胰腺癌细胞株PANC-1中肿瘤干细胞的体外耐药性研究

目的:根据肿瘤干细胞学说理论,探讨胰腺癌耐药的新机制。方法:通过胰腺癌肿瘤干细胞表面标记途径和侧群(side population,SP)细胞途径,分离出人胰腺癌细胞株PANC-1中的SP/NSP(非SP)细胞及CD44+CD24+/CD44-CD24-细胞亚群,用MTT检测上述各亚群细胞在体外对化疗药物耐受的差异,用AnnexinV-PI双染法检测2种肿瘤细胞的抗凋亡能力,并采用实时荧光定量PCR检测两者耐药基因ABCG2、ABCB1和PLK-1表达的差异。结果:人胰腺癌细胞株PANC-1中的SP细胞比例为(7.64±0.96)%,CD44+CD24+细胞比例为(2.60±0.96)%。相对于NSP和CD44-CD24-细胞而言,SP和CD44+CD24+细胞具有更强的化疗耐受能力(P0.01)和抗凋亡能力(P0.01);荧光定量RT-PCR结果均提示,SP和CD44+CD24+细胞高表达耐药基因ABCG2、ABCB1和PLK1。结论:人胰腺癌细胞株PANC-1中SP及CD44+CD24+细胞具有更强的化疗耐受能力,研究胰腺癌肿瘤干细胞可为克服胰腺癌化疗敏感性差的现状提供新的实验基础和理论依据。     

肝癌SMMC-7721细胞中侧群细胞干细胞标记的表达

目的 分选肝癌细胞株SMMC-7721中的侧群(SP)细胞,并分析其干细胞标记的表达.方法 采用流式细胞荧光激活分选(FACS)技术将SMMC-7721细胞分为SP细胞和非侧群(NSP)细胞两个亚群,以实时荧光定量聚合酶链反应(real-time PCR)技术和流式细胞术对两个亚群细胞干细胞标记mRNA和蛋白表达进行分析.结果 SMMC-7721细胞株中分选出的SP细胞比例为(9.2±0.2)%.SP细胞ABCG2、CD133、Oct4、Sox2和NANOG等干细胞标记mRNA的表达水平分别是NSP细胞的7.132倍、4.985倍、8.642倍、5.095倍和5.164倍,差异均有统计学意义(P＜0.01);ABCG2、CD133、Oct4、Sox2和NANOG蛋白在肝癌SP细胞中的含量分别为(92.65±3.92)%、(12.75±1.62)%、(17.35±2.31)%、(9.57±1.71)%和(28.39±5.28)%,在NSP细胞中的含量分别为(0.26±0.06)%、(2.51±0.17)%、(1.74±0.38)%、(1.52±0.41)%和(3.37±1.02)%,差异有统计学意义(P＜0.01).结论 肝癌SMMC-7721细胞中的SP细胞可能富集了肝癌干细胞,联合应用多种干细胞标记筛选肝癌SP细胞可能会获得纯化的肝癌干细胞.

Abstract：

Objective To study the expression of stem cell markers in side population cells sorted from SMMC-7721 cell line. Methods Fluorescence-activated cell sorting (FACS) was used to sort side population (SP) cells and non-SP (NSP) cells from SMMC-7721 cell line. Real-time polymerase chain reaction (PCR) and flow cytometry (FCM) were used to evaluate the expression of several stem cell markers such as ABCG2, CD133, Oct4, Sox2 and NANOG in SP cells and NSP cells. Results FACS analysis indicated that (9.2 ±0. 2)% of the SMMC-7721 cells were SP cells. Real-time PCR analysis suggested that ABCG2, CD133, Oct4, Sox2 and NANOG were expressed in the SP cells at higher levels than the NSP cells by about 7. 132, 4. 985, 8. 642, 5.095 and 5. 164 folds, respectively ( P ＜0. 01 ). FCM analysis revealed that the expression of ABCG2, CD133, Oct4, Sox2 and NANOG proteins in SP cells was (92. 65 ±3.92)%, (12.75 ±1.62)%, (17.35 ±2.31)%, (9.57 ± 1.71)% and (28.39 ±5.28)% respectively,while in NSP cells that was (0. 26 ±0. 06)%, (2. 51 ±0. 17)%, ( 1.74 ±0. 38)%, ( 1.52 ±0. 41 )% and ( 3.37 ± 1.02) % respectively ( P ＜ 0. 01 ). Conclusion The SP cells sorted from SMMC-7721 cell line may enrich tumor stem cells. Purified liver cancer stem cells may be obtained by screening SP cells using a variety of stem cell markers.     

膀胱癌细胞株T24中侧群细胞的分离和功能鉴定

目的 观察膀胱癌细胞株T24中是否存在侧群(SP)细胞及其比例,并鉴定其功能.方法 利用双波长流氏细胞仪(FACS)检测T24中SP细胞的比例,并证实这些SP细胞是否具有癌干细胞的特点.结果 T24中SP细胞占34.7%;与非侧群(NSP)细胞比较,SP细胞有更强的生长增殖能力和克隆形成能力(P＜0.05),表达更高的ATP结合转运蛋白G超家族成员2(ABCG2)和干性基因,对放化疗有更强的抵抗能力,有更多的细胞处于G_0/G_1期(87.4%比63.3%,P＜0.05);分选后的sP和NSP细胞经过约10 d的常规培养,SP细胞中NSP的比例占76.2%,而NSP细胞中SP的比例只占2.6%.结论 膀胱癌细胞株T24中存在很高比例的SP细胞,而且这些SP细胞有癌干细胞的特点.     

细胞株源人前列腺肿瘤干细胞的分选与鉴定

目的:论证人前列腺癌(prostate cancer,PCa)细胞株中是否存在干细胞亚群。方法:分别用免疫表型法和侧群(side population,SP)细胞法从5种人PCa细胞株(Du145、IA8、LNCaP、TSU-PrL和PC-3)中富集类干细胞,再应用软琼脂克隆形成试验初步验证类干细胞亚群的体外生长方式及成瘤能力。选择LNCaP源SP细胞(LNCaP/SP),依次采用免疫细胞化学技术、Transwell、MTT以及裸鼠致瘤试验,分别检测其干细胞标记物的表达情况、鉴定其体外增殖和侵袭能力以及动物体内的致瘤和转移潜能。结果:5种细胞株中均难以分选出免疫表型为CD133+CD44+的细胞亚群。除PC-3外,其余4株细胞可分选出呈现典型克隆性生长特点的SP细胞。体外克隆形成率在IA8、LNCaP和TSU-PrL源SP细胞与非侧群(non-side population,NSP)细胞间有显著性差异(P<0.05)。与LNCaP/NSP相比,LNCaP/SP的体外增殖和侵袭能力显著增强,同时阳性表达整合素α2、Nanog、CD44、OCT4以及ABCG2等5种干细胞标记物。而且,LNCaP/SP的皮下成瘤率、骨转移率及瘤体体积亦显著高于LNCaP/NSP(P<0.01)。结论:SP分选法更适合富集人PCa细胞株中类干细胞,LNCaP/SP细胞是PCa细胞株LNCaP中的肿瘤干细胞(cancer stem cell,CSC)。     

小干扰RNA逆转Wnt/β-catenin通路对肝癌耐药细胞株HepG2/ADM的影响研究

目的 了解沉默β-catenin基因对肝癌耐药细胞HepG2的影响。方法 实验分为5组：正常肝细胞（LO2）组、HepG2组、HepG2/ADM组、SiNC-HepG2/ADM阴性转染组和Siβ-catenin-HepG2/ADM转染组;细胞免疫荧光技术检测各组β-catenin的表达情况;设计并筛选出抑制效率最高的Siβ-catenin;Western-blot及RT-PCR技术检测各组β-catenin、P-gp、MRP1的mRNA和蛋白的表达水平;MTT法观察各组对阿霉素（ADM）、氟尿嘧啶（5-FU）、环磷腺苷（VCR）和奥沙利铂（OHP）的敏感性;流式细胞仪检测各组细胞凋亡。结果 50 mol/L的ctnnb1-001在HepG2/ADM中抑制效率最高：78.86%（P<0.05）,选其为Si-β-catenin;细胞免疫荧光显示HepG2/ADM中β-catenin荧光最强,转染Si-β-catenin后荧光显著减弱;β-catenin、P-gp和MRP1 mRNA和蛋白在HepG2/ADM组表达较高,mRNA表达分别为：0.92±0.03、7.98±0.43和4.56±0.12（P<0.05）,蛋白表达分别为：1.128±0.214、1.678±0.344和1.405±0.212（P<0.05）;转染Siβ-catenin至HepG2/ADM中,β-catenin、P-gp和MRP1在mRNA及蛋白水平均不同程度表达减少,mRNA表达分别为：0.47±0.03、0.66±0.054和0.74±0.03（P<0.05）,蛋白表达分别为：0.787±0.032、0.797±0.055和1.390±0.050（P<0.05）;Siβ-catenin-HepG2/ADM转染组较HepG2/ADM组对ADM、5-FU、VCR和OHP的耐药系数（RI）分别为0.61、0.55、0.30、0.55,对化疗药物敏感性显著增强（P<0.05）;Siβ-catenin-HepG2/ADM转染组凋亡率（28.05±0.35）%,较其他组明显增加（P<0.05）。结论 Wnt/β-catenin通路在HepG2中异常激活,其中β-catenin可能正性调控肝癌耐药基因P-gp和MRP1,Si-β-catenin能一定程度阻断Wnt通路,并能一定程度逆转肝癌细胞株HepG2的耐药性和增强化疗敏感性,增加凋亡。     

小干扰RNA逆转Wnt/β-catenin通路对肝癌耐药细胞株HepG2/ADM的影响研究

目的 了解沉默β-catenin基因对肝癌耐药细胞HepG2的影响。方法 实验分为5组：正常肝细胞（LO2）组、HepG2组、HepG2/ADM组、SiNC-HepG2/ADM阴性转染组和Siβ-catenin-HepG2/ADM转染组；细胞免疫荧光技术检测各组β-catenin的表达情况；设计并筛选出抑制效率最高的Siβ-catenin；Western-blot及RT-PCR技术检测各组β-catenin、P-gp、MRP1的mRNA和蛋白的表达水平；MTT法观察各组对阿霉素（ADM）、氟尿嘧啶（5-FU）、环磷腺苷（VCR）和奥沙利铂（OHP）的敏感性；流式细胞仪检测各组细胞凋亡。结果 50 mol/L的ctnnb1-001在HepG2/ADM中抑制效率最高：78.86%（P<0.05），选其为Si-β-catenin；细胞免疫荧光显示HepG2/ADM中β-catenin荧光最强，转染Si-β-catenin后荧光显著减弱；β-catenin、P-gp和MRP1 mRNA和蛋白在HepG2/ADM组表达较高，mRNA表达分别为：0.92±0.03、7.98±0.43和4.56±0.12（P<0.05），蛋白表达分别为：1.128±0.214、1.678±0.344和1.405±0.212（P<0.05）；转染Siβ-catenin至HepG2/ADM中，β-catenin、P-gp和MRP1在mRNA及蛋白水平均不同程度表达减少，mRNA表达分别为：0.47±0.03、0.66±0.054和0.74±0.03（P<0.05），蛋白表达分别为：0.787±0.032、0.797±0.055和1.390±0.050（P<0.05）；Siβ-catenin-HepG2/ADM转染组较HepG2/ADM组对ADM、5-FU、VCR和OHP的耐药系数（RI）分别为0.61、0.55、0.30、0.55，对化疗药物敏感性显著增强（P<0.05）；Siβ-catenin-HepG2/ADM转染组凋亡率（28.05±0.35）%，较其他组明显增加（P<0.05）。结论 Wnt/β-catenin通路在HepG2中异常激活，其中β-catenin可能正性调控肝癌耐药基因P-gp和MRP1，Si-β-catenin能一定程度阻断Wnt通路，并能一定程度逆转肝癌细胞株HepG2的耐药性和增强化疗敏感性，增加凋亡。     

人肝癌细胞系MHCC97H中侧群细胞初探

目的 从人肝癌细胞系MHCC97H中分选侧群细胞,探索其形态、表型和功能特征.方法 利用流式细胞仪从MHCC97H中分选得到侧群细胞,用相差显微镜和透射电镜观察其形态学特点;Western blotting观察其ABCG2表达;用流式细胞仪评估侧群细胞与干细胞基因CD133、CD117的关系;用克隆形成实验和NOD/SCID接种实验分别观察侧群细胞体外增殖和体内成瘤情况.结果 侧群细胞体积较小,呈圆形或短梭形;侧群细胞的细胞器如线粒体、内质网等明显少于非侧群细胞;流式检测显示侧群细胞中含有CD133、CD117阳性细胞;Western blotting显示侧群细胞和非侧群细胞中均有ABCG2表达,两者无明显差异;平板克隆形成实验显示SP具有较强的克隆形成能力;体内移植实验显示2000个SP细胞即能在体内形成肿瘤.结论 肝癌细胞系MHCC97H中侧群细胞具有肿瘤干细胞样细胞的表型和特性;ABCG2可能不是决定肝癌侧群细胞表型的主要因素.     

醛糖还原酶相似基因-1与人肝癌细胞耐药性关系的实验研究

目的探讨醛糖还原酶相似基因-1（ARL-1基因）与人肝癌细胞耐药性的关系。方法线性化的ARL-1表达载体DNA转染至人肝癌细胞系（HepG-2细胞）作为实验组。含有活性醛基的表阿霉素、丝裂霉素作为实验用药，不含活性醛基的氟尿嘧啶作为阳性对照。通过乳酸脱氢酶（LDH）的测定、MTT法和流式细胞仪研究ARL-1基因在肝癌细胞耐药性形成中的作用。结果ARL-1基因成功转染至人肝癌细胞HepG-2，加入表阿霉素和丝裂霉素后，实验组的细胞毒性水平和细胞凋亡率明显低于对照组（P〈0．05），耐药水平明显上升。加入氟尿嘧啶后，实验组及对照组的耐药水平无明显差异。结论高表达的ARL-1基因明显降低含有活性醛基化疗药物的细胞毒性和抑制其所诱导的细胞凋亡，增强人肝癌细胞的耐药性。     

肠癌SW-620肿瘤细胞SP亚群研究

目的 观察肠癌细胞株SW-620中肿瘤干细胞相关的SP(side population)亚群比例,分选出相关的SP和NSP细胞,并鉴别相关的SP是否具有干细胞某些生物学特性.方法 制备SW-620细胞悬液,经Hoechst33342和PI染色(其中对照组同时加入拮抗剂维拉帕米),流式细胞仪分析SP亚群.通过流式细胞仪分选出肠癌细胞株SW-620中SP细胞.克隆形成实验和噻唑蓝(MTT)实验检测SP和NSP细胞增殖能力的差异.裸鼠体内成瘤对比实验验证SP细胞和NSP细胞致瘤性.结果 SP亚群占SW-620细胞的1.0%左右,维拉帕米阻断后减少为0.01%左右.流式细胞仪分选出SP细胞7.5×105个,大部分为NSP细胞.克隆形成实验和MTT证实SP细胞和NSP细胞增殖能力有差异.裸鼠体内成瘤实验发现:1×105个SP细胞可以致裸鼠成瘤,1×105个NSP细胞及以下数量级别细胞不能致裸鼠成瘤.结论 人肠癌细胞株SW-620中存在SP亚群细胞,维拉帕米可抑制染料外排而明显减少SP细胞的比例.可以通过流式细胞仪分选出SP细胞,通过克隆形成实验和MTT实验检测SP和NSP细胞增殖能力有差异.裸鼠成瘤对比实验证实SP比NSP细胞更具致瘤性.     

中药复方肝癌-1号逆转肝癌多药耐药的实验研究

目的 分析中药复方肝癌-1号逆转阿霉素（ADM）诱导的HepG2／ADM细胞的多药耐药性的机制。方法 以亲本细胞HepG2为对照，MTT法观察肝癌-1号对HepG2／ADM细胞的毒性作用；流式细胞仪检测肝癌1号作用后细胞表面p-糖蛋白（P-gp）表达阳性率；逆转录聚合酶链式反应检查多药耐药基因（MDR1）mRNA表达水平；MTT法观察肝癌-1号处理后的HepG2／ADM细胞对阿霉素、表阿霉素、氟尿嘧啶耐药的逆转作用。结果 肝癌-1号〈50μmol／L对HepG2／ADM细胞无明显毒性，半数抑制率（IC50）为75μmol／L；50μmol／L的肝癌-1号可部分抑制HepG2／ADM细胞P-gp合成及MDR1 mRNA的表达，可逆转HepG2／ADM的耐药性，对阿霉素、表阿霉素、氟尿嘧啶的逆转倍数分别为3．94（P〈0．01），1．72（P〈0．05），1．67（P〈0．05）。结论 肝癌-1号通过抑制HepG2／ADM耐药细胞MDR1 mRNA的表达及P-gp合成，能部分逆转HepG2／ADM的耐药性。     

Induction of chondrogenesis and superficial zone protein accumulation in synovial side population cells by BMP‐7 and TGF‐β1

Synovial cells are known to contain a sub‐population of cells with multipotent differentiating capacity including chondrocytes. However, the stem/progenitor cells in synovial cells have not been well characterized. Stem/progenitor cells can exclude Hoechst 33342 dye, and the cell fraction with this property is called “side population (SP).” SP cells are present in many adult tissues. The aim of this investigation was to identify, isolate, and characterize SP cells from bovine synovium. Hoechst dye efflux and fluorescence activated cell sorting showed that synovial cells contained 0.60% SP cells. In the presence of verapamil, an inhibitor of ABC transporters critical for the dye efflux property, the SP cell fraction was not observed, indicating the critical role of ABC transporters. Isoforms of ABC transporters (ABCG2 and ABCB1 mRNA) were highly expressed in SP cells derived from the synovial cells by real‐time RT‐PCR analysis. Bone morphogenetic protein‐7 (BMP‐7) induced type II collagen mRNA expression characteristic of chondrogenesis in articular cartilage with both SP and non‐SP cells. In addition, expression of SZP mRNA, a marker of the surface layer of articular cartilage, was significantly up‐regulated by BMP‐7, and the protein accumulation of SZP was stimulated by both BMP‐7 and TGF‐β1. Thus, synovial cells contain ABC transporter‐dependent SP cells. These findings demonstrate that side population cells of synovium differentiate toward an articular chondrocyte phenotype of the surface layer and have direct implications for tissue engineering and regeneration of articular cartilage. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:485–492, 2008     

趋化因子受体在肝癌细胞中的表达及其意义

目的 观察趋化因子受体(CCR1 CCR7 CXCB3 CXCR4)在人肝癌细胞系(Hep3BHepG2 HLE和HLF)的表达,并探讨其作用机制.方法 采用逆转录-聚合酶链反应(RT-PCR)、细胞免疫组织化学、流式细胞仪测定趋化因子受体在肝癌细胞表面的表达,通过肝癌细胞Hep3B体外侵袭实验即重组大鼠巨噬细胞激动蛋白(CCL3)-1对Hep3B细胞穿透人工重组基底膜能力的影响来检测趋化因子受体在肝癌侵袭转移时所起的作用,并通过激光共聚焦显微镜观察趋化因子受体在肝癌细胞侵袭转移过程中的作用机制.结果 通过RT-PCR,流式细胞术可检测到CCR1 mR-NA及蛋白在肝癌细胞中表达,免疫组织化学显示CCR1在肝癌细胞包膜和胞质内都有表达.细胞侵袭实验提示Hep3B在CCL3的刺激下,实验组Hep-3B细胞通过基底膜的数量(213±12)多于对照组细胞通过基底膜的数量(102±11),差异有统计学意义(P<0.01).运用激光共聚焦检测到实验组胞内钙离子的浓度增加到.结论 肝癌细胞系(Hep3B HepG2 HLE和HLF)的表面的表达CCRl,且CCR1在肝癌细胞侵袭转移中发挥重要的作用,其作用机制与细胞内钙离子浓度有很关.     

Increased Expression of ABCB6 Enhances Protoporphyrin IX Accumulation and Photodynamic Effect in Human Glioma

Background

Glioma recurrence usually occurs close to the tumor resection margins as a result of residual infiltrating glioma cells. 5-aminolevulinic acid (ALA) fluorescence-guided resection of gliomas has been demonstrated to enhance discrimination of tumor tissue and to improve survival. ALA-based photodynamic therapy is an effective albeit still experimental adjuvant treatment option for gliomas. However, insufficient protoporphyrin IX (PpIX) accumulation may limit the benefits of fluorescence-guided resection and photodynamic therapy.

Methods

We investigated the expression of the ATP-binding cassette transporter ABCB6, which regulates porphyrin synthesis, in surgical specimens from human gliomas and manipulated ABCB6 in human glioma cell lines.

Results

Our findings demonstrated that expression levels of ABCB6 were greatly elevated in human gliomas compared with normal brain tissues and correlated with World Health Organization histologic grade. A previously undescribed finding was that ABCB6 mRNA expression in solidly fluorescing tumor tissues was higher than that in vaguely fluorescing tumors, suggesting that ABCB6 may be at least in part responsible for PpIX accumulation in glioma cells. Accordingly, ABCB6 overexpression in glioma cell lines caused a marked increase in intracellular levels of PpIX, and was more sensitive to ALA-induced photodynamic therapy—events that could be prevented by silencing ABCB6 via siRNA treatment.

Conclusions

Our findings indicate a crucial role of ABCB6 in ALA metabolism and accumulation of PpIX in glioma. ABCB6 overexpression is a potential approach to enhance accumulation of PpIX for optimizing the subjective discrimination of vague fluorescence and improving the efficacy of ALA-based photodynamic therapy.     

Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations

BACKGROUND: The prostate epithelial stem cell has been proposed as the primary origin of neoplastic change in prostate cancer. However, the isolation and characterization of unexpanded prostate epithelial stem cells have proven problematic. METHODS: A prostate epithelial side population (SP) has been isolated utilizing a modified Hoechst 33342 dye efflux assay from both benign and malignant prostate tissue. CD45(-ve), integrin alpha2(+ve) Hoechst 33342 SP and NSP cells were isolated by FACS, immunophenotyped and functionally characterized in 3D culture. RESULTS: FACS analysis revealed a verapamil sensitive SP accounting for 0.93 +/- 0.12% and 0.57 +/- 0.11% of the total epithelial population from both benign and malignant prostates. The benign SP phenotype revealed a heterogeneous cell population consisting predominantly of small basal cells containing minimal cytoplasm. Conversely, the malignant SP was of undetermined acinar origin and with a complete loss of expression of the CDK2 inhibitor p21(WAF1/Cip1). In vitro androgen-enhanced 3D culture of the benign and malignant SP cells led to the production of spheroids which had acinus like morphology and expressed primitive and basal cell markers. Incorporation of the CD133 marker isolated a further SP sub-fraction accounting for 0.037 +/- 0.01% of epithelial cells. CONCLUSIONS: Our observations are consistent with the Hoechst 33342 dye efflux assay isolating a stem cell enriched population which can be further sub-fractionated by CD133 selection. Moreover, the loss of the CDK inhibitor in malignancy is consistent with the hypothesis that neoplastic change originates in the stem cell compartment.     

siRNA下调THBS4对人骨肉瘤MG63增殖的影响

目的探讨小干扰RNA(small interfering RNA,siRNA)抑制血小板反应蛋白4(thrombospondin 4,THBS4)基因的表达对骨肉瘤MG-63细胞增殖的影响及其分子机制。方法将合成的THBS4 siRNA转染到人成骨细胞株MG63,CCK-8法观察对细胞增殖的影响,荧光定量PCR检测THBS4及TGF-beta1的mRNA表达,Western-blotting检测THBS4及TGF-beta1的蛋白表达。结果 Thbs4 siRNA转染后实验组48 h,72 h OD值高于阴性对照组(P0.05),差异具有显著性;Thbs4 mRNA和蛋白的表达水平明显低于阴性对照组和空白对照组(P0.01);Thbs4 siRNA转染后MG63细胞中TGF-beta1 mRNA及蛋白的表达水平提高(P0.01)。结论 THBS4 siRNA转染MG63后能够促进细胞增殖,其机制可能是激活TGF-beta1信号通路。     

RNA干扰对肝癌细胞巨噬细胞移动抑制因子基因表达的抑制作用

目的 观察特异性小干扰RNA(siRNA)对肝癌细胞巨噬细胞移动抑制因子(MIF)基因表达的抑制作用.方法 脂质体方法将siRNA转染肝癌细胞PLC、Hep3B.定量RT-PCR、Western blot检测MIF mRNA和蛋白、MAPK信号分子的表达.噻唑蓝(MTY)比色法、体外细胞侵袭实验检测细胞增殖和对重组基底膜(matrigel)穿透能力.结果 100 nmol/L的MIF siRNA使MIF mR-NA表达下调81.3%和89.1%,蛋白水平降低69.50%、72.31%,与对照组比较其差异有统计学意义(P均＜0.01).细胞增殖率下降16.79%和47.14%(P均＜0.05).穿透matrigel的细胞数为51.00±11.27和18.56±4.72,与对照组比较差异有统计学意义(P均＜0.05).磷酸化MAPK下调.结论 MIF siRNA有效抑制MIF表达及肝癌细胞增殖和迁移,可能部分通过抑制MAPK磷酸化起作用.     

Notch1 Contributes to Chemoresistance to Gemcitabine and Serves as an Unfavorable Prognostic Indicator in Pancreatic Cancer

Background

Pancreatic cancer (PC) carries frequent chemoresistance and extremely dismal prognosis. The underlying mechanisms remain to be further elucidated. We here report the role of Notch1 in gemcitabine resistance and its prognostic significance in PC.

Methods

A small interfering RNA (siRNA) specifically targeting Notch1 was transiently transfected into three PC cell lines (AsPC-1, BxPC-3, and MIA PaCa-2), followed by examination of chemosensitivity to gemcitabine. On the other hand, Notch1 expression was evaluated immunohistochemically and correlated with clinicopathological and prognostic variables.

Results

Successful knockdown of Notch1 by specific siRNA induced increased chemosensitivity to gemcitabine in all three cell lines. Immunohistochemical staining revealed that Notch1 was highly expressed in PC tissues (54.8 %), in contrast to that in para-tumor tissues (16.4 %). In addition, Notch1 positivity was significantly correlated with early-term metastasis and shortened overall survival. Multivariate Cox regression identified Notch1 as an independent prognostic factor.

Conclusions

Notch1 contributes to chemoresistance to gemcitabine, and serves as a significant indicator of unfavorable prognosis in PC.