摘要
目的探讨转化生长因子βⅡ型受体免疫球蛋白(Ig)G重链Fc段(TGFβRⅡ/IgG Fc)嵌合蛋白基因转染改善小鼠环孢素A(CsA)肾毒性细胞凋亡的作用。方法雌性美国癌症研究所(ICR)小鼠予低钠饮食,CsA组和干预组每日皮下注射CsA。在给药的第1天和第7天,CsA组在胫骨肌处注射携带lacZ基因的质粒(pCAGGS-lacZ),干预组注射pCAGGS-TGFβRⅡ/IgGFc,给药2或3周后处死。血浆和肾组织中转化生长因子(TGF)-β1蛋白水平由酶联免疫吸附试验(ELISA)和免疫组织化学法测得,肾组织中TGF-β1 mRNA水平用Northern印迹法测定。利用过碘酸雪夫反应(PAS)和Masson三色染色半定量评分评估肾小管损害和肾间质纤维化程度。采用末端脱氧核糖转移酶介导的生物素化脱氧尿嘧啶缺刻标记技术(TUNEL法)检测肾小管间质细胞凋亡。结果CsA给药2周后,血浆、肾脏TGF-β1表达及其mRNA水平均达到最高,显著高于对照组(P值分别<0.05、0.01);小管间质病变也逐渐出现,PAS染色半定量评分和Masson套染计算肾皮质胶原容积均显著高于对照组(P值均<0.01);小管间质凋亡细胞百分比显著高于对照组(P<0.01)。CsA给药3周后,血浆TGF-β1表达降至正常,肾脏TGF-β1表达及其mRNA水平虽有所下降,但仍显著高于对照组(P值分别<0.01、0.05);小管间质病变较2周时明显加重(P<0.01);而小管间质凋亡细胞表达进一步升高(P<0.01)。干预2周组小鼠的血浆及肾组织TGF-β1表达均较CsA 2周组显著下降(P值分别<0.01、0.05),而TGF-β1 mRNA水平无明显改变;Masson套染计算肾皮质胶原容积显著低于CsA2周组(P<0.05);小管间质凋亡细胞百分比显著低于CsA2周组(P<0.01)。干预3周组的血浆、肾脏TGF-β1表达及其mRNA水平与CsA3周组的差异均无统计学意义(P值均>0.05);小管间质病变持续减轻,PAS染色半定量评分和Masson套染计算肾皮质胶原容积显著低于CsA3周组(P值均<0.01);小管间质凋亡细胞百分比显著低于CsA3周组(P<0.01)。小管间质损伤程度和间质纤维化程度均与凋亡细胞百分比呈正相关(r=0.804、0.906,P值均<0.01)。结论TGFβRⅡ/IgG Fc基因转染有效抑制了CsA引起的小管间质病变。细胞过度凋亡参与了慢性CsA肾病的发病,且与小管间质损伤及纤维化程度密切相关。TGF-β1在这一过程中发挥了重要作用,TGFβRⅡ/IgG Fc的干预治疗可有效抑制上述改变。
Objective To study the inhibitory effect of TGFβRⅡ / IgG Fc, a soluble chimeric protein containing the extracellular domain of human TGFβtype Ⅱ receptor and Fc fragment of IgG1, on cell apoptosis in Cyclosporine A （CsA） nephrotoxicity in mice. Methods Subcutaneous injection of CsA （25 mg·kg^-1·d^-1） was given daily to female mice maintained on low sodium diet. Three days before transfection, bupivacaine was injected into the bilateral tibial anterior muscles. On day 1 and 7, expression vectors carrying cDNA for either TGFβRⅡ/IgG Fc or β-galactosidase （50 μg） were transfected into the skeletal muscles by electroporation. CsA injection was continued until sacrifice at 2 or 3 weeks. The concentration of total TGF-β in plasma and the expression of TGF-β in renal tissues were determined by a sandwish enzyme linked immunosorbent assay and immunohistochemistry method, respectively. The levels of TGF-β mRNA were determined by Northern blot Analysis. The degrees of tubulointerstitial injury were semiquantitatively evaluated on periodic-acid-Schiff stain and the extents of interstitial fibrosis were determined using Masson＇s trichrom staining. Renal apoptotic cells were detected by TUNEL method. Results The plasma, renal TGF-β1 levels as well as TGF-β1 mRNA in renal tissues peaked 2 weeks＇ after CsA administration, significantly higher than the normal levels （P〈 0.05 or P〈0.01 ）; tubulointerstitial alterations also became appreciable. The percentage of apoptotic cells in the tubulointerstitial areas were significantly elevated （P〈0.01）. At 3 weeks, the plasma TGF-β1 levels decreased to the normal levels renal TGF-β1 levels and TGF-β1 mRNA expression were also decreased, but still higher than the baseline levels （P〈0.05）; and the tubulointerstitial alterations were intensified obviously （ P〈0.01 ）. The percentage of apoptotic cells in the tubulointerstitial areas was further increased compared to those at 2 weeks（ P〈0.01 ）. At 2 weeks, the plasma and renal TGF-β1 levels were significantly reduced （P〈0.05）; however, the renal TGF-β1 mRNA had no significant changes when compared to the CsA group at 2 weeks. The TGFβR Ⅱ/IgG Fc intervention markedly suppressed the histological alterations （P〈0.05）. The percentage of apoptotic cells in the tubulointerstitial areas was significantly reduced compared to the CsA group at 2 weeks. At 3 weeks, the plasma and renal TGF-β1 levels as well as TGF-β1 mRNA in renal tissues were similar to those of the CsA group at 3 weeks; the histological alterations remained markedly suppressed by the intervention （P〈0.01）. The degree of tubulointerstitial injury and the extent of interstitial fibrosis were correlated with the percentage of apoptotic cells （r=0.804 and r= 0.906, P〈0.01）. Conclusion Introduction of TGFβR Ⅱ/IgG Fc by gene can effectively abrogate CsA-induced tubulointerstitial alterations. Excessive celt apoptosis may take part in the pathogenesis of CsA nephrotoxicity; it may also be related to both the degree of tubulointerstitial injury and the extent of interstitial fibrosis. TGF-β1 may play an important role during the above process and introduction of TGFβRⅡ/IgG Fc by gene transfer can effectively abrogate CsA-induced tubulointerstitial alterations. （Shanghai Med J, 2009, 32： 230-235）
出处
《上海医学》
CAS
CSCD
北大核心
2009年第3期230-235,I0004,共7页
Shanghai Medical Journal
关键词
环孢素A
转化生长因子-Β
凋亡
基因治疗
Cyclosporine A
Transforming growth factor
Apoptosis
Gene theraphy
