The Atomic Force Microscope Cell Dna, In The Field Of Drug Action

Objective:Use atomic force microscope (AFM) to investigate the influence on morphology of tumor cell DNA taken by static magnetic field (SMF) or SMF with anticancer drugs, to study the possible biological mechanisms of synergistic anticancer effects.Methods:1. After the different concentrations of poly-lysine were dropped directly or coated on freshly cleaved mica, the surface topography of mica was observed by AFM.2. After the mica were coated by different concentrations of poly-lysine, observe the deposition and exhibition of cellular DNA on it by AFM; also observe the adsorption and deposition effects of DNA on the surface of mica after the mica were modified by poly-lysine in different ways.3. Observe DNA molecules by AFM that extracted by different DNA extraction kit, to select the better one.4. Observe DNA molecules by AFM after K562 cells were exposed to SMF for different durations.5. Visualize the micro-morphology of DNA molecules after K562 cells were exposed to SMF or SMF with anticancer drugs, to study the relations between synergistic anticancer effects and DNA damage.Results:1. Particles and aggregates would appear on the surface of mica while poly-lysine were directly dropped on it; If the mica were coated with poly-lysine, the number and size of particles or aggregates would be reduced obviously. When the mica was treated with poly-lysine of same concentration, the roughness average (Ra) was smaller in coating than dropping. When the concentration became lower, the roughness of mica became smaller until the poly-lysine concentration was reduced to 0.1μg/mL.2. When the mica was treated with 1μg/mL poly-lysine, a lot of DNA accumulated on the surface of the mica. The amount of DNA was so large that many of them became winded and gathered. In addition, many residual granular impurities could be viewed on the mica. The quantity of DNA was decreased while reducing the concentration of poly-lysine. When the mica was coated with 0.5μg/mL poly-lysine, the linear DNA appeared gradually and impurities decreased obviously. When the poly-lysine concentration was reduced to 0.1μg/mL, DNA stretched well and deposited on the surface of mica in the form of linear monomer, the propotion of Linear DNA was very large and no impurities could be viewed. When the mica was coated with 0.01μg/mL poly-lysine, the mica also remained absorbability for DNA, but DNA became winded and DNA cross-linking could be viewed. When dropped on bare mica directly, DNA was crossed and gathered. When poly-lysine was dropped on mica directly, extensibility of DNA was not good, some of them crossed and gathered to netted structure. When the mica was coated with poly-lysine, DNA dispersed well, a mono-linear structure of DNA could be viewed.3. Compared with DNA in the kit from Aidlab Biotechnologies, the length of DNA would be longer, the surface of DNA would be more smooth, and impurities was less in kit came from TIANGEN BIOTECH.4. When K562 cells were exposed to SMF for 12h, the nodes of DNA-DNA increased, a small quantity of DNA cross linking appeared; when exposed to SMF for 24h, DNA fragments increased obviously, the length was shorter, DNA broke partly; when the cells were exposed to SMF for 36h, DNA molecular broke entirely and DNA fragments crossed each other to form flake polymers in different sizes.5. (1) When K562 cells were exposed to SMF and/or 25ng/ml ADM for 12h, the morphology of DNA changed. In the SMF group, the nodes of DNA-DNA increased, some DNA crossed and some of them existed in clintheriform; In the ADM group, DNA winded partly, a few of them crossed together; In SMF+ADM group, the morphology of DNA changed significantly, a mass of DNA crossed and winded to become slabby aggregates.(2) When K562 cells were exposed to SMF for 24h, DNA fragments increased obviously, The length of DNA became shorter, part of DNA broke down; when K562 cells were incubated with Taxol alone, most DNA winded, a few of them crossed slightly and surface was rough; In SMF+ Taxol group, DNA damage were aggravated, most of DNA crossed to slabby polymers, a few of them winded.(3) When K562 cells were exposed to SMF for 12h, branches appeared in most DNA molecular, the length of DNA became shorter, the nodes of DNA-DNA increased, the DNA cross linking appeared and became thicker; When cells were incubated with 0.4mg/ml cyclophosphamidum(CP) for 12h, most of DNA crossed to net structure on substrate, part of DNA wind each other; In SMF+CP group, the length of DNA was shortened significantly, nodes of DNA-DNA increased, part of DNA crossed on substrate, some DNA molecules represent in "poly direct links" shape.Conclusions:1 Compared with dropping poly-lysine directly, Poly-lysine distributed more uniformly on the mica and the roughness was smaller when the mica was coated; the roughness of mica became smaller until the poly-lysine was reduced to 0.1μg/mL and the flatness was very well.2 DNA molecule extended well while mice surface was coated by poly-lysine (0.1μg/mL); compared with bare mica and poly-lysine dropping mica, The surface firmly secures DNA and DNA stretched well when mice coated by poly-lysine.3 DNA molecules extracted by TIANamp Genomic DNA Kit are more suitable for the research of AFM.4 When K562 cells were exposed to SMF, cellular DNA presented breakage, crosslink, aggregation and DNA damaged seriously as exposed times prolong.5. DNA presented breakage and cross-link after cell exposed to SMF; DNA appeared to bend when anticarcinogen was used and the height of DNA increased; Compared to SMF or anticarcinogen group, DNA damaged more critically when SMF and anticarcinogen combined.