| [Background]Colorectal cancer (CRC) is one of the higher incidence of gastrointestinal cancer, the world’smost common malignancies in fourth place, the mortality rate has leapt to second place in themalignant tumors in our malignant third. Relatively low due to the rate of early diagnosis ofcolorectal cancer and found that more than in the middle and late postoperative recurrence andmetastasis, and therefore early diagnosis of colorectal cancer has become the focus of research inrecent years.In the morphological diagnosis of the tumor is still relying on the pathologists personalexperience and the ability to identify tumor cells, the lack of an objective to determine theindicators and data. Therefore, to explore a new, able to observe the morphology, more objective,more accurate and earlier idenitifcation of precancerous lesions and early cancer changes ofmethods and techniques, with signiifcant academic value and clinical significance.The atomic force microscope (AFM) is a nanometer or sub-nanometer ultra-high resolutionanalytical instruments for precise imaging of cell ultrastructure and mechanical properties. It wasin1986by Binning, etc. from the basis of the scanning tunneling microscope developed rfom,and then won the Nobel Pirze in Physics. To its other microscopy techniques do not have theadvantage of imaging:1. Specimens can be observed very wide range of specimens can be metal,semi-metals and insulators, can be observed using atomic force microscopy;2has a very highresolution can be clearly observed in the morphology of the atomic and molecular structure andmechanical characteirstics;3. imaging environment requirements, both in the air imaging,imaging in the other environment; short imaging time can be timely and accuratethree-dimensional topography of the specimen; five probes such as modified can be used todistinguish specific molecules and their mutual interaction, so in recent years been widely usedin vairous ifelds including clinical medicine, including, in particular the observation of tumormechanical properties and ultrastructure of the cells, showing good applicaiton prospects.However, observational study of the morphology characteirstics of the colorectal cancer cell lineis rarely reported.[Objecitve]In this study, light microscopy and atomic force microscopy contrast observation ofcolon cancer cell line HT-29(HT-29) and5-FU-resistant colon cancer cell lines HCT-8/5-FU(HCT-8/5-FU)topography change; contrast observed atfer5-FU treatment, characteirstics ofdifferent stages of HT-29and the dynamic morphology of the HCT-8/5-FUtwo cell lines; explore atomic force microscopy topography detection in colorectal cancer cellsthe applicaitonof the value of the correlation between the changes in tumor morphology of comparaitve analysisof subjective judgment in an ordinary optical microscope and atomic force microscopy analysisof objective value. Explore new avenues for precancerous lesions and early stage of colorectalcancer diagnosed pathologically; topography change to determine the resistance or not such as toprovide a new idea based on colorectal cancer cells to chemotherapy drugs.[Methods]The expeirment was divided into three parts:LA cell impirnt method and cell crawling plate method of normal colorectal epithelial cellsand colon cancer cell line HT-29producer, and then use the inverted phase contrast microscopeand atomic force microscopy comparaitve observation of normal colon epithelial cells and coloncancer cell line HT-29cell morphology characteirstics.2.By cell culture and drug-resistant strains of the establishment (HCT-8/5-FU) cellcrawl-chip legal system piece, using inverted phase contrast microscope and atomic forcemicroscopy of HT-29and HCT-8/5-FUtwo cell strain morphology were compared.3.5ug/ml5-FU dealt with separately and HCT-8/5-FU2h and6h atfer the colon cancer cellline HT-29by inverted phase contrast microscope and atomic force microscopy on the shape ofthe different itmes of the two cell groups in the treatment of chemotherapy drugs The physicalcharacteristics were compared.[Results]LA light microscope observation of normal colon epithelial cells and colon cancer cell lineHT-29, only to see the different shapes and sizes, there is no objective values. Atomic forcemicroscopy analysis: colorectal cancer cell line HT-29cells in diameter,25.684士6.265, theaverage roughness of94.800士11.375, the average peak height of11.764士3.278, the averagelevel of depression-24.863士8.386and the surface area of difference2.511士1.254; normalcolorectal epithelial cells: the diameter of11.482士2.314, the average roughness of13士.1056.307, the average peak height of7士士.8272.673, the average level of depression-18.8377.265difference between the surface area of1.894士0.675; two groups have a statistically significantdifference (P <0.05) quantiifable assessment.2. light microscope to observe the colorectal cancer cell line HT-29and resistant strainsHCT-8/5-FU of, only to see the different shapes and sizes, there is no objective values. Atomicforce microscopy analysis: drug-resistant strains HCT-8/5-FU cell diameter of30.175士5.247,with an average roughness of98.750士10.460, the average peak height of16.856士2.673, theaverage level of depression-30.524士7.265and the surface area difference of5.278士1.175；colon cancer cell line HT-29cells diameter25.684士6.265, with an average roughness of94.800 士11.375, the average peak height of11.764士3.278, the average level of depression-24.863士8.386and the surface area difference2士.5111.254; the two groups statistically significantdifference (P <0.05), quantitative assessment.3.The cell number of colorectal cancer cell line HT-29and resistant strains HCT-8/5-FU ofordinary light microscope for2,6hours by5-FU reduced, mild changes in cell size, there is noobjective values. Atomic force microscopy analysis: colorectal cancer cell line HT-29at Oh,2h,6h dynamic observation process士, the cell diameter of25.6846.265,31.356士5.018,49士.0734.147, the average roughness of94.800士11.375,98.175士10.269士,115.2349.656, the averagepeak height of11士.764士3.278,15.6782.768,26.359士2.051, the average level of depression-24-.863士8.386,-29.476±6.712,35.317士6.018difference between the surface area of2.511士1.254,2.959士0.976,6.768士0.873ifve objective values showed signiifcant change (P <0.05);resistant strains HCT-8/5-FU cells by5-FU treatment afterOh,2h,6h, the cell diameterof30.175±5.247,31±.9794.317,35.073±4.205, with an average roughness of98.750士10.460,99.265士9.475,101.670士9.015, the average peak height16.856士2.673,17.569士2-.113,19.478士1.964, the average level of depression-30.524士7士.265，-31.2636.055,32.456士5.798, the surface area difference5.278士1.175,5.924士1.013,6.567士0.927ifve objecitvevalue no significant difference (P>0.05). In addiiton, both cell lines in5-FU treatment Oh and6hof the cell diameter, the average roughness, average peak height of the average depression leveland the surface area difference between the ifve objective value and there was significantdifference (P <0-.05);5FU, dealing with the2h ifve objecitve numerical two groups nosigniifcant difference (P>0.05).[Conclusion]1.Atomic force microscope scanning to observe the normal colorectal epithelial cells andcolon cancer cell line HT-29cell morphology characteirstics can be drawn rfom the objecitvevalue of (ordinary optical microscope observation of less than the ifne morphology data) willhelp quantified to determine the benign and malignant cells. Prompted the atomic forcemicroscope topography observed can be objectively determined for colorectal cancer.2.The atomic force microscope topography features quantitative analysis ofnon-drug-resistant strains HT-29and drug-resistant strains HCT-8/5-FU fine topography featureshelp to distinguish drug-resistant or not from topography change.3AFM scans observed5ug/ml5-FU processing found and HCT-8/5-FU cell line HT-29,before processing by5-FU and2,6hours atfer processing, morphology characteirstics ofdynamic value changes, help change the topography to determine whether tumor cells resistantor sensitive to chemotherapy drugs. |
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