生物芯片和纳米中药对肝胆肿瘤早期诊治的研究
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摘要
第一部分生物芯片早期诊断肝胆肿瘤的探讨
PartⅠMeDIP芯片分析和建立胆管癌差异甲基化谱
目的：利用全基因组启动子区CpG岛甲基化位点检测芯片技术，分析人胆管癌细胞株与人正常胆管上皮细胞株间的基因甲基化差异，建立胆管癌基因差异甲基化谱,为寻找特异性胆管癌早期诊断标志物奠定。
方法：采用NimbleGen HG18CpG Promoter芯片（Roche，Germany）分别检测人胆管癌细胞株TFK-1和人正常胆管上皮细胞株BEC全基因组启动子区CpG岛甲基化位点，并分析比较两细胞株间的差异甲基化位点。并应用MolecularAnnotation System （MAS）软件分析差异甲基化位点对应的基因的功能。采用亚硫酸氢盐测序法（BSP）检测HOX基因甲基化水平。采用荧光定量PCR和western-blot法检测目的基因在细胞水平的表达情况。免疫组化检测目的基因在胆管癌和癌旁组织中的表达差异。甲基化PCR（MSP）检测甲基转移酶抑制剂干预前后，目的基因甲基化的变化情况。
结果:1．MeDIP芯片结果显示：相比于BEC细胞株TFK-1细胞株中有2103个CpG岛表现为差异性高甲基化；
2．在所有差异性高甲基化CpG岛中有97个基因属于HOX家族基因，这些基因涉及细胞分化、周期改变、粘附、侵袭与转移以及血管生成等多个肿瘤发生机制；
3．SignalMap软件分析这97个HOX家族基因，发现甲基化率最高的前15位基因是HOXA5、HOXA2、HOXA11、HOXB4和HOXD13。BSP证实其各自的甲基化率为：HOXA5（95.38%）、HOXA2（94.29%）、HOXA11（91.67%）、HOXB4（90.56%）和HOXD13（94.38%）；
4．PCR和Western-blotting结果显示：在肝癌、胆管癌、胰腺癌和大肠癌等消化道肿瘤细胞株中，胆管癌细胞株TFK-1中HOXA5表达量最低。免疫组化显示：相比于癌旁和正常胆管组织而言，胆管癌中HOXA5表达量明显降低。MSP证实：在甲基转移酶抑制剂干预后，HOXA5在TFK-1中甲基化程度明显降低。
结论：1．MeDIP芯片是筛选胆管癌早期诊断标志物的有效方法之一；
2．DNA甲基化可能是HOXA5在胆管癌中表达降低的重要原因，且HOXA5高甲基化可作为胆道肿瘤的早期诊断标志物之一。
PartⅡ无透镜显微镜联合细胞诊断芯片集成系统的构建
目的：微流控芯片实验室（Lab-on-a-chip）技术越来越多地用于细胞毒性检测和药物测试。
该技术主要利用蚀刻技术到以各种材料制作的芯片上的通道和孔洞构成的“网络”构建微型实验室。实际应用时，压力以精细控制的方式推动钠升的体积流过通道，从而实现在单个集成的系统上进行样品处理、混合、稀释、电泳、定点追踪以及色谱分析、染色和检测。本实验的目的是利用德国IBMT研究所先进的芯片实验室，构建一种方便携带使用、成本低廉、快速分析、样本和试剂消耗少的细胞诊断芯片。
方法：本实验利用接触式光学平板印刷技术的原理构建了一种无透镜荧光显微镜/光学显微镜联合细胞芯片集成系统。生物样本被放置在一个包含有一个1.5μl漏斗状的腔及底部为1μm厚的硅氮化合物的MEMS芯片内。再将芯片装载在一个5兆彩色CMOS成像元件阵列上，最后在两者间覆盖一个光滤片。利用此系统培养鼠源性纤维细胞L929和人胆管癌细胞TFK-1。同时，分别用ErythrosineB和FITC进行细胞染色处理后，再比较此系统与传统荧光显微镜的成像效果。
结果：1．该系统监测L929细胞及TFK-1细胞影像的对比度和清晰度与4x物镜下的影像基本一致；
2．定点追踪观察单个或少量细胞形态变化，效果良好；
3．L929经红染或绿色荧光染色后，该系统显示细胞形态清晰。
结论：该组件可用于动态定点监测细胞形态变化和细胞的荧光图像，为后期诊断芯片的进一步开发提供重要的参考数据。
第二部分雷公藤内酯醇新型固体纳米粒治疗肝胆肿瘤的实验研究
PartⅠ地西他滨和丙戊酸钠单用对人胆管癌生长抑制作用研究
目的：探讨地西他滨（DAC）和丙戊酸钠（VPA）单用对人胆管癌细胞株TFK-1、QBC939、HUCCT、CCLP1细胞增殖的影响，并探讨其可能的机制。
方法：1．采用CCK8法检测DAC、VPA单独使用时对细胞生长的抑制率；
2．应用流式细胞仪检测DAC、VPA单独使用后细胞周期的变化；
3．经Hochst33342/PI染色，光镜下观察经DAC、VPA单独使用后细胞形态的变化，同时应用流式细胞术检测处理后细胞凋亡情况；
4．光镜下观察经DAC、VPA单独使用细胞120h后，细胞分化的情况；
5．构建GFP-LC3质粒并转染细胞，再加入DAC、VPA单独使用处理72小时，荧光显微镜观察细胞自噬情况；
6．体内实验采用裸鼠TFK-1细胞皮下种植瘤模型，观察DAC、VPA单独使用对瘤体生长及生存率的影响。
结果：1．DAC、VPA单独使用对人胆管癌细胞的增殖抑制作用呈时间和剂量依赖性；
2．流式细胞术检测显示，随着药物浓度的增加和作用时间的延长，胆管癌细胞呈现明显的G2/M或G0/G1期阻滞；
3．经DAC、VPA单独使用后，光镜下可见凋亡的细胞呈明显的胞体固缩、核固缩、核碎裂及凋亡小体形成。流式细胞术结果表明：细胞凋亡呈剂量依赖性；
4．光镜下观察到DAC、VPA单独使用120h后，细胞形态呈树突状突起；
5．荧光显微镜下观察到DAC、VPA单独使用后，细胞有明显的自噬现象；
6．体内试验证明：当应用DAC或VPA后，总给药时间为2周时，其对裸鼠TFK-1细胞皮下移植瘤的生长有明显的抑制作用。而且，治疗组的裸鼠生存期较对照组明显延长。
结论：体外实验和体内实验均证实， DAC或VPA对胆管癌细胞均具有明显的生长抑制作用。
PartⅡ雷公藤内酯醇新型固体纳米粒对肝癌生长抑制的研究
目的：1．观察雷公藤内酯醇新型固体纳米粒（TP-SLN）经尾静脉注射给药对SPF级BALB/C小鼠的急性毒性研究；
2．观察TP-SLN对肝癌细胞株H22体外和荷瘤体内的抗肿瘤作用研究
方法：1．雷公藤内酯醇（TP）和TP-SLN分别作用于H22细胞24h、48h和72h后，CCK-8法检测细胞的存活率；
2．SPF级BALB/c小鼠50只(雌雄各半),按照0.65mg/kg,0.773mg/kg,0.919mg/kg,1.189mg/kg,1.300mg/kg单次尾静脉注射给予TP-SLN，共5个剂量组。观察小鼠单次给药的症状体征，统计死亡率、体重等相应指标的变化;
3．建立H22细胞BALB/c小鼠皮下移植瘤模型，分为生理盐水空白对照组，空白固体脂质纳米粒组，雷公藤内酯醇（TP）组，TP-SLN组，顺铂阳性对照组，隔日尾静脉给药后观察记录肿瘤的体积、体重、一般生长活动状况。12天后处死所有小鼠，剥离肿瘤组织进行称重。
结果：1．体外实验中，TP-SLN对H22细胞生长抑制呈时间和剂量依赖性，作用明显且强于TP；
2．TP-SLN对于SPF级BALB/c小鼠尾静脉注射途径而言，其LD50值为0.891mg/kg，95%的可置信区间是0.814mg/kg—0.971mg/kg。并且在该剂距范围内，TP-PM对于BALB/c小鼠而言，死亡率呈现良好剂量效应关系；观察发现：TP-PM对于SPF级BALB/c小鼠而言,在注射后的4hrs内小鼠均未出现死亡，随着时间的延长，部分小鼠症状逐渐加重抖动，共济失调，进而活动减少，活动抑制直至死亡; BALB/c小鼠死亡时间集中在24-48hrs，96hrs后绝大部分存活动物恢复正常的运动、呼吸等，体重上升。
3．与生理盐水空白对照组比较，TP、TP-SLN及顺铂均引起肿瘤体积下降和体重减轻。抑瘤率分别为22.4%、49.2%、51.5%。TP、TP-SLN与生理盐水空白对照组相比，小鼠的生活状态（体重、反应能力等）均有一定的改善。顺铂组生活状态无明显改善。
结论：与TP相比，TP-SLN体内、外对肝癌细胞的抑制增强且稳定、持久，副作用减弱。
PartⅢ地西他滨联合丙戊酸钠增强TP-SLN对人胆管癌细胞生长抑制作用的初步研究
目的：探讨DAC联合VPA能否增强TP-SLN对人胆管癌细胞杀伤作用。
方法：DAC联合VPA预处理TFK-1细胞3天后，再加入TP-SLN处理24h、48h和72h，采用CCK-8法检测细胞存活率。
结果：与未预处理组相比，地西他滨联合丙戊酸钠预处理后，TP-SLN对人胆管癌细胞增值抑制作用明显增强
结论：地西他滨联合丙戊酸钠预处理增强TP-SLN对人胆管癌细胞增值抑制效应。
第三部分应用无透镜显微镜联合细胞诊断芯片集成系统初探纳米中药对胆管癌细胞作用机制
目的：应用无透镜显微镜联合细胞诊断芯片集成系统观察纳米中药与胆管癌细胞间相互作用
方法：应用无透镜显微镜联合细胞诊断芯片集成系统培养TFK-1细胞72小时后，培养基中加入TP-SLN，采集处理24h、48h和72h后的细胞图像
结果：无透镜显微镜联合细胞诊断芯片集成系统动态观察到纳米药物与细胞的相互作用后，细胞形态发生变化，但纳米材料荧光信号很弱。
结论：无透镜显微镜联合细胞诊断芯片集成系统可应用于观察纳米药物与细胞相互作用过程。
1. Exploration to early diagnosis of cholangiocarcinoma by biochip
PartⅠAnalysis and establishment of methylation profiles ofcholangiocarcinoma by MeDIP chip
Aims: To analyze gene methylation differences between human cholangiocarcinoma cellline and normal bile duct epithelial cell line by genome-wide promoter region CpGisland methylation sites microarray technology and establish differentiallymethylated spectrum of cholangiocarcinoma so as to lay basis of early diagnose tocholangiocarcinoma.
Methods: NimbleGen HG18CpG Promoter chip (Roche, Germany) were used to detecthuman cholangiocarcinoma cell lines TFK-1and normal human bile duct epithelialcells BEC genome-wide promoter CpG island methylation sites,. AnnotationSystem (MAS) software was applied to analysis the function of the gene,which hasdifferentially methylated loci. bisulfite sequencing (BSP) was used to detect HOXgene methylation level. PCR and western-blot method were applied to detect targetgene expression at the cellular level. Immunohistochemical detection were used toexamine target gene expression differences in cholangiocarcinoma and adjacenttissues. Methylation PCR (MSP) was used to detect the target gene methylationchanges before and after methyltransferase inhibitor intervention
Results:1.There were2103CpG islands difference hypermethylation performancebetween BEC cells and TFK-1cells.
2.There were97genes belonging to the HOX family genes among all thedifference methylation of CpG islands,which involved in multiple tumor mechanism,such as cell differentiation, the cycle of change, adhesion, invasion andmetastasis and angiogenesis
3. SignalMap software was used to Analyze of the97HOX family genes, the top15highest methylation rates of genes are HOXA5, HOXA2, HOXA11, HOXB4and HOXD13et al. BSP confirmed their respective methylation: HOXA5(95.38%), HOXA2(94.29%), of HOXA11(91.67%), upregulation of HOXB4(90.56%) and HOXD13(94.38%);
4. PCR and Western-blotting results showed that: among liver cancer, bile ductcancer, colorectal cancer cell lines, the expression of HOXA5ofcholangiocarcinoma cells was the lowest. Immunohistochemistry showed that:compared to adjacent normal bile duct, bile duct cancer HOXA5expression wassignificantly reduced. The MSP confirmed: After methyltransferase inhibitorsintervention, HOXA5in TFK-1significantly reduced the degree of methylation.
Conclusion:1.MeDIP chip is one of the effective method of screening early diagnosticmarker of cholangiocarcinoma;
2.DNA methylation may be important reason for HOXA5expression decreased incholangiocarcinoma, and HOXA5hypermethylation can be used as one of earlydiagnostic marker for biliary tract tumors
PartⅡConstruction of on-chip integrated lensless microscopy modulefor cell-based sensors
Aims: Lab-on-a-chip systems are increasingly applied in cell-based assays for toxicologyand drug testing. In this paper, by use of the advanced trchnology of IBMTinstitute (Germany), we worked together to construct an on-chip integrated lenslessmicroscopy module using a direct projection method for optical monitoring of the shadow images of adherent growing mammalian cells.
Methods:We present an on-chip integrated lensless fluorescence imaging moduleapplying the principle of contact/proximate optical lithography. The biologicalsamples or solutions are sustained in disposable sterilized microfluidic chipswith1μ m thick silicon nitride (Si3N4) membranes. These chips are assembledon the surface of a5megapixel colored CMOS image sensor array with1.75μmpixel size, which is coated with an additional interference filter. The functionis demonstrated by the growth monitoring of L929and TFK-1cells cultured incavity chips with Si3N4substrate for2days and by checking the colorimetricstaining of cells with a compromised membrane.
Results:1. The pixel resolution is comparable with a4×objective microscope.
2. It is possible for point-of-care applications to observe the morphology ofsingle cell.
3. The image quality of the module with a Si3N4-chip for L929cells is good.
Conclusion: the module can be used for point-of-care observation.It can be offered someimportant informations for further development of dignosis chip.
2Research on treatment to hepatobiliary tumors by TP-SLN
PartⅠEffect of valproic acid or Decitabine on inhibition growth ofhuman cholangiocarcinoma cells and its mechanism
Aims： To investigate the effect of valproic acid or Decitabine on inhibition growth ofhuman cholangiocarcinoma cells in vitro and in vivo and its possible mechanism.
Methods： cell growth inhibition rates were determined by CCK-8assay; Cell cycle andcell apoptosis were analyzed by flow cytometry after treated with various concentrations.Cell autophagy was observed under fluorescence microscope. The effect of valproic acid (VAP) or DAC on growth of cholangiocarcinoma in vivo was determined incholangiocarcinoma cells mice xenograft model.
Results：The growth inhibition rate of cholangiocarcinoma cells in VPA or or DAC-treatedgroup decreased in a time-and dose-dependent manner. After treated with VPA or DAC,cell cycle of cholangiocarcinoma cells was arrested at G2/M or G0/G1phase. The apoptosisrate was significantly higher in treated group than control group. Cell autophagy wasobserved after treated with VPA or DAC in cholangiocarcinoma cells. The growth ofimplanted tumor of cholangiocarcinoma cells was significantly slowed down after micewere treated with300mg/kg/6times a week VPA or0.8mg/kg/6times a week DAC for2weeks. Conclusion VPA or DAC can inhibit growth of cholangiocarcinoma in vitro and invivo, which is probably related with cell cycle arrest, cell apoptosis, and partially withautophagy.
PartⅡInhibition of the New Triptolide-loaded Solid Lipid Nanoparticlesto H22Hepatoma Cells
Aims：1. To investigate the acute toxicity and degree of the animal's death of triptolideloaded polymeric micelles via the tail vein injection in SPF grade BALB/cmice, then calculate its LD50, which will provide dose design basis for thelong-term toxicology experiment.
2.To investigate the anti-tumor effects of triptolide-loaded solid lipidnanoparticles(TP-SLN) on the H22hepatoma cell line, and provide the basis forfurther clinical application as anticancer drugs.
Method：1. H22cells were incubated with triptolide (TP) and TP-SLN for24h,48h and72h. Cell viability was then measured by the CCK-8assay．
2.50SPF grade BALB/c mice (half male half female) are given TP-PM inaccordance with the0.65mg/kg,0.773mg/kg,0.919mg/kg,1.189mg/kg,1.300mg/kg by single tail vein injection, a total of five dose groups. Thecorresponding indicators of change the signs with single tail vein injectionand symptoms in mice were observed, mortality, body weight, etc wascounted.
3. H22tumor-bearing mouse models were then established． The H22tumor-bearing mice were randomly divided into saline group，SLN(freeTP)group， TP group， TP-SLN group, cisplatin group. Drugs wereadministered by tail vein every other day and tumor volume, body weightgeneral activity status were observed after administration．12days later, allmice were sacrificed. The tumor tissue were weighed, and then detected theexpression of VEGF and p53by immunohistochemistry.
Results：1.in vitro,TP-SLN was more effective than TP, and the inhibitory effect increasedin a time—dependent and dose—dependent manner.
2.The triptolide loaded polymer micelles for SPF grade of BALB/c mice tailvein injection route, The LD50value is0.891mg/kg,95%of the confidenceinterval is0.814mg/kg-0.971mg/kg. Also in this agent pitch range, triptolideloaded polymeric micelles for BALB/c mice, the mortality showed gooddose-response relationship; we also observed: The triptolide polymer micellesfor SPF grade of BALB/c mice were not death4hrs after injection,, but withthe extension of time, some mice’s symptoms were gradually worsened;trembling, ataxia, activities poor, inactive until death; the BALB/c mice’sdeath period is concentrated in the24-48hrs. most of96hrs after survivinganimals to restore normal movement, breathing,etc, weight gain.
3.In vivo, compared with the saline group, TP, TP-SLN and cisplatin all causeda decline in tumor volume and weight loss. The tumors were inhibited by 22.4%,49.2%and51.5%, respectively. Also, the living conditions (bodyweight, response capability, etc.) of TP and TP-SLN group have someimprovement. But in the cisplatin group, no significant improvement wasobserved. TP-SLN group had more P53expression rates than TP group, butfewer at the level of expression of VEGF.
Conclusions：Compared with TP, TP-SLN has a more potent anti-tumor effect, which isstable, long-lasting. side effects are ruduced at the same time.
PartⅢDecitabine and valprete enhanced effect of TP-SLN on growthinhibition of cholangiocarcinoma cells
Aims: To investigate whether Decitabine and valprete enhanced effect of TP-SLN ongrowth inhibition of cholangiocarcinoma cells or not.
Method: After pretreated on TFK-1cells by DAC and VPA for3days,TP-SLN was usedto treat for24h、48h and72h。Cell growth inhibition rates were determined by CCK-8assayResults: Compared to unpretreated group,the pretreated group can enhance effect ofTP-SLN on growth inhibition of cholangiocarcinoma cells
Conclusion: Decitabine and valprete enhanced effect of TP-SLN on growth inhibition ofcholangiocarcinoma cells
3on-chip integrated lensless microscopy module applied to explorethe mechanism of effect of nanodrugs on cholangiocarcinoma cells.
Aims: To explore the mechanism of effect of nanodrugs on cholangiocarcinoma cells byon-chip integrated lensless microscopy module.
Methods: TFK-1cells were cultured by on-chip integrated lensless microscopy module for 72h. The cells were then treated by TP-SLN.
Results: the morphology changes can be observed by on-chip integrated lenslessmicroscopy module when TP-SLN were used to treat cholangiocarcinoma cells.
Conclusions: on-chip integrated lensless microscopy module can be used to explore themechanism of effect of nanodrugs on cholangiocarcinoma cells.
PartⅠMeDIP芯片分析和建立胆管癌差异甲基化谱
目的：利用全基因组启动子区CpG岛甲基化位点检测芯片技术，分析人胆管癌细胞株与人正常胆管上皮细胞株间的基因甲基化差异，建立胆管癌基因差异甲基化谱,为寻找特异性胆管癌早期诊断标志物奠定。
方法：采用NimbleGen HG18CpG Promoter芯片（Roche，Germany）分别检测人胆管癌细胞株TFK-1和人正常胆管上皮细胞株BEC全基因组启动子区CpG岛甲基化位点，并分析比较两细胞株间的差异甲基化位点。并应用MolecularAnnotation System （MAS）软件分析差异甲基化位点对应的基因的功能。采用亚硫酸氢盐测序法（BSP）检测HOX基因甲基化水平。采用荧光定量PCR和western-blot法检测目的基因在细胞水平的表达情况。免疫组化检测目的基因在胆管癌和癌旁组织中的表达差异。甲基化PCR（MSP）检测甲基转移酶抑制剂干预前后，目的基因甲基化的变化情况。
结果:1．MeDIP芯片结果显示：相比于BEC细胞株TFK-1细胞株中有2103个CpG岛表现为差异性高甲基化；
2．在所有差异性高甲基化CpG岛中有97个基因属于HOX家族基因，这些基因涉及细胞分化、周期改变、粘附、侵袭与转移以及血管生成等多个肿瘤发生机制；
3．SignalMap软件分析这97个HOX家族基因，发现甲基化率最高的前15位基因是HOXA5、HOXA2、HOXA11、HOXB4和HOXD13。BSP证实其各自的甲基化率为：HOXA5（95.38%）、HOXA2（94.29%）、HOXA11（91.67%）、HOXB4（90.56%）和HOXD13（94.38%）；
4．PCR和Western-blotting结果显示：在肝癌、胆管癌、胰腺癌和大肠癌等消化道肿瘤细胞株中，胆管癌细胞株TFK-1中HOXA5表达量最低。免疫组化显示：相比于癌旁和正常胆管组织而言，胆管癌中HOXA5表达量明显降低。MSP证实：在甲基转移酶抑制剂干预后，HOXA5在TFK-1中甲基化程度明显降低。
结论：1．MeDIP芯片是筛选胆管癌早期诊断标志物的有效方法之一；
2．DNA甲基化可能是HOXA5在胆管癌中表达降低的重要原因，且HOXA5高甲基化可作为胆道肿瘤的早期诊断标志物之一。
PartⅡ无透镜显微镜联合细胞诊断芯片集成系统的构建
目的：微流控芯片实验室（Lab-on-a-chip）技术越来越多地用于细胞毒性检测和药物测试。
该技术主要利用蚀刻技术到以各种材料制作的芯片上的通道和孔洞构成的“网络”构建微型实验室。实际应用时，压力以精细控制的方式推动钠升的体积流过通道，从而实现在单个集成的系统上进行样品处理、混合、稀释、电泳、定点追踪以及色谱分析、染色和检测。本实验的目的是利用德国IBMT研究所先进的芯片实验室，构建一种方便携带使用、成本低廉、快速分析、样本和试剂消耗少的细胞诊断芯片。
方法：本实验利用接触式光学平板印刷技术的原理构建了一种无透镜荧光显微镜/光学显微镜联合细胞芯片集成系统。生物样本被放置在一个包含有一个1.5μl漏斗状的腔及底部为1μm厚的硅氮化合物的MEMS芯片内。再将芯片装载在一个5兆彩色CMOS成像元件阵列上，最后在两者间覆盖一个光滤片。利用此系统培养鼠源性纤维细胞L929和人胆管癌细胞TFK-1。同时，分别用ErythrosineB和FITC进行细胞染色处理后，再比较此系统与传统荧光显微镜的成像效果。
结果：1．该系统监测L929细胞及TFK-1细胞影像的对比度和清晰度与4x物镜下的影像基本一致；
2．定点追踪观察单个或少量细胞形态变化，效果良好；
3．L929经红染或绿色荧光染色后，该系统显示细胞形态清晰。
结论：该组件可用于动态定点监测细胞形态变化和细胞的荧光图像，为后期诊断芯片的进一步开发提供重要的参考数据。
第二部分雷公藤内酯醇新型固体纳米粒治疗肝胆肿瘤的实验研究
PartⅠ地西他滨和丙戊酸钠单用对人胆管癌生长抑制作用研究
目的：探讨地西他滨（DAC）和丙戊酸钠（VPA）单用对人胆管癌细胞株TFK-1、QBC939、HUCCT、CCLP1细胞增殖的影响，并探讨其可能的机制。
方法：1．采用CCK8法检测DAC、VPA单独使用时对细胞生长的抑制率；
2．应用流式细胞仪检测DAC、VPA单独使用后细胞周期的变化；
3．经Hochst33342/PI染色，光镜下观察经DAC、VPA单独使用后细胞形态的变化，同时应用流式细胞术检测处理后细胞凋亡情况；
4．光镜下观察经DAC、VPA单独使用细胞120h后，细胞分化的情况；
5．构建GFP-LC3质粒并转染细胞，再加入DAC、VPA单独使用处理72小时，荧光显微镜观察细胞自噬情况；
6．体内实验采用裸鼠TFK-1细胞皮下种植瘤模型，观察DAC、VPA单独使用对瘤体生长及生存率的影响。
结果：1．DAC、VPA单独使用对人胆管癌细胞的增殖抑制作用呈时间和剂量依赖性；
2．流式细胞术检测显示，随着药物浓度的增加和作用时间的延长，胆管癌细胞呈现明显的G2/M或G0/G1期阻滞；
3．经DAC、VPA单独使用后，光镜下可见凋亡的细胞呈明显的胞体固缩、核固缩、核碎裂及凋亡小体形成。流式细胞术结果表明：细胞凋亡呈剂量依赖性；
4．光镜下观察到DAC、VPA单独使用120h后，细胞形态呈树突状突起；
5．荧光显微镜下观察到DAC、VPA单独使用后，细胞有明显的自噬现象；
6．体内试验证明：当应用DAC或VPA后，总给药时间为2周时，其对裸鼠TFK-1细胞皮下移植瘤的生长有明显的抑制作用。而且，治疗组的裸鼠生存期较对照组明显延长。
结论：体外实验和体内实验均证实， DAC或VPA对胆管癌细胞均具有明显的生长抑制作用。
PartⅡ雷公藤内酯醇新型固体纳米粒对肝癌生长抑制的研究
目的：1．观察雷公藤内酯醇新型固体纳米粒（TP-SLN）经尾静脉注射给药对SPF级BALB/C小鼠的急性毒性研究；
2．观察TP-SLN对肝癌细胞株H22体外和荷瘤体内的抗肿瘤作用研究
方法：1．雷公藤内酯醇（TP）和TP-SLN分别作用于H22细胞24h、48h和72h后，CCK-8法检测细胞的存活率；
2．SPF级BALB/c小鼠50只(雌雄各半),按照0.65mg/kg,0.773mg/kg,0.919mg/kg,1.189mg/kg,1.300mg/kg单次尾静脉注射给予TP-SLN，共5个剂量组。观察小鼠单次给药的症状体征，统计死亡率、体重等相应指标的变化;
3．建立H22细胞BALB/c小鼠皮下移植瘤模型，分为生理盐水空白对照组，空白固体脂质纳米粒组，雷公藤内酯醇（TP）组，TP-SLN组，顺铂阳性对照组，隔日尾静脉给药后观察记录肿瘤的体积、体重、一般生长活动状况。12天后处死所有小鼠，剥离肿瘤组织进行称重。
结果：1．体外实验中，TP-SLN对H22细胞生长抑制呈时间和剂量依赖性，作用明显且强于TP；
2．TP-SLN对于SPF级BALB/c小鼠尾静脉注射途径而言，其LD50值为0.891mg/kg，95%的可置信区间是0.814mg/kg—0.971mg/kg。并且在该剂距范围内，TP-PM对于BALB/c小鼠而言，死亡率呈现良好剂量效应关系；观察发现：TP-PM对于SPF级BALB/c小鼠而言,在注射后的4hrs内小鼠均未出现死亡，随着时间的延长，部分小鼠症状逐渐加重抖动，共济失调，进而活动减少，活动抑制直至死亡; BALB/c小鼠死亡时间集中在24-48hrs，96hrs后绝大部分存活动物恢复正常的运动、呼吸等，体重上升。
3．与生理盐水空白对照组比较，TP、TP-SLN及顺铂均引起肿瘤体积下降和体重减轻。抑瘤率分别为22.4%、49.2%、51.5%。TP、TP-SLN与生理盐水空白对照组相比，小鼠的生活状态（体重、反应能力等）均有一定的改善。顺铂组生活状态无明显改善。
结论：与TP相比，TP-SLN体内、外对肝癌细胞的抑制增强且稳定、持久，副作用减弱。
PartⅢ地西他滨联合丙戊酸钠增强TP-SLN对人胆管癌细胞生长抑制作用的初步研究
目的：探讨DAC联合VPA能否增强TP-SLN对人胆管癌细胞杀伤作用。
方法：DAC联合VPA预处理TFK-1细胞3天后，再加入TP-SLN处理24h、48h和72h，采用CCK-8法检测细胞存活率。
结果：与未预处理组相比，地西他滨联合丙戊酸钠预处理后，TP-SLN对人胆管癌细胞增值抑制作用明显增强
结论：地西他滨联合丙戊酸钠预处理增强TP-SLN对人胆管癌细胞增值抑制效应。
第三部分应用无透镜显微镜联合细胞诊断芯片集成系统初探纳米中药对胆管癌细胞作用机制
目的：应用无透镜显微镜联合细胞诊断芯片集成系统观察纳米中药与胆管癌细胞间相互作用
方法：应用无透镜显微镜联合细胞诊断芯片集成系统培养TFK-1细胞72小时后，培养基中加入TP-SLN，采集处理24h、48h和72h后的细胞图像
结果：无透镜显微镜联合细胞诊断芯片集成系统动态观察到纳米药物与细胞的相互作用后，细胞形态发生变化，但纳米材料荧光信号很弱。
结论：无透镜显微镜联合细胞诊断芯片集成系统可应用于观察纳米药物与细胞相互作用过程。
1. Exploration to early diagnosis of cholangiocarcinoma by biochip
PartⅠAnalysis and establishment of methylation profiles ofcholangiocarcinoma by MeDIP chip
Aims: To analyze gene methylation differences between human cholangiocarcinoma cellline and normal bile duct epithelial cell line by genome-wide promoter region CpGisland methylation sites microarray technology and establish differentiallymethylated spectrum of cholangiocarcinoma so as to lay basis of early diagnose tocholangiocarcinoma.
Methods: NimbleGen HG18CpG Promoter chip (Roche, Germany) were used to detecthuman cholangiocarcinoma cell lines TFK-1and normal human bile duct epithelialcells BEC genome-wide promoter CpG island methylation sites,. AnnotationSystem (MAS) software was applied to analysis the function of the gene,which hasdifferentially methylated loci. bisulfite sequencing (BSP) was used to detect HOXgene methylation level. PCR and western-blot method were applied to detect targetgene expression at the cellular level. Immunohistochemical detection were used toexamine target gene expression differences in cholangiocarcinoma and adjacenttissues. Methylation PCR (MSP) was used to detect the target gene methylationchanges before and after methyltransferase inhibitor intervention
Results:1.There were2103CpG islands difference hypermethylation performancebetween BEC cells and TFK-1cells.
2.There were97genes belonging to the HOX family genes among all thedifference methylation of CpG islands,which involved in multiple tumor mechanism,such as cell differentiation, the cycle of change, adhesion, invasion andmetastasis and angiogenesis
3. SignalMap software was used to Analyze of the97HOX family genes, the top15highest methylation rates of genes are HOXA5, HOXA2, HOXA11, HOXB4and HOXD13et al. BSP confirmed their respective methylation: HOXA5(95.38%), HOXA2(94.29%), of HOXA11(91.67%), upregulation of HOXB4(90.56%) and HOXD13(94.38%);
4. PCR and Western-blotting results showed that: among liver cancer, bile ductcancer, colorectal cancer cell lines, the expression of HOXA5ofcholangiocarcinoma cells was the lowest. Immunohistochemistry showed that:compared to adjacent normal bile duct, bile duct cancer HOXA5expression wassignificantly reduced. The MSP confirmed: After methyltransferase inhibitorsintervention, HOXA5in TFK-1significantly reduced the degree of methylation.
Conclusion:1.MeDIP chip is one of the effective method of screening early diagnosticmarker of cholangiocarcinoma;
2.DNA methylation may be important reason for HOXA5expression decreased incholangiocarcinoma, and HOXA5hypermethylation can be used as one of earlydiagnostic marker for biliary tract tumors
PartⅡConstruction of on-chip integrated lensless microscopy modulefor cell-based sensors
Aims: Lab-on-a-chip systems are increasingly applied in cell-based assays for toxicologyand drug testing. In this paper, by use of the advanced trchnology of IBMTinstitute (Germany), we worked together to construct an on-chip integrated lenslessmicroscopy module using a direct projection method for optical monitoring of the shadow images of adherent growing mammalian cells.
Methods:We present an on-chip integrated lensless fluorescence imaging moduleapplying the principle of contact/proximate optical lithography. The biologicalsamples or solutions are sustained in disposable sterilized microfluidic chipswith1μ m thick silicon nitride (Si3N4) membranes. These chips are assembledon the surface of a5megapixel colored CMOS image sensor array with1.75μmpixel size, which is coated with an additional interference filter. The functionis demonstrated by the growth monitoring of L929and TFK-1cells cultured incavity chips with Si3N4substrate for2days and by checking the colorimetricstaining of cells with a compromised membrane.
Results:1. The pixel resolution is comparable with a4×objective microscope.
2. It is possible for point-of-care applications to observe the morphology ofsingle cell.
3. The image quality of the module with a Si3N4-chip for L929cells is good.
Conclusion: the module can be used for point-of-care observation.It can be offered someimportant informations for further development of dignosis chip.
2Research on treatment to hepatobiliary tumors by TP-SLN
PartⅠEffect of valproic acid or Decitabine on inhibition growth ofhuman cholangiocarcinoma cells and its mechanism
Aims： To investigate the effect of valproic acid or Decitabine on inhibition growth ofhuman cholangiocarcinoma cells in vitro and in vivo and its possible mechanism.
Methods： cell growth inhibition rates were determined by CCK-8assay; Cell cycle andcell apoptosis were analyzed by flow cytometry after treated with various concentrations.Cell autophagy was observed under fluorescence microscope. The effect of valproic acid (VAP) or DAC on growth of cholangiocarcinoma in vivo was determined incholangiocarcinoma cells mice xenograft model.
Results：The growth inhibition rate of cholangiocarcinoma cells in VPA or or DAC-treatedgroup decreased in a time-and dose-dependent manner. After treated with VPA or DAC,cell cycle of cholangiocarcinoma cells was arrested at G2/M or G0/G1phase. The apoptosisrate was significantly higher in treated group than control group. Cell autophagy wasobserved after treated with VPA or DAC in cholangiocarcinoma cells. The growth ofimplanted tumor of cholangiocarcinoma cells was significantly slowed down after micewere treated with300mg/kg/6times a week VPA or0.8mg/kg/6times a week DAC for2weeks. Conclusion VPA or DAC can inhibit growth of cholangiocarcinoma in vitro and invivo, which is probably related with cell cycle arrest, cell apoptosis, and partially withautophagy.
PartⅡInhibition of the New Triptolide-loaded Solid Lipid Nanoparticlesto H22Hepatoma Cells
Aims：1. To investigate the acute toxicity and degree of the animal's death of triptolideloaded polymeric micelles via the tail vein injection in SPF grade BALB/cmice, then calculate its LD50, which will provide dose design basis for thelong-term toxicology experiment.
2.To investigate the anti-tumor effects of triptolide-loaded solid lipidnanoparticles(TP-SLN) on the H22hepatoma cell line, and provide the basis forfurther clinical application as anticancer drugs.
Method：1. H22cells were incubated with triptolide (TP) and TP-SLN for24h,48h and72h. Cell viability was then measured by the CCK-8assay．
2.50SPF grade BALB/c mice (half male half female) are given TP-PM inaccordance with the0.65mg/kg,0.773mg/kg,0.919mg/kg,1.189mg/kg,1.300mg/kg by single tail vein injection, a total of five dose groups. Thecorresponding indicators of change the signs with single tail vein injectionand symptoms in mice were observed, mortality, body weight, etc wascounted.
3. H22tumor-bearing mouse models were then established． The H22tumor-bearing mice were randomly divided into saline group，SLN(freeTP)group， TP group， TP-SLN group, cisplatin group. Drugs wereadministered by tail vein every other day and tumor volume, body weightgeneral activity status were observed after administration．12days later, allmice were sacrificed. The tumor tissue were weighed, and then detected theexpression of VEGF and p53by immunohistochemistry.
Results：1.in vitro,TP-SLN was more effective than TP, and the inhibitory effect increasedin a time—dependent and dose—dependent manner.
2.The triptolide loaded polymer micelles for SPF grade of BALB/c mice tailvein injection route, The LD50value is0.891mg/kg,95%of the confidenceinterval is0.814mg/kg-0.971mg/kg. Also in this agent pitch range, triptolideloaded polymeric micelles for BALB/c mice, the mortality showed gooddose-response relationship; we also observed: The triptolide polymer micellesfor SPF grade of BALB/c mice were not death4hrs after injection,, but withthe extension of time, some mice’s symptoms were gradually worsened;trembling, ataxia, activities poor, inactive until death; the BALB/c mice’sdeath period is concentrated in the24-48hrs. most of96hrs after survivinganimals to restore normal movement, breathing,etc, weight gain.
3.In vivo, compared with the saline group, TP, TP-SLN and cisplatin all causeda decline in tumor volume and weight loss. The tumors were inhibited by 22.4%,49.2%and51.5%, respectively. Also, the living conditions (bodyweight, response capability, etc.) of TP and TP-SLN group have someimprovement. But in the cisplatin group, no significant improvement wasobserved. TP-SLN group had more P53expression rates than TP group, butfewer at the level of expression of VEGF.
Conclusions：Compared with TP, TP-SLN has a more potent anti-tumor effect, which isstable, long-lasting. side effects are ruduced at the same time.
PartⅢDecitabine and valprete enhanced effect of TP-SLN on growthinhibition of cholangiocarcinoma cells
Aims: To investigate whether Decitabine and valprete enhanced effect of TP-SLN ongrowth inhibition of cholangiocarcinoma cells or not.
Method: After pretreated on TFK-1cells by DAC and VPA for3days,TP-SLN was usedto treat for24h、48h and72h。Cell growth inhibition rates were determined by CCK-8assayResults: Compared to unpretreated group,the pretreated group can enhance effect ofTP-SLN on growth inhibition of cholangiocarcinoma cells
Conclusion: Decitabine and valprete enhanced effect of TP-SLN on growth inhibition ofcholangiocarcinoma cells
3on-chip integrated lensless microscopy module applied to explorethe mechanism of effect of nanodrugs on cholangiocarcinoma cells.
Aims: To explore the mechanism of effect of nanodrugs on cholangiocarcinoma cells byon-chip integrated lensless microscopy module.
Methods: TFK-1cells were cultured by on-chip integrated lensless microscopy module for 72h. The cells were then treated by TP-SLN.
Results: the morphology changes can be observed by on-chip integrated lenslessmicroscopy module when TP-SLN were used to treat cholangiocarcinoma cells.
Conclusions: on-chip integrated lensless microscopy module can be used to explore themechanism of effect of nanodrugs on cholangiocarcinoma cells.
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[6] CARTERBZ,MAKDH, SCHOBERWD, et al. Triptolide induces caspase-dependentcell death mediated via the mitochondrial pathway in leukemic cells[J] Blood,2006,108(2):630-637.
[7] Liu J, Shen M, Yue Z, et al. Triptolide inhibits colon-rectal cancer cells proliferationby induction of G1phase arrest through upregulation of p21. Phytomedicine.2012Jun15;19(8-9):756-62.
[8] Chen L, Liu Q, Huang Z, et al. Tripchlorolide induces cell death in lung cancer cellsby autophagy. Int J Oncol.2012Apr;40(4):1066-70.
[9] Zhang H, Zhu W, Su X, et al. Triptolide inhibits proliferation and invasion ofmalignant glioma cells.[J] J Neurooncol.2012Aug;109(1):53-62.
[10] Clawson KA, Borja-Cacho D, Antonoff MB, et al. Triptolide and TRAIL combinationenhances apoptosis in cholangiocarcinoma.[J] J Surg Res.2010Oct;163(2):244-9.
[11] YANG S, CHEN J, GUO Z, et al. Triptolide inhibits the growth and metastasis ofsolid tumors.[J] Mol Cancer Ther,2003,2(1):65-72
[12]王建新，张志荣.固体脂质纳米粒的研究进展[J].中国药学杂志,2001,36(2):73-76
[13] Xue M, Zhao Y, Li XJ, et al. Comparison of toxicokinetic and tissue distribution oftriptolide-loaded solid lipid nanoparticles vs free triptolide in rats. Eur J Pharm Sci.2012Nov20;47(4):713-7.
[14] Muller RH, Mader K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drugdelivery-a review of the state of the art[J].Eur J Pharm Biopharm,2000,50(1):161-177.
[15] Huang M, Zhang H, Liu T, et al.Triptolide inhibits MDM2and induces apoptosis inacute lymphoblastic leukemia cells through a p53-independent pathway. Mol CancerTher.2013Feb;12(2):184-94.
[16] Xiaowen H, Yi S. Triptolide sensitizes TRAIL-induced apoptosis in prostate cancercells via p53-mediated DR5up-regulation. Mol Biol Rep.2012Sep;39(9):8763-70.
[17] Johnson SM, Wang X, Evers BM. Triptolide Inhibits Proliferation and Migration ofColon Cancer Cells by Inhibition of Cell Cycle Regulators and Cytokine Receptors.
[J]. J Surg Res.2011Jun15;168(2):197-205.
[1] Khan SA, Emadossadaty S, Ladep N, et al. Rising trends in Cholangiocarcino-ma: is the ICD classification system misleading US?[J]. Hepatol,2012，56:848-854.
[2] Ramacciato G, Nigri G, Bellagamba R, et al. Univariate and multivariate analysis ofprognostic factors in the surgical treatment of hilar cholangiocarcinoma [J]. Am Surg,2010，76:1260-1268.
[3] Nuzzo G, Giuliante F, Ardito F, et al. Intrahepatic cholangiocarcinoma:prognostic factors after liver resection [J]. Updates Surg,2010，62:11-19.
[4] Oki.Y, Aoki E,Issa JPJ. Decitabine-bedside to bench [J]. Crit Rev OncolHematol,2007,61:140-152.
[5] Griffiths EA, Gore SD. DNA methyltransferase and histone deacetylase inhibitors inthe treatment of myelodysplastic syndromes [J]. Semin Hematol,2008,45(1):23-30.
[6] Ding WJ, Fang JY, Chen XY, et al. The expression and clinical significance ofDNA methyltransferase proteins in human gastric cancer [J]. Dig Dis Sci,2008,53:2083–2089.
[7] Schmiedel BJ, Arelin V, Gruenebach F, et al. Azacytidine impairs NK cellreactivity while decitabine augments NK cell responsiveness toward stimulation [J].Int J Cancer,2011,128:2911–2922.
[8] Fahy J, Jeltsch A, Arimondo PB. DNA methyltransferase inhibitors in cancer: achemical and therapeutic patent overview and selected clinical studies [J]. ExpertOpin Ther Pat,2012,22(12):1427-1442.
[9] Dawson MA, Kouzarides T. Cancer epigenetics: from mechanism to therapy [J].Cell,2012,150(1):12-27.
[10] Saito Y, Kanai Y, Nakagawa T, et al. Increased protein expression of DNAmethyltransferase (DNMT)1is significantly correlated with the malignant potentialand poor prognosis of human hepatocellular carcinomas [J]. Int J Cancer,2003,105:527–532.
[11] Girault I, Tozlu S, Lidereau R, et al. Expression analysis of DNA methyltransferases1,3A, and3B in sporadic breast carcinomas [J]. Clin Cancer Res,2003,9:4415–4422.
[12] Liu QY, Chen DW, Xie LP, et al. Decitabine, independent of apoptosis, exerts itscytotoxic effects on cell growth in melanoma cells [J]. Environ Toxicol Pharmacol,2011,32(3):423-429.
[13] Ghanim V, Herrmann H, Heller G, et al.5-azacytidine and decitabine exertproapoptotic effects on neoplastic mast cells: role of FAS-demethylation and FASre-expression, and synergism with FAS-ligand [J]. Blood,2012,119(18):4242-52.
[14] VHodges-Gallagher L, Valentine CD, Bader SE, et al. Inhibition of histonedeacetylase enhances the anti-proliferative action of antiestrogens on breast cancercells and blocks tamoxifen-induced proliferation of uterine cells [J]. Breast CancerRes Treat,2006,105:297–309.
[15] Zheng S, Chang S, Lu J et al. Characterization of9-nitrocamptothecinliposomes:anticancer properties and mechanisms on hepatocellular carcinoma in vitroand in vivo [J]. PLoS One,2011,6(6): e21064
[16] Schnekenburger M, Grandjenette C, Ghelfi J, et al. Sustained exposure to the DNAdemethylating agent,2'-deoxy-5-azacytidine, leads to apoptotic cell death in chronicmyeloid leukemia by promoting differentiation, senescence, and autophagy [J].Biochem Pharmacol,2011,81(3):364-378.
[17] Yi TZ, Li J, Han X, et al. DNMT inhibitors and HDAC inhibitors regulate E-cadherinand Bcl-2expression in endometrial carcinoma in vitro and in vivo [J].Chemotherapy,2012,58(1):19-29.
1. Khan SA, Emadossadaty S, Ladep N, et al. Rising trends in cholangiocarcinoma: is theICD classification system misleading US? J Hepatol2012，56:848-54
2. Ramacciato G, Nigri G, Bellagamba R, et al. Univariate and multivariate analysis ofprognostic factors in the surgical treatment of hilar cholangiocarcinoma. Am Surg2010，76:1260-8
3. Nuzzo G, Giuliante F, Ardito F, et al. Intrahepatic cholangiocarcinoma: prognosticfactors after liver resection. Updates Surg2010，62:11-19
4. Belmaker RH. Bipolar disorder. N. Engl. J. Med.2004,351:476–486
5. Shabbeer S, Kortenhorst MS, Kachhap S, et al. Multiple molecular pathways explainthe anti-proliferative effect of valproic acid on prostate cancer cells in vitro and in vivo.Prostate2007,67:1099–110.
6. Kuendgen A, Gattermann N. Valproic acid for the treatment of myeloid malignancies.Cancer2007,110:943–54.7Jenuwein T,Allis CD. Translating the histone code. Science2001,293:10748Makoto Koyama, Yasuyuki lzutani, Ahmed E. Goda, et al. Histone deacetylaseinhibitors and15-deoxy-Delta12,14-prostaglandin J2synergistically induce apoptosis.Clin Cancer Res2010,16:2320-2332.9Blaheta RA, Michaelis M, Natsheh I, et al. Valproic acid inhibits adhesion ofvincristine-and cisplatin-resistant neuroblastoma tumour cells to endothelium. Br JCancer2007,96:1699–706.10Hodges-Gallagher L, Valentine CD, Bader SE, et al. Inhibition of histone deacetylaseenhances the anti-proliferative action of antiestrogens on breast cancer cells and blockstamoxifen-induced proliferation of uterine cells. Breast Cancer Res Treat2006,105:297–309.
11Landreville, S, et al. Histone deacetylase inhibitors induce growth arrest anddifferentiation in uveal melanoma. Clin Cancer Res,2012,18:408-416
12Neri, P, et al. In vivo anti-myeloma activity and modulation of gene expression profileinduced by valproic acid, a histone deacetylase inhibitor. Br J Haematol,2008,143(4):p.520-31.
7Jurkowska RZ, Jurkowski TP, Jeltsch A. Structure and function of mammalian DNA206–222(2011).
8Klose RJ, Bird AP. Genomic DNA methylation: the mark and its mediators. TrendsBiochem. Sci.31(2),89–97(2006).
9Boyes J, Bird A. DNA methylation inhibits transcription indirectly via a methyl-Cpgbinding-protein. Cell64(6),1123–1134(1991).
10Bannister AJ, Kouzarides T. Regulation of chromatin by histone modifications. Cell
11Kouzarides T. Chromatin modifications and their function. Cell128(4),693–705(2007).
6Wade PA. Methyl CpG binding proteins: coupling chromatin architecture to generegulation. Oncogene20(24),3166–3173(2001).
7Herman JG, Baylin SB. Gene silencing in cancer in association with promoterhypermethylation. N. Engl. J. Med.349(21),2042–2054(2003).
8Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumorspromoted by DNAhypomethylation. Science300(5618),455(2003).
9Tsai HC, Baylin SB. Cancer epigenetics: linking basic biology to clinical medicine.Cell Res.21(3),502–517(2011).
10Ropero S, Fraga MF, Ballestar E et al. A truncating mutation of HDAC2in humancancers confers resistance to histone deacetylase inhibition. Nat. Genet.38(5).
11Hamamoto R, Furukawa Y, Morita M et al. SMYD3encodes a histonemethyltransferase involved in the proliferation of cancer cells. Nat. Cell Biol.6(8),731–740(2004).
12Chuang JC, Yoo CB, Kwan JM et al. Comparison of biological effects of non-nucleoside DNA methylation inhibitors versus5-aza-2′-deoxycytidine. Mol.Cancer Ther.4(10),1515–1520(2005).
13Baud MGJ, Leiser T, Haus P et al. Defining the mechanism of action and enzymaticselectivity of psammaplin Aagainst its epigenetic targets. J. Med. Chem. doi:
141021/jm2016182(2012)(Epub ahead of print).
15Richon VM, Sandhoff TW, Rifkind R A, Marks PA. Histone deacetylase inhibitorselectively induces p21WAF1expression and gene-associated histone acetylation. Proc.NatlAcad. Sci. USA97(18),10014–10019(2000).
16Graham JS, Kaye SB, Brown R. The promises and pitfalls of epigenetic therapies insolid tumours. Eur. J. Cancer45(7),1129–1136(2009).
17Galanis E, Jaeckle K A, Maurer MJ et al. Phase II trial of vorinostat in recurrentglioblastoma multiforme: a north central cancer treatment group study. J. Clin. Oncol.27(12),2052–2058(2009).
18Thaler F, Minucci S. Next generation histone deacetylase inhibitors: the answer to thesearch for optimized epigenetic therapies? Expert Opin. Drug Disc.6(4),393–404(2011).
19Shi X, Kachirskaia I, Yamaguchi H et al. Modulation of p53function bySET8-mediated methylation at lysine382. Mol. Cell27(4),636–646(2007).
20Clevers H. The cancer stem cell: premises, promises and challenges. Nat. Med.313–319(2011).
2. Shaib, Y. and H.B. El-Serag, The epidemiology of cholangiocarcinoma. SeminLiver Dis,2004.24(2): p.115-25.
3. Khan, S.A., et al., Rising trends in cholangiocarcinoma: is the ICD classificationsystem misleading us? J Hepatol,2012.56(4): p.848-54.
4. Nagino, M., et al., Evolution of Surgical Treatment for PerihilarCholangiocarcinoma: A Single-Center34-Year Review of574ConsecutiveResections. Ann Surg,2012.
5. Kitiyakara, T. and R.W. Chapman, Chemoprevention and screening in primarysclerosing cholangitis. Postgrad Med J,2008.84(991): p.228-37.
6. Wiencke, K. and K.M. Boberg, Current consensus on the management of primarysclerosing cholangitis. Clin Res Hepatol Gastroenterol,2011.35(12): p.786-91.
7. Beuers, U., EASL Recognition Awardee2009: Prof. Raoul Poupon. J Hepatol,2009.51(4): p.617-9.
8. Chapman, R., et al., Diagnosis and management of primary sclerosing cholangitis.Hepatology,2010.51(2): p.660-78.
9. Sano, T., et al., One hundred two consecutive hepatobiliary resections for perihilarcholangiocarcinoma with zero mortality. Ann Surg,2006.244(2): p.240-7.
10. Shaib, Y.H., et al., Endoscopic and surgical therapy for intrahepaticcholangiocarcinoma in the united states: a population-based study. J ClinGastroenterol,2007.41(10): p.911-7.
11. Kozarek, R.A., Inflammation and carcinogenesis of the biliary tract: update onendoscopic treatment. Clin Gastroenterol Hepatol,2009.7(11Suppl): p. S89-94.
12. Blechacz, B. and G.J. Gores, Cholangiocarcinoma: advances in pathogenesis,diagnosis, and treatment. Hepatology,2008.48(1): p.308-21.
13. Ustundag, Y. and Y. Bayraktar, Cholangiocarcinoma: a compact review of theliterature. World J Gastroenterol,2008.14(42): p.6458-66.
14. Ramacciato, G., et al., Univariate and multivariate analysis of prognostic factors inthe surgical treatment of hilar cholangiocarcinoma. Am Surg,2010.76(11): p.1260-8.
[1] Fareid Asphahani and Miqin Zhang,“Cellular impedance biosensors for drugscreening and toxin detection,” Analyst, vol.132, no.9, pp.835–841,2007.
[2] Gregorz T. A. Kovacs,“Electronic sensors with living cellular components,” in Proc.of the IEEE, vol.91, No.6, pp.915–929,2003.
[3] Xiquan Cui, Lap Man Lee, Xin Heng, Weiwei Zhong, Paul W. Sternberg, DemetriPsaltis, Changhui Yang,“Lensless high-resolution on-chip optofluidic microscopesfor Caenorhabditis elegans and cell imaging,” in Proc. of the National Academy ofSciences, vol.105, no.31, pp.10670–10675,2008.
[4] Aydogan Ozcan, and Utkan Demirci,“Ultra wide-field lens-free monitoring of cellson-chip,” Lab Chip, Issue8, pp.98–106,2008.
[5] SangJun Moon, Hasan Onur Keles, Aydogan Ozcan, Ali Khademhosseini, EdwardHaggstrom, Daniel Kuritzkes, Utkan Demirci,“Integrating microfluidics andlensless imaging for point-of-care testing,” Biosensors and Bioelectronics, vol.24,pp.3208-3214,2009.
[6] Sungkyu Seo, Tingwei Su, Anthony Erlinger, and Aydogan Ozcan,“Multi-colorLUCAS: Lensfree On-chip Cytometry Using Tunable Monochromatic Illuminationand Digital Noise Reduction,” Cellular and Molecular Bioengineering, vol.1, nos.2–3, pp.146–156,2008.
[7] Chang K. Choi, Anthony E. English, Seung-Ik Jun, Kenneth D. Kihm and Philip D.Rack,“An endothelial cell compatible biosensor fabricated using optically thinindium tin oxide silicon nitride electrodes,” Biosensors&Bioelectronics, vol.22,Issue11, pp.2585–2590,2007。
[1] Krosch TC, Sangwan V, Banerjee S,et al. Triptolide-mediated cell death inneuroblastoma occurs by both apoptosis and autophagy pathways and results ininhibition of nuclear factor-kappa B activity.[J] Am J Surg.2013Apr;205(4):387-96.
[2] Brinker A M, Ma J, Lipsky P E, et a.l Medicinal chemistry and pharmacology ofgenus Tripterygium (Celastraceae)[J]. Phytochemisty,2007,68(6):1819
[3] Naito S. et al. Growth and metastasis of tumor cells isolated from a human renal cellcarcinoma implanted into different organa of nude mice. Cancer Reserch,1986,46:4109-15.
[4] Fromowitz FB, Viola MV, Chao S, et al. Ras p21expression in the progression ofbreast cancer [J]. Hum Pathol,1987,18(12):1268-75.
[5] Ebina T, Ogama N, Shimanuki H, et al. Life-prolonging effect of immunocellBAK(BRM-acticated killer) therapy for advanced solid cancer patients; prognosticsignificance of serum immunosuppressive acidic protein levels [J]. Cancer ImmunolImmunother,2003,52(9):555-560.
[6] CARTERBZ,MAKDH, SCHOBERWD, et al. Triptolide induces caspase-dependentcell death mediated via the mitochondrial pathway in leukemic cells[J] Blood,2006,108(2):630-637.
[7] Liu J, Shen M, Yue Z, et al. Triptolide inhibits colon-rectal cancer cells proliferationby induction of G1phase arrest through upregulation of p21. Phytomedicine.2012Jun15;19(8-9):756-62.
[8] Chen L, Liu Q, Huang Z, et al. Tripchlorolide induces cell death in lung cancer cellsby autophagy. Int J Oncol.2012Apr;40(4):1066-70.
[9] Zhang H, Zhu W, Su X, et al. Triptolide inhibits proliferation and invasion ofmalignant glioma cells.[J] J Neurooncol.2012Aug;109(1):53-62.
[10] Clawson KA, Borja-Cacho D, Antonoff MB, et al. Triptolide and TRAIL combinationenhances apoptosis in cholangiocarcinoma.[J] J Surg Res.2010Oct;163(2):244-9.
[11] YANG S, CHEN J, GUO Z, et al. Triptolide inhibits the growth and metastasis ofsolid tumors.[J] Mol Cancer Ther,2003,2(1):65-72
[12]王建新，张志荣.固体脂质纳米粒的研究进展[J].中国药学杂志,2001,36(2):73-76
[13] Xue M, Zhao Y, Li XJ, et al. Comparison of toxicokinetic and tissue distribution oftriptolide-loaded solid lipid nanoparticles vs free triptolide in rats. Eur J Pharm Sci.2012Nov20;47(4):713-7.
[14] Muller RH, Mader K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drugdelivery-a review of the state of the art[J].Eur J Pharm Biopharm,2000,50(1):161-177.
[15] Huang M, Zhang H, Liu T, et al.Triptolide inhibits MDM2and induces apoptosis inacute lymphoblastic leukemia cells through a p53-independent pathway. Mol CancerTher.2013Feb;12(2):184-94.
[16] Xiaowen H, Yi S. Triptolide sensitizes TRAIL-induced apoptosis in prostate cancercells via p53-mediated DR5up-regulation. Mol Biol Rep.2012Sep;39(9):8763-70.
[17] Johnson SM, Wang X, Evers BM. Triptolide Inhibits Proliferation and Migration ofColon Cancer Cells by Inhibition of Cell Cycle Regulators and Cytokine Receptors.
[J]. J Surg Res.2011Jun15;168(2):197-205.
[1] Khan SA, Emadossadaty S, Ladep N, et al. Rising trends in Cholangiocarcino-ma: is the ICD classification system misleading US?[J]. Hepatol,2012，56:848-854.
[2] Ramacciato G, Nigri G, Bellagamba R, et al. Univariate and multivariate analysis ofprognostic factors in the surgical treatment of hilar cholangiocarcinoma [J]. Am Surg,2010，76:1260-1268.
[3] Nuzzo G, Giuliante F, Ardito F, et al. Intrahepatic cholangiocarcinoma:prognostic factors after liver resection [J]. Updates Surg,2010，62:11-19.
[4] Oki.Y, Aoki E,Issa JPJ. Decitabine-bedside to bench [J]. Crit Rev OncolHematol,2007,61:140-152.
[5] Griffiths EA, Gore SD. DNA methyltransferase and histone deacetylase inhibitors inthe treatment of myelodysplastic syndromes [J]. Semin Hematol,2008,45(1):23-30.
[6] Ding WJ, Fang JY, Chen XY, et al. The expression and clinical significance ofDNA methyltransferase proteins in human gastric cancer [J]. Dig Dis Sci,2008,53:2083–2089.
[7] Schmiedel BJ, Arelin V, Gruenebach F, et al. Azacytidine impairs NK cellreactivity while decitabine augments NK cell responsiveness toward stimulation [J].Int J Cancer,2011,128:2911–2922.
[8] Fahy J, Jeltsch A, Arimondo PB. DNA methyltransferase inhibitors in cancer: achemical and therapeutic patent overview and selected clinical studies [J]. ExpertOpin Ther Pat,2012,22(12):1427-1442.
[9] Dawson MA, Kouzarides T. Cancer epigenetics: from mechanism to therapy [J].Cell,2012,150(1):12-27.
[10] Saito Y, Kanai Y, Nakagawa T, et al. Increased protein expression of DNAmethyltransferase (DNMT)1is significantly correlated with the malignant potentialand poor prognosis of human hepatocellular carcinomas [J]. Int J Cancer,2003,105:527–532.
[11] Girault I, Tozlu S, Lidereau R, et al. Expression analysis of DNA methyltransferases1,3A, and3B in sporadic breast carcinomas [J]. Clin Cancer Res,2003,9:4415–4422.
[12] Liu QY, Chen DW, Xie LP, et al. Decitabine, independent of apoptosis, exerts itscytotoxic effects on cell growth in melanoma cells [J]. Environ Toxicol Pharmacol,2011,32(3):423-429.
[13] Ghanim V, Herrmann H, Heller G, et al.5-azacytidine and decitabine exertproapoptotic effects on neoplastic mast cells: role of FAS-demethylation and FASre-expression, and synergism with FAS-ligand [J]. Blood,2012,119(18):4242-52.
[14] VHodges-Gallagher L, Valentine CD, Bader SE, et al. Inhibition of histonedeacetylase enhances the anti-proliferative action of antiestrogens on breast cancercells and blocks tamoxifen-induced proliferation of uterine cells [J]. Breast CancerRes Treat,2006,105:297–309.
[15] Zheng S, Chang S, Lu J et al. Characterization of9-nitrocamptothecinliposomes:anticancer properties and mechanisms on hepatocellular carcinoma in vitroand in vivo [J]. PLoS One,2011,6(6): e21064
[16] Schnekenburger M, Grandjenette C, Ghelfi J, et al. Sustained exposure to the DNAdemethylating agent,2'-deoxy-5-azacytidine, leads to apoptotic cell death in chronicmyeloid leukemia by promoting differentiation, senescence, and autophagy [J].Biochem Pharmacol,2011,81(3):364-378.
[17] Yi TZ, Li J, Han X, et al. DNMT inhibitors and HDAC inhibitors regulate E-cadherinand Bcl-2expression in endometrial carcinoma in vitro and in vivo [J].Chemotherapy,2012,58(1):19-29.
1. Khan SA, Emadossadaty S, Ladep N, et al. Rising trends in cholangiocarcinoma: is theICD classification system misleading US? J Hepatol2012，56:848-54
2. Ramacciato G, Nigri G, Bellagamba R, et al. Univariate and multivariate analysis ofprognostic factors in the surgical treatment of hilar cholangiocarcinoma. Am Surg2010，76:1260-8
3. Nuzzo G, Giuliante F, Ardito F, et al. Intrahepatic cholangiocarcinoma: prognosticfactors after liver resection. Updates Surg2010，62:11-19
4. Belmaker RH. Bipolar disorder. N. Engl. J. Med.2004,351:476–486
5. Shabbeer S, Kortenhorst MS, Kachhap S, et al. Multiple molecular pathways explainthe anti-proliferative effect of valproic acid on prostate cancer cells in vitro and in vivo.Prostate2007,67:1099–110.
6. Kuendgen A, Gattermann N. Valproic acid for the treatment of myeloid malignancies.Cancer2007,110:943–54.7Jenuwein T,Allis CD. Translating the histone code. Science2001,293:10748Makoto Koyama, Yasuyuki lzutani, Ahmed E. Goda, et al. Histone deacetylaseinhibitors and15-deoxy-Delta12,14-prostaglandin J2synergistically induce apoptosis.Clin Cancer Res2010,16:2320-2332.9Blaheta RA, Michaelis M, Natsheh I, et al. Valproic acid inhibits adhesion ofvincristine-and cisplatin-resistant neuroblastoma tumour cells to endothelium. Br JCancer2007,96:1699–706.10Hodges-Gallagher L, Valentine CD, Bader SE, et al. Inhibition of histone deacetylaseenhances the anti-proliferative action of antiestrogens on breast cancer cells and blockstamoxifen-induced proliferation of uterine cells. Breast Cancer Res Treat2006,105:297–309.
11Landreville, S, et al. Histone deacetylase inhibitors induce growth arrest anddifferentiation in uveal melanoma. Clin Cancer Res,2012,18:408-416
12Neri, P, et al. In vivo anti-myeloma activity and modulation of gene expression profileinduced by valproic acid, a histone deacetylase inhibitor. Br J Haematol,2008,143(4):p.520-31.
7Jurkowska RZ, Jurkowski TP, Jeltsch A. Structure and function of mammalian DNA206–222(2011).
8Klose RJ, Bird AP. Genomic DNA methylation: the mark and its mediators. TrendsBiochem. Sci.31(2),89–97(2006).
9Boyes J, Bird A. DNA methylation inhibits transcription indirectly via a methyl-Cpgbinding-protein. Cell64(6),1123–1134(1991).
10Bannister AJ, Kouzarides T. Regulation of chromatin by histone modifications. Cell
11Kouzarides T. Chromatin modifications and their function. Cell128(4),693–705(2007).
6Wade PA. Methyl CpG binding proteins: coupling chromatin architecture to generegulation. Oncogene20(24),3166–3173(2001).
7Herman JG, Baylin SB. Gene silencing in cancer in association with promoterhypermethylation. N. Engl. J. Med.349(21),2042–2054(2003).
8Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumorspromoted by DNAhypomethylation. Science300(5618),455(2003).
9Tsai HC, Baylin SB. Cancer epigenetics: linking basic biology to clinical medicine.Cell Res.21(3),502–517(2011).
10Ropero S, Fraga MF, Ballestar E et al. A truncating mutation of HDAC2in humancancers confers resistance to histone deacetylase inhibition. Nat. Genet.38(5).
11Hamamoto R, Furukawa Y, Morita M et al. SMYD3encodes a histonemethyltransferase involved in the proliferation of cancer cells. Nat. Cell Biol.6(8),731–740(2004).
12Chuang JC, Yoo CB, Kwan JM et al. Comparison of biological effects of non-nucleoside DNA methylation inhibitors versus5-aza-2′-deoxycytidine. Mol.Cancer Ther.4(10),1515–1520(2005).
13Baud MGJ, Leiser T, Haus P et al. Defining the mechanism of action and enzymaticselectivity of psammaplin Aagainst its epigenetic targets. J. Med. Chem. doi:
141021/jm2016182(2012)(Epub ahead of print).
15Richon VM, Sandhoff TW, Rifkind R A, Marks PA. Histone deacetylase inhibitorselectively induces p21WAF1expression and gene-associated histone acetylation. Proc.NatlAcad. Sci. USA97(18),10014–10019(2000).
16Graham JS, Kaye SB, Brown R. The promises and pitfalls of epigenetic therapies insolid tumours. Eur. J. Cancer45(7),1129–1136(2009).
17Galanis E, Jaeckle K A, Maurer MJ et al. Phase II trial of vorinostat in recurrentglioblastoma multiforme: a north central cancer treatment group study. J. Clin. Oncol.27(12),2052–2058(2009).
18Thaler F, Minucci S. Next generation histone deacetylase inhibitors: the answer to thesearch for optimized epigenetic therapies? Expert Opin. Drug Disc.6(4),393–404(2011).
19Shi X, Kachirskaia I, Yamaguchi H et al. Modulation of p53function bySET8-mediated methylation at lysine382. Mol. Cell27(4),636–646(2007).
20Clevers H. The cancer stem cell: premises, promises and challenges. Nat. Med.313–319(2011).