【作者】 王鹏；

【导师】 刘鲁明； 于尔辛； 陈震；

【作者基本信息】 复旦大学 ， 中西医结合临床， 2008， 博士

【摘要】 目的探索胰腺癌中医"证"与病机可能的关系,探寻清胰化积方(QYHJ)治疗胰腺癌的可能作用机制。方法第一部分1、建立不同中医证型(脾虚型、湿热型和血瘀型)pancO2胰腺癌皮下移植瘤模型,评价不同证型对肿瘤生长的影响。2、建立不同中医证型(脾虚型、湿热型和血瘀型)pancO2胰腺癌皮下移植瘤模型,观察不同证型胰腺癌对抗胰腺癌中药QVHJ治疗的反应性。第二部分1、采用real-time PCR和western blot检测QYHJ治疗后胰腺肿瘤细胞SKImRNA和蛋白的表达变化。2、采用Real-time PCR对SKI mRNA在四种人胰腺癌细胞系SW1990、BxPC3、PANC-1和PC3中的差异性表达进行半定量分析。3、采用RT-PCR技术,检测SKI相关TGF-β信号通路关键分子mRNA在四种人胰腺癌细胞系SW1990、BxPC3、PANC-1和PC3中的表达。4、设计针对SKI基因cds区的Negative、siRNA1、siRNA2、siRNA3序列,设计合成互补单链DNA片段,以H1启动子后的BamHI(GGATCC)和EcoRI(GAATTC)为插入位点,克隆进真核表达载体pSIH1-H1-copGFPshRNA Vector,重组载体PCR扩增回收及DNA序列分析进行鉴定。5、应用脂质体转染方法将重组载体pSIH-negative和pSIH-siRNA分别导入293T细胞,Real-time PCR筛选有效的SKI干扰序列。6、参照SBI Lentivector Expression System操作手册,包装产生含有pSIH-siRNA3和pSIH-negative穿梭质粒的假病毒颗粒,分别感染SW1990和BxPC3细胞,建立干扰SKI基因的稳定细胞株以及对照细胞株。7、通过Real-time PCR和Western blot方法对细胞RNAi后SKI表达情况进行鉴定。8、WST(water-soluble tetrazolium salt)法测定SKI RNAi对人胰腺癌细胞体外增殖的影响,并绘制细胞增殖曲线。9、标准PI染色流式细胞计数法(flow cytometry,FCM)检测SKI RNAi对人胰腺癌细胞细胞周期分布的影响。10、体外运动实验(wound healing assay)检测SKI RNAi对人胰腺癌细胞运动能力的影响。11、体外侵袭实验(transwell)检测SKI RNAi对人胰腺癌细胞体外侵袭能力的影响。12、建立裸鼠皮下移植瘤模型,探讨SKI RNAi对人胰腺癌细胞体内肿瘤生长的影响。13、通过尾静脉注射人胰腺癌细胞,探讨SKI RNAi对人胰腺癌细胞体内肝转移的影响。14、RT-PCR和real-time PCR检测SKI RNAi后TGFβ信号通路下游反应性靶基因PAI-1 mRNA和CTGF mRNA的表达,同时观检测其他下游反应性靶基因表达情况。15、RT-PCR检测SKI RNAi后TGF-β信号通路关键分子TβRⅡ、TβRI、smad2、3、4、7的表达情况,以及通过western blot检测smad3磷酸化水平。16、Real-time PCR检测与细胞增殖、侵袭、转移相关基因表达的变化。17、流式细胞仪检测TGFB信号通路阻断后SKI RNAi对细胞周期分布的影响。18、体外侵袭实验检测TGFβ信号通路阻断后SKI RNAi对细胞侵袭能力的影响。19、建立裸鼠人胰腺癌皮下移植瘤模型,评价SKI不同表达人胰腺癌细胞对QYHJ治疗的反应性。结果第一部分1、不同的中医证型pancO2细胞体内成瘤率均为100%,但三模型组肿瘤生长速度均要低于对照组,尤其湿热组表现明显(瘤重与对照组比较,下降了31.7%)。2、脾虚组与湿热组肿瘤在应用QYHJ治疗后肿瘤体积与瘤重均有降低。血瘀组肿瘤体积与瘤重在应用QYHJ后并没有表现出明显的抑瘤作用。第二部分l、经QVHJ治疗后SW1990人胰腺癌细胞SKI mRNA和蛋白表达量与未经QYHJ治疗比较,分别下降了39.6%和41.3%。2、SKI在四种人胰腺癌细胞中均有表达,其中BxPC3、PANC-1和PC3细胞SKI mRNA表达量分别是SW1990细胞的0.9、1.4和0.7倍。3、SKI相关TGFβ信号通路关键分子TβRⅠ、TβRⅡ、smad2、smad3、smad7在四种人胰腺癌细胞系(SW1990、BxPC3、PANC-1和PC3)均有表达,除外smad4在BxPC3中不表达、TβRⅡ在PC3中不表达。4、重组载体pSIH-negative pSIH-siRNAl、pSIH-siRNA2和pSIH-siRNA3经PCR扩增回收电泳及DNA测序证明其序列的正确性。5、Real-time PCR检测发现转染pSIH-siRNAl、pSIH-siRNA2和pSIH-siRNA3细胞SKI mRNA表达水平分别是转染pSIH-negative细胞的55.0%、54.0%和45.0%,我们选择pSIH-siRNA3和pSIH-negative进行后续实验。6、包装产生含有pSIH-siRNA3和pSIH-negative穿梭质粒的假病毒颗粒,分别感染SW1990和BxPC3细胞,建立干扰SKI基因的稳定细胞株(SW1990/SKIRNAi和BxPC3/SKI RNAi)以及对照细胞株(SW1990/con RNAi和BxPC3/con RNAi)。7、SW1990/con RNAi和SW1990/SKI RNAi细胞SKI mRNA表达水平分别是亲代细胞的105%和46%,SKI蛋白表达水平分别是亲代细胞的123%和30%:BxPC3/con RNAi和BxPC3/SKI RNAi细胞SKI mRNA表达水平分别是亲代细胞的94%和38%,SKI蛋白表达水平分别是亲代细胞的90%和16%。8、SKI RNAi增加了SW1990和BxPC3细胞对TGF-D1的敏感性,两种人胰腺癌细胞在不同浓度TGF-β1(0、25、50、100、200、300pM)的诱导下,细胞体外增殖明显减慢。9、SKI RNAi后细胞经TGF-β1诱导,GO-G1期细胞比列明显增加,在SW1990细胞中由原来的40.4%上升到62.9%(P＜0.05);在BxPC3细胞中由原来的20.0%上升到66.6%(P＜0.05)。10、在TGF-β1诱导下,SW1990和BxPC3细胞体外运动能力明显增强,但SW1990/SKI RNAi细胞经TGFBl诱导24小时后,与SW1990/con RNAi细胞相比,伤口面积降低了54.3%(P＜0.05);BxPC3/SKI RNAi细胞经TGFβ1诱导12小时后,与BxPC3/con RNAi细胞相比,伤口面积降低了57.2%(P＜0.05)。11、在TGF-β1诱导下,SW1990和BxPC3胞体外侵袭能力明显增强,但SW1990/SKI RNAi细胞经TGFβ1诱导48小时后,与SW1990/con RNAi细胞相比,侵袭细胞数增加了36.3%(P＜0.05);BxPC3/SKI RNAi细胞经TGFβ1诱导24小时后,与BxPC3/con RNAi细胞相比,侵袭细胞数增加了43.3%(P＜0.05)。12、裸鼠皮下移植瘤在SKI RNAi后生长明显减慢,SW1990在接种后d31时肿瘤平均体积和瘤重分别下降至对照组的55.8%(P＜0.01)和58.4%(P＜0.05);BxPC3在接种后8周时肿瘤平均体积和瘤重分别下降至对照组的53.7%(P＜0.01)和46.7%(P＜0.05)。13、细胞经尾静脉注射后后4周BxPC3、BxPC3/con RNAi和BxPC3/SKI RNAi组肝脏转移发生率分别为83.3%(5/6)、71.4%(5/7)和100%(6/6),而平均肝脏转移灶数目分别为15.8、18.0和43.6,BxPC3/SKI RNAi与BxPC3/con RNAi组比较,增加了1.4倍(P＜0.01)。SW1990、SW1990/conRNAi和SW1990/SKI RNAi细胞在尾经脉注射后两月均未发现有肝脏转移。14、SW1990和BxPC3细胞在SKI RNAi后PAI-1和CTGF mRNA表达量明显上升,而且维持时间延长;同时伴有c-jun、JunB、p21 mRNA的表达上调和c-myc、CDC25A mRNA表达下调。15、SKI RNAi并不会对TβRⅡ、TβRⅠ、smad2、3、4、7的表达产生影响,但在SKIRNAi后磷酸化smad3水平明显增加。16、SKI RNAi在体内、外上调p21、MMP-2、MMP-9和VEGF,同时下调CyclinD1的表达(均P＜0.05)。17、TβRI/ALK5激酶抑制剂SB431542可阻断SKI RNAi后TGF-β诱导的G1期阻滞。18、TpRFALK5激酶抑制剂SB431542可阻断SKI RNAi后TGF-β诱导的促侵袭作用。19、SW1990裸小鼠皮下移植瘤给予QYHJ治疗后瘤重抑制率为29.6-32.2%,但SKI RNAi后SW1990裸小鼠皮下移植瘤予QYHJ治疗后瘤重抑制率为16.0%。结论1、胰腺癌证与瘤之间可能是一种因瘤致证的关系,证的存在是机体对肿瘤存在所表现出的一种反应,因此要针对胰腺癌本质病机的治疗。2、SKI下调在体内外具有抑制肿瘤生长作用,该作用的发挥主要通过影响smad3磷酸化、进而增强TGF-β转录活性的诱导作用,恢复胰腺癌细胞对TGF-β诱导细胞周期阻滞作用的反应性来实现的,QYHJ可通过下调SKI表达抑制了胰腺肿瘤的生长,SKI可能是QYHJ治疗胰腺癌的一个重要作用靶点之一。3、SKI不同表达可导致胰腺肿瘤对QYHJ治疗反应性的差异,通过对SKI表达情况的检测在一定程度上可用于预测肿瘤对治疗的反应,用于筛选潜在有效的适用人群。更多还原

【Abstract】 ObjectiveTo study the relationship between traditional Chinese medicine（TCM） syndrome and TCM pathogenesis in pancreatic cancer.And to explore the potential mechanism of Qingyihuaji formula（QYHJ） in the treatment of pancreatic cancer.MethodsPartⅠ:Three subcutaneous transplanted panc02 tumor models with different TCM syndrome types,including spleen deficiency,damp-heat and blood stasis,in the nude mice were established.The effect of each TCM syndrome on subcutaneous transplanted tumor were evaluated and the response of anti-pancreatic cancer TCM formular named QYHJ on subcutaneous transplanted tumor with defferent TCM syndrome were also studied.PartⅡ:The expression changes of SKI mRNA and protein in pancreatic carcinoma subcutaneous tumor treated with QYHJ were detected by real-time PCR and western blot.Then the expression of SKI and related TGF-βsignaling pathway components in four human pancreatic cancer cell lines,including SW1990,BxPC3, PANC-1and PC3 were also analysed by semi-quantitative real-time RT-PCR. SW1990 and BxPC3 cell lines were chosen for the following RNAi study.Three different small interfering RNA for human SKI（siRNA1 siRNA2 and siRNA3） and one negative siRNA were designed.Then,the primer pairs were annealed and subcloned into the BamH I（GGATCC） and EcoRI（GAATTC） sites of pSIH1-H1-copGFP shRNA Vector.The recombinant vectors were identified by PCR and automated sequencing analysis and then transfected into 293T cells by Lipofectamine^TM 2000 Transfection Reagent.Real-time PCR were used for selection of the most effective SKI silencing sequence.Next,according to the instruction of SBI Lentivector Expression System,a lentiviral expression construct and pPACK packaging plasmid mix were co-transfected into 293T cells.Then viral particles were collected and the titer were determined.The recombinant lentivector were used for infecting target cells（SW1990 and BxPC3）.The expression of SKI after SKI RNAi or negative RNAi were identified by real-time PCR and western blotting.Cell growth,cell cycle distribution,migration and invasion in vitro were detected respectively by WST,FCM with PI staining,wound healing assay and in vitro invasion assay.To examine the effects of SKI RNAi on tumor growth,we performed the in vivo assay using an subcutaneous transplanted tumor model in the nude mice. in vivo metastasis model were also established through tail vein injection with tumor cells and the effect of SKI expression status on metastasis were also evaluated.Next, molecular basis underlying the involvement of SKI in the phenotypes of human pancreatic cancer cells were explored.Firstly,the induction of specific TGFβ-responsive targeted genes were detected by RT-PCR and real-time PCR.Then the expression of TGFβsignaling pathway components and proliferative-,invasive-and metastatic-related genes after SKI RNAi were also examined by RT-PCR and western blot.Finally,using a selective TβR-I kinase inhibitor,SD-431542,we evaluate the effect of SKI RNAi on in vitro proliferative and invasive potential after blocking TGF-βsignaling.After determination of the function and its potential molecular mechanism,we studied the effect of QYHJ on subcutaneous transplanted tumor with defferent SKI expression status.ResultsPartⅠ:The incidence of tumor with different TCM syndrome type were all 100%,however,subcutaneous transplanted tumor grew slowly than that with no TCM syndrome,especial in damp-heat group（compared with control group,the tumor weight inhibitory rate decreased 31.7%）.The volume and weight of tumor with spleen deficiency and damp-heat syndrome decreased after administration of QYHJ.But this didn’t happened in blood stasis group. PartⅡ:Expression of SKI mRNA and protein in SW1990 subcutaneous transplanted tumor after treatment of QYHJ decreased 39.6%and 41.3%of that in the untreated respectively.The levels of SKI mRNA in BxPC3、PANC-1 and PC3 were 0.9-,1.4- and 0.7-fold respectively in comparison to that in SW1990.The expresssion of SKI related TGF-βsignaling pathway components,including TβRⅡ, TβRⅠ,smad2,smad3,smad4 and smad7 were all detected in the four human pancreatic cancer cell lines,except for smad4 in BxPC3 and TβRⅡin PC3.Then SW1990 and BxPC3 pancreatic cancer cell lines were chosen for further SKI RNAi study.The expression of SKI mRNA in pSIH-siRNA1,pSIH-siRNA2 and pSIH-siRNA3 transfectants were 55.0%,54.0%and 45.0%respectively of that in pSIH-negative transfectant（P＜0.01）.The pSIH-siRNA3 and pSIH-negative were selected for the subsequent studies.The recombinant lentivector were used for infecting SW1990 and BxPC3 cells.Then two stable transfection cells （SW1990/SKI RNAi and BxPC3/SKI RNAi） were created and SW1990/con RNAi and BxPC3/con RNAi were used as control.The expression of SKI mRNA and protein in SW1990/con RNAi and SW1990/SKI RNAi were 105%,123%and 46%, 30%respectively of that in parental cells.Similarly,the expression of SKI mRNA and protein in BxPC3/con RNAi and BxPC3/SKI RNAi were 94%,90%and 38%, 16%respectively of that in parental cells.SKI RNAi sensesized the response of SW1990 and BxPC3 cell to TGF-β1 and reduced the cell growth in vitro induced by TGF-β1 with different concentration.Flow cytometric（FCM） analyses revealed that the percentage of cells in G1-phase was dramatically increased from 40.4%to 62.9% in SW990 cell line and from 20.2%to 66.6%in BxPC3 cell line when SKI RNAi induced by TGF-β1.SKI RNAi transfectants were much higher migrative and invasive in comparison to parental or mock transfected cells.Tumors formed by SW1990/SKI RNAi and BxPC3/SKI RNAi cells were much smaller than those formed by parental and control vector-transfected cells at the end of the in vivo assays（P＜0.05）.Tail vein injection of SKI RNAi BxPC3 into nude mice resulted in a significant increase of liver metastatic colonizations.Analysis of specific TGF-β-responsive target gene expression detected by real-time PCR showed that the expression of PAI-1 and CTGF mRNA were up-regulated dramatically and sustained for much longer time.Accompanied with PAI-1 and CTGF up-regulation,the expression of other TGF-β-responsive target genes also changed,with c-jun,JunB and p21 mRNA up-regulated and c-myc and CDC25A mRNA down-regulated.SKI RNAi had no influence on the expression of TGF-βsignaling pathway components, including TβRⅡ,TβRⅠ,smad2,smad3,smad4 and smad7,however,SKI RNAi resulted in a rapid and sustained increase in phosphorylated Smad3 in the two cells. The expression of proliferatory,invasive and metastatic related gene were also examined,with result of up-regulation of p21,MMP-2,MMP-9,VEGF and down-regulation of cyclin D1.Blocking TβR-I/ALK5 activity using a selective kinase inhibitor,SD-431542,strongly conteract the effect of G1-phase arrest and invasion promoting induced by SKI RNAi.After determination that down-regulation of SKI can result in decreased tumor growth,we examined the response of QYHJ on subcutaneous transplanted pancreatic cancer with different SKI expression status. The result showed that the tumor weight inhibitory rate of QYHJ on subcutaneous transplanted tumor formed by SW1990 or SW1990/con RNAi were 29.6%and 32.2%repectively,while it was 16.0%when the tumors were formed by SW1990/SKI RNAiconclusionTCM syndrome originating from tumor might be the relationship between tumor and TCM syndrome.The presence of TCM syndromes might be reaction of body to the existence of tumor.So the treatment of pancreatic cancer should aim at the essential TCM pathogenesis underlying the TCM syndrome.Down-regulation of SKI expression can inhibit the growth of pancreatic cancer both in vitro and in vivo. This effect is mainly achieved through increasing the level of phosphorylated smad3, enhancing the transcriptional activity induced by TGFβsignaling,thus restored the response of pancreatic cancer cell to the TGFβinduced cell cycle arrest.QYHJ inhibits growth of pancreatic cancer by down-regulating SKI expression.SKI is one of the potential targets of QYHJ in the treatment of pancreatic cancer Also,different expression status of SKI results in the difference of response of pancreatic cancer to QYHJ treatment.So SKI expression status detected before treatment might be used for predicting the response of pancreatic cancer to QYHJ treatment and screening for potential suitable population.更多还原