【作者】 陈超；

【导师】 周序珑； 蒋建伟；

【作者基本信息】 暨南大学 ， 病理学， 2008， 硕士

【摘要】 目的:观察促肝细胞生长素（pHGF）和结缔组织生长因子（CTGF）反义寡核苷酸（ASODN）对人肾小管上皮细胞HKC上皮-间质细胞转分化,及对大鼠肾间质纤维化的作用。方法:细胞实验:用巨噬细胞趋化因子-1和马兜铃酸I联合用药诱导建立HKC上皮-间质细胞转分化模型。分别给与pHGF、CTGF ASODN和（pHGF+CTGF ASODN）联合用药。流式细胞仪检测12h、24h时HKC细胞凋亡率;RT-PCR方法检测α-SMA的mRNA表达水平;细胞免疫组化检测HKC细胞α-SMA、TGF-β₁、FN的蛋白表达水平。动物实验:48只清洁级Sprague-Dawley（SD）大鼠随机分为假手术空白对照组、手术模型对照组、CTGF错义寡核苷酸对照组（CTGF MSODN）、pHGF组、CTGFASODN组、（pHGF+CTGF ASODN）联合用药组。以上各组造模后给予不同处理,饲养14d,然后处死大鼠。碱水解比色法检测新鲜湿重大鼠肾组织胶原定量;Masson染色观察肾皮质胶原形态学改变;RT-PCR检测大鼠肾组织α-SMA的mRNA表达水平;免疫组化检测大鼠肾肾皮质α-SMA、TGF-β₁、FN蛋白表达水平。结果:细胞试验:流式细胞仪检测:24 h模型组细胞凋亡率9.1%,浓度为15、150、1500 ng/ml的pHGF细胞凋亡率为3.3%—4.4%,与模型组比较,有显著性差异,p＜0.01;CTGF ASODN无显著差异,p＞0.05;RT-PCR方法:模型组的α-SMA/GAPDH比值为0.91±0.002,pHGF组、CTGF ASODN组和联合用药组α-SMA/GAPDH比值分别为0.68±0.015、0.61±0.005、0.65±0.010。与模型对照组比较,三者均有统计学意义,p＜0.05。但协同拮抗作用经金正均概率和法分析:Q=0.572,＜0.85,二者为拮抗作用;免疫组化检测:pHGF、CTGF ASODN和联合用药均降低α-SMA、TGF-β₁、FN蛋白水平。动物试验:碱水解比色法:湿重肾组织胶原含量UUO模型组均值为4.85±0.201μg/mg,pHGF组均值为3.76±0.134μg/mg,CTGF ASODN组均值为3.34±0.306μg/mg,联合用药组为4.06±0.179μg/mg,CTGF MSODN错义寡核苷酸对照组为4.91±0.172。与UUO模型组比较,pHGF组、CTGF ASODN组及联合用药组均有显著性差异,p＜0.01。但协同拮抗作用,经金正均概率和法分析:pHGF和CTGF ASODN两种药物的Q=0.349,＜0.85,二者为拮抗作用;②Masson染色:与UUO模型对照组相比,pHGF、CTGF ASODN及联合用药组的肾脏着色绿染的胶原纤维面积明显减少;③RT-PCR检测:UUO模型组α-SMA/β-actin比值为1.31±0.049,CTGF ASODN组为0.77±0.022,有统计学意义,p＜0.05;④免疫组化:pHGF、CTGF ASODN及二者联合用药均明显降低α-SMA、TGF-β₁、FN蛋白表达水平。结论:pHGF和CTGF ASODN在离体试验和在体试验,二者均抑制肾小管上皮细胞的上皮-间质细胞转分化,抑制肾脏间质纤维化。但二者联合用药,没有协同作用。更多还原

【Abstract】 Objective:To explore the roles of hepatocyte growth-promoting factor（pHGF） and connective tissue growth factor（CTGF） antisense oligodeoxynucleotide（ASODN） in epithelial-to-mesenchymal transition（EMT） of human kidney epithelial cell lines（HKC） and in the process of rat renal interstitial fibrosis.Methods:Cell experiment:Epithelial-to-mesenchymal transition of human kidney epithelial cell lines（HKC） were induced by MCP-1 and AAⅠ,then administered pHGF,CTGF antisense oligodeoxynucleotide（ASODN） respectively and synergistically.Cell apoptosis andα-SMA mRNA were detected by flow cytometry,andα-smooth muscle actin（α-SMA） mRNA was detected by reverse transcriptase-polymerase chain reaction（RT-PCR）,α-SMA,Fibronectin（FN） and transforming growth factor-β₁ （TGF-β₁） of HKC cells were assessed by indirect enzyme immunohistochemistry. Animal experiment:48 Sprague-Dawley（SD） rats were randomly divided to sham-operation group（SOR）,unilateral ureteral obstruction group（UUO）,pHGF treated group（pHGF）,CTGF ASODN treated group,CTGF missense oligodeoxynucleotide （MSODN） treated group and Drug combination group（pHGF and CTGF ASODN）.All groups of rats were treated with different drugs and raised in the following days after unilateral ureteral obstruction,then killed in the 14th day.The degree of renal fibrosis was assessed by measuring cortex hydroxyproline content, The Collagen distribution was evaluated by Masson stain,α-SMA mRNA was detected by RT-PCR,andα-SMA,FN,TGF-β₁ in the renal cortex were examined by immunohistochemistry.Results:Cell experiment:apoptosis rate for cells treated with 5、150、1500 ng/ml pHGF was 3.3%—4.4%on 24 h by flow cytometry,but increased to 9.1%in model group.The rate was significantly lower in treated groups than that in pHGF group（p＜0.01）,but there was no difference between model group and CTGF ASODN group.The rate ofα-SMA/GAPDH in Model group,pHGF,CTGF ASODN and Drug combination group was 0.91±0.002,0.68±0.015,0.61±0.005,0.65±0.010 respectively by RT-PCR.α-SMA mRNA level in treated groups was lower than that in model group（p＜0.01）. Antagonistic effect existed in pHGF and CTGF ASODN valuated by Jin Zhengjun’s Q-value Probability Statistics（Q=0.572,＜0.85）.Proteins ofα-SMA,FN,TGF-β₁ in HKC cells were decreased in pHGF,CTGF ASODN and combined group,detected by immunohistochemistry,compared with that in model group.Animal experiment:Renal tissue hydroxyproline content in UUO model group, pHGF group,CTGF ASODN group,Drug combination group and CTGF MSODN group was 4.85±0.201,3.76±0.134,3.34±0.306,4.06±0.179 and 4.91±0.172μg/mg respectively.Compared wtih UUO model group,pHGF group CTGF ASODN group and Drug combination group had higher content of hydroxyproline（p＜0.01）.But there was opposite effects in pHGF and CTGF ASODN valuated by Jin Zhengjun’s Q-value Probability Statistics（Q =0.349,＜0.85）.Fibrosis degree was weaker in pHGF group,CTGF ASODN group and Drug combination group than that in model group assessed by Masson stain.Rate ofα-SMA/β-actin mRNA in UUO group and CTGF ASODN group was 1.31±0.049,0.77±0.022 by RT-PCR,respectively.The rate in CTGF ASODN group was lower than that in UUO model group（p＜0.05）,α-SMA, FN,TGF-β₁ expressed in the renal cortex were weaker in pHGF group,CTGF ASODN group and Drug combination group,compared with that in UUO model group and CTGF MSODN group detected by immunohistochemistry.Conclusions:pHGF and CTGF ASODN antagonize EMT of HKC and inhibited fibrosis development in rat renal interstitium induced by unilateral ureteral obstruction, respectively,but there was no synergistic effect when treated with both.更多还原