compared with the rats in group M and P, rats in group I showed significant therapeutic effects: decreased the degree of hepatocyte necrosis and degeneration and the number of inflammatory cells around lobular veins, reduced the disposition of collagen fibers and reduced the levels of ALT and AST in serum(P<0.01). Picrosirius staining confirmed the expression of collagen type I、III was significantly suppressed (P<0.01)and the immunohistochemistry confirmed positive levels forα-SMA and TIMP-1 were decreased significantly in group I (P<0.01).
Conclusions: rIL-10 plasmid DNA tail vein injection can prevent efficiently the progression of experimental rat hepatic fibrosis induced by pig serum, the therapeutic effects of rIL-10 plasmid injection on hepatic fibrosis may be primarily exerted by making rIL-10 transcription and expression mainly in liver, inhibiting the expression ofα-SMA and TIMP-1 and promoting collagen type I、III resolution.
方法采用RT-巢式PCR和分子克隆技术构建大鼠IL-10真核表达质粒PcDNA3-rIL-10,通过受体介导的脂质体转染试剂转染大鼠正常肝细胞BRL,RT-PCR及ELISA法检测转染后BRL细胞表达分泌型rIL-10基因的情况,MTT法及流式细胞术观察rIL-10重组质粒的转染对BRL细胞的影响;尾静脉快速大容量注射法将PcDNA3-rIL-10转移至大鼠体内,RT-PCR、ELISA和免疫组织化学法检测rIL-10在大鼠体内的分布及表达情况;30只清洁级SD大鼠被随机分为正常对照组(N组),纤维化模型组(M组), rIL-10质粒治疗组(I组),空质粒对照组(P组)。N组大鼠腹腔注射生理盐水,每周2次,每次0.5m L/只,共8周;其余各组大鼠腹腔注射猪血清,每周2次,每次0.5m L/只,共8周;从第5周开始, N组和M组大鼠尾静脉注射林格氏液作为试剂对照,I组大鼠尾静脉注射pcDNA3-rIL-10质粒, P组大鼠尾静脉注射pcDNA3.0空质粒,每周1次。所有大鼠于实验第8周末处死,收集肝组织和血清备用。HE染色检测肝组织病理形态学改变,生化检测血清丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)的含量、苦味酸-天狼星红染色检测各组肝组织中I、III胶原的改变,SP免疫组织化学检测各组肝组织中α-SMA、MMP-13、TIMP-1的表达。结果重组质粒DNA酶切和DNA测序证实真核表达载体PcDNA3-rIL-10构建成功;RT-PCR法及ELISA法证实转染rIL-10重组质粒的BRL细胞高表达分泌型rIL-10基因及rL-10的表达对质粒转染后肝细胞具有一定的保护作用; RT-PCR和免疫组织化学法证实PcDNA3-rIL-10尾静脉注射后主要在肝组织中转录及表达,ELISA法检证实血浆中rIL-10浓度可通过质粒的重复注射使其保持在一个稳态;肝病理组织学证实猪血清诱导大鼠肝纤维化模型构建成功,与P组和M组大鼠比较,I组大鼠肝细胞变性坏死程度减轻、汇管区及其周围炎细胞的浸润减少,胶原纤维沉积明显下降,显著降低肝组织HAI积分(P<0.01)。I组血清ALT和AST水平显著降低(P<0.01),天狼猩红染色显示肝组织中I、III型胶原的沉积明显减少(P<0.01),免疫组化结果显示肝组织中α-SMA和TIMP1的表达显著降低(P<0.01)。
结论rIL-10质粒DNA静脉注射能有效地阻止猪血清诱导的大鼠肝纤维化的进展,具有一定的治疗作用;其机制可能是rIL-10质粒DNA静脉注射可使rIL-10基因在肝组织中高表达及其能有效抑制肝组织中α-SMA和TIMP1的表达和促进肝组织中I、III型胶原的降解。
Aims: to investigate the therapeutic effects of rIL-10 plasmid DNA in large volume in short time tail vein injection on hepatic fibrosis in rat and its mechanisms.
Methods: RT-nest-PCR and gene recombination technique were used to construct rat interleukin-10 eukaryotic expression vector (pcDNA3-rIL-10). The reconstituted plasmid pcDNA3-rIL-10 and the empty vector pcDNA3 were transfected respectively into BRL cells by asialoglycoprotein receptor mediated liposome PEIjet-gal. The expression of rIL-10 gene was detected by RT-PCR and ELISA analysis. The influence of the transfection of reconstituted plasmid pcDNA3-rIL-10 on BRL cells was detected by MMT and flow cytometric analysis. Reconstituted plasmid pcDNA3-rIL-10 was rapidly injected into rat in large volume in a short time through tail vein. RT-PCR, ELISA and immunohistochemistry analysis were used to detect the expression and distribution of rIL-10 gene in rat. Thirty SD rats were divided randomLy into control group (group N), fibrosis model group (group M), rIL-10 gene therapy group (group I) and empty vector control group (group P). Rats in group N were treated with 0.5mL normal sodium twice a week for 8 wk via intraperitoneal injection, and rats in other groups were treated with 0.5mL pig serum twice a week for 8 wk. At the beginning of the 5th wk, rats in group N and M were injected weekly with Ringer,s solution, rats in group I were injected weekly with the rIL-10 recombinant plasmid pcDNA3-rIL-10 and rats in group P were injected weekly with empty vector pcDNA3 via tail vein. At the end of the 8th wk, all rats were put to death and then collected sample of liver tissue and serum. The grading and staging of hepatic fibrosis were measured by HE staining. The change of ALT and AST in serum was measured by biochemistry analysis. The change of collagen type I、III was measured by Picrosirius staining and the expression ofα-SMA、MMP-13 and TIMP-1 in liver tissue were measured by S-P immunohistochemistry.
Results: DNA sequencing and restriction endonuclease digestion confirmed that eukaryotic expression vector pcDNA3-rIL-10 was constructed successfully. RT-PCR and ELISA assay confirmed that the BRL cells transfected with plasmid pcDNA3-rIL-10 expressed highly secretary type rIL-10 protein and the expression of rIL-10 show a few protection effect on RBL cells transfected. RT-PCR and immunohistochemistry confirmed the presence of the rIL-10 transcription and expression mainly in liver and ELISA assay confirmed maintaining a stable expression of rIL-10 in serum can be assessed by repeated administration. The liver pathohistology analysis confirmed experimental rat hepatic fibrosis model induced by pig serum was established successfully. Compared with the rats in group M and P, rats in group I showed significant therapeutic effects: decreased the degree of hepatocyte necrosis and degeneration and the number of inflammatory cells around lobular veins, reduced the disposition of collagen fibers and reduced the levels of ALT and AST in serum(P<0.01). Picrosirius staining confirmed the expression of collagen type I、III was significantly suppressed (P<0.01)and the immunohistochemistry confirmed positive levels forα-SMA and TIMP-1 were decreased significantly in group I (P<0.01).
Conclusions: rIL-10 plasmid DNA tail vein injection can prevent efficiently the progression of experimental rat hepatic fibrosis induced by pig serum, the therapeutic effects of rIL-10 plasmid injection on hepatic fibrosis may be primarily exerted by making rIL-10 transcription and expression mainly in liver, inhibiting the expression ofα-SMA and TIMP-1 and promoting collagen type I、III resolution.
引文
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