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  本文選題：動態(tài)壓應(yīng)力 + 前軟骨干細胞　； 參考：《華中科技大學(xué)》2012年博士論文

【摘要】：第一部分動態(tài)壓應(yīng)力對前軟骨干細胞Sox9和細胞外基質(zhì)表達的影響 目的 研究間斷性動態(tài)壓應(yīng)力對體外培養(yǎng)的大鼠前軟骨干細胞Sox9及軟骨特異性細胞外基質(zhì)(Ⅱ型膠原和aggrecan)表達的影響。 方法 1.分離原代大鼠前軟骨干細胞并接種于培養(yǎng)基中； 2.細胞免疫磁珠分選純化體外培養(yǎng)的前軟骨干細胞(PSCs); 3.將體外培養(yǎng)的PSCs隨機分為4組：對照組、壓力1天組、壓力3天組和壓力7天組,使用自行設(shè)計制備的細胞壓力裝置對細胞進行間斷性(1d刺激12h,分別1d,3d和7d)的壓應(yīng)力刺激(90mmHg),未壓力組為對照組； 4.采用熒光實時定量聚合酶鏈反應(yīng)檢測各組細胞的Sox9和Ⅱ型膠原和aggrecan的基因表達情況； 5. Western-blot檢測細胞Sox9和Ⅱ型膠原蛋白表達水平并進行分析。結(jié)果 1.倒置相差顯微鏡下見細胞形態(tài)多為為三角形或多角形,生長良好。 2.前軟骨干細胞在90mmHg強度的動態(tài)壓應(yīng)力培養(yǎng)條件下,壓力組各時間點Sox9、aggrecan和Ⅱ型膠原的基因表達水平明顯增加并高于對照組(P0.05)； 3.1d壓力組Sox9和Ⅱ型膠原蛋白表達與對照組無明顯差異(P0.05),3d和7d時間點的蛋白表達增加并高于對照組(P0.05)； 4.Sox9和Ⅱ型膠原基因和蛋白表達水平以及aggrecan基因表達都隨著壓應(yīng)力刺激時間增加而增加。 結(jié)論 間斷性動態(tài)壓應(yīng)力可以明顯增加大鼠前軟骨干細胞Sox9、Ⅱ型膠原和aggrecan的表達,可能會促進前軟骨干細胞向軟骨細胞分化。 第二部分動態(tài)壓應(yīng)力對胰島素樣生長因子-1誘導(dǎo)前軟骨干細胞Sox9和細胞外基質(zhì)表達的影響 目的 研究動態(tài)壓應(yīng)力對胰島素樣生長因子-1誘導(dǎo)前軟骨干細胞Sox9和細胞外基質(zhì)表達的影響。 方法 1.常規(guī)體外培養(yǎng)前軟骨干細胞并分離純化； 2.將純化后的前軟骨干細胞接種于細胞瓶中,隨機分為4組：對照組、壓力組、IGF-1組合壓力復(fù)合IGF-1組。壓力組予以90mmHg強度的壓應(yīng)力,IGF-1組予以IGF-1,終濃度為100ng/ml；對照組不施加壓力,也不加入IGF-1； 3.光鏡下觀察細胞形態(tài)改變以及阿利新藍染色和Ⅱ型膠原免疫組化染色； 4.實時熒光定量PCR方法檢測前軟骨干細胞中Sox9和軟骨特異性細胞外基質(zhì)基因表達情況。 結(jié)果 1.分選后的前軟骨干細胞呈三角形或梭形,折光性好；誘導(dǎo)后細胞由梭形逐漸變?yōu)槎嘟切位蝾悎A形,細胞突起逐漸變短。 2.阿利新藍和Ⅱ型膠原免疫組化染色顯示IGF-1組陽性反應(yīng),壓力組呈弱陽性而壓力復(fù)合IGF-1組介于兩者之間； 3.壓力組、IGF-1組合復(fù)合組中Sox9、和Ⅱ型膠原的基因表達水平明顯增加并高于對照組(P0.05)；壓力復(fù)合IGF-1組中Sox9、aggrecan和Ⅱ型膠原基因表達低于IGF-1組,有統(tǒng)計學(xué)差異(P0.05)。 結(jié)論 長期間斷恒定的動態(tài)壓應(yīng)力對IGF-1誘導(dǎo)前軟骨干細胞Sox9和細胞外基質(zhì)表達有抑制作用,可能會抑制IGF-1調(diào)控的軟骨分化。 第三部分ERK信號通路在前軟骨干細胞響應(yīng)力學(xué)信號傳導(dǎo)中的作用 目的 探討ERK信號通路在前軟骨干細胞響應(yīng)力學(xué)信號轉(zhuǎn)導(dǎo)中的作用。 方法 1.常規(guī)體外培養(yǎng)前軟骨干細胞并分離純化； 2.將純化后的前軟骨干細胞接種于細胞瓶中,隨機分為2組：對照組和壓力組,壓力組予以90mmHg強度的壓應(yīng)力,持續(xù)24h；每組分為兩個小組：空白組和抑制劑組,抑制劑組中加入PD98059,終濃度為20μmol/L；共4個組。 3.實時熒光定量PCR方法檢測前軟骨干細胞中Sox9和Ⅱ型膠原基因表達情況； 4. Western-blot檢測細胞Sox9和Ⅱ型膠原蛋白表達水平并進行分析。 結(jié)果 1.前軟骨干細胞在90mmHg強度的動態(tài)壓應(yīng)力培養(yǎng)條件下,壓力組pERK蛋白表達水平要高于對照組,有統(tǒng)計學(xué)差異(P0.05)；2. qPCR和Western-blot顯示Sox9和Ⅱ型膠原的基因和蛋白表達水平明顯增加并高于對照組(P0.05)；但壓應(yīng)力誘導(dǎo)的Sox9和Ⅱ型膠原的基因表達水平升高可被PD98059抑制。 3.加入PD98059后,對照組和壓力組中Sox9和Ⅱ型膠原的基因和蛋白表達水平明顯均明顯下降,但壓力組其表達下降更多,均具有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 ERK抑制劑可降低動態(tài)壓應(yīng)力誘導(dǎo)前軟骨干細胞Sox9和Ⅱ型膠原表達水平,提示ERK信號通路可能參與了細胞響應(yīng)力學(xué)信號轉(zhuǎn)導(dǎo)過程。
[Abstract]:Part 1 the effect of dynamic compressive stress on the expression of Sox9 and extracellular matrix in anterior cartilaginous stem cells
objective
The effects of intermittent dynamic compressive stress on the expression of Sox9 and cartilage specific extracellular matrix (collagen II and aggrecan) in cultured rat anterior cartilaginous stem cells were studied.
Method
1. the primary rat anterior cartilaginous stem cells were isolated and inoculated in the medium.
2. ex vivo cultured soft cells (PSCs) were purified by cell immunomagnetic beads.
3. the PSCs in vitro was randomly divided into 4 groups: control group, pressure 1 day group, pressure 3 day group and pressure 7 day group, using self designed cell pressure device to perform intermittent (1D stimulation 12h, 1D, 3D and 7D) pressure stress stimulation (90mmHg), and unstressed group as control group.
4. fluorescence real-time quantitative polymerase chain reaction was used to detect the expression of Sox9 and type II collagen and aggrecan in each group.
5. the expression levels of Sox9 and type II collagen were detected by Western-blot.
1. under inverted phase contrast microscope, the morphology of cells was mostly triangular or polygonal, and grew well.
The gene expression level of Sox9, aggrecan and type II collagen in the stress group at every time point of the 2. anterior soft diaphysis cells was higher than that of the control group (P0.05) under the condition of dynamic compressive stress culture of 90mmHg strength.
The expression of Sox9 and type II collagen in 3.1d pressure group was not significantly different from that in the control group (P0.05), and the protein expression at 3D and 7d time points increased significantly (P0.05).
4.Sox9 and type II collagen gene and protein expression levels and aggrecan gene expression increased with the increase of compressive stress stimulation time.
conclusion
Intermittent dynamic pressure stress can significantly increase the expression of Sox9, type II collagen and aggrecan in the pre rat cartilage stem cells, which may promote the differentiation of pre cartilage stem cells into chondrocytes.
The second part is the effect of dynamic compressive stress on the expression of Sox9 and extracellular matrix induced by insulin-like growth factor -1 in anterior cartilaginous stem cells.
objective
Objective to investigate the effect of dynamic compressive stress on the expression of Sox9 and extracellular matrix induced by insulin-like growth factor -1 in anterior cartilaginous stem cells.
Method
1. routinely cultured chondrocytes were isolated and purified in vitro.
2. the purified anterior soft diaphysis cells were inoculated into the cell bottle and were randomly divided into 4 groups: control group, pressure group, IGF-1 combined pressure compound IGF-1 group. Pressure group gave 90mmHg compressive stress, IGF-1 group was IGF-1, final concentration was 100ng/ml; control group did not exert pressure, nor did IGF-1;
3. the morphological changes of cells and alcian blue staining and type II collagen immunohistochemical staining were observed under light microscope.
4. real-time fluorescence quantitative PCR was used to detect the expression of Sox9 and cartilage specific extracellular matrix genes in anterior cartilage stem cells.
Result
After 1. separation, the soft diaphysis cells were triangular or spindle shaped, and the refraction was good. After induction, the cells gradually changed from spindle shape to polygonal or round, and the cell protruded gradually became shorter.
2. immunohistochemical staining of Alcian blue and type II collagen showed positive reaction in group IGF-1, while weak in pressure group and IGF-1 in pressure group.
3. in the 3. pressure group, the gene expression level of Sox9 and type II collagen in the composite group of IGF-1 was significantly increased and higher than that in the control group (P0.05), and the expression of Sox9, aggrecan and type II collagen gene in the pressure compound IGF-1 group was lower than that in the IGF-1 group, and there was a statistically significant difference (P0.05).
conclusion
The long discontinuous and constant dynamic pressure stress inhibits the expression of Sox9 and extracellular matrix of cartilage stem cells before IGF-1, and may inhibit the differentiation of cartilage regulated by IGF-1.
The third part is the role of ERK signaling pathway in response to mechanical signal transduction in anterior soft core cells.
objective
Objective to investigate the role of ERK signaling pathway in response to mechanical signal transduction in anterior soft core cells.
Method
1. routinely cultured chondrocytes were isolated and purified in vitro.
2. the purified anterior soft diaphysis cells were inoculated into the cell bottle and were randomly divided into 2 groups: the control group and the pressure group. The pressure group gave 90mmHg compressive stress and continued 24h; each group was divided into two groups: the blank group and the inhibitor group, the inhibitor group was added to the PD98059 and the final concentration was 20 mu mol/L; a total of 4 groups were found.
3. real-time fluorescent quantitative PCR was used to detect the expression of Sox9 and type II collagen in anterior cartilaginous stem cells.
4. the expression levels of Sox9 and type II collagen were detected by Western-blot.
Result
The expression level of pERK protein in stress group was higher than that of control group under dynamic pressure stress culture condition of 90mmHg strength, and there was statistical difference (P0.05). 2. qPCR and Western-blot showed that the gene and protein expression level of Sox9 and type II collagen increased obviously and higher than that of control group (P0.05), but the compressive stress induced Sox9 and type II type (P0.05) were higher than those of control group. Elevated expression level of collagen can be inhibited by PD98059.
After 3. PD98059, the gene and protein expression level of Sox9 and type II collagen in the control group and the stress group were obviously decreased, but the expression of the pressure group decreased more, all of them were statistically significant (P0.05).
conclusion
ERK inhibitors can reduce the expression of Sox9 and type II collagen in the cartilage stem cells before the dynamic compressive stress, suggesting that the ERK signaling pathway may be involved in the signal transduction of cell response mechanics.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R681.3

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本文編號：2051816