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Effects of Three Dimensional Clinostat -Simulated Weightlessness on the Biological Characteristics of Human Periodontal Ligament Fibroblasts

Author: WangLei
Tutor: CaoJun；ZuoAiRong
School: Fourth Military Medical University
Course: Clinical Stomatology
Keywords: Weightlessness simulation 3D gyrator Human periodontal ligament fibroblasts Proliferation Apoptosis Cytoskeletal Collagen type I RT-PCR
CLC: R85
Type: Master's thesis
Year: 2011
Downloads: 24
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Abstract

Microgravity environment can cause musculoskeletal system, cardiovascular system, immune system, nervous - the endocrine system, self-balancing system and other system changes, such as bone loss, muscle atrophy and cardiovascular system disorders, orthostatic tolerance decreased, recognized known function decreased. Microgravity environment of the periodontal tissue to see what impact it? In the present study, we intend to use the three-dimensional rotation (three dimensional clinostat, TDC) to simulate the weightless environment from such a weightless environment on human periodontal ligament fibroblasts The impact of the biological properties of the cells (human periodontal ligament fibroblasts, hPDLFs) start, this issue was discussed. Objective: To observe the impact of weightlessness the environment hPDLFs morphology and function simulation work discussed in this special environment, individual life periodontal tissue (such as astronauts and space tourists) tolerated this kind of environment. Methods: in vitro 4-7 substituting hPDLFs, inoculated in 20ml flasks adherent 24h, were randomly divided into the rotary group and control group. The use of three-dimensional rotation (three dimensional clinostat, TDC) to simulate the weightless environment set (4-10) r / min random speed, the gravity level at (10 ^{-3 -10 ^{for -4 ) g. The rotation group hPDLFs the TDC, respectively, 48h, 72h and 96h, the control group, cells were cultured in a normal environment. Then the following detection and comparative analysis of the differences between the two groups of cells. 1 cell morphology detection: HE staining, rhodamine - phalloidin immunofluorescence staining observed between the two groups of cell morphology and cell changes of the actin cytoskeleton. 2 cell function testing: use of cell counting, MTT cell viability testing, flow cytometry, Hoechst 33258 nuclear staining and RT-PCR technique, the two groups were compared between cells proliferation, apoptosis and type I collagen mRNA expression in cell function differences. : TDC simulation of weightlessness on hPDLFs cell morphology affect HE staining: 48h culture, the rotation group and the control group hPDLFs showed a spindle or polygonal nucleus centered cell body is plump, uniform cytoplasm, does not appear intracytoplasmic particles increased, the intracellular vacuoles nuclear bifurcation phenomenon of nuclear fragmentation. Rotary group cell nucleus area reduced by about 12% (P lt; 0.05) than the control group;, the morphology of the rotation group and control group showed no significant difference for 72h and 96h. Rhodamine - phalloidin cyclic peptide immunofluorescence staining: 48h culture, the slewing group actin cytoskeleton structure is slightly fuzzy, slightly disordered arrangement than the control group. For 72h and 96h, the actin cytoskeleton structure of the rotation group and the control group showed a clear and regular bundle structure, evenly distributed. 2 TDC simulate weightlessness on hPDLFs cell function affects cell counting instrument count: culture after 48h and 72h, the rotary and control groups hPDLFs cells quantity no significant difference (P gt; 0.05); culture 96h after the rotation group and the control group cell count result there are significant differences in the the rotary group the number of cells was significantly higher (P lt; 0.01). MTT cell viability test: 48h culture, the rotary cell viability of the control group had no significant difference (P gt; 0.05); for 72h and 96h, the rotary cell viability was significantly higher (P lt; 0.01). Cell cycle: culture after 48h and 72h, the percentage of cases result of the rotation group, with the S phase of the cell cycle of the control group to the total cycle was no significant difference (P gt; 0.05); simulated weightlessness 96h after training rotating group of cell cycle S share percentage of the total cycle cases higher than that of the control group (P lt; 0.05). Hoechst 33258 nuclear staining: cultured 48h, 72h and 96h, the rotary and control groups hPDLFs nucleus showed diffuse uniformly fluorescent, smooth rounded edge of the nucleus does not appear nuclear condensation, nuclear fragmentation, stain dense particles massive apoptosis a small autofluorescence. RT-PCR: cultivation after 48h and 72h, the rotary group hPDLFs collagen type I mRNA expression compared with the control group did not change significantly (P gt; 0.05); culture 96h after the the rotation group hPDLFs collagen type I mRNA expression somewhat elevated (P lt; 0.05 ). The conclusion: exposure to the microgravity environment of the early (48h), the nucleus smaller hPDLFs showed morphological changes of the actin cytoskeleton structure slightly disordered, but not cell lysis, apoptosis enhanced cell activity decreased destructive changes. Extend (72h, 96h) exposure time in the microgravity environment hPDLFs cell morphology returned to normal, and gradually showed cell viability, proliferation and expression of type I collagen enhanced functional status. The results suggest that hPDLFs has tolerated the microgravity environment of short-term (96h), but they exhibit functional activities enhanced the periodontal tissue structure will produce what kind of impact, further research to explore.}}

Effects of Three Dimensional Clinostat -Simulated Weightlessness on the Biological Characteristics of Human Periodontal Ligament Fibroblasts

Abstract

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