| Objective:To observation human oral squamous cell,AFM was applied to describing atomic-resolution three-dimensional image of the sample surface in this experimental.This experiment is intended to in-depth analysis on oral squamous cell membrane surface structure of different degree anemia, measuring different characteristics of the image of the cell surface, so as to further study the relation of the membrane protein structure and hunman health. We tride to develop predictors of human health assessment basis.Methods:1 Criteria of groupingFour groups : control Hb : male120~160g/L,female (nonpregnant) 110~150g/L; mild anemia group Hb:90~109g/L;moderate anemia group Hb:60~89g/L;severe anemia group Hb:30~59g/L.2 Inclusion criteria(1)from twenty ages to thirtyfive ages(2)no oral diseases(3)no local affective factors(4)no experience of being in touch with radiations and poison(5)no smoking(6)Agree with cooperation3 AFM sample preparationTo remove impurities in mouth,subjects were toldto rinse mouth with mouthwash and distiller respectively for three times.I scrapped the cheek oral mucosa with a sterile wooden and washed 2nd scraping the material into the 5mL centrifuge tube with 2% formaldehyde solution.It was fixed about 20 min. And then placed tube in centrifuge tubes to 1 000 r / min centrifugation for 10 min, discard supernatant, repeated again. The cell suspension was gently applied to the processed clean prepared slide , and placed in clean bench to dry.4 The right way to operate AFMWe Operated AFM at room temperature in the atmosphere ,and selected the tap (Tapping) mode, set all parameters. Took a sample of dried slices and lightly loaded on the AFM stage. We selected more distributed cells and put in the central field of vision, close to the probe position. We adjusted of the laser point scanning probe manually, And ensure the laser spot, center of the screen dots, probe tip had three points coincident with.We scaned 10 cells on each samples, with scanning range from 500nm to 90μm, scanning frequency of 0.5 and 2 Hz.We selected light and dark color junction on the cell surface to image respectively .And area imaging. 5μm region were used to analyze , applicating the latest version of the AFM image analysis software Nanoscope5.31r1 to process images and the recorded numbers of Ra (average roughness) on height and amplitude image.The two parameters were collected.5 All experimental data were indicated in form of x±s. All numbers were analyzed using SPSS 13.0 statistical software. On the Height image and Amplitude image, the average roughness (Ra) values were analyzed statistically. All data were put to the normal distribution test. If meet the normal distribution and homogeneity of variance, the difference were compared using the single factor analysis .The test standardαis equal to 0.05.Results:1 Pathological cytology resultsCompared squamous epithelial cells of normal oral,mild anemia, moderate anemia and severe anemia patients,with hematoxylin eosin stain stain, significant changes were not observed.2 AFM image resultsAFM was applicated to observe oral squamous epithelial cells of healthy people and anemia patients. And cells were mostly flat, oval or polygonal. there was a small bright spots in the central area of image . We determined the bright area just was the nucleus region.We found that squamous epithelial cells of control group generally had smooth and full surface, ordering a density level. The bulge-shaped depression formed between the gap is more uniform. While cells'surface of the mild, the moderate, and the severe anemia groups has gradually increase the roughness images. Especially we saw cell-like worm-eaten Hole on surface of the severe anemia patients'cell.Figure of 5μm on both height and amplitude imagine were flattened, and got the average roughness Ra values.. Compared with healthy people,the mild, moderate, and severe anemia had significantly increasing Ra values.3 Statistical analysis resultsAll data were divided into four groups: control group, mild anemia group, moderate anemia group, severe anemia group,and used SPSS 13.0 software for processing. All experimental data were indicated in form of x±s. All numbers were analyzed using SPSS 13.0 statistical software. On the height map (Height) and amplitude map (Amplitude), the average roughness Ra values were analyzed statistically.The two Ra values of control group were 18.8411±0.427,11.011±0.106; mild anemia group 39.4683±0.863,14.164±0.563; moderate anemia group was 67.5262±1.062,17.344±0.673; severe anemia group was 106.6947±1.915,22.984±0. 763. Values differ significantly. average roughness Ra Values of 4 groups were analyzed using SPSS 13.0 satistical software.The average roughness Ra values of the surface membrane of cells were measured and analyzed statistically using SPSS 11.0 software, the results of the four, the average surface roughness of the membrane have significant difference.The results had statistical signification(P <0.05).Conclusion: Compared with the control group, the anemia have different surface structure of oral squamous cell membrane under AFM. As anemia become more and more serious, Ra values increase.On Height map imagine of the severe anemia group whole cells, we can see structures, such as worm-eaten like Hole.So According to the ultrastructure change of oral squamous cell membrane surface on AFM imaging, a prediction of human health effects can be early detected made.Roughness Ra values cell surface showed levels of anemia. |
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