【作者】 刘蔚；

【导师】 刘晓城；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 慢性肾脏疾病(chronic kidney disease,CKD)的进展是一个不可逆转的过程,最终导致终末期肾病(end stage renal disease,ESRD)。肾间质纤维化(renal interstitial fibrosis,RIF)几乎是所有肾脏疾病进展到终末期肾功能衰竭的共同病理途径。大量动物和临床试验表明,肾功能的恶化趋势取决于肾小管间质损伤的程度和范围。肾小管间质病变程度是反映肾功能下降严重程度和判断预后最重要的指标。目前全世界罹患终末期肾功能衰竭的患者以每年7%的速度递增,给家庭及社会带来了沉重的经济负担。进一步研究肾间质纤维化的分子机制,探索有效的防治措施,对延缓ESRD的进程意义重大。肾间质纤维化是以肾小管萎缩,间质成纤维细胞增生及细胞外基质(extracellular matrix,ECM)过度积聚为特征的病理改变。在肾脏疾病时,肾间质内浸润的炎症细胞特别是单核细胞及活化的肾小管上皮细胞合成分泌的促纤维化因子,如TGF-β1、内皮素及血小板衍生生长因子(PDGF)等,以旁分泌方式作用于成纤维细胞,使之活化增殖,不仅数量增加,还会转分化为肌成纤维细胞(MyoF),表达标志性蛋白α-SMA,同时分泌大量的细胞外基质(ECM),包括Ⅲ型胶原及FN等。因此抑制肾成纤维细胞的活化增殖,降低ECM的分泌,在阻止甚至逆转肾间质纤维化过程中起至关重要的作用。甲磺酸伊马替尼是一种Abl酪氨酸激酶抑制剂,它能特异性抑制BCR-ABL+细胞酪氨酸激酶的活性,能与三磷酸腺苷(ATP)或底物竞争位于激酶催化中心的结合位点,使之因无ATP而失去磷酸的来源,阻止磷酸基团向酪氨酸残基转移,从而抑制酪氨酸激酶的磷酸化,造成信号传导抑制。除p210BCR-ABL外,它也能抑制p185BCL-ABL、V-ABL、C-ABL以及血小板源性生长因子(PDGF),干细胞因子(c-kit)受体的酪氨酸激酶的活性。初步观察显示,甲磺酸伊马替尼(Imatinib mesylate)在骨髓和肺纤维化防治方面显示出良好的效果,有可能为CML以外的其他酪氨酸激酶相关疾病带来新的治疗前景。甲磺酸伊马替尼对肾间质纤维化的作用如何目前并不清楚。因此,我们以小鼠单侧输尿管梗阻(UUO)动物模型为研究对象,观察甲磺酸伊马替尼对梗阻侧肾脏的病理改变影响,用酸水解-比色法测定肾组织胶原的含量;用免疫组化法观察肾组织TGF-β、α-SMA、PAI-1、PCNA和FN的表达,用RT-PCR法测定梗阻侧肾组织α-SMA、Vimentin、FN和ColⅢmRNA的表达水平,用Western杂交分析法检测α-SMA和Vimentin蛋白表达情况。另外,本研究还以体外培养的小鼠swiss-3T3细胞为研究对象,观察STI571对TGF-β1诱导小鼠成纤维细胞活化增殖及细胞外基质表达的影响,以探讨甲磺酸伊马替尼对肾间质纤维化的影响及可能机制,为临床防治肾间质纤维化寻找新的途径。第一部分Abl酪氨酸激酶抑制剂对单侧输尿管梗阻小鼠肾脏间质纤维化的影响目的:研究甲磺酸伊马替尼STI571对单侧输尿管梗阻(UUO)小鼠肾间质纤维化的防治作用。方法: 48只雄性清洁级昆明种小鼠随机分为4组:假手术组,模型组,小剂量治疗组(80mg/kg/d),大剂量治疗组(160mg/kg/d)。假手术组仅游离左侧输尿管后即缝合皮肤, UUO组和STI571治疗组进行左侧输尿管双结扎,治疗组在UUO的基础上每天以STI571 80、160mg/Kg灌胃。假手术组, UUO组和甲磺酸伊马替尼治疗组于术后第8,11天分别处死6只小鼠。肾组织行PAS及Masson染色,光镜下观察肾脏病理改变。采用百分比评分法评估肾间质纤维化程度,计算400×HP下炎性细胞计数;用酸水解-比色法测定肾组织胶原的含量;用免疫组化技术检测肾脏纤维连结蛋白(FN)的沉积。利用逆转录-聚合酶链反应(RT-PCR)观察FN和Ⅲ型胶原(ColⅢ) mRNA表达。结果:从外观上看,与UUO组相比较,甲磺酸伊马替尼治疗组在对应时间点上梗阻侧肾脏的肿大及积水程度较轻,色泽较红润。STI571治疗组的肾间质纤维化定量分析在术后11天显著低于UUO组(P<0.05),且不同剂量组之间存在显著差异(P<0.05)。随着梗阻时间延长,UUO组和STI571治疗组炎性细胞浸润逐渐增多,但两组无明显差异。UUO组和STI571治疗组梗阻肾FN染色随梗阻时间延长而逐渐增加,术后11天STI571治疗组的表达较UUO组明显减低,统计学上有显著性差异(p<0.05),且不同剂量组之间存在显著差异(P<0.05)。UUO组和STI571治疗组FN和ColⅢA1mRNA表达随梗阻时间延长而逐渐增加,在对应时间点上FN和ColⅢA1mRNA表达较UUO组明显减低,统计学上有显著性差异(p<0.05),且不同剂量组之间存在显著差异(P<0.05)。结论:甲磺酸伊马替尼可显著减轻UUO小鼠梗阻侧肾脏间质纤维化,下调纤维连结蛋白(FN)和Ⅲ型胶原(ColⅢ)的表达,减少肾间质细胞外基质的沉积,对UUO小鼠肾间质纤维化有一定防治作用。第二部分STI571缓解小鼠梗阻性肾病肾间质纤维化的机制研究目的:对甲磺酸伊马替尼STI571减轻UUO小鼠肾间质纤维化的机理进行初步探讨。方法: 48只雄性清洁级昆明种小鼠随机分为4组:假手术组,模型组,小剂量治疗组(80mg/kg/d),大剂量治疗组(160mg/kg/d)。假手术组仅游离左侧输尿管后即缝合皮肤, UUO组和STI571治疗组进行左侧输尿管双结扎,治疗组在UUO的基础上每天以STI571 80、160mg/Kg灌胃。假手术组, UUO组和甲磺酸伊马替尼治疗组于术后第8,11天分别处死6只小鼠。用免疫组化技术检测TGF-β1、PAI-1、α-SMA和间质PCNA的表达。利用逆转录-聚合酶链反应(RT-PCR)观察α-SMA、Vimentin mRNA表达。利用Western Blot方法观察α-SMA和Vimentin蛋白表达情况。结果:1)假手术组TGF-β1、PAI-1仅有少量表达,随着梗阻时间延长,UUO组和STI571治疗组TGF-β1、PAI-1的表达逐渐增多,术后8～11天STI571治疗组TGF-β1、PAI-1的表达虽低于对应时间点的UUO组,但均无显著差异(P>0.05)。2)假手术组α-SMA仅有微量表达,随着梗阻时间延长,UUO组和STI571治疗组α-SMA的表达逐渐增多,术后8～11天STI571治疗组α-SMA的表达显著低于对应时间点的UUO组(P<0.05),且术后11天大剂量治疗组α-SMA的表达显著低于小剂量组(P<0.05)。3)假手术组肾小管上皮细胞、肾间质细胞胞核内有部分细胞核表达PCNA,随着梗阻时间延长,模型组表达PCNA的细胞核数明显增加(P<0.01)。用药组表达较模型组明显减少(P<0.05)。4)RT-PCR和Western-blot检测结果显示测定随着梗阻时间的延长,UUO组和STI571治疗组α-SMA、Vimentin在mRNA和蛋白表达水平上逐渐上升,STI571治疗组两者的表达显著低于对应时间点的UUO组(P<0.05),两剂量组间差异显著(P<0.05)。结论:甲磺酸伊马替尼STI571可能通过抑制肾间质成纤维细胞增殖活化,从而下调肾组织α-SMA表达水平,减少肌成纤维细胞的聚集,减少肾间质细胞外基质的沉积,并最终改善肾间质纤维化。同时该作用独立于TGF-β1和纤溶系统的变化。第三部分Abl酪氨酸激酶抑制剂STI571对TGF-β1激活小鼠成纤维细胞及诱导细胞外基质表达的影响目的:研究Abl酪氨酸激酶抑制剂(PKI)STI571对转化生长因子TGF-β1致组织纤维化作用的影响,探讨其抗纤维化的潜在作用机制。方法:体外培养小鼠成纤维细胞株(3T3-swiss)。用MTT法测定PKI STI571对TGFβ1诱导的3T3细胞增殖效应的影响。利用逆转录-聚合酶链反应(RT-PCR)观察PKI STI571对TGFβ1诱导的α-平滑肌肌动蛋白(α-SMA)、纤维连结蛋白(FN)和Ⅲ型胶原(ColⅢ) mRNA表达的影响。利用Western Blot方法观察PKI STI571对TGFβ1诱导的α-SMA蛋白表达的影响。结果:TGF-β1明显诱导3T3细胞增殖,而PKI STI571可以阻断该效应。TGF-β1能显著增加3T3细胞α-SMA、FN和ColⅢmRNA表达;与TGF-β1刺激组相比, PKI STI571处理组α-SMA、FN和ColⅢmRNA表达量显著减少,而α-SMA mRNA、α-SMA蛋白表达量无显著下降。结论:Abl酪氨酸激酶抑制剂STI571可抑制TGF-β1诱导的成纤维细胞活化增殖和细胞外基质(ECM)合成,但不能阻断TGF-β1诱导的α-SMA表达增加。更多还原

【Abstract】 Section one The effects of Abl protein tyrosine kinase on renal interstitial fibrosis lesions in UUO miceOBJECTIVE: To evaluate the effect of Imatinib mesylate (ST571) on the process of renal interstitial fibrosis following unilateral ureteral obstruction in mice.METHODS: 48 male mice were randomly assigned to four groups: Shame operation group, UUO group, and UUO receiving STI571 treatment daily. The mice of treatment group were treated with ST571 (80、160mg/Kg/d) after operation through intragastric administration. The mice of shame operation group were just separated left ureter while the mice of UUO group and treatment group were performed operation by left ureter double ligature. Mice of each group were killed at 8,11 days after operation respectively. Morphological changes of renal tissue were observed by PAS and Masson stain and were investigated through light microscope. The method of percentage evaluation was used to evaluate the degree of renal fibrosis. The number of the inflammatory cells which accumulate in renal interstitial was calculated. The content of total collagen from each group was examined by acidolysis and shade selection. Immunohistochemistry method was performed to investigate the protein expression of fibronectin(FN) in kidney. Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of FN and ColⅢmRNA.RESULTS:1 Compared with UUO group, the degree of swelling and hydrocele of the obstructive renal in the treatment group was milder. The color of latter was redder.2 The degree of renal fibrosis in treatment group was much lower than that in UUO group at 11 days after surgery (p<0.05). There was significant difference between the two treatment group. (p<0.05)3 In both UUO and treatment groups, the number of the inflammatory cells which accumulated in renal interstitial increased markedly in time-dependent manner from 8 to 11 days after operation. There was no significant difference between UUO group and treatment group.4 The expression of FN and ColⅢincreased in time-dependent manner in both UUO group and treatment group, while, it was significantly lower in treatment group at 11 days after surgery (p<0.05). There was significant difference between the two treatment groups. (p<0.05)CONCLUTION: Compared to UUO group, Imatinib mesylate significantly ameliorated the histological changes of the UUO renal tissue. The expression of FN were decreased significantly in treatment group.It could reduce the deposition of extracellular matrix in the interstitium.It played a protective role in the renal interstitial fibrosis. Section two The mechanisms of STI571 to ameliorate the degree of renal interstitial fibrosis in experimental obstructive nephropathy in miceObjective: To explore the mechanism of Abl protein tyrosine kinase inhibitor to ameliorate the degree of renal interstitial fibrosis.METHODS: 48 male mice were randomly assigned to four groups: Shame operation group, UUO group, and UUO receiving STI571 treatment daily. The mice of treatment group were treated with STI571 (80、160mg/Kg/d) after operation through intragastric administration. The mice of shame operation group were just separated left ureter while the mice of UUO group and treatment group were performed operation by left ureter double ligature. Mice of each group were killed at 8,11 days after operation respectively. Immunohistochemistry method was performed to investigate the protein expression of TGF-β1、PAI-1、α-SMAand PCNA in the interstitium. Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression ofα-SMA and Vimentin mRNA. Western-blot was performed to examine the expression ofα-SMA and Vimentin protein.RESULTS:1 The results of immunohistochemistry showed that there was little expression of TGF-β1、PAI-1 in shame operation group .The expression of TGF-β1、PAI-1 increased in time-dependent manner in both UUO and treatment group. But there was no significant difference between UUO group and treatment group. (p>0.05).2 There was little expression ofα-SMA in shame operation group .The expression ofα-SMA increased in time-dependent manner in both UUO and treatment group while it was significantly less in the latter (p<0.05). There was significant difference between the two treatment group at 11 days after surgery. (p<0.05)3 The expression of PCNA increased in time-dependent manner in both UUO and treatment group while they were significantly less in the latter (p<0.05). 4 The results of RT-PCR and Western-blot showed that the expression ofα-SMA and vimentin mRNA or protein increased since operation. The expression ofα-SMA and vimentin mRNA or protein in treatment group markedly lower than UUO group (p<0.05 ).Conclusion: Imatinib mesylate could suppress the activation and proliferation of fibroblasts in the interstitium, downregulate the expression ofα-SMA in the interstitium, reduce the accumulation of myofibroblast and deposition of ECM, ameliorate renal interstitial fibrosis. Meanwhile it did not accompany with the change of expression of TGFβ1 and the fibronolysis system.Section three Effects of protein tyrosine kinase inhibitor STI571 on TGF-β1 induced activation and extracellular matrix expression in mouse fibroblastsObjective: To study the effects of protein tyrosine kinase inhibitor STI571 on TGF-β1 induced fibrotic response in the mouse fibroblasts, so as to investigate its effects on preventing tubulointerstitial fibrosis.Methodology: In cultured mouse fibroblasts cell line (3T3-swiss),the effects of PKI STI571 on activation and proliferation induced by TGF-β1 were observed by MTT assay. The effects of PTKI STI571 on the level of TGF-β1 inducedα-smooth muscle actin (α-SMA), fibronectin (FN), and collagen type III m RNA expression was observed by reverse trancriptase–polymerase chain reaction (RT-PCR).the effects of PKI STI571 on the level ofα-SMA protein expression induced by TGF-β1 was observed by Western Blot.Results:TGF-β1 stimulated the NIH/3T3 cells proliferation and it can be blocked by STI571. In NIH/3T3 cells, TGF-β1 enhancedα-SMA, FN and ColⅢmRNA expression. The level of FN and ColⅢmRNA expression in STI571 treated groups were significantly decreased compared with TGF-β1 stimulated group. But level ofα-SMA mRNA andα-SMA protein expression in STI571 treated groups were not significantly decreased.Conclusion: Protein tyrosine kinase inhibitor STI571 may inhibit TGF-β1-induced cell proliferation and extracellular matrix sythesis in the mouse fibroblasts, but it can not block theα-SMA expression induced by TGF-β1.更多还原