本文選題：失重模擬　切入點(diǎn)：三維回轉(zhuǎn)器　出處：《第四軍醫(yī)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：微重力環(huán)境可引起肌肉骨骼系統(tǒng)、心血管系統(tǒng)、免疫系統(tǒng)、神經(jīng)-內(nèi)分泌系統(tǒng)、自主平衡系統(tǒng)等各個(gè)系統(tǒng)的改變,如骨丟失、肌肉萎縮、心血管系統(tǒng)失調(diào)、立位耐受性下降、認(rèn)知功能降低等等。那么,微重力環(huán)境對(duì)牙周組織會(huì)造成怎樣的影響呢?在本研究中我們擬采用三維回轉(zhuǎn)器(three dimensional clinostat,TDC)來(lái)模擬失重環(huán)境,從這種失重環(huán)境對(duì)人牙周膜成纖維細(xì)胞(human periodontal ligament fibroblasts, hPDLFs)生物學(xué)性能的影響入手,對(duì)這一問(wèn)題進(jìn)行了探討。 目的: 觀察模擬失重環(huán)境對(duì)hPDLFs形態(tài)和功能的影響,初步探討在這一特殊環(huán)境中工作、生活的個(gè)體(如宇航員、太空旅游者)的牙周組織對(duì)這種環(huán)境的耐受性。 方法: 取體外培養(yǎng)的4-7代hPDLFs,接種于20ml培養(yǎng)瓶貼壁24h,隨機(jī)分為回轉(zhuǎn)組和對(duì)照組。運(yùn)用三維回轉(zhuǎn)器(three dimensional clinostat,TDC)模擬失重環(huán)境,設(shè)定(4-10)r/min隨機(jī)轉(zhuǎn)速,所產(chǎn)生的重力水平在(10~(-3)-10~(-4) )g間�；剞D(zhuǎn)組hPDLFs在TDC中分別培養(yǎng)48h、72h和96h,對(duì)照組細(xì)胞分別在常規(guī)環(huán)境中培養(yǎng)。然后進(jìn)行以下檢測(cè),比較分析兩組間細(xì)胞的差異性。 1細(xì)胞形態(tài)學(xué)檢測(cè):利用HE染色、羅丹明-鬼筆環(huán)肽免疫熒光染色觀察兩組間細(xì)胞形態(tài)及細(xì)胞微絲骨架的變化。 2細(xì)胞功能檢測(cè):利用細(xì)胞計(jì)數(shù)法、MTT細(xì)胞活力檢測(cè)、流式細(xì)胞術(shù)、Hoechst 33258核染色及RT-PCR技術(shù),比較兩組細(xì)胞間增殖、凋亡及I型膠原mRNA的表達(dá)等細(xì)胞功能的差異。 結(jié)果: 1 TDC模擬失重對(duì)hPDLFs細(xì)胞形態(tài)的影響 HE染色:培養(yǎng)48h后,回轉(zhuǎn)組和對(duì)照組hPDLFs均呈梭形或多角形,細(xì)胞核大,居中,胞體豐滿,胞漿均勻,未出現(xiàn)胞漿內(nèi)顆粒增多、胞內(nèi)空泡、核分叉、核碎裂等現(xiàn)象。但回轉(zhuǎn)組細(xì)胞的胞核面積較對(duì)照組縮小約12%(P0.05);培養(yǎng)72h和96h后,回轉(zhuǎn)組細(xì)胞形態(tài)與對(duì)照組比較無(wú)明顯差異。 羅丹明-鬼筆環(huán)肽免疫熒光染色:培養(yǎng)48h后,回轉(zhuǎn)組微絲骨架結(jié)構(gòu)略顯模糊,排列較對(duì)照組稍顯紊亂。培養(yǎng)72h和96h后,回轉(zhuǎn)組和對(duì)照組微絲骨架結(jié)構(gòu)均呈現(xiàn)清晰而有規(guī)律的束狀結(jié)構(gòu),分布均勻。 2 TDC模擬失重對(duì)hPDLFs細(xì)胞功能的影響 細(xì)胞計(jì)數(shù)儀計(jì)數(shù):培養(yǎng)48h和72h后,回轉(zhuǎn)組與對(duì)照組hPDLFs細(xì)胞數(shù)量無(wú)明顯差異(P0.05);培養(yǎng)96h后,回轉(zhuǎn)組與對(duì)照組細(xì)胞計(jì)數(shù)結(jié)果有顯著差異,回轉(zhuǎn)組細(xì)胞數(shù)量明顯高于對(duì)照組(P0.01)。 MTT細(xì)胞活力檢測(cè):培養(yǎng)48h后,回轉(zhuǎn)組與對(duì)照組細(xì)胞活力無(wú)明顯差異(P0.05);培養(yǎng)72h和96h后,回轉(zhuǎn)組細(xì)胞活力明顯高于對(duì)照組(P0.01)。 細(xì)胞周期:培養(yǎng)48h和72h后,回轉(zhuǎn)組與對(duì)照組細(xì)胞周期S期所占總周期的百分比例結(jié)果無(wú)明顯差異(P0.05);模擬失重培養(yǎng)96h后,回轉(zhuǎn)組細(xì)胞周期S期所占總周期的百分比例高于對(duì)照組(P0.05)。 Hoechst 33258核染色:培養(yǎng)48h、72h和96h后,回轉(zhuǎn)組和對(duì)照組hPDLFs細(xì)胞核均呈彌散均勻熒光,細(xì)胞核邊緣光滑圓潤(rùn),未出現(xiàn)細(xì)胞核固縮、核碎裂、濃染致密的顆粒塊狀凋亡小體熒光。 RT-PCR:培養(yǎng)48h和72h后,回轉(zhuǎn)組hPDLFsⅠ型膠原mRNA表達(dá)較對(duì)照組變化不明顯(P0.05);培養(yǎng)96h后,回轉(zhuǎn)組hPDLFsⅠ型膠原mRNA表達(dá)有所升高(P0.05)。 結(jié)論: 暴露于微重力環(huán)境的初期(48h),hPDLFs表現(xiàn)出細(xì)胞核變小、微絲骨架結(jié)構(gòu)稍顯紊亂的形態(tài)學(xué)變化,但未出現(xiàn)細(xì)胞裂解、凋亡增強(qiáng)、細(xì)胞活性下降等破壞性變化。隨著在微重力環(huán)境中暴露時(shí)間的延長(zhǎng)(72h、96h),hPDLFs細(xì)胞形態(tài)恢復(fù)正常,并且逐漸表現(xiàn)出細(xì)胞活力、增殖能力及Ⅰ型膠原表達(dá)增強(qiáng)的功能狀態(tài)。這一結(jié)果提示,hPDLFs對(duì)短期(96h)的微重力環(huán)境具有耐受性,但其所表現(xiàn)出的功能活動(dòng)增強(qiáng)的情況會(huì)對(duì)牙周組織結(jié)構(gòu)產(chǎn)生何種影響,尚需進(jìn)一步的研究進(jìn)行探討。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R85

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 熊江輝,李瑩輝,聶捷琳,丁柏,張曉鈾,黃增明,畢蕾;槲皮素對(duì)模擬微重力條件下體外培養(yǎng)大鼠心肌細(xì)胞骨架的影響[J];動(dòng)物學(xué)報(bào);2003年01期

2 楊芬,李瑩輝,戴鐘銓,聶捷琳,熊江輝;回轉(zhuǎn)模擬失重對(duì)心肌成纖維細(xì)胞Ⅰ型膠原代謝的影響[J];動(dòng)物學(xué)報(bào);2004年02期

3 劉順利,王錦玲,林順漲;尾部懸吊8wk對(duì)大鼠耳石及顳骨、聽骨、股骨鈣含量的影響[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2002年02期

4 譚雄進(jìn),孫永建,王前,鄭磊;模擬失重對(duì)不同發(fā)育階段大鼠骨代謝的影響[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2002年07期

5 孫喜慶,姚永杰,楊長(zhǎng)斌,馮岱雅,蔣昌林,梁文彬;21d頭低位臥床期間第一周和最后一周下體負(fù)壓鍛煉對(duì)立位耐力和心功能的影響[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2002年02期

6 談?wù)\,蔣昌林,汪娜,梁文彬,姜世忠;21d頭低位臥床對(duì)人體左心舒張功能的影響[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2002年03期

7 袁林天,文玲英,羅亞寧,胡沛臻,蔣維中,吳興裕;尾懸吊大鼠牙體、牙髓、牙周組織的變化[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2003年04期

8 劉朝霞,馬鐵民,楊鴻慧,吳大蔚,汪德生,張淑靜;模擬失重對(duì)心肌細(xì)胞間縫隙連接蛋白CX43表達(dá)及分布的影響[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2003年06期

9 楊芬,李瑩輝,馬永潔,鐘萍,宋錦萍,戴鐘銓;回轉(zhuǎn)模擬失重對(duì)心肌成纖維細(xì)胞生長(zhǎng)因子及ERK信號(hào)傳導(dǎo)的影響[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2003年S1期

10 郭芮,胡敏,孫振宇,薛京偉;模擬失重對(duì)大鼠下頜骨、腰椎和股骨組織結(jié)構(gòu)的影響[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2005年03期

，

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