BMP7对TGFβ 1诱导人近端肾小管上皮细胞转分化及细胞外基质分泌的影响

BMP7对TGFβ 1诱导人近端肾小管上皮细胞转分化及细胞外基质分泌的影响

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精品参考文献资料英文缩略词表

a-SMA(a-smooth muscle actin) a一平滑肌肌动蛋白

BMP-7(bone moiphogenctic protein-7) 骨形态发生蛋白一7

Col I(type I collagen) I型胶原

DEPC(diethyl pyrocaeoonate) 焦碳酸二乙酯

E-cadhcrin B钙粘连素

ECM(extmcellular matrix) 细胞外基质

ELISA(enzyme linked immunosorbent assay) 酶联免疫吸附实验

EMT(epithelial-to-mesenchymal transition) 蚴胞·间充质转分化CrGF(connectivc tissue growth factor) 结缔组织生长因子

fN(h'bronectin) 纤维连接蛋白

HK-2(human renal proximal tubular epithelial cells) 人近端肾小管上皮细胞

MET(mesenehymal-to-epithclial transition) 间充质细胞．E皮细胞转化

MMP-2(malri metalloproteinase-2) 基质金属蛋白酶-2

MFB(myofibroblast) 肌成纤维细胞

MCP-l(monocyte chcmoatWactant protein-I) 单核细胞趋化蛋白．1

"IGF冯,(transforming growth factor一01) 转化生长因子B l

Tm(mbmommrstitial fibrosis) 肾小管间质纤维化

TIMP-I(tissue inlu'bitor of the matri metalloproteinase-1) 组织基质金属蛋白酶抑制物．1 TEC(r砌tubular epithelial cell) 肾小管E皮细胞

PAI-1(plasminogen activator inlu'bitor-1) 纤溶酶原激活物抑制剂一1

PBS(phosphate buffered saline) 磷酸盐缓冲液

RT-PCR(reverse Iranscription-polymerase chain system) 反转录一聚合酶链式反

应SDS(sodium dodecyl sulphate) 十二烷基磺酸钠

I兀Jo(unilateral ureteral obstruction) 单侧输尿管梗阻

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BMP-7对TG椰1诱导人近端肾小管上皮细胞

转分化及细胞外基质分泌的影响

摘要

目的：观察骨形态发生蛋8-7(洲)对转化生长因子-81(TGF-B1)诱导人近端肾小

管上皮细胞(HK-2)向间充质细胞转分化(D仉’)及细胞外基质(ECM)分泌的影响，探

讨其逆转肾间质纤维化的可能机

制。

方法：将HK-2细胞在体外与TGF-B1或TGF-B1+BMP-7共培养，分成对照组，不同浓度TGF-B1诱导组，TGF-B1不同时间诱导组，3ng／ml TGF-B1加不同浓度BMP-7组。相差显

微镜观察细胞形态的改变；间接免疫荧光测定a-SMA、E-cadherin的表达；RT-PCR检

测a-SMA、E-cadherin、Col I a1、FN、PAI-1 mRNA的表达；Western B lot检测a-SMA、E,odherin

蛋白的表达；ELISA方法测定细胞E清液Col I、FN的表达。

结果：(1)随着TGF-B1浓度的增加及作用时间的延长，HK-2细胞形态逐渐发生改变，由立方形铺路石样转变为梭形长条样；在3ng／ml TGF-81诱导48h后再加A．200ng／na BMP-7组中，可见到已转变为梭形长条样的细胞大部分逆转回原来的形态。(2)免疫荧光检测显示，正常HK-2细胞表达F_,cadhadn、而无a-SMA表达，经3ng／ml TGF-B1刺激4811后，细胞浆

中a-SMA表达明显增强，细胞质膜E-eadherin表达显著减弱；去除TGF-BI，再加入200ng

／ml BMP-7干预48h，能明显抑制a-．SMA的表达、恢复E-eadherin的表达。(3)TGF-81在

lOng／ml 内呈剂量依赖性匕调a-SMAmRNA的表达。在3ng／ml TGF冯I诱导下，

a-SMAmRNA表达

随作用时间的延长而增加，E．eadherin mRNA表达随作用时间的延长而逐渐减少。在3ng／

ml TGF-BI与BMP-7共同干预下，a-SMA mRNA及蛋白的表达随着BMP-7(100 ng／ml

枷O ng／m1)的浓度增加而减少，400 ng压llI BMP-7即可显著减少a-SMA mRNA及蛋

白的表达

(P

的浓度增加而逐渐增加，400 ng／ml BMP-7即可显著增加F．,-odherin mRNA及蛋白的表

达

(咖．01)。(4)在3ng／ml TGF-St诱导下，随刺激时间的延长，Col I al、FN mRNA表达

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逐渐增加：BMP-7呈剂量依赖性抑制Col I、FN的表达，400 ng／IIll BMP-7即可显著抑制Col I、FN的表达(P

结论：(1)TGF-B1呈剂量和时间依赖性匕调a-SMA的表达、抑制E-cadherin的表达，诱导HK-2细胞转分化，并促进Col I、FN的合成。(2)BMP-7可呈剂量依赖性地抑制TGF-Bl诱导下a-SMAmRNA和蛋白的表达，并通过再诱导E-cadhcdn mRNA和蛋白的表达抑制、

逆转TGF-B1诱导的EMT。(3)BMP-7可呈剂量依赖性抑制TGF-Bl诱导下HK-2细胞外基

质成分C以I、FNmRNA和蛋白的表达；抑制PAI-1mRNA的表达，即BMP-7可抑制硎l

诱导下肾小管上皮细胞ECM的合成、促进ECM降解。关键词：骨形态发生蛋白质类转化生长因子B上皮细胞转分化细胞外基质

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Effects of BMP-7 on

Epithelial-to-Mensenchymal Transition and Extraceilular

Matrix Accumulation in Human Renal Proximal Epithelial Cells Induced by TGF-Ih

Objective：To investigate the effects of bone morphogcnic protein(BMP)-7 on

epithelial-to-mesenchymal昀nsi60n㈣and t he expression 0f 6bIoIlec面(FN)and t ype collagen

I(Col I)in human renal proximal tubular ce 郴-2)induced by transforming growth factor-B1

(TGF-B1)，and to explore the possible mc《?Jlanisms of BMP-7 for the inlffoifion and rwemal of 嗽lal intcmtitial fibrosis．

Methods：HK-2 cells、)l眦treated with TGF-BI or a combination of TGF-Bx and BMP-7．HK-2 cells divided into the following groups according to different f

础Ol'S：Control group,TGF-B1(different

time and concentration)group,3ng／ml TGF-81+BMP-7(different concentration)group．Morphological clI．anges wefc assessed by phase contrast micrcsoopy．The expression of a-SMA and E-cadherin were arlalysed by indirect immunofluorescencc J王T-PCR and Western Blot

respectively．The mRNA levels of extracelluar n强由敦components(Col I al and Frq)and plasminogen activator inlu'bitior(PAI)-1 were measured by RT-PCR．．The∞o'etion of Col I and

FN protein was quantitated by ELISA．

Results：(1)TGF-81 trealment of confluent HK-2 cells resulted in distinct morphological changes in a t ime and dose-dependent manner．The control cells displayed t)『piCal cobblestone morphology of epithelial cells．3ng／ml TGF-Bx induced profound morphologic changes after 48h,with cells becoming elongated in shape,but addition of 200neghn BMP-7 for 48h restored the epithelial morphology of t he H K-2 ceils．(2)Indirect i mmunofluorescem七staining showed t hat expression of a-SMA could not be seen in control cells but upregulated and enhanced by 3ng／ml"I'GF-B1 after 48h．Incubafion with 200ng／ml BMP-7 for 48h dramatically abrogated TGF-Bl-induced a-SMA expression．Treatment of 3ng／ml TGF-fll resulted in distinct loss of Ecadherin staining in the plasma membrane of HK-2 cells but subsequent treatment with 200ng／n：ll BMP-7 largely restored the

E-cadherin protein staining．(3)The expression of a-SMAmRNAwere upregulated by TGF-B1 in a dose-dependent mallner maximally at the concentration of 10ng／ml TGF-81．The 3ng／ml TGF-B1一induced levels of a-S；MA mRNA and protein were increased while E．cadherin decreased in

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protein expression in a dose-dependent manner ．400 ng ／ml BMP-7

could significantly block the expression of a-SMA mRNA

and protein induced by 3ng ／ml TGF-B ：(P

口<0．01，400 ng ／ml

BMP-7+TGF-B1VS ． TGF-BI alone)．(4)3ng ／ml TGF-B1

could increase the mRNAexpression levels of FN and Col I al in a

血珊州魄'ed 锄t manner ．BMP-7 could inlu'bit the expression of ECM

components(FN,Col I)

induced by TGF-81 in a dose-depedent manner ：400ng ／ml BMP-7 could supress the expression of

FN and Col I significantly(P<0．01)．固3ng ／ml TGF-81 could increase the expression of PAI-1

mRNA(P<0．01 VS ．contr01)．BMP-7 could inlu'bit the expression of PAI-1 mRNA induced by TGF431 in a dose-dependent

manner ．400ng ／ml BMP-7 could inhibit the expression of PAI-1 mRNA significantly(P<0．01)．

Condusiom ：(1)TGF-81

can induce EMT and produce ECM components in assodafion with all

inlu'bition of E ．．cadherin expression,increase of a-SM&Col I and FN expression and

loss of

epithelial cell morphology ．(2)BMP-7 Call b lock a nd revel 辩TGF-Brinduced EMT by s upressing the expression of a -SMA and restoring the expression

of E-cadherin in a 由孵也脚lllallner_(3)

BMP-7 Call prevent aocumulation of ECM components

and enhance degradation of ECM

components induced

by TGF-B1 through reducing the expression of Col I,FN and PAl-1． Key words ：Bone morphogenetic proteins ；Transforming growth factor beta ；Epithelial cells ； Transition ；Extracellar matrix

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