Effect of intraoperative blood salvage on erythrocyte morphosis observed under atomic force microscope

AIM: To investigate the effect of intraoperative blood salvage (IOBS) on the morphosis of erythrocytes with atomic force microscopic (AFM) observation. METHODS: Blood samples from the patients with spinal operation were collected before operation (T1) and 1 h after IOBS (T4). Unprocessed blood (T2) and processed blood (T3) in the cell saver were also measured. An AFM with nanometer resolution was used to examine the ultrastructure of membrane surface of the erythrocytes in the samples. RESULTS: The percentage of heteromorphous erythrocytes in T1 samples was the lowest. There were significant differences in AFM images and height profile of a single erythrocyte between salvaged blood and venous blood. AFM images at 1 μm range showed that the distribution of the particles on the erythrocyte membrane surface in unprocessed blood in cell saver was significantly different from the other blood samples. CONCLUSION: There are significant changes in the AFM images of erythrocytes and the ultrastructure of the membrane surface in salvaged blood.     

Effects of artesunate on membrane surface morphology of human gastric cancer SGC-7901 cells observed under atomic force microscope

AIM: To detect the membrane surface morphology of cancer cells treated with artesunate (ART) under atomic force microscope (AFM). METHODS: Human gastric cancer SGC-7901 cells were cultured and treated with different concentrations of ART for 24 h. The membrane surface morphology, three-dimensional structures and ultrastructural changes of SGC-7901 cells were observed under AFM. The apoptosis of SGC-7901 cells was detected by flow cytometry. RESULTS: The AFM images revealed that the cell nuclear area was full and the surface of cell membrane was flat and smooth in control group. Compared with control group, the cell nuclear area collapsed and suffered atrophy, and the membrane surface had more pores in ART treatment groups. More pores were observed and the diameter of the pores was increased with the increase in ART concentration. The cell membrane ultrastructure showed that the particles in control group had an intensive distribution, some were cord-shaped and some gathered into a mass. The particles in ART treatment groups were fewer and distributed sparsely. Moreover, there were obvious lacunae in the surface of cell membrane. Quantitive measurement found that the height of cell nucleus area was decreased, and the surface root mean square roughness (Rq) and the surface average roughness (Ra) became smaller in ART treatment groups compared with control group. The apoptotic rates of the cells in ART treatment groups were increased with the increase in ART concentration, and were all significantly higher than that in control group. CONCLUSION: Our findings emphasize the significance of AFM in exploring the changes of the membrane surface morphology at nano-scale resolution following the treatment with anticancer drugs, which could not only identify the specific characteristics of morphological changes of the tumor cells, but also provide micromorphological references to reveal the mechanisms of anticancer drugs.     

Observation of molecular interaction between gold nanoparticle and VEGF165 with atomic force microscope

AIM: To investigate the molecular interaction between gold nanoparticles (GNP) and vascular endothelial growth factor 165(VEGF₁₆₅) under atomic force microscope (AFM). METHODS: Before and after incubation with VEGF₁₆₅, GNP were screened by integrated tools including AFM, ultraviolet-visible absorption spectroscopy and particle size analysis under near-physiological condition. In addition, GNP at different concentrations were incubated with VEGF₁₆₅, then added to starved human umbilical vein endothelial cells(HUVECs). The ultrastructural changes of HUVECs surface were examined by AFM. The effects of GNP on the growth of HUVECs were assessed by MTT assay. RESULTS: After treated with VEGF₁₆₅, the GNP absorption peak revealed a slight red shift, and the size distribution of GNP was increased from 20 nm to 30 nm. By AFM imaging, the diameter of GNP was (22.05±1.52) nm in average while the average diameter of GNP-VEGF₁₆₅ was (33.91±2.61) nm. Binding of GNP and VEGF₁₆₅, and the formation of GNP-VEGF₁₆₅ core-shell complex were indicated by the AFM imaging. AFM screening showed the changes of ultrastructure on HUVECs surface. The group of VEGF₁₆₅ displayed the signs of cell proliferation. Granulation of cell surface, increase in cell-to-cell contact, formation of pseudopodia and appearance of membranes pores were all observed. The proliferation of HUVECs was inhibited by GNP with MTT assay. CONCLUSION: GNP bind to VEGF₁₆₅ through chemical bonds to block or inactivate the receptor binding sites of VEGF₁₆₅. Therefore, GNP inhibit VEGF₁₆₅-induced proliferation of HUVECs.     

The abnormalities of microenvironment in myelodysplastic syndrome

Myelodysplastic syndrome(MDS)is considered as a preleukemic course, characteristic of hypercel ular marrow and pancytopenia.Many studies have demonstrated that defects occur in the heamtopoietic cels from patients with MDS.Recently, many abnormal changes in apoptosis, proliferation, ability of hematopoietic support, cytokine secret ion, clonal origin of stromal cells and angiogenesis have also been re vealed in the bone marrow microenvironment of MDS patients.     

Application of multiple gene methylations in plasma for diagnosis of lung cancer

AIM: To determine the aberrant methylation status in the gene promoter regions of CDH13, RASSF1A, DLEC1, SEPT9and RUNX3by detecting the plasma specimens and the value of their combined detection for diagnosis of lung cancers. METHODS: Nest methylation specific PCR (nMSP) was used to detect the promoter methylation status of the 5 genes in the plasma from 106 normal controls， lung cancer tissues, lung benign tissues and the plasma from 106 patients with lung cancers. Multiple displacement amplification (MDA) was used to amplify modified genomic DNA to solve the problem of insufficient of plasma DNA template. RESULTS: The positive rates of promoter methylation of CDH13, RASSF1A, DLEC1, SEPT9and RUNX3in the lung cancer tissues were 51.9%, 44.3%, 54.7%, 36.8%, 24.5%, respectively, and those in the plasma were 46.2%, 41.5%, 50.9%, 31.1%, 19.8%, respectively. The results of the Kappa consistency check showed that the lung cancer tissues and the plasma had obviously coherence in the methylation status of the 5 gene promoter regions. Combination of DLEC1, CDH13, RASSF1A, and SEPT9 had a higher diagnostic efficiency than the others, as their ACC value was 0.8208 and youden index was 0.6415 (with the sensitivity of 81.13% and the specificity of 83.02%). CONCLUSION: Combination detection of promoter methylation of lung cancer-related genes in the plasma is expected to apply to the early diagnosis of lung cancer.