Research On Early Diagnosis And Treatment Of Hepatobiliary Tumors By Biochip And Nano-traditional Chinese Medicine

1. Exploration to early diagnosis of cholangiocarcinoma by biochipPart I Analysis and establishment of methylation profiles of cholangiocarcinoma by MeDIP chipAims:To analyze gene methylation differences between human cholangiocarcinoma cell line and normal bile duct epithelial cell line by genome-wide promoter region CpG island methylation sites microarray technology and establish differentially methylated spectrum of cholangiocarcinoma so as to lay basis of early diagnose to cholangiocarcinoma.Methods:NimbleGen HG18CpG Promoter chip (Roche, Germany) were used to detect human cholangiocarcinoma cell lines TFK-1and normal human bile duct epithelial cells BEC genome-wide promoter CpG island methylation sites,. Annotation System (MAS) software was applied to analysis the function of the gene,which has differentially methylated loci, bisulfite sequencing (BSP) was used to detect HOX gene methylation level. PCR and western-blot method were applied to detect target gene expression at the cellular level. Immunohistochemical detection were used to examine target gene expression differences in cholangiocarcinoma and adjacent tissues. Methylation PCR (MSP) was used to detect the target gene methylation changes before and after methyltransferase inhibitor interventionResults:1.There were2103CpG islands difference hypermethylation performance between BEC cells and TFK-1cells.2.There were97genes belonging to the HOX family genes among all the difference methylation of CpG islands,which involved in multiple tumor mechanism,such as cell differentiation, the cycle of change, adhesion, invasion and metastasis and angiogenesis3. 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 duct cancer, colorectal cancer cell lines, the expression of HOXA5of cholangiocarcinoma cells was the lowest. Immunohistochemistry showed that: compared to adjacent normal bile duct, bile duct cancer HOXA5expression was significantly reduced. The MSP confirmed:After methyltransferase inhibitors intervention, HOXA5in TFK-1significantly reduced the degree of methylation.Conclusion:1.MeDIP chip is one of the effective method of screening early diagnostic marker of cholangiocarcinoma;2.DNA methylation may be important reason for HOXA5expression decreased in cholangiocarcinoma, and HOXA5hypermethylation can be used as one of early diagnostic marker for biliary tract tumors PartⅡ Construction of on-chip integrated lensless microscopy module for cell-based sensorsAims:Lab-on-a-chip systems are increasingly applied in cell-based assays for toxicology and drug testing. In this paper, by use of the advanced trchnology of IBMT institute (Germany), we worked together to construct an on-chip integrated lensless microscopy 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 module applying the principle of contact/proximate optical lithography. The biological samples or solutions are sustained in disposable sterilized microfluidic chips with1μm thick silicon nitride (Si3N4) membranes. These chips are assembled on the surface of a5megapixel colored CMOS image sensor array with1.75μm pixel size, which is coated with an additional interference filter. The function is demonstrated by the growth monitoring of L929and TFK-1cells cultured in cavity chips with Si3N4substrate for2days and by checking the colorimetric staining 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 of single 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 some important informations for further development of dignosis chip. 2Research on treatment to hepatobiliary tumors by TP-SLNPart I Effect of valproic acid or Decitabine on inhibition growth of human cholangiocarcinoma cells and its mechanismAims:To investigate the effect of valproic acid or Decitabine on inhibition growth of human cholangiocarcinoma cells in vitro and in vivo and its possible mechanism.Methods:cell growth inhibition rates were determined by CCK-8assay; Cell cycle and cell 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 in cholangiocarcinoma cells mice xenograft model.Results:The growth inhibition rate of cholangiocarcinoma cells in VPA or or DAC-treated group 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 apoptosis rate was significantly higher in treated group than control group. Cell autophagy was observed after treated with VPA or DAC in cholangiocarcinoma cells. The growth of implanted tumor of cholangiocarcinoma cells was significantly slowed down after mice were 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 in vivo, which is probably related with cell cycle arrest, cell apoptosis, and partially with autophagy. PartⅡ Inhibition of the New Triptolide-loaded Solid Lipid Nanoparticles to H22Hepatoma CellsAims:1. To investigate the acute toxicity and degree of the animal’s death of triptolide loaded polymeric micelles via the tail vein injection in SPF grade BALB/c mice, then calculate its LD50, which will provide dose design basis for the long-term toxicology experiment.2.To investigate the anti-tumor effects of triptolide-loaded solid lipid nanoparticles(TP-SLN) on the H22hepatoma cell line, and provide the basis for further 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 in accordance 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. The corresponding indicators of change the signs with single tail vein injection and symptoms in mice were observed, mortality, body weight, etc was counted.3. H22tumor-bearing mouse models were then established. The H22tumor-bearing mice were randomly divided into saline group, SLN(free TP)group, TP group, TP-SLN group, cisplatin group. Drugs were administered by tail vein every other day and tumor volume, body weight general activity status were observed after administration.12days later, all mice were sacrificed. The tumor tissue were weighed, and then detected the expression of VEGF and p53by immunohistochemistry.Results:1.in vitro,TP-SLN was more effective than TP, and the inhibitory effect increased in a time—dependent and dose—dependent manner.2.The triptolide loaded polymer micelles for SPF grade of BALB/c mice tail vein injection route, The LD50value is0.891mg/kg,95%of the confidence interval is0.814mg/kg-0.971mg/kg. Also in this agent pitch range, triptolide loaded polymeric micelles for BALB/c mice, the mortality showed good dose-response relationship; we also observed:The triptolide polymer micelles for SPF grade of BALB/c mice were not death4hrs after injection,, but with the extension of time, some mice’s symptoms were gradually worsened; trembling, ataxia, activities poor, inactive until death; the BALB/c mice’s death period is concentrated in the24-48hrs. most of96hrs after surviving animals to restore normal movement, breathing,etc, weight gain.3.1n vivo, compared with the saline group, TP, TP-SLN and cisplatin all caused a decline in tumor volume and weight loss. The tumors were inhibited by 22.4%,49.2%and51.5%, respectively. Also, the living conditions (body weight, response capability, etc.) of TP and TP-SLN group have some improvement. But in the cisplatin group, no significant improvement was observed. TP-SLN group had more P53expression rates than TP group, but fewer at the level of expression of VEGF.Conclusions:Compared with TP, TP-SLN has a more potent anti-tumor effect, which is stable, long-lasting. side effects are ruduced at the same time. Part Ⅲ Decitabine and valprete enhanced effect of TP-SLN on growth inhibition of cholangiocarcinoma cellsAims:To investigate whether Decitabine and valprete enhanced effect of TP-SLN on growth inhibition of cholangiocarcinoma cells or not.Method:After pretreated on TFK-1cells by DAC and VPA for3days,TP-SLN was used to treat for24h、48h and72h。Cell growth inhibition rates were determined by CCK-8assayResults:Compared to unpretreated group,the pretreated group can enhance effect of TP-SLN on growth inhibition of cholangiocarcinoma cellsConclusion:Decitabine and valprete enhanced effect of TP-SLN on growth inhibition of cholangiocarcinoma cells 3on-chip integrated lensless microscopy module applied to explore the mechanism of effect of nanodrugs on cholangiocarcinoma cells.Aims:To explore the mechanism of effect of nanodrugs on cholangiocarcinoma cells by on-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 lensless microscopy module when TP-SLN were used to treat cholangiocarcinoma cells.Conclusions:on-chip integrated lensless microscopy module can be used to explore the mechanism of effect of nanodrugs on cholangiocarcinoma cells.