·論著·

PAX2穩(wěn)轉(zhuǎn)細(xì)胞系構(gòu)建及對(duì)細(xì)胞遷移和侵襲能力的作用研究

李里，宮亮，吳玉斌

作者單位：121000遼寧省錦州市，遼寧醫(yī)學(xué)院附屬第一醫(yī)院風(fēng)濕免疫科(李里)，耳鼻喉科(宮亮)；中國(guó)醫(yī)科大學(xué)附屬盛京醫(yī)院小兒腎臟風(fēng)濕免疫科(吳玉斌)

通信作者：吳玉斌，110004 遼寧省沈陽(yáng)市，中國(guó)醫(yī)科大學(xué)附屬盛京醫(yī)院小兒腎臟風(fēng)濕免疫科；E-mail：772652284@qq.com

【摘要】目的 探討PAX2穩(wěn)轉(zhuǎn)細(xì)胞系構(gòu)建及生物學(xué)作用。方法選擇處于對(duì)數(shù)生長(zhǎng)期大鼠腎小管上皮細(xì)胞，實(shí)驗(yàn)分為穩(wěn)轉(zhuǎn)組、空載組、對(duì)照組。穩(wěn)轉(zhuǎn)組采用陽(yáng)離子脂質(zhì)體轉(zhuǎn)導(dǎo)將pEGFP-PAX2轉(zhuǎn)入大鼠腎小管上皮細(xì)胞，空載組采用同樣方法配置空載質(zhì)粒轉(zhuǎn)染溶液，對(duì)照組不添加質(zhì)粒轉(zhuǎn)染溶液。經(jīng)G418篩選，建立穩(wěn)定轉(zhuǎn)染細(xì)胞系。通過(guò)細(xì)胞劃痕實(shí)驗(yàn)及Transwell實(shí)驗(yàn)檢測(cè)PAX2穩(wěn)轉(zhuǎn)細(xì)胞系的細(xì)胞遷移率及細(xì)胞侵襲作用。結(jié)果應(yīng)用G418篩選14 d，穩(wěn)轉(zhuǎn)組大鼠腎小管上皮細(xì)胞有明顯克隆長(zhǎng)出，建立PAX2基因穩(wěn)定轉(zhuǎn)染腎小管上皮細(xì)胞系。穩(wěn)轉(zhuǎn)組在熒光顯微鏡下可見(jiàn)綠色熒光，基因融合表達(dá)獲得成功。穩(wěn)轉(zhuǎn)組細(xì)胞PAX2條帶密度與β-action吸光度比值為(1.00±0.04)，空載組為(0.83±0.03)，對(duì)照組為(0.85±0.04)，3組吸光度比值比較，差異有統(tǒng)計(jì)學(xué)意義(F=398.7，P<0.05)；其中穩(wěn)轉(zhuǎn)組吸光度比值高于空載組和對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。細(xì)胞劃痕后18 h，對(duì)照組、空載組和穩(wěn)轉(zhuǎn)組細(xì)胞遷移率分別為(39.34±5.34)%、(40.56±7.54)%和(83.72±7.12)%，3組細(xì)胞遷移率比較，差異有統(tǒng)計(jì)學(xué)意義(F=431.8，P<0.05)；其中穩(wěn)轉(zhuǎn)組細(xì)胞遷移率高于對(duì)照組和空載組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。各組細(xì)胞培養(yǎng)48 h后，對(duì)照組、空載組和穩(wěn)轉(zhuǎn)組遷移細(xì)胞分別為(23.33±3.63)、(22.00±8.14)、(45.67±7.14)，3組遷移細(xì)胞數(shù)比較，差異有統(tǒng)計(jì)學(xué)意義(F=248.5，P<0.05)；其中穩(wěn)轉(zhuǎn)組遷移細(xì)胞數(shù)高于對(duì)照組和空載組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論pEGFP-PAX2質(zhì)粒成功轉(zhuǎn)染大鼠腎小管上皮細(xì)胞，并成功篩選出高效穩(wěn)定表達(dá)PAX2的穩(wěn)轉(zhuǎn)細(xì)胞系。PAX2穩(wěn)轉(zhuǎn)增加了腎小管上皮細(xì)胞的遷移及侵襲能力。

【關(guān)鍵詞】PAX2基因；細(xì)胞侵襲；細(xì)胞運(yùn)動(dòng)；腎小管；上皮細(xì)胞；大鼠

基金項(xiàng)目：遼寧省博士科研啟動(dòng)基金計(jì)劃資助項(xiàng)目(20141136)

【中圖分類號(hào)】R 692.6

收稿日期：(2015-07-02；修回日期：2015-09-12)

李里，宮亮，吳玉斌.PAX2穩(wěn)轉(zhuǎn)細(xì)胞系構(gòu)建及對(duì)細(xì)胞遷移和侵襲能力的作用研究[J].中國(guó)全科醫(yī)學(xué)，2015，18(35)：4325-4329.[www.chinagp.net]

Li L，Gong L，Wu YB.Study on the construction of PAX2 stably transfected cell lines and their effects on cell migration and invasion ability[J].Chinese General Practice，2015，18(35)：4325-4329.

Study on the Construction of PAX2 Stably Transfected Cell Lines and Their Effects on Cell Migration and Invasion AbilityLILi，GONGLiang，WUYu-bin.DepartmentofRheumatologyandImmunology，theFirstAffiliatedHospitalofLiaoningMedicalUniversity，Jinzhou121000，China

Abstract【】ObjectiveTo investigate the construction and biological effects of PAX2 stably transfected cell lines.MethodsRat renal tubular epithelial cells at exponential phase were selected，and the cells were divided into stably transfected group，empty-loading group and control group.For stably transfected group，cationic liposome transduction was conducted to transfer pEGFP-PAX2 into rat renal tubular epithelial cells；for empty-loading group，cationic liposome transduction was also conducted with empty-loading plasmid transfection solution；for control group，no plasmid transfection solution was added.By G418 selection，stably transfected cell lines were constructed.Cell wound scratch assay and Tanswell test were conducted to measure cell migration rate and cell invasion of PAX2 stably transfected cell lines.ResultsG418 selection was undertaken for 14 days，the cells of stably transfected group had obvious cloning growth，and PAX2 stably transfected cell lines were constructed.Green fluorescence was observed under fluorescence microscope in stably transfected group，and successful gene fusion expression was made.The ratio of the PAX2 strip density to the absorbancy of β-action was (1.00±0.04) for stably transfected group，(0.83±0.03)for empty-loading group and (0.85±0.04)for control group.The three groups were significantly different in optical density ratio(F=398.7，P<0.05)；the stably transfected group was higher than empty-loading group in optical density ratio(P<0.05).At 18 hours after cell scratch was made，the cell migration rates of control group，empty-loading group and stably transfected group were(39.34±5.34)%，(40.56±7.54)% and(83.72±7.12)%，with significant differences among the three groups(F=431.8，P<0.05)and the stably transfected group higher than control group and empty-loading group(P<0.05).At 48 hours of culture，the numbers of migrated cells of control group，empty-loading group and stably transfected group were(23.33±3.63)，(22.00±8.14)and(45.67±7.14)，with significant differences among the three groups(F=248.5，P<0.05)and stably transfected group higher than control group and empty-loading group(P<0.05).ConclusionpEGFP-PAX2 plasmid is successfully transfected into rat renal tubular epithelial cells，and stably transfected cell lines with highly effective and stable expression of PAX2 are successfully constructed.The stable transfection of PAX2 increases migration and invasion of renal tubular epithelial cells.

【Key words】PAX2 gene；Cell invasion；Cell movement；Kidney tubules;Epithelial cells;Rats

PAX2(Paired Box2)基因是胚胎發(fā)育基因，在胚胎發(fā)育過(guò)程中發(fā)揮重要的調(diào)控作用。尤其在腎臟，其是腎臟發(fā)育過(guò)程中前腎、中腎和后腎重要的調(diào)控基因。PAX2基因主要通過(guò)誘導(dǎo)腎小管上皮細(xì)胞轉(zhuǎn)分化發(fā)揮調(diào)控作用[1-3]。有學(xué)者[4-7]發(fā)現(xiàn)在病變大鼠體內(nèi)出現(xiàn)PAX2基因重新表達(dá)，并可能調(diào)控轉(zhuǎn)分化作用。在體外研究中發(fā)現(xiàn)PAX2基因轉(zhuǎn)染腎小管上皮細(xì)胞后出現(xiàn)纖維表型標(biāo)志物的增加以及上皮細(xì)胞表型標(biāo)志物的減少[8]。判定細(xì)胞出現(xiàn)上皮細(xì)胞轉(zhuǎn)分化的標(biāo)志除了出現(xiàn)表型的轉(zhuǎn)變外還表現(xiàn)為細(xì)胞侵襲及遷移能力的增加。本研究將首先構(gòu)建PAX2穩(wěn)轉(zhuǎn)細(xì)胞系，進(jìn)一步探討PAX2穩(wěn)轉(zhuǎn)細(xì)胞系細(xì)胞侵襲及遷移能力，為臨床疾病治療提供實(shí)驗(yàn)依據(jù)。

1材料與方法

1.1材料大鼠腎小管上皮細(xì)胞株(NRK52E)，購(gòu)自上海中科院；pEGFP-PAX2質(zhì)粒(含Neo抗性基因)，購(gòu)自Clontech 公司；Transwell細(xì)胞小室，購(gòu)自Corning公司。

1.2方法

1.2.1pEGFP-PAX2穩(wěn)定轉(zhuǎn)染腎小管上皮細(xì)胞系的構(gòu)建及篩選選擇處于對(duì)數(shù)生長(zhǎng)期大鼠腎小管上皮細(xì)胞，其生長(zhǎng)狀態(tài)良好。分為穩(wěn)轉(zhuǎn)組、對(duì)照組、空載組。將pEGFP-grp78質(zhì)粒4 μg加入無(wú)血清培養(yǎng)液245 μl，再加入Lipofectamine 2000，將上述液體混合，在室溫靜置30 min。同樣方法配置空載質(zhì)粒轉(zhuǎn)染溶液。對(duì)照組不加人質(zhì)粒轉(zhuǎn)染溶液。在無(wú)血清無(wú)抗生素的培養(yǎng)液中將腎小管上皮細(xì)胞洗滌2次，再將上述轉(zhuǎn)染液與細(xì)胞加入培養(yǎng)孔中，在5% CO237 ℃培養(yǎng)箱中培養(yǎng)6 h后，將轉(zhuǎn)染液棄除，再加入胎牛血清繼續(xù)培養(yǎng)。細(xì)胞傳代，再用G418的選擇培養(yǎng)基篩選，篩選14 d后pEGFP-PAX2重組質(zhì)粒轉(zhuǎn)染組有克隆形成，建立穩(wěn)定轉(zhuǎn)染細(xì)胞系。

1.2.2穩(wěn)定轉(zhuǎn)染細(xì)胞系鑒定在熒光顯微鏡下觀察各組細(xì)胞，藍(lán)色激光激發(fā)，隨機(jī)選一視野，發(fā)出綠色熒光的細(xì)胞并攝像。PAX2 mRNA的表達(dá)檢測(cè)：在24孔板中加入PBS漂洗2次，取1×106細(xì)胞加Trizol 1.0 ml，按試劑盒操作說(shuō)明用三氯甲烷分離、采用異丙醇RNA沉淀、將RNA再溶解，提取細(xì)胞總RNA。按照試劑盒說(shuō)明書配制20 μl RT反應(yīng)體系；PCR反應(yīng)：引物設(shè)計(jì)：上游：5′-CAACGGTGAGAAGAGGAAACGAG-3′，下游：5′-TAATGCTGCTGGGTGAAGGTGTC-3′，β-actin作為內(nèi)參照，按照試劑盒說(shuō)明配制50 μl PCR反應(yīng)體系，結(jié)束后進(jìn)行瓊脂凝膠電泳檢測(cè)并攝像；經(jīng)圖像分析儀測(cè)定吸光度值，即檢測(cè)基因產(chǎn)物吸光度值與內(nèi)參照吸光度的比值為A值。以對(duì)照組和空載組的腎小管上皮細(xì)胞作為對(duì)照。

1.2.3細(xì)胞劃痕試驗(yàn)細(xì)胞鋪板：前1天將培養(yǎng)瓶中細(xì)胞用0.25%胰蛋白酶消化，接種1×105細(xì)胞至6孔培養(yǎng)板中，當(dāng)細(xì)胞融合至80%～90%時(shí)開(kāi)始進(jìn)行實(shí)驗(yàn)。棄去培養(yǎng)基，應(yīng)用200 μl pipette tip使培養(yǎng)板出現(xiàn)劃痕，應(yīng)用培養(yǎng)基清洗細(xì)胞表面2次，再加入培養(yǎng)基中，在鏡下觀察10、18 h，并于100倍鏡下拍照記錄。并計(jì)算各組遷移率。遷移率用來(lái)描述細(xì)胞遷移修復(fù)速度，具體計(jì)算方法：遷移率=(0 h細(xì)胞劃痕寬度-10 h細(xì)胞劃痕寬度或18 h細(xì)胞劃痕寬度)/0 h細(xì)胞劃痕寬度×100%。

1.2.4Transwell實(shí)驗(yàn)將50 μl的Matrigel(1 μg/μl)平鋪在Transwell的8 μm孔徑的聚碳酸酯微孔濾膜上，37 ℃放置1 h，室溫干燥過(guò)夜。將600 μl含20%胎牛血清的NIH3T3細(xì)胞的上清加入Transwell下室，將200 μl對(duì)照組、空載組和穩(wěn)轉(zhuǎn)組細(xì)胞懸液加入上室，每種細(xì)胞設(shè)3個(gè)復(fù)孔，培養(yǎng)48 h后取出小室，將膜上層未穿越細(xì)胞拭去，用多聚甲醇固定30 min，蘇木精染色15 min，PBS洗去多余蘇木精，鏡下計(jì)數(shù)穿到膜背細(xì)胞數(shù)。計(jì)數(shù)3個(gè)不同視野，取平均值(×200)。

2結(jié)果

2.1穩(wěn)定轉(zhuǎn)染細(xì)胞系的構(gòu)建及篩選應(yīng)用G418篩選14 d，穩(wěn)轉(zhuǎn)組大鼠腎小管上皮細(xì)胞有明顯克隆長(zhǎng)出，建立了PAX2基因穩(wěn)定轉(zhuǎn)染腎小管上皮細(xì)胞系(見(jiàn)圖1)。

圖1　PAX2基因轉(zhuǎn)染腎小管上皮細(xì)胞的G418篩選(×200)

Figure 1G418 selection of renal tubular epithelial cells after PAX2 gene transfection

2.2穩(wěn)定轉(zhuǎn)染細(xì)胞系鑒定

2.2.1各組細(xì)胞綠色熒光表達(dá)穩(wěn)轉(zhuǎn)組及空載組在熒光顯微鏡下均可見(jiàn)綠色熒光，對(duì)照組未見(jiàn)綠色熒光(見(jiàn)圖2)，基因融合表達(dá)獲得成功。

2.2.2各組細(xì)胞PAX2 mRNA表達(dá)穩(wěn)轉(zhuǎn)組細(xì)胞PAX2條帶密度與β-action吸光度比值為(1.00±0.04)，空載組為(0.83±0.03)，對(duì)照組為(0.85±0.04)。3組吸光度比值比較，差異有統(tǒng)計(jì)學(xué)意義(F=398.7，P<0.05)；其中穩(wěn)轉(zhuǎn)組吸光度比值高于空載組和對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)圖3)。

2.3PAX2穩(wěn)定轉(zhuǎn)染后腎小管上皮細(xì)胞運(yùn)動(dòng)遷移改變細(xì)胞劃痕后10 h，對(duì)照組、空載組和穩(wěn)轉(zhuǎn)組細(xì)胞遷移率分別為(23.00±3.10)%、(24.33±4.02)%和(43.12±7.13)%。3組細(xì)胞遷移率比較，差異有統(tǒng)計(jì)學(xué)意義(F=501.6，P<0.05)。細(xì)胞劃痕后18 h，對(duì)照組、空載組和穩(wěn)轉(zhuǎn)組細(xì)胞遷移率分別為(39.34±5.34)%、(40.56±7.54)%和(83.72±7.12)%。3組細(xì)胞遷移率比較，差異有統(tǒng)計(jì)學(xué)意義(F=431.8，P<0.05)；其中穩(wěn)轉(zhuǎn)組細(xì)胞遷移率高于對(duì)照組和空載組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)圖4)。

2.4PAX2穩(wěn)定轉(zhuǎn)染對(duì)腎小管上皮細(xì)胞侵襲能力的影響各組細(xì)胞培養(yǎng)48 h后，計(jì)數(shù)膜下表面的細(xì)胞數(shù)。對(duì)照組、空載組和穩(wěn)轉(zhuǎn)組遷移細(xì)胞分別為(23.33±3.63)、(22.00±8.14)、(45.67±7.14)，3組遷移細(xì)胞數(shù)比較，差異有統(tǒng)計(jì)學(xué)意義(F=248.5，P<0.05)；其中穩(wěn)轉(zhuǎn)組遷移細(xì)胞數(shù)高于對(duì)照組和空載組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)圖5)。

圖3　3組細(xì)胞PAX2 mRNA表達(dá)的RT-PCR電泳圖

Figure 3RT-PCR eelectrophoretogram of PAX2 mRNA expression of the three groups

3討論

PAX2基因?yàn)榕咛グl(fā)育基因，既往研究表明PAX2在病理腎臟出現(xiàn)重新表達(dá)，認(rèn)為其可能通過(guò)調(diào)控腎小管上皮細(xì)胞轉(zhuǎn)分化調(diào)控疾病發(fā)生，但具體機(jī)制不清楚。腎小管上皮細(xì)胞轉(zhuǎn)分化發(fā)生中重要的一步即細(xì)胞出現(xiàn)了遷移及侵襲能力的增強(qiáng)[9]，故本研究通過(guò)建立PAX2穩(wěn)轉(zhuǎn)細(xì)胞系，探討PAX2轉(zhuǎn)染是否導(dǎo)致細(xì)胞的侵襲及遷移能力增強(qiáng)，為進(jìn)一步探討PAX2基因調(diào)控腎小管上皮細(xì)胞轉(zhuǎn)分化提供依據(jù)。

圖2　熒光顯微鏡觀察pEGFP-PAX2在腎小管上皮細(xì)胞中的表達(dá)(×200)

圖4　3組0 h、10 h、18 h細(xì)胞遷移率比較

圖5　3組細(xì)胞侵襲能力比較(×200)

本實(shí)驗(yàn)應(yīng)用脂質(zhì)體介導(dǎo)轉(zhuǎn)染法將PAX2質(zhì)粒轉(zhuǎn)染入大鼠腎小管上皮細(xì)胞，G418是目前廣泛應(yīng)用的穩(wěn)定轉(zhuǎn)染篩選試劑[10]，本實(shí)驗(yàn)應(yīng)用該試劑進(jìn)行了大鼠腎小管上皮細(xì)胞篩選，獲得了穩(wěn)定轉(zhuǎn)染PAX2基因的腎小管上皮細(xì)胞系。通過(guò)熒光顯微鏡觀察及RT-PCR鑒定，穩(wěn)定轉(zhuǎn)染PAX2組熒光表達(dá)明顯增加，PAX2 mRNA表達(dá)也明顯增加，其表達(dá)較對(duì)照組及空載組差異有統(tǒng)計(jì)學(xué)意義，證明穩(wěn)定轉(zhuǎn)染細(xì)胞系構(gòu)建成功。同時(shí)也表明脂質(zhì)體介導(dǎo)轉(zhuǎn)染法可以成功進(jìn)行基因的穩(wěn)定表達(dá)轉(zhuǎn)染。

細(xì)胞遷移實(shí)驗(yàn)是通過(guò)細(xì)胞受傷后的自動(dòng)修復(fù)能力來(lái)評(píng)估，如單層細(xì)胞在受傷24 h內(nèi)，其只靠細(xì)胞移動(dòng)來(lái)愈合修復(fù)，并不會(huì)出現(xiàn)反應(yīng)性修復(fù)增生[11]，故本實(shí)驗(yàn)應(yīng)用細(xì)胞劃痕實(shí)驗(yàn)評(píng)估細(xì)胞遷移運(yùn)動(dòng)能力。結(jié)果表明穩(wěn)轉(zhuǎn)組細(xì)胞遷移速度較空載組及對(duì)照組明顯增快，表明過(guò)表達(dá)PAX2可明顯增加腎小管上皮細(xì)胞遷移能力。本試驗(yàn)通過(guò)目前較為常用的Transwell實(shí)驗(yàn)探討細(xì)胞侵襲能力，細(xì)胞在含有Matrigel的聚碳酸酯微孔中穿過(guò)，是在模擬體內(nèi)細(xì)胞穿過(guò)細(xì)胞外基質(zhì)的過(guò)程，可以反映細(xì)胞的侵襲能力[12]，本研究結(jié)果表明穩(wěn)轉(zhuǎn)組穿透的細(xì)胞數(shù)較空載組及對(duì)照組明顯增加，說(shuō)明PAX2可明顯增加細(xì)胞侵襲能力。

本文價(jià)值：

本研究首次通過(guò)構(gòu)建PAX2穩(wěn)轉(zhuǎn)細(xì)胞系探討其生物學(xué)作用，發(fā)現(xiàn)PAX2轉(zhuǎn)染可以增加腎小管上皮細(xì)胞的遷移及侵襲能力，這可能是PAX2基因誘導(dǎo)腎小管上皮細(xì)胞轉(zhuǎn)分化的重要機(jī)制，為臨床疾病的治療提供實(shí)驗(yàn)依據(jù)。

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(本文編輯：賈萌萌)