全反式维甲酸对体外培养阿霉素致肾小球足细胞损伤的作用及其分子机制

发布时间：2018-05-14 10:55

本文选题：全反式维甲酸 + 足细胞损伤　；参考：《广西医科大学》2014年博士论文

【摘要】：目的：通过检测肾小球足细胞凋亡指数、nephrin、podocin、TGF-β1和α-SMA的表达，探讨全反式维甲酸（ATRA）对体外培养阿霉素（ADR）致肾小球足细胞损伤的作用。方法：将体外培养肾小球足细胞随机分成4个组：空白对照组（NC），阿霉素致足细胞损伤组（AI），ATRA干预的空白对照组（AC），ATRA干预的阿霉素致足细胞损伤组（AA）。AI组和AA组肾小球足细胞铺满孔底后首先置于含有0.15ug/ml阿霉素的培养基中孵育24小时构建足细胞损伤模型。阿霉素孵育24小时后各组肾小球足细胞均换液，NC组和AI组肾小球足细胞继续置于正常培养基中培养，AC组和AA组肾小球足细胞则置于含有0.1uM ATRA的培养基中继续培养。ATRA干预后24小时，收集肾小球足细胞进行形态学和分子生物学检测。倒置显微镜和电子显微镜下观察肾小球足细胞形态和超微结构的改变，流式细胞仪检测肾小球足细胞凋亡指数，MTT法检测并绘制肾小球足细胞生长曲线，蛋白质印迹法（Western-blot）检测各组肾小球足细胞转化生长因子-β1（transforming growth factor-β1，TGF-β1）、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、nephrin和podocin的蛋白表达，逆转录实时定量聚合酶链反应（Real time RT-PCR）检测肾小球足细胞TGF-β1、α-SMA、nephrin和podocin的mRNA表达。结果：（1）与NC组比较，AI组肾小球足细胞排列紊乱、稀疏，胞体四周失去应有的折光性，电镜下肾小球足细胞足突融合、回缩，部分细胞足突消失。ATRA干预后肾小球足细胞大部分呈梭形或多边形、排列较整齐，细胞轮廓清楚；电镜下肾小球足细胞萎缩，足突融合、回缩不明显。（2）NC组和AC组肾小球足细胞呈现近似“S”型的细胞生长曲线。AI组肾小球足细胞在阿霉素干预24小时后细胞生长均受到明显抑制；AA组肾小球足细胞生长抑制现象较AI组肾小球足细胞明显减轻。（3）与NC组相比，AI组肾小球足细胞凋亡率显著增加（P0.05）。与AI组相比，AA组肾小球足细胞凋亡率显著降低（P0.05）。（4）AI组肾小球足细胞TGF-β1、α-SMA蛋白及其mRNA表达均较NC组显著升高（P0.05）。AC组和AA组肾小球足细胞TGF-β1、α-SMA蛋白及其mRNA表达均较AI组显著降低（P0.05）。（5）AI组肾小球足细胞nephrin、podocin蛋白及其mRNA表达均较NC组显著降低（P0.05）。AC组和AA组肾小球足细胞nephrin、podocin蛋白及其mRNA表达均较AI组显著升高（P0.05）。（6）AI组肾小球足细胞nephrin和podocin蛋白表达分别与α-SMA、TGF-β1蛋白表达呈显著负相关（P0.05）。AI组肾小球足细胞α-SMA蛋白表达与TGF-β1蛋白表达呈显著正相关（P0.05）。AA组肾小球足细胞凋亡指数与TGF-β1蛋白表达呈显著正相关（P0.05）。结论： ATRA可能通过抑制体外培养的肾小球足细胞凋亡和转分化，促进足细胞分化，从而减轻阿霉素所致的足细胞损伤。目的：通过检测肾小球足细胞基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）、维甲酸受体-α（RAR-α）、维甲酸受体-β（RAR-β）和维甲酸受体-γ（RAR-γ）等的表达，，探讨全反式维甲酸减轻体外培养阿霉素致肾小球足细胞损伤的分子机制。方法：将体外培养的肾小球足细胞随机分成3个组：空白对照组（NC），阿霉素致足细胞损伤组（AI），ATRA干预的阿霉素致足细胞损伤组（AA）。肾小球足细胞损伤模型的构建及各组肾小球足细胞的干预方法同第一部分。倒置显微镜和扫描电子显微镜下观察肾小球足细胞形态和超微结构改变；MTT法检测各组肾小球足细胞活力；明胶酶谱法检测各组肾小球足细胞MMP-2、MMP-9的酶活性；Western-blot检测各组肾小球足细胞MMP-2、MMP-9、RAR-α、RAR-β、RAR-γ、α-SMA和TGF-β1的蛋白表达；Real-time RT-PCR检测各组肾小球足细胞MMP-2、MMP-9、RAR-α、RAR-β、RAR-γ的mRNA表达。结果：（1）与NC组比较，电镜下AI组足细胞萎缩，足突融合、回缩、部分消失；AA组肾小球足细胞萎缩，足突融合、回缩不明显。（2）与NC组相比，AI组肾小球足细胞活力显著减弱（P0.05）；与AI组相比，AA组肾小球足细胞活力显著增强（P0.05）。（3）与NC组相比，AI组肾小球足细胞MMP-2、MMP-9的蛋白及其mRNA表达均显著降低（P0.05），而TGF-β1蛋白表达显著升高（P0.05）；与AI组相比，AA组肾小球足细胞MMP-2、MMP-9的蛋白及其mRNA表达均显著升高（P0.05），而TGF-β1蛋白表达显著降低（P0.05）。（4）AI组肾小球足细胞RAR-α、RAR-γ的蛋白及其mRNA表达均较NC组显著降低（P0.05），AA组肾小球足细胞RAR-α、RAR-γ的蛋白和mRNA表达均较AI组明显升高（P0.05）。（5）AI组肾小球足细胞RAR-β蛋白及其mRNA表达较NC组降低，但差异无显著性（P0.05）； AA组肾小球足细胞RAR-β蛋白及其mRNA表达较AI组升高，但差异无显著性（P0.05）。（6）与NC组相比，AI组肾小球足细胞MMP-2和MMP-9酶活性均显著减弱（P0.05）。与AI组相比，AA组肾小球足细胞MMP-2和MMP-9酶活性均显著增强（P0.05）。（7）AI组肾小球足细胞RAR-α、RAR-γ蛋白表达与MMP-2和MMP-9蛋白表达均呈显著正相关（P均0.05），与TGF-β1和α-SMA蛋白表达均呈显著负相关（P＜0.05）。AI组肾小球足细胞RAR-β蛋白表达与MMP-2、MMP-9、TGF-β1和α-SMA蛋白表达均无显著相关性（P0.05）。AI组肾小球足细胞MMP-2和MMP-9蛋白表达与肾小球足细胞活力呈显著正相关（P0.05），与TGF-β1和α-SMA蛋白表达呈显著负相关（P均0.05）。结论：在体外培养的阿霉素致足细胞损伤中，ATRA可能通过与RAR-α和RAR-γ结合调控肾小球足细胞MMP-2和MMP-9表达增加，从而减轻肾小球足细胞损伤。
[Abstract]:Objective : To investigate the effect of all - trans retinoic acid ( ATRA ) on the damage of cultured rat glomerular cells in vitro by detecting the expression of apoptosis index , nephrin , podocin , TGF - 尾1 and 伪 - SMA . Methods : After 24 hours of incubation with adriamycin , the cells were cultured in normal medium . The expression of TGF - 尾1 , 伪 - SMA , nephrin and podocin were detected by reverse microscopy and electron microscope . The mRNA expression of TGF - 尾1 , 伪 - SMA , nephrin and podocin was detected by reverse transcription - real time RT - PCR .

Results : ( 1 ) Compared with NC group , in AI group , the cells of glomerulus were disordered , sparse , and the surrounding of the cells lost their proper refractive index . Under electron microscope , the cells of glomerulus were fused , retracted , and some of them disappeared . All of them were fusiform or polygonal after ATRA treatment .
Under electron microscope , there was no obvious atrophy of the glomerulus under the electron microscope , the fusion of the foot and the retraction were not obvious . ( 2 ) The cells of the NC and AC groups showed approximately " S " type cell growth curve , and the cell growth was significantly inhibited after 24 hours after the intervention of adriamycin in AI group .
( 4 ) The expression of nephrin , podocin protein and its mRNA were significantly lower in the AI group than in the AI group ( P0.05 ) .

Conclusion : ATRA may inhibit the apoptosis and differentiation of the cultured human mesangial cells in vitro , promote the differentiation of the cells , thereby reducing the damage of the foot cells caused by adriamycin .

Objective : To investigate the molecular mechanism of all - trans retinoic acid to reduce the damage of adriamycin - induced glomerular - foot cells in vitro by detecting the expression of matrix metalloproteinase - 2 ( MMP - 2 ) , matrix metalloproteinase - 9 ( MMP - 9 ) , retinoic acid receptor - 伪 ( RAR伪 ) , retinoic acid receptor - 尾 ( RAR尾 ) and retinoic acid receptor - 纬 ( RAR纬 ) .
MTT assay was used to detect the cell viability of each group .
The enzyme activity of MMP - 2 and MMP - 9 was detected by gelatin zymography method .
Western - blot was used to detect the expression of MMP - 2 , MMP - 9 , RAR伪, RAR尾 , RAR纬 , 伪 - SMA and TGF - 尾1 in each group .
Real - time RT - PCR was used to detect the mRNA expression of MMP - 2 , MMP - 9 , RAR伪 , RAR尾 , RAR纬 mRNA in each group .

Results : ( 1 ) Compared with NC group , AI in AI group was atrophy , foot process fusion , retraction and partial disappearance .
( 2 ) Compared with NC group , the cell viability was significantly decreased ( P0.05 ) .
Compared with the control group , the expression of MMP - 2 , MMP - 9 and MMP - 9 decreased significantly ( P0.05 ) , but the expression of TGF - 尾1 increased significantly ( P0.05 ) .
Compared with AI group , the expressions of MMP - 2 , MMP - 9 and MMP - 9 were significantly increased in AA group ( P0.05 ) .
The expressions of MMP - 2 and MMP - 9 were significantly correlated with the expressions of MMP - 2 , MMP - 9 , TGF - 尾1 and 伪 - SMA in AI group ( P < 0.05 ) . The expressions of MMP - 2 and MMP - 9 were positively correlated with the expression of MMP - 2 , MMP - 9 , TGF - 尾1 and 伪 - SMA in AI group ( P0.05 ) .

Conclusion : ATRA may regulate the expression of MMP - 2 and MMP - 9 through the combination of RAR伪and RAR纬 in the injury of adriamycin - induced foot cells cultured in vitro .

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

【参考文献】