周期性牽拉應(yīng)力下三維培養(yǎng)的成骨樣細(xì)胞的生物力學(xué)效應(yīng)及其機(jī)制的研究
發(fā)布時(shí)間：2019-06-18 19:07
【摘要】：1 成骨樣細(xì)胞的三維培養(yǎng)及牽拉應(yīng)力下形態(tài)學(xué)改變 目的:研究克隆的鼠顱骨成骨樣細(xì)胞(colonal murine osteoblast-like cell line MC3T3-E1)三維培養(yǎng)方法及在特定的周期性牽拉應(yīng)力刺激下形態(tài)學(xué)改變。骨組織工程的關(guān)鍵在于創(chuàng)建類似活體生理環(huán)境的細(xì)胞體外培養(yǎng)體系。以往的體外細(xì)胞培養(yǎng)為單層、即二維細(xì)胞培養(yǎng),這與細(xì)胞在活體的生存環(huán)境有很大差異。目前,建立三維細(xì)胞培養(yǎng)體系是骨組織工程的研究熱點(diǎn),不同的組織細(xì)胞對(duì)三維結(jié)構(gòu)載體的要求條件存在差異。本實(shí)驗(yàn)選擇克隆的鼠顱骨成骨樣細(xì)胞作為種子細(xì)胞,創(chuàng)建三維結(jié)構(gòu)培養(yǎng)體系,研究細(xì)胞培養(yǎng)過程中的細(xì)胞演變,并進(jìn)一步在動(dòng)力條件下觀察細(xì)胞的形態(tài)學(xué)改變,為進(jìn)一步探討成骨細(xì)胞受到機(jī)械牽拉應(yīng)力刺激后的分子生物學(xué)效應(yīng)及信號(hào)傳導(dǎo)機(jī)制奠定基礎(chǔ),從而指導(dǎo)臨床中骨折的治療。 方法: 以明膠海綿(2cmx2cmx0.25cm)作為MC3T3-E1 的三維培養(yǎng)支架,明膠海綿來源于純化的豬皮膚膠原蛋白(Gelfoam; Upjohn, Kalamazoo, MI), 每塊明膠海綿種植細(xì)胞懸液100μl,細(xì)胞數(shù)目為1.25×10~5個(gè)。生物牽拉裝置(Bio-Stretch System; ICCT Technologies, Inc., Canada)包括一個(gè)程序控制的脈沖計(jì)時(shí)器、控制器、雙向調(diào)節(jié)直流電源和一套帶有平臺(tái)的螺線電導(dǎo)管。放有明膠海綿的培養(yǎng)皿置于每一個(gè)電導(dǎo)管的前方。明膠海綿的一端固定于皿底,另一端用塑料夾包裹一個(gè)金屬棒。供電后通過線圈的電流產(chǎn)生磁場(chǎng),吸引金屬棒朝線圈方向移動(dòng),拉伸明膠海綿。斷電后,明膠海綿回縮為原來的長(zhǎng)度。膠原海綿拉伸度為5%、作用頻率為60cycles/min、作用時(shí)間為15min/h。牽拉后2d、4d、6d、8d、10d 分別從牽拉組和對(duì)照組中各取3 個(gè)樣本,行細(xì)胞計(jì)數(shù)。將對(duì)照組和牽拉組各時(shí)相的每快明膠海綿均勻切成四份,布安氏液(Bouin’s fluid)固定48h,然后按以下程序制作切片:脫水、石蠟包埋:石蠟切片為6μm;脫蠟、蘇木精伊紅染色。HE 染色光鏡下觀察其形態(tài)學(xué)改變。所得數(shù)據(jù)采用SAS6.12統(tǒng)計(jì)軟件進(jìn)行兩樣本t 檢驗(yàn)。
[Abstract]:1 three-dimensional culture of osteoblast-like cells and morphological changes under traction stress objective: to study the three-dimensional culture method of cloned rat skull osteoblast-like cells (colonal murine osteoblast-like cell line MC3T3-E1) and the morphological changes under specific periodic traction stress stimulation. The key of bone tissue engineering is to create a cell culture system similar to the physiological environment in vivo. In the past, cell culture in vitro was monolayer, that is, two-dimensional cell culture, which was very different from the living environment of cells in vivo. At present, the establishment of three-dimensional cell culture system is the research focus of bone tissue engineering, and the requirements of different tissue cells for three-dimensional structure carrier are different. In this experiment, the cloned rat skull osteoblast-like cells were selected as seed cells, the three-dimensional structure culture system was established, the cell evolution in the process of cell culture was studied, and the morphological changes of cells were further observed under dynamic conditions, which laid a foundation for further exploring the molecular biological effects and signal transduction mechanism of osteoblasts stimulated by mechanical traction stress, so as to guide the treatment of fracture in clinic. Methods: gelatin sponge (2cmx2cmx0.25cm) was used as three-dimensional culture scaffold of MC3T3-E1. Gelatin sponge was derived from purified pig skin collagen (Gelfoam; Upjohn, Kalamazoo, MI), 100 渭 l per piece of gelatin sponge, and the number of cells was 1.25 脳 10 ~ 5. Biological traction device (Bio-Stretch System; ICCT Technologies, Inc., Canada) includes a programmed pulse timer, controller, bidirectional regulating DC power supply and a set of solenoid conduit with platform. Petri dishes with gelatin sponges are placed in front of each catheter. One end of the gelatin sponge is fixed to the bottom of the dish, and the other end is wrapped in a plastic clip over a metal rod. After power supply, the magnetic field is generated through the current of the coil to attract the metal rod to move in the direction of the coil and stretch the gelatin sponge. After the power is cut off, the gelatin sponge retracts to the original length. The tensile degree of collagen sponge was 5%, the action frequency was 60 cycles / min, and the action time was 15 min / h. Three samples were taken from the traction group and the control group on the 2nd day, 4th day, 6th day, 8th day and 10th day, respectively, and the cells were counted. Each fast gelatin sponge in the control group and the traction group was evenly cut into four parts and fixed with (Bouin's fluid) for 48 hours, and then the sections were made according to the following procedure: dehydration, paraffin embedding: paraffin section 6 渭 m, dewaxing, hematoxylin eosin staining. He staining was used to observe the morphological changes. Two samples t test was carried out by SAS6.12 statistical software.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R329.2
本文編號(hào)：2501714
[Abstract]:1 three-dimensional culture of osteoblast-like cells and morphological changes under traction stress objective: to study the three-dimensional culture method of cloned rat skull osteoblast-like cells (colonal murine osteoblast-like cell line MC3T3-E1) and the morphological changes under specific periodic traction stress stimulation. The key of bone tissue engineering is to create a cell culture system similar to the physiological environment in vivo. In the past, cell culture in vitro was monolayer, that is, two-dimensional cell culture, which was very different from the living environment of cells in vivo. At present, the establishment of three-dimensional cell culture system is the research focus of bone tissue engineering, and the requirements of different tissue cells for three-dimensional structure carrier are different. In this experiment, the cloned rat skull osteoblast-like cells were selected as seed cells, the three-dimensional structure culture system was established, the cell evolution in the process of cell culture was studied, and the morphological changes of cells were further observed under dynamic conditions, which laid a foundation for further exploring the molecular biological effects and signal transduction mechanism of osteoblasts stimulated by mechanical traction stress, so as to guide the treatment of fracture in clinic. Methods: gelatin sponge (2cmx2cmx0.25cm) was used as three-dimensional culture scaffold of MC3T3-E1. Gelatin sponge was derived from purified pig skin collagen (Gelfoam; Upjohn, Kalamazoo, MI), 100 渭 l per piece of gelatin sponge, and the number of cells was 1.25 脳 10 ~ 5. Biological traction device (Bio-Stretch System; ICCT Technologies, Inc., Canada) includes a programmed pulse timer, controller, bidirectional regulating DC power supply and a set of solenoid conduit with platform. Petri dishes with gelatin sponges are placed in front of each catheter. One end of the gelatin sponge is fixed to the bottom of the dish, and the other end is wrapped in a plastic clip over a metal rod. After power supply, the magnetic field is generated through the current of the coil to attract the metal rod to move in the direction of the coil and stretch the gelatin sponge. After the power is cut off, the gelatin sponge retracts to the original length. The tensile degree of collagen sponge was 5%, the action frequency was 60 cycles / min, and the action time was 15 min / h. Three samples were taken from the traction group and the control group on the 2nd day, 4th day, 6th day, 8th day and 10th day, respectively, and the cells were counted. Each fast gelatin sponge in the control group and the traction group was evenly cut into four parts and fixed with (Bouin's fluid) for 48 hours, and then the sections were made according to the following procedure: dehydration, paraffin embedding: paraffin section 6 渭 m, dewaxing, hematoxylin eosin staining. He staining was used to observe the morphological changes. Two samples t test was carried out by SAS6.12 statistical software.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R329.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 荊鑫,趙劍,祝云利,潘欣,周維江,吳海山;pcDNA3-EGFP基因轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞的實(shí)驗(yàn)研究[J];徐州醫(yī)學(xué)院學(xué)報(bào);2001年06期
，本文編號(hào)：2501714
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2501714.html
最近更新
教材專著
