【作者】 陆良生；

【导师】 肖现民； 李凯； 董岿然； 陈莲；

【作者基本信息】 复旦大学 ， 儿科学， 2009， 博士

【摘要】 研究背景肾母细胞瘤是儿童最常见的肾脏原发性恶性肿瘤,约占儿童所有恶性肿瘤的8%,目前被认为是一种胚胎发育性肿瘤。Wnt信号通路是一条在生物进化中极为保守的通路,参与了胚胎发育过程中的诸多领域,受到严格的调节。近年来研究发现,Wnt信号通路在肿瘤的发生中也具有重要的意义,在众多人类肿瘤发生中均存在Wnt信号通路的广泛活化,Wnt信号通路与肿瘤的关系正日益受到众多学者的关注。因此,从调控胚胎发育和肿瘤发生的共同途径Wnt信号通路研究入手,能否揭示肾母细胞瘤发生的可能机制?能否在Wnt信号通路中寻找到新的抗癌分子靶位以提高肾母细胞瘤治疗的临床疗效?这些研究对寻找新的治疗方法和提高肾母细胞瘤患儿的生存率,具有重要的临床价值和意义,而目前国内外尚未见相关报道。研究目的本研究通过体外培养肾母细胞瘤细胞株SK-NEP-1,检测Wnt信号通路相关基因及蛋白在肾母细胞瘤中的表达情况;观察不同浓度的sFRP-1(secreted Frizzled Related Protein-1)干预Wnt信号通路对细胞增殖的影响;通过RNAi技术敲减Wnt信号通路中β-catenin基因,观察对SK-NEP-1细胞生长及Wnt信号通路的影响;检测17β-雌二醇(E2)和双酚A(Bisphenol A,BPA)干预对SK-NEP-1细胞生长及Wnt信号通路的影响;本实验将初步探讨Wnt信号通路在肾母细胞瘤发生发展过程中的作用及可能机制,为肾母细胞瘤靶向治疗寻找新的靶点并提供科学理论依据。材料与方法一、SK-NEP-1的培养鉴定及sFRP-1蛋白干预对细胞增殖的影响:在含15%胎牛血清和1%青链霉素的McCoys 5A培养基中培养肾母细胞瘤SK-NEP-1细胞,观察细胞形态,绘制生长曲线;采用Envision两步法检测细胞中Vimentin、β-catenin、WT1、CK蛋白的表达;RT-PCR方法检测Wnt信号通路中WNT4、sFRP1、CTNNB1、CCND1、MYC基因的表达,并与肾脏胚胎细胞293细胞比较;观察不同浓度sFRP-1干预Wnt通路对SK-NEP-1增值的影响:将SK-NEP-1常规培养扩增后分为:对照组;低剂量组sFRP-1(100～500ng/mE);高剂量组sFRP-1(10μg/mL);低剂量sFRP-1抗体组(100～500ng/mL);高剂量sFRP-1抗体组(10μg/mL),分别在干预后0、24、48、72hr用CCK-8试剂检测比较各组光吸收度值(AV)。二、β-catenin特异性siRNA对SK-NEP-1生长的影响及与Wnt通路的关系:采用HiPerFect转染试剂,经转染优化实验,将Qiagen设计的两种不同序列β-catenin特异性siRNA(small interfering RNA)瞬时转染到SK-NEP-1细胞中,实验分为4组:未转染组(UT);阴性对照组(NS);siRNA1组(S1);siRNA2组(S2),分别在转染后0、24、48、72hr用CCK-8试剂检测比较各组光吸收度值(AV);在转染24hr后检测各组中CTNNB1、CCND1、MYC基因的表达水平;转染48hr后免疫荧光法检测各组中βcatenin表达水平;转染48hr后流式细胞仪AnnexinV-FITC/PI双染法检测各组细胞凋亡率差异。三、E2、BPA对SK-NEP-1生长的影响及与Wnt通路的关系:将SK-NEP-1细胞常规培养扩增后分为:对照组;加不同浓度17β-雌二醇组(E2);加不同浓度双酚A组(BPA),分别在干预后48hr用CCK-8检测比较各组光吸收度值(AV);干预48hr后real time PCR检测各组中CTNNB1、CCND1、MYC的基因表达水平;干预48hr后免疫荧光法检测各组中β-catenin表达水平;流式细胞仪AnnexinV-FITC/PI双染法检测无血清培养24hr各组细胞凋亡率差异。结果一、SK-NEP-1的培养鉴定及sFRP-1蛋白干预对细胞增殖的影响:SK-NEP-1为悬浮细胞,半贴壁生长,细胞倍增时间大约为24小时;免疫组化结果显示SK-NEP-1中Vimentin、WT1、β-catenin、CK均呈阳性表达;RT-PCR显示SK-NEP-1中sFRP1;CTNNB1;CCND1;MYC基因均有表达,且CTNNB1呈现高表达状态,提示SK-NEP-1细胞中有Wnt通路的激活。与胚胎细胞293相比较,两种细胞中sFRP-1均呈现高表达,而WNT4无明显表达。外源性sFRP-1蛋白干预Wnt通路的结果显示:低浓度sFRP-1(100～500ng/mL)及高浓度sFRP1抗体干预SK-NEP-1对细胞增殖无明显作用,而sFRP-1在高浓度(10μg/mL)时,干预24小时后对照组AV值为干预组的1.23倍,p＜0.05;干预48小时后对照组AV值为干预组的1.25倍,p＜0.05,干预组对细胞增殖有明显抑制作用,而在干预72小时后抑制作用减弱,对照组AV值为干预组的1.1倍,p＞0.05。二、β-catenin特异性siRNA对SK-NEP-1生长的影响及与Wnt通路的关系:转染优化实验表明,SK-NEP-1细胞浓度在4.0×10~5/mLL～1.0×10~6/mL之间,24孔板体系每孔100ul transfection complexes含6ul Hiperfect转染试剂可以获得满意的转染效率,而β-catenin特异性siRNA1和siRNA2干预浓度在100nmol/L时可以获得有效的抑制效应。分别在SK-NEP-1细胞中瞬时转染100nmol/L的S1和S2后24hr,转染组细胞数量较对照组明显减少,CCK-8检测各组AV值,UT组的AV值分别是S1组和S2组的1.25倍(p＜0.01)和1.23倍(p＜0.01);48hr后UT组的AV值分别是S1组和S2组的1.48倍(p＜0.01)和1.43倍(p＜0.01);72hr后UT组的AV值分别是S1组和S2组的1.27倍(p＜0.05)和1.1倍(p＞0.05),转染24、48hr,细胞增殖明显受到抑制。转染24hr后S1和S2组β-catenin的mRNA分别被敲减了67%和73%,S1、S2组与UT组比较,下游基因CCND1、MYC的mRNA表达水平也明显下调,CCND1组间比较p＜0.01;MYC组间比较p＜0.05;而UT组与NS组比较,β-catenin、CCND1、MYC基因的p值均＞0.05(Oneway,Bonferroni法)。转染48hr后免疫荧光检测各组中β-catenin表达,采用积分光密度/面积(IOD/Area)值分析比较各组β-catenin荧光强度,S1、S2组较UT、NS组明显减少,p＜0.01(Nemenyi-Wilcoxson-Wilcox法)。干预48hr后流式细胞仪AnnexinV-FITC/PI双染法检测各组细胞凋亡率,S1、S2组凋亡率较NS组增加,p＜0.05。三、E2、BPA对SK-NEP-1生长的影响及与Wnt通路的关系:免疫组化检测SK-NEP-1中无明显雌激素受体(ER)表达。CCK-8检测对照组与不同浓度E2、BPA干预组光吸收度值(AV)结果显示,5nmol/L的17β-雌二醇及5μmol/L的双酚A对SK-NEP-1有促增殖作用(p＜0.05)。检测干预48hr后各组β-catenin及下游基因CCND1、MYC的mRNA的表达差异,各组间均无区别(p＞0.05),免疫荧光检测干预48hr后β-catenin在各组细胞中表达,采用积分光密度/面积(IOD/Area)分析比较各组β-catenin荧光强度,各组之间无明显区别(p＞0.05)。流式细胞仪Annexin V-FITC/PI双染法检测各组细胞凋亡率无明显区别(p＞0.05)。结论1.肾母细胞瘤细胞株SK-NEP-1中存在Wnt通路的激活,其中sFRP-1、β-catenin基因在mRNA水平高表达,而Wnt-4基因无明显表达。2.肾脏在发育过程中,后肾胚胎细胞在向上皮细胞分化过程中,Wnt通路调节异常,sFRP-1高表达抑制了Wnt-4的正常表达,导致后肾的间质细胞向上皮细胞转化障碍,持续增殖,可能是肾母细胞瘤发生机制之一。3.加入低浓度sFRP-1(100～500ng/mL)或高浓度的sFRP-1抗体(10μg/mL)对SK-NEP-1细胞增殖无影响,而加入高浓度sFRP-1(10μg/mL)可以抑制SK-NEP-1细胞的增殖,表明高浓度的sFRP-1对SK-NEP-1增殖起抑制作用。4.瞬时转染β-catenin特异性siRNA可以抑制SK-NEP-1细胞增殖,促进细胞凋亡,细胞β-catenin蛋白表达降低,通路下游相关基因MYC及CCND1表达下调,表明β-catenin的激活在肾母细胞瘤发生发展过程中起着重要作用。5.SK-NEP-1中无明显雌激素受体的表达,但是17β-雌二醇及环境内分泌干扰物双酚A可以促进SK-NEP-1细胞的增殖。6.17β-雌二醇及环境内分泌干扰物BPA干预SK-NEP-1对Wnt通路β-catenin及下游基因的表达均无明显影响。更多还原

【Abstract】 Backgrounds Nephroblastoma is the most common primary malignant tumor of kidney in children.It accounts for about 8 percent of all pediatric malignant tumors and it is confirmed as a kind of embryonal tumor.Wnt signal pathway is a conservative pathway in organic evolutions.It participates in the embryonal development of many organs and is accommodated strictively.In recent years,wnt signal pathway has been found to play a key role in tumorigenesis.The extensive activation of wnt signal pathway is common in many tumors and the relationship between wnt signal pathway and tumor is becoming more and more attractive. Therefore,wnt signal pathway,the common pathway of embryonal development and tumorigenesis,maybe become the clue to reveal the mechanism of nephroblastoma.If any new anticancer target would be found in wnt signal pathway,it could be significant to improve the therapeutic effect and survival rate of nephroblastoma.Up to now,such kind of research has not been reported in the world.Purpose We cultured nephroblastoma cell line SK-NEP-1 in vitro and detected the expression of wnt related genes and proteins to confirm the activation of wnt signal pathway.After that,we investigated the effect of sFRP-1 on the proliferation of SK-NEP-1 and the role of siRNAs againstβ-catenin on the proliferation and apoptosis of SK-NEP-1 respectively.The expression level of downstream genes and proteins concerned with wnt signal pathway in each group was detected by real time PCR to explor the relationship ofβ-catenin and the proliferation of nephroblastoma.We also observed the effect of 17β-estradiol and bisphenol A on the proliferation and apoptosis of SK-NEP-1 and detected the expression change of downstream genes and proteins concerned with wnt signal pathway.We would try to explore the role of wnt signal pathway on nephroblastoma cell line SK-NEP-1 and its possible mechanism.It would conduce to find new targets for targeted therapy of Wilms’tumor and provide the scientific evidence.Materials and Methods1.Nephroblastoma cell line SK-NEP-1 culture and detection of wnt signal pathway’s activation:SK-NEP-1 cell line was cultured in McCoys 5A medium with 15%fetal bovine serum and 1%mycillin under routine condition.Growth curve of SK-NEP-1 was obtained by viable cell counting at each time-point with hemocytometer and trypan blue exclusion.The expression of Vimentin,β-catenin, WT1 and CK in cell line were tested by Envision immunohistochemistry.The RT-PCR was applied to detect the mRNA level of WNT4、sFRP1、CTNNB1、CCND1、MYC genes in SK-NEP-1 and 293 cells respectively.The effect of sFRP-1 on Wnt signal pathway and the proliferation of SK-NEP-1 cells was tested by CCK-8 kit.Cells were divided into control group,low-dose group of sFRP-1(100～500ng/mL),high-dose group of sFRP-1(10μg/mL),high-dose group of Anti sFRP-1 (10μg/mL).The sFRP-1 at different concentrations were added to complete growth medium and the absorbance value(AV) was tested at 0、24、48、72hours after interference respectively.2.Transient transfection with siRNAs againstβ-catenin into SK-NEP-1 cell: Transfecection of siRNA againstβ-catenin was implemented by HiPerFect Transfection Reagant according to the manufacturer’s instructions.Cells were typically seeded in six-well plates(400,000 cells per well) before transfection. Transfection complexes were prepared in serum-flee medium(McCoys 5A). Immediately before transfection,complete growth medium was removed from all wells,and wells were rinsed with PBS.Cells were typically exposed to transfecion complexes for 6 hours before replacement of complete growth medium depending on the particular experiment.The concentration of each siRNAs within HiPerFect is indicated in each experiment.Cells were divided into four groups:no transfection group(UT),negative control group(NS),siRNA1 group(S1),siRNA2 group (S2).The effect of siRNAs againstβ-catenin on the proliferation and apoptosis of SK-NEP-1 were tested by CCK-8 kit and flow cytometry at each time-point respectively.The change of wnt signal pathway were detected by real time PCR and immunofluorescence.3.The effect of 17β-estradiol and bisphenol A on wnt signal pathway and the proliferation of SK-NEP-1 cells:Cells were divided into three groups and different concentrations of 17β-estradiol and bisphenol A were added into each group respectively.The AV value at 48 hours was tested to discriminate the effective concentration of 17β-estradiol and bisphenol A on the proliferation of SK-NEP-1. Their effect on apoptosis of SK-NEP-1 was tested by flow cytometry.The change of wnt signal pathway was detected by real time PCR and immunofluorescence.Results1.Nephroblastoma cell line SK-NEP-1 culture and detection of wnt signal pathway’s activation:SK-NEP-1 cell line was suspension cell and its double time was about 24 hours.The expression of Vimentin,β-catenin,WT1 and CK were all detected by Envision immunohistochemistry.The RT-PCR confirmed the activation of Wnt signal pathway and that the mRNA level of WNT4 in SK-NEP-1 was very low whereas sFRP-1 and CTNNB1 were extremely high,which were similar to 293 cell line.The result of sFRP-1 on wnt signal pathway and proliferation of SK-NEP-1 cells showed that sFRP-1 at low concentrations of 100 to 500 ng/mL and Anti sFRP-1 at high-dose concentration had no effect on proliferation.Whereas sFRP-1 at high concentrations of 10ug/mL could inhibit the growth of SK-NEP-1.The AV value of control group was 1.23 times more than sFRP-1 group at 24 hours time-point(p＜0.05) and at 48 hours time-point it was 1.25 times(p＜0.05).72hours later the AV value between groups had no difference((P＞0.05,Oneway,respectively).2.Transient transfection with siRNAs againstβ-catenin into SK-NEP-1 cell: Optimization of transfection indicated that the satisfactory transfection efficiency (＞70%) could be obtained with cell density at 4.0×10~5/mL～1.0×10~6/mL and 6ul Hiperfect Transfection Reagant per 100ul transfection complexes in 24 orifice plate system.Growth inhibition was obvious when the working concentration of siRNAs againstβ-catenin reached 100 nM.24 hours after transfection,the AV value of UT was 1.25 times of S1 and 1.23 times of S2 respectively(p＜0.01)and after 48 hours it was 1.48 times of S1 and 1.43 times of S2 respectively(p＜0.01).72hours later the AV value of UT was 1.27 times of S1(p＜0.05) and 1.1 times of S2(P＞0.05,Oneway, respectively).100 nM siRNA1 and siRNA2 againstβ-catenin at 24hours inhibited theβ-catenin mRNA by 67%and 73%(P＜0.05,Oneway,respectively).Real time PCR showed the down-regulated expression of CCND1 and MYC in S1 and S2 groups compared with UT group(p＜0.05).Immunofluorescence disclosed the shutdown ofβ-catenin translation in siRNA groups.48hours after transfection,IOD/Area analysis showed the decrease value of S1 and S2 groups compared with UT and NS group (p＜0.01).Apoptosis of SK-NEP-1 increased in siRNA1 and siRNA2 groups compared with UT group(p＜0.05).3.The effect of estradiol and bisphenol A on proliferation of SK-NEP-1 cells and wnt signal pathway:The expression of ER was negative in SK-NEP-1 detected by Envision immunohistochemistry.17β-estradiol at concentration of 5 nM,and bisphenol A at concentration of 5μM had effect on the proliferation of SK-NEP-1 cells(P＜0.05,Oneway,respectively).Real time PCR showed that the expression of CCND1 and MYC had no difference between groups(P＜0.05,Oneway,respectively). Immunofluorescence found a stable level ofβ-catenin in three groups.IOD/Area analysis showed no difference between goups(p＞0.05).There was also no difference of apoptosis between groups(p＞0.05).Conclusions1.There is activation of wnt signal pathway in SK-NEP-1 cell line.The expression level of sFRP-1 andβ-catenin is high and Wnt-4 is not obvious in SK-NEP-1 cell.2.During the differentiation from embryonic cell to epithelial cell in human metanephron,malmoduration of wnt signal pathway would be probably one of the mechanism leading to the tumorigenesis.That means the inhibition of Wnt-4 would lead to dysdifferentiation.The activation of sFRP-1 andβ-catenin would result in the continuous proliferation of SK-NEP-1 cell.3.The addition of low-dose sFRP-1 and high-dose Anti sFRP-1 has no effect on the proliferation and downstream gene regulation,but high concentration of sFRP-1 can inhibit the proliferation of SK-NEP-1 cell.This implicate that high-dose sFRP-1 would be the inhibitor of wnt signal pathway.4.Growth inhibition and down-regulated expression of CCND1 and MYC are the result of siRNAs againstβ-catenin,and it also promotes the apoptosis of SK-NEP-1 cell.The activation ofβ-catenin act as a key role in the development of nephroblastoma.5.No estradiol receptor(ER) is expressed in SK-NEP-1.17β-estradiol at concentrations of 5 nM and bisphenol A at concentration of 5 uM have effect on the proliferation of SK-NEP-1 cell.6.There is no change of the wnt signal pathway after the interference of estradiol and bisphenol A.更多还原