本文選題：全反式維甲酸 + 足細(xì)胞損傷��； 參考：《廣西醫(yī)科大學(xué)》2014年博士論文

【摘要】：目的：通過檢測(cè)腎小球足細(xì)胞凋亡指數(shù)、nephrin、podocin、TGF-β1和α-SMA的表達(dá)，探討全反式維甲酸（ATRA）對(duì)體外培養(yǎng)阿霉素（ADR）致腎小球足細(xì)胞損傷的作用。方法：將體外培養(yǎng)腎小球足細(xì)胞隨機(jī)分成4個(gè)組：空白對(duì)照組（NC），阿霉素致足細(xì)胞損傷組（AI），ATRA干預(yù)的空白對(duì)照組（AC），ATRA干預(yù)的阿霉素致足細(xì)胞損傷組（AA）。AI組和AA組腎小球足細(xì)胞鋪滿孔底后首先置于含有0.15ug/ml阿霉素的培養(yǎng)基中孵育24小時(shí)構(gòu)建足細(xì)胞損傷模型。阿霉素孵育24小時(shí)后各組腎小球足細(xì)胞均換液，NC組和AI組腎小球足細(xì)胞繼續(xù)置于正常培養(yǎng)基中培養(yǎng)，AC組和AA組腎小球足細(xì)胞則置于含有0.1uM ATRA的培養(yǎng)基中繼續(xù)培養(yǎng)。ATRA干預(yù)后24小時(shí)，收集腎小球足細(xì)胞進(jìn)行形態(tài)學(xué)和分子生物學(xué)檢測(cè)。倒置顯微鏡和電子顯微鏡下觀察腎小球足細(xì)胞形態(tài)和超微結(jié)構(gòu)的改變，流式細(xì)胞儀檢測(cè)腎小球足細(xì)胞凋亡指數(shù)，MTT法檢測(cè)并繪制腎小球足細(xì)胞生長(zhǎng)曲線，蛋白質(zhì)印跡法（Western-blot）檢測(cè)各組腎小球足細(xì)胞轉(zhuǎn)化生長(zhǎng)因子-β1（transforming growth factor-β1，TGF-β1）、α-平滑肌肌動(dòng)蛋白（α-smooth muscle actin，α-SMA）、nephrin和podocin的蛋白表達(dá)，逆轉(zhuǎn)錄實(shí)時(shí)定量聚合酶鏈反應(yīng)（Real time RT-PCR）檢測(cè)腎小球足細(xì)胞TGF-β1、α-SMA、nephrin和podocin的mRNA表達(dá)。 結(jié)果：（1）與NC組比較，AI組腎小球足細(xì)胞排列紊亂、稀疏，胞體四周失去應(yīng)有的折光性，電鏡下腎小球足細(xì)胞足突融合、回縮，部分細(xì)胞足突消失。ATRA干預(yù)后腎小球足細(xì)胞大部分呈梭形或多邊形、排列較整齊，細(xì)胞輪廓清楚；電鏡下腎小球足細(xì)胞萎縮，足突融合、回縮不明顯。（2）NC組和AC組腎小球足細(xì)胞呈現(xiàn)近似“S”型的細(xì)胞生長(zhǎng)曲線。AI組腎小球足細(xì)胞在阿霉素干預(yù)24小時(shí)后細(xì)胞生長(zhǎng)均受到明顯抑制；AA組腎小球足細(xì)胞生長(zhǎng)抑制現(xiàn)象較AI組腎小球足細(xì)胞明顯減輕。（3）與NC組相比，AI組腎小球足細(xì)胞凋亡率顯著增加（P0.05）。與AI組相比，AA組腎小球足細(xì)胞凋亡率顯著降低（P0.05）。（4）AI組腎小球足細(xì)胞TGF-β1、α-SMA蛋白及其mRNA表達(dá)均較NC組顯著升高（P0.05）。AC組和AA組腎小球足細(xì)胞TGF-β1、α-SMA蛋白及其mRNA表達(dá)均較AI組顯著降低（P0.05）。（5）AI組腎小球足細(xì)胞nephrin、podocin蛋白及其mRNA表達(dá)均較NC組顯著降低（P0.05）。AC組和AA組腎小球足細(xì)胞nephrin、podocin蛋白及其mRNA表達(dá)均較AI組顯著升高（P0.05）。（6）AI組腎小球足細(xì)胞nephrin和podocin蛋白表達(dá)分別與α-SMA、TGF-β1蛋白表達(dá)呈顯著負(fù)相關(guān)（P0.05）。AI組腎小球足細(xì)胞α-SMA蛋白表達(dá)與TGF-β1蛋白表達(dá)呈顯著正相關(guān)（P0.05）。AA組腎小球足細(xì)胞凋亡指數(shù)與TGF-β1蛋白表達(dá)呈顯著正相關(guān)（P0.05）。 結(jié)論： ATRA可能通過抑制體外培養(yǎng)的腎小球足細(xì)胞凋亡和轉(zhuǎn)分化，促進(jìn)足細(xì)胞分化，從而減輕阿霉素所致的足細(xì)胞損傷。 目的：通過檢測(cè)腎小球足細(xì)胞基質(zhì)金屬蛋白酶-2（MMP-2）、基質(zhì)金屬蛋白酶-9（MMP-9）、維甲酸受體-α（RAR-α）、維甲酸受體-β（RAR-β）和維甲酸受體-γ（RAR-γ）等的表達(dá)，，探討全反式維甲酸減輕體外培養(yǎng)阿霉素致腎小球足細(xì)胞損傷的分子機(jī)制。方法：將體外培養(yǎng)的腎小球足細(xì)胞隨機(jī)分成3個(gè)組：空白對(duì)照組（NC），阿霉素致足細(xì)胞損傷組（AI），ATRA干預(yù)的阿霉素致足細(xì)胞損傷組（AA）。腎小球足細(xì)胞損傷模型的構(gòu)建及各組腎小球足細(xì)胞的干預(yù)方法同第一部分。倒置顯微鏡和掃描電子顯微鏡下觀察腎小球足細(xì)胞形態(tài)和超微結(jié)構(gòu)改變；MTT法檢測(cè)各組腎小球足細(xì)胞活力；明膠酶譜法檢測(cè)各組腎小球足細(xì)胞MMP-2、MMP-9的酶活性；Western-blot檢測(cè)各組腎小球足細(xì)胞MMP-2、MMP-9、RAR-α、RAR-β、RAR-γ、α-SMA和TGF-β1的蛋白表達(dá)；Real-time RT-PCR檢測(cè)各組腎小球足細(xì)胞MMP-2、MMP-9、RAR-α、RAR-β、RAR-γ的mRNA表達(dá)。 結(jié)果：（1）與NC組比較，電鏡下AI組足細(xì)胞萎縮，足突融合、回縮、部分消失；AA組腎小球足細(xì)胞萎縮，足突融合、回縮不明顯。（2）與NC組相比，AI組腎小球足細(xì)胞活力顯著減弱（P0.05）；與AI組相比，AA組腎小球足細(xì)胞活力顯著增強(qiáng)（P0.05）。（3）與NC組相比，AI組腎小球足細(xì)胞MMP-2、MMP-9的蛋白及其mRNA表達(dá)均顯著降低（P0.05），而TGF-β1蛋白表達(dá)顯著升高（P0.05）；與AI組相比，AA組腎小球足細(xì)胞MMP-2、MMP-9的蛋白及其mRNA表達(dá)均顯著升高（P0.05），而TGF-β1蛋白表達(dá)顯著降低（P0.05）。（4）AI組腎小球足細(xì)胞RAR-α、RAR-γ的蛋白及其mRNA表達(dá)均較NC組顯著降低（P0.05），AA組腎小球足細(xì)胞RAR-α、RAR-γ的蛋白和mRNA表達(dá)均較AI組明顯升高（P0.05）。（5）AI組腎小球足細(xì)胞RAR-β蛋白及其mRNA表達(dá)較NC組降低，但差異無顯著性（P0.05）； AA組腎小球足細(xì)胞RAR-β蛋白及其mRNA表達(dá)較AI組升高，但差異無顯著性（P0.05）。（6）與NC組相比，AI組腎小球足細(xì)胞MMP-2和MMP-9酶活性均顯著減弱（P0.05）。與AI組相比，AA組腎小球足細(xì)胞MMP-2和MMP-9酶活性均顯著增強(qiáng)（P0.05）。（7）AI組腎小球足細(xì)胞RAR-α、RAR-γ蛋白表達(dá)與MMP-2和MMP-9蛋白表達(dá)均呈顯著正相關(guān)（P均0.05），與TGF-β1和α-SMA蛋白表達(dá)均呈顯著負(fù)相關(guān)（P＜0.05）。AI組腎小球足細(xì)胞RAR-β蛋白表達(dá)與MMP-2、MMP-9、TGF-β1和α-SMA蛋白表達(dá)均無顯著相關(guān)性（P0.05）。AI組腎小球足細(xì)胞MMP-2和MMP-9蛋白表達(dá)與腎小球足細(xì)胞活力呈顯著正相關(guān)（P0.05），與TGF-β1和α-SMA蛋白表達(dá)呈顯著負(fù)相關(guān)（P均0.05）。 結(jié)論：在體外培養(yǎng)的阿霉素致足細(xì)胞損傷中，ATRA可能通過與RAR-α和RAR-γ結(jié)合調(diào)控腎小球足細(xì)胞MMP-2和MMP-9表達(dá)增加，從而減輕腎小球足細(xì)胞損傷。
[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 偽 - SMA . Methods : After 24 hours of incubation with adriamycin , the cells were cultured in normal medium . The expression of TGF - 尾1 , 偽 - SMA , nephrin and podocin were detected by reverse microscopy and electron microscope . The mRNA expression of TGF - 尾1 , 偽 - 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 - 偽 ( RAR偽 ) , retinoic acid receptor - 尾 ( RAR尾 ) and retinoic acid receptor - 緯 ( RAR緯 ) .
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偽, RAR尾 , RAR緯 , 偽 - 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偽 , RAR尾 , RAR緯 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 偽 - 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 偽 - SMA in AI group ( P0.05 ) .

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

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692

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