三维回转模拟失重对人牙周膜成纤维细胞生物学性能的影响

发布时间：2018-03-11 22:37

  本文选题：失重模拟　切入点：三维回转器　出处：《第四军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：微重力环境可引起肌肉骨骼系统、心血管系统、免疫系统、神经-内分泌系统、自主平衡系统等各个系统的改变,如骨丢失、肌肉萎缩、心血管系统失调、立位耐受性下降、认知功能降低等等。那么,微重力环境对牙周组织会造成怎样的影响呢?在本研究中我们拟采用三维回转器(three dimensional clinostat,TDC)来模拟失重环境,从这种失重环境对人牙周膜成纤维细胞(human periodontal ligament fibroblasts, hPDLFs)生物学性能的影响入手,对这一问题进行了探讨。 目的: 观察模拟失重环境对hPDLFs形态和功能的影响,初步探讨在这一特殊环境中工作、生活的个体(如宇航员、太空旅游者)的牙周组织对这种环境的耐受性。 方法: 取体外培养的4-7代hPDLFs,接种于20ml培养瓶贴壁24h,随机分为回转组和对照组。运用三维回转器(three dimensional clinostat,TDC)模拟失重环境,设定(4-10)r/min随机转速,所产生的重力水平在(10~(-3)-10~(-4) )g间。回转组hPDLFs在TDC中分别培养48h、72h和96h,对照组细胞分别在常规环境中培养。然后进行以下检测,比较分析两组间细胞的差异性。 1细胞形态学检测:利用HE染色、罗丹明-鬼笔环肽免疫荧光染色观察两组间细胞形态及细胞微丝骨架的变化。 2细胞功能检测:利用细胞计数法、MTT细胞活力检测、流式细胞术、Hoechst 33258核染色及RT-PCR技术,比较两组细胞间增殖、凋亡及I型胶原mRNA的表达等细胞功能的差异。 结果: 1 TDC模拟失重对hPDLFs细胞形态的影响 HE染色:培养48h后,回转组和对照组hPDLFs均呈梭形或多角形,细胞核大,居中,胞体丰满,胞浆均匀,未出现胞浆内颗粒增多、胞内空泡、核分叉、核碎裂等现象。但回转组细胞的胞核面积较对照组缩小约12%(P0.05);培养72h和96h后,回转组细胞形态与对照组比较无明显差异。 罗丹明-鬼笔环肽免疫荧光染色:培养48h后,回转组微丝骨架结构略显模糊,排列较对照组稍显紊乱。培养72h和96h后,回转组和对照组微丝骨架结构均呈现清晰而有规律的束状结构,分布均匀。 2 TDC模拟失重对hPDLFs细胞功能的影响 细胞计数仪计数:培养48h和72h后,回转组与对照组hPDLFs细胞数量无明显差异(P0.05);培养96h后,回转组与对照组细胞计数结果有显著差异,回转组细胞数量明显高于对照组(P0.01)。 MTT细胞活力检测:培养48h后,回转组与对照组细胞活力无明显差异(P0.05);培养72h和96h后,回转组细胞活力明显高于对照组(P0.01)。 细胞周期:培养48h和72h后,回转组与对照组细胞周期S期所占总周期的百分比例结果无明显差异(P0.05);模拟失重培养96h后,回转组细胞周期S期所占总周期的百分比例高于对照组(P0.05)。 Hoechst 33258核染色:培养48h、72h和96h后,回转组和对照组hPDLFs细胞核均呈弥散均匀荧光,细胞核边缘光滑圆润,未出现细胞核固缩、核碎裂、浓染致密的颗粒块状凋亡小体荧光。 RT-PCR:培养48h和72h后,回转组hPDLFsⅠ型胶原mRNA表达较对照组变化不明显(P0.05);培养96h后,回转组hPDLFsⅠ型胶原mRNA表达有所升高(P0.05)。 结论: 暴露于微重力环境的初期(48h),hPDLFs表现出细胞核变小、微丝骨架结构稍显紊乱的形态学变化,但未出现细胞裂解、凋亡增强、细胞活性下降等破坏性变化。随着在微重力环境中暴露时间的延长(72h、96h),hPDLFs细胞形态恢复正常,并且逐渐表现出细胞活力、增殖能力及Ⅰ型胶原表达增强的功能状态。这一结果提示,hPDLFs对短期(96h)的微重力环境具有耐受性,但其所表现出的功能活动增强的情况会对牙周组织结构产生何种影响,尚需进一步的研究进行探讨。
[Abstract]:The microgravity environment can cause musculoskeletal system, cardiovascular system, immune system, nerve endocrine system, the system of self balancing system changes, such as bone loss, muscle atrophy, cardiovascular system dysfunction, orthostatic tolerance decreased, reduced cognitive function and so on. Then, what is the impact of microgravity on periodontal tissue? In this study, we adopted three-dimensional clinostat (three dimensional clinostat, TDC) to simulate weightlessness, from this weightlessness on human periodontal ligament fibroblasts (human periodontal ligament fibroblasts, hPDLFs) with the effect of biological properties, to explore this problem.
Objective:
To observe the effect of simulated weightlessness environment on the morphology and function of hPDLFs, we preliminarily explore the tolerance of periodontal tissue to individuals living in this special environment, such as astronauts and space travelers.
Method:
In vitro cultured 4-7 hPDLFs, inoculated in 20ml culture flask adherent 24h, were randomly divided into rotary group and control group. Using 3D clinostat (three dimensional, clinostat, TDC) of simulated weightlessness (4-10 r/min), set the random speed generated by the gravity level (10~ (-3) -10~ (-4) g). Group hPDLFs rotary incubation 48h in TDC, 72h and 96h, the control group cells were cultured in normal environment. Then the following test, comparative analysis of differences between the two groups of cells.
1 cell morphologic detection: using HE staining, observe the changes between the two groups in cell morphology and actin cytoskeleton of Luo Danming - phalloidin staining.
2 cell function detection: cell counting, MTT cell viability assay, flow cytometry, Hoechst 33258 nuclear staining and RT-PCR technology were used to compare cell function difference among two groups, including cell proliferation, apoptosis and I collagen mRNA expression.
Result:
The effect of 1 TDC simulated weightlessness on the morphology of hPDLFs cells
HE staining: after 48h culture, rotary group and control group hPDLFs showed fusiform or polygonal, large nucleus, cell centered, plump body, uniform cytoplasm, no granules within the cytoplasm increased, intracellular vacuoles, nuclear bifurcation, nuclear fragmentation phenomenon. But the nuclear area of rotating group than the control group cells a decrease of about 12% (P0.05); the culture of 72h and 96h, no significant difference was found between the rotary cell morphology in group and control group.
Luo Danming - phalloidin staining: after 48h culture, rotary group cytoskeleton structure slightly blurred, arranged slightly disordered. Compared with the control group, the culture of 72h and 96h, the rotary group and the control group showed clear and cytoskeleton structure like structure, regular distribution.
The effect of 2 TDC simulated weightlessness on the function of hPDLFs cells
Counting cell count: after cultured 48h and 72h, there was no significant difference in the number of hPDLFs cells between the rotary group and the control group (P0.05). After training 96h, there was a significant difference in the cell count between the rotary group and the control group, and the number of cells in the rotation group was significantly higher than that in the control group (P0.01).
MTT cell viability assay: after cultured 48h, there was no significant difference in cell viability between the rotary group and the control group (P0.05). After training 72h and 96h, the cell viability of the rotation group was significantly higher than that of the control group (P0.01).
Cell cycle: after cultured 48h and 72h, there was no significant difference in percentage of S cycle between the gyration group and the control group (P0.05). After simulated weightlessness culture 96h, the percentage of S cycle in the gyration group was higher than that in the control group (P0.05).
Hoechst 33258 nuclear staining: after cultured 48h, 72h and 96h, the nucleus of hPDLFs cells in the rotary group and the control group showed uniform fluorescence. The nuclei were smooth and round, and no nuclear shrinkage, nuclear fragmentation, dense and dense granular apoptotic bodies were observed.
After RT-PCR: cultured 48h and 72h, the expression of hPDLFs type I collagen mRNA in the gyration group was not significantly different from that in the control group (P0.05). After training 96h, the expression of hPDLFs collagen I mRNA increased in the rotation group (P0.05).
Conclusion:
Early exposure to microgravity environment (48h), hPDLFs showed smaller nuclei, slightly morphological changes of cytoskeleton structure disorder, but there was no cell lysis, cell apoptosis increased, decreased activity and other destructive changes. As the exposure time prolonged in microgravity (72h, 96h), hPDLFs cell shape recovery normal, and gradually showed cell viability, proliferation and expression of type I collagen function enhanced. The results suggest that hPDLFs (96h) on the short-term microgravity environment have tolerance, but its functional activities showed enhanced conditions will affect the structure of periodontal tissue, further studies discussion is needed.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R85

【参考文献】

相关期刊论文 前10条

1 熊江辉,李莹辉,聂捷琳,丁柏,张晓铀,黄增明,毕蕾;槲皮素对模拟微重力条件下体外培养大鼠心肌细胞骨架的影响[J];动物学报;2003年01期

2 杨芬,李莹辉,戴钟铨,聂捷琳,熊江辉;回转模拟失重对心肌成纤维细胞Ⅰ型胶原代谢的影响[J];动物学报;2004年02期

3 刘顺利,王锦玲,林顺涨;尾部悬吊8wk对大鼠耳石及颞骨、听骨、股骨钙含量的影响[J];第四军医大学学报;2002年02期

4 谭雄进,孙永建,王前,郑磊;模拟失重对不同发育阶段大鼠骨代谢的影响[J];第一军医大学学报;2002年07期

5 孙喜庆,姚永杰,杨长斌,冯岱雅,蒋昌林,梁文彬;21d头低位卧床期间第一周和最后一周下体负压锻炼对立位耐力和心功能的影响[J];航天医学与医学工程;2002年02期

6 谈诚,蒋昌林,汪娜,梁文彬,姜世忠;21d头低位卧床对人体左心舒张功能的影响[J];航天医学与医学工程;2002年03期

7 袁林天,文玲英,罗亚宁,胡沛臻,蒋维中,吴兴裕;尾悬吊大鼠牙体、牙髓、牙周组织的变化[J];航天医学与医学工程;2003年04期

8 刘朝霞,马铁民,杨鸿慧,吴大蔚,汪德生,张淑静;模拟失重对心肌细胞间缝隙连接蛋白CX43表达及分布的影响[J];航天医学与医学工程;2003年06期

9 杨芬,李莹辉,马永洁,钟萍,宋锦萍,戴钟铨;回转模拟失重对心肌成纤维细胞生长因子及ERK信号传导的影响[J];航天医学与医学工程;2003年S1期

10 郭芮,胡敏,孙振宇,薛京伟;模拟失重对大鼠下颌骨、腰椎和股骨组织结构的影响[J];航天医学与医学工程;2005年03期

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