模擬失重條件下對骨髓體外成骨能力及承重骨影響的研究
發(fā)布時間：2018-08-07 19:54
【摘要】： 空間飛行過程中，宇航人員暴露于失重環(huán)境下，將導致骨骼系統(tǒng)發(fā)生結構和功能的變化，主要表現(xiàn)為骨量丟失，骨骼脫礦，骨力學性能下降，出現(xiàn)負鈣平衡。這種失重導致的骨骼系統(tǒng)變化，在重力生理學中稱為失重性骨質稀少。失重性骨質稀少是人類長期太空停留和探索其它星球的主要障礙之一。雖然經(jīng)過大量研究，但目前對失重性骨喪失的機理仍缺乏深刻認識，所以，深入探討失重性骨喪失的發(fā)生機理，發(fā)展行之有效的對抗措施是目前重力生理學的重要任務。已有的研究表明，失重性骨喪失主要發(fā)生在承重骨。其機制主要是抑制了局部成骨作用，而對破骨細胞活性可能影響不大。在此過程中，由骨的細胞分泌并在局部發(fā)揮作用的一些調節(jié)蛋白和細胞因子被認為起著重要作用，但這類物質在失重條件下的確切作用及其機制并不是十分清楚。 本研究采用后肢去負荷大鼠模型，通過細胞培養(yǎng)觀察了4w模擬失重后對骨基質細胞數(shù)及骨髓體外成骨能力的影響；比較2w和4w模擬失重后對骨局部調節(jié)蛋白和細胞因子的影響作用，以及骨形態(tài)學的變化。主要結果與發(fā)現(xiàn)如下： 1．4w尾吊對大鼠股骨基質細胞數(shù)的影響 本實驗通過細胞培養(yǎng)進行原代細胞培養(yǎng)計數(shù)，細胞計數(shù)法和MTT法繪制生長曲線，，結果顯示：S組培養(yǎng)系的股骨基質細胞數(shù)明顯減少，約是C組細胞數(shù)的50％。S組和C組細胞生長曲 第四軍醫(yī)大學碩士學位論文 一 線和倍增時問相似，表明S組和C組細胞增咱速度相似。提示。lw模擬失重大 鼠股骨基質細胞數(shù)減少。 2．4W 尾吊對大鼠股骨骨髓體外成骨能力的影響 本實驗通過細胞培養(yǎng) 檢測原代和傳代培養(yǎng)細胞，ALP活性及體外礦化小結形成量。結果顯示：原代 培養(yǎng)中S組ALP活性和小結形成量同C組相比較顯著下降（50％），說明骨髓成 骨祖細胞減少，導致分化的成骨細胞數(shù)減少。傳代培養(yǎng)中細胞接種密度相同， S組竹P活性和小結形成數(shù)較C明顯降低（約50們，表明去負荷對骨髓基質中 的成骨祖細胞有抑制作用。 3．OPNr，OC，BMP和 TGF－日在 ZW，4W尾吊大鼠承重骨的變化 本實驗通過 原位雜交的方法檢測了股骨和骨髓 OPN，OC，BMP和 TGF－6。結果發(fā)現(xiàn)：S組 OPN，OC，BMP和 TGF－p在骨細胞和骨髓中含量顯著低于 C組，S組 4W低于 ZW。 提示模擬失重可能抑制了成骨細胞分泌OPN，影響細胞在骨面的粘附。同樣可 能抑制了 OC的產(chǎn)生，影響骨質的礦化。另外抑制了 BMP和 TGF－p的產(chǎn)生及促 成骨作用的發(fā)揮。 4．ALP和 ACP在 ZW，4W尾吊大鼠承重骨的變化 本實驗通過特殊染色的 方法檢測了股骨ALP和 ACP。結果顯示尾吊大鼠股骨中 ALP顯著降低，ACP顯 著增高。提示模擬失重后，大鼠成骨作用降低，破骨作用增強。 5．承重骨形態(tài)學變化 本實驗通過三色染色的方法觀察股骨形態(tài)學變 化，顯示骨基質鈣化障礙，原有沉積鈣鹽脫鈣明顯，提示模擬失重使大鼠鈣 鹽代謝障礙。 總之，本工作利用細胞培養(yǎng)的方法，觀察到模擬失重對骨基質細胞數(shù)減 少和骨髓體外成骨能力下降的影響；通過原位雜交的方法觀察BMP、TGF－p、 OPN和 OC在模擬失重中承重骨分泌量減少的變化；通過特殊染色方法觀察骨 質中 ALP降低和 ACP增高及承重骨中鈣鹽代謝的形態(tài)學變化，對進一步探討 失重性骨喪失的機理具有參考意義。
[Abstract]:During space flight, the space flight personnel exposed to the weightlessness environment will lead to changes in the structure and function of the skeletal system, which mainly include bone loss, bone demineralization, bone mechanical performance decline, and negative calcium balance. This kind of weightlessness results in skeletal system change, which is called weightless bone scarcity in heavy stress physiology. Scarce is one of the main obstacles for human long space stay and exploration of other planets. Although a lot of research has been done, the mechanism of weightless bone loss is still lack of deep understanding. Therefore, it is an important task for gravity physiology to explore the mechanism of weightless bone loss and to develop effective countermeasures. Studies have shown that weightless bone loss mainly occurs in the load-bearing bone. Its mechanism is mainly to inhibit the local osteogenesis and may not affect the activity of osteoclast. In this process, some regulatory proteins and cytokines secreted by bone cells and part of the role play an important role in the process, but these substances are in weightless strips. The exact role and mechanism of this component are not very clear.
The effect of 4W simulated weightlessness on the number of bone stromal cells and bone marrow in vitro after weightlessness was observed by cell culture, and the effects of 2W and 4W on bone local regulatory proteins and cytokines after weightlessness were compared, and the changes of bone morphology were compared. The main results were as follows:
The effect of 1.4W tail suspension on the number of stromal cells in the femur of rats was carried out by cell culture to count the primary cell culture, cell count method and MTT method to draw the growth curve. The results showed that the number of femur stroma cells in the S group was significantly reduced, which was about the 50%.S and C cell growth of the C group.
Master's degree thesis of The Fourth Military Medical University
One
The similarity between the line and doubling time indicated that the cells in group S and group C were similar in speed, suggesting that.Lw simulation was not significant.
The number of stromal cells in the femur of the rat was reduced.
Effects of 2.4W tail suspension on osteogenic potential of femoral bone in rats in vitro
The ALP activity and mineralization nodule formation in primary and passage cells were detected.
ALP activity and nodule formation in S group decreased significantly compared with C group (50%), indicating bone marrow formation.
The number of osteoblasts decreased as the number of bone progenitor cells decreased.
In group S, P activity and nodule formation were significantly lower than those in C (about 50), indicating that the load was in bone marrow stroma.
The osteoblast progenitor cells have inhibitory effect.
3.OPNr, OC, BMP and TGF - day in ZW, 4W tail suspended rats bearing bone changes in this experiment
OPN, OC, BMP and transforming growth factor - 6 were detected by in situ hybridization. The results showed that: S group
The contents of OPN, OC, BMP and transforming growth factor - P in bone cells and bone marrow were significantly lower than those in group C, while 4W in group S was lower than ZW..
It is suggested that simulated weightlessness may inhibit the secretion of OPN from osteoblasts and affect the adhesion of cells to the bone surface.
It can inhibit the production of OC, affect the mineralization of bone, and inhibit the production and promotion of BMP and TGF P.
The exertion of osteogenesis.
4.ALP and ACP changes in the weight bearing bone of ZW and 4W tail suspended rats.
Methods detection of ALP and ACP. in the femur showed that ALP in the femur of tail suspended rats was significantly lower than that in ACP rats.
The results showed that after simulated weightlessness, the osteogenesis of rats decreased and the osteoclast increased.
5. the morphological changes of the weight-bearing bone were observed by trichrome staining.
It showed that the calcification of bone matrix was impaired and the calcium deposition was decalcified.
Salt metabolism disorder.
In conclusion, cell culture was used to observe the reduction of bone marrow stromal cells by simulated weightlessness.
The effects of BMP and TGF P on bone marrow in vitro were observed by in situ hybridization.
OPN and OC changes in the weight loss of the bearing bone in simulated weightlessness; bone is observed by special staining.
Morphological changes of ALP and ACP in the bone and calcium metabolism in the bearing bone were further explored.
The mechanism of weightless bone loss is of reference significance.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2002
【分類號】：R852.22
本文編號：2171180
[Abstract]:During space flight, the space flight personnel exposed to the weightlessness environment will lead to changes in the structure and function of the skeletal system, which mainly include bone loss, bone demineralization, bone mechanical performance decline, and negative calcium balance. This kind of weightlessness results in skeletal system change, which is called weightless bone scarcity in heavy stress physiology. Scarce is one of the main obstacles for human long space stay and exploration of other planets. Although a lot of research has been done, the mechanism of weightless bone loss is still lack of deep understanding. Therefore, it is an important task for gravity physiology to explore the mechanism of weightless bone loss and to develop effective countermeasures. Studies have shown that weightless bone loss mainly occurs in the load-bearing bone. Its mechanism is mainly to inhibit the local osteogenesis and may not affect the activity of osteoclast. In this process, some regulatory proteins and cytokines secreted by bone cells and part of the role play an important role in the process, but these substances are in weightless strips. The exact role and mechanism of this component are not very clear.
The effect of 4W simulated weightlessness on the number of bone stromal cells and bone marrow in vitro after weightlessness was observed by cell culture, and the effects of 2W and 4W on bone local regulatory proteins and cytokines after weightlessness were compared, and the changes of bone morphology were compared. The main results were as follows:
The effect of 1.4W tail suspension on the number of stromal cells in the femur of rats was carried out by cell culture to count the primary cell culture, cell count method and MTT method to draw the growth curve. The results showed that the number of femur stroma cells in the S group was significantly reduced, which was about the 50%.S and C cell growth of the C group.
Master's degree thesis of The Fourth Military Medical University
One
The similarity between the line and doubling time indicated that the cells in group S and group C were similar in speed, suggesting that.Lw simulation was not significant.
The number of stromal cells in the femur of the rat was reduced.
Effects of 2.4W tail suspension on osteogenic potential of femoral bone in rats in vitro
The ALP activity and mineralization nodule formation in primary and passage cells were detected.
ALP activity and nodule formation in S group decreased significantly compared with C group (50%), indicating bone marrow formation.
The number of osteoblasts decreased as the number of bone progenitor cells decreased.
In group S, P activity and nodule formation were significantly lower than those in C (about 50), indicating that the load was in bone marrow stroma.
The osteoblast progenitor cells have inhibitory effect.
3.OPNr, OC, BMP and TGF - day in ZW, 4W tail suspended rats bearing bone changes in this experiment
OPN, OC, BMP and transforming growth factor - 6 were detected by in situ hybridization. The results showed that: S group
The contents of OPN, OC, BMP and transforming growth factor - P in bone cells and bone marrow were significantly lower than those in group C, while 4W in group S was lower than ZW..
It is suggested that simulated weightlessness may inhibit the secretion of OPN from osteoblasts and affect the adhesion of cells to the bone surface.
It can inhibit the production of OC, affect the mineralization of bone, and inhibit the production and promotion of BMP and TGF P.
The exertion of osteogenesis.
4.ALP and ACP changes in the weight bearing bone of ZW and 4W tail suspended rats.
Methods detection of ALP and ACP. in the femur showed that ALP in the femur of tail suspended rats was significantly lower than that in ACP rats.
The results showed that after simulated weightlessness, the osteogenesis of rats decreased and the osteoclast increased.
5. the morphological changes of the weight-bearing bone were observed by trichrome staining.
It showed that the calcification of bone matrix was impaired and the calcium deposition was decalcified.
Salt metabolism disorder.
In conclusion, cell culture was used to observe the reduction of bone marrow stromal cells by simulated weightlessness.
The effects of BMP and TGF P on bone marrow in vitro were observed by in situ hybridization.
OPN and OC changes in the weight loss of the bearing bone in simulated weightlessness; bone is observed by special staining.
Morphological changes of ALP and ACP in the bone and calcium metabolism in the bearing bone were further explored.
The mechanism of weightless bone loss is of reference significance.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2002
【分類號】：R852.22
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本文編號：2171180
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