动态压应力对IGF-1诱导前软骨干细胞软骨分化的影响和ERK在细胞响应力学信号转导的作用

发布时间：2018-06-22 05:42

本文选题：动态压应力 + 前软骨干细胞　；参考：《华中科技大学》2012年博士论文

【摘要】：第一部分动态压应力对前软骨干细胞Sox9和细胞外基质表达的影响目的研究间断性动态压应力对体外培养的大鼠前软骨干细胞Sox9及软骨特异性细胞外基质(Ⅱ型胶原和aggrecan)表达的影响。方法 1.分离原代大鼠前软骨干细胞并接种于培养基中； 2.细胞免疫磁珠分选纯化体外培养的前软骨干细胞(PSCs); 3.将体外培养的PSCs随机分为4组：对照组、压力1天组、压力3天组和压力7天组,使用自行设计制备的细胞压力装置对细胞进行间断性(1d刺激12h,分别1d,3d和7d)的压应力刺激(90mmHg),未压力组为对照组； 4.采用荧光实时定量聚合酶链反应检测各组细胞的Sox9和Ⅱ型胶原和aggrecan的基因表达情况； 5. Western-blot检测细胞Sox9和Ⅱ型胶原蛋白表达水平并进行分析。结果 1.倒置相差显微镜下见细胞形态多为为三角形或多角形,生长良好。 2.前软骨干细胞在90mmHg强度的动态压应力培养条件下,压力组各时间点Sox9、aggrecan和Ⅱ型胶原的基因表达水平明显增加并高于对照组(P0.05)； 3.1d压力组Sox9和Ⅱ型胶原蛋白表达与对照组无明显差异(P0.05),3d和7d时间点的蛋白表达增加并高于对照组(P0.05)； 4.Sox9和Ⅱ型胶原基因和蛋白表达水平以及aggrecan基因表达都随着压应力刺激时间增加而增加。结论间断性动态压应力可以明显增加大鼠前软骨干细胞Sox9、Ⅱ型胶原和aggrecan的表达,可能会促进前软骨干细胞向软骨细胞分化。第二部分动态压应力对胰岛素样生长因子-1诱导前软骨干细胞Sox9和细胞外基质表达的影响目的研究动态压应力对胰岛素样生长因子-1诱导前软骨干细胞Sox9和细胞外基质表达的影响。方法 1.常规体外培养前软骨干细胞并分离纯化； 2.将纯化后的前软骨干细胞接种于细胞瓶中,随机分为4组：对照组、压力组、IGF-1组合压力复合IGF-1组。压力组予以90mmHg强度的压应力,IGF-1组予以IGF-1,终浓度为100ng/ml；对照组不施加压力,也不加入IGF-1； 3.光镜下观察细胞形态改变以及阿利新蓝染色和Ⅱ型胶原免疫组化染色； 4.实时荧光定量PCR方法检测前软骨干细胞中Sox9和软骨特异性细胞外基质基因表达情况。结果 1.分选后的前软骨干细胞呈三角形或梭形,折光性好；诱导后细胞由梭形逐渐变为多角形或类圆形,细胞突起逐渐变短。 2.阿利新蓝和Ⅱ型胶原免疫组化染色显示IGF-1组阳性反应,压力组呈弱阳性而压力复合IGF-1组介于两者之间； 3.压力组、IGF-1组合复合组中Sox9、和Ⅱ型胶原的基因表达水平明显增加并高于对照组(P0.05)；压力复合IGF-1组中Sox9、aggrecan和Ⅱ型胶原基因表达低于IGF-1组,有统计学差异(P0.05)。结论长期间断恒定的动态压应力对IGF-1诱导前软骨干细胞Sox9和细胞外基质表达有抑制作用,可能会抑制IGF-1调控的软骨分化。第三部分ERK信号通路在前软骨干细胞响应力学信号传导中的作用目的探讨ERK信号通路在前软骨干细胞响应力学信号转导中的作用。方法 1.常规体外培养前软骨干细胞并分离纯化； 2.将纯化后的前软骨干细胞接种于细胞瓶中,随机分为2组：对照组和压力组,压力组予以90mmHg强度的压应力,持续24h；每组分为两个小组：空白组和抑制剂组,抑制剂组中加入PD98059,终浓度为20μmol/L；共4个组。 3.实时荧光定量PCR方法检测前软骨干细胞中Sox9和Ⅱ型胶原基因表达情况； 4. Western-blot检测细胞Sox9和Ⅱ型胶原蛋白表达水平并进行分析。结果 1.前软骨干细胞在90mmHg强度的动态压应力培养条件下,压力组pERK蛋白表达水平要高于对照组,有统计学差异(P0.05)；2. qPCR和Western-blot显示Sox9和Ⅱ型胶原的基因和蛋白表达水平明显增加并高于对照组(P0.05)；但压应力诱导的Sox9和Ⅱ型胶原的基因表达水平升高可被PD98059抑制。 3.加入PD98059后,对照组和压力组中Sox9和Ⅱ型胶原的基因和蛋白表达水平明显均明显下降,但压力组其表达下降更多,均具有统计学意义(P0.05)。结论 ERK抑制剂可降低动态压应力诱导前软骨干细胞Sox9和Ⅱ型胶原表达水平,提示ERK信号通路可能参与了细胞响应力学信号转导过程。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R681.3

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