机械牵伸对大鼠TSCs MGF表达的影响及其对TSCs分化增殖的调控作用

发布时间：2018-04-29 00:00

本文选题：机械牵伸 + 力生长因子　；参考：《第三军医大学》2017年硕士论文

【摘要】：前言肌腱病(Tendinopathy)是一种常见的多种因素引起的肌腱功能障碍的综合征,其治疗方式多样,但总体治疗效果有限,究其原因在于肌腱病的发病机制尚未完全阐明[1-3]。肌腱干细胞(tendon stem cells,TSCs)作为肌腱来源的特殊干细胞在维持肌腱正常生理功能及肌腱病病理生理过程中都扮演重要角色[4,5]。如何调控TSCs正常分化为肌腱细胞修复肌腱微损伤,对于提高肌腱病的防治水平具有重要意义。目前已研究证实TSCs可受多种因素调控,如机械应力[6-11]、炎症因子[11-15]、皮质醇激素[16]、细胞因子[14,17-21]等。在众多调控因素中,机械应力是肌腱病发病始动因素,对于TSCs调控作用重要性不言而喻。力生长因子(Mechano growth factor,MGF),也称为机械生长因子,广泛存在于骨、骨骼肌、肌腱、脑等组织[22-25],是在受到机械应力、电刺激或者组织损伤时产生的一种胰岛素样生长因子-1的选择性剪接异构体[24-27]。自从MGF被发现以来,其独特的E肽结构及其特有的功能吸引着众多学者的关注,现已证实MGF对多种细胞的增殖、迁移及分化有重要作用[28-36]。另外有研究发现肌腱组织经机械应力刺激后,MGF表达会显著升高,且反应时间明显早于IGF-1[37-41]。然而,TSCs作为肌腱病发病过程中的“明星细胞”,在受到机械牵伸应力作用后MGF表达水平如何变化以及MGF对其增殖分化及迁移能力是否起到一定的调节作用却未见相关报道。同时,机械牵伸和MGF对于TSCs的分化调控是单独作用还是共同作用?是相互协同作用还是拮抗作用呢?这一系列问题均亟待解决。因此,本课题研究目的主要是:1.验证机械牵伸对TSCs分化的调控及对MGF表达的影响;2.明确MGF对TSCs增殖、分化、迁移的影响及调控机制;3.阐明MGF在机械牵伸条件下对TSCs分化调控的影响及其机制。方法第一部分:进行大鼠TSCs分离培养鉴定;通过Real-time PCR、Western Blot分别从转录和翻译水平检测不同机械牵伸条件处理后TSCs中成骨、成脂及成肌腱分化的相关基因mRNA及蛋白表达水平,同时检测TSCs中MGF mRNA表达水平。第二部分:通过CCK8及实时无标记动态细胞分析技术检测TSCs经不同浓度MGF处理后,细胞增殖变化情况;通过Transwell、划痕试验及激光共聚焦显微镜检测不同浓度MGF处理对TSCs迁移能力及细胞骨架变化的影响;通过Western Blot检测TSCs经不同浓度MGF及MAPK/ERK通路抑制剂PD98059处理后,成腱分化相关基因蛋白表达变化。第三部分:通过Real-time PCR和Western Blot分别检测机械牵伸与MGF共同处理TSCs后,成肌腱、成骨及成脂三系分化相关基因mRNA及蛋白相对表达变化。结果第一部分:1.4%1Hz组牵伸48h成腱分化指标TNC和成骨分化指标RUNX2相对表达量较静态培养组均显著升高(P0.05);4%2Hz组各牵伸时间点TNC相对表达量较静态培养组均明显升高(P0.05),同时牵拉24h时CEBPα表达受到显著抑制(P0.05),RUNX2相对表达量较对照组无明显差异(P0.05);8%1Hz组牵拉24h RUNX2表达水平显著高于静态培养组(P0.05),同时成肌腱分化及成脂肪分化相关基因表达较静态培养组无显著变化(P0.05);8%2Hz组牵拉48h时CEBPα表达水平显著高于静态培养组(P0.05),同时RUNX2相对表达量较静态培养组无显著差异(P0.05),TNC相对表达量较静态培养组显著降低(P0.05)。2.不同条件机械牵伸会引起TSCs中MGF mRNA相对表达量出现不同水平的增加。4%2Hz及8%1Hz组牵伸12小时即可见MGF mRNA水平显著升高(P0.05),牵伸24h时MGF mRNA表达量达到顶峰,差异有统计学意义(P0.05)。第二部分:1.CCK-8结果显示:TSCs经5ng/ml MGF处理后,24小时开始OD值即出现显著升高,与其余各组相比差异有统计学意义(P0.05),但其余各组间吸光度无明显差异(P0.05)。RTCA结果显示:经1ng/ml及5ng/ml MGF处理后,TSCs细胞指数较其余各组均升高,差异有统计学意义(P0.05),但两组间无显著性差异(P0.05);经10ng/ml、25ng/ml和50ng/ml MGF及5ng/ml MGF+10μg/ml PQ401处理后,细胞指数较对照组显著降低(P0.05);5ng/mlMGF+50μM PD98059组较其余各组,细胞指数明显降低,差异有统计学意义(P0.05)。2.划痕实验结果显示:经1ng/ml、5ng/ml和10ng/ml MGF处理后,TSCs迁移能力较对照组均明显加快,其中5ng/ml MGF处理组TSCs的迁移速度最快。Transwell结果显示:TSCs经过不同浓度MGF处理后,TSCs迁移数量均有显著增加,OD值显著升高(P0.05),其中以5ng/ml和7.5ng/ml组最为明显;另外5ng/ml MGF+50μM PD98059组与5ng/ml MGF组相比,TSCs迁移数量明显减少,OD值显著降低,差异有统计学意义(P0.05);而5ng/ml MGF+10μg/ml PQ401组细胞形态发生明显改变,细胞迁移数量最少,OD值最低。激光共聚焦显微镜观察可见:经0、1.0、2.5、5.0、7.5及10.0ng/ml MGF处理的TSCs,细胞骨架连续性良好,荧光强,细胞成典型长梭形,肌动蛋白纤维排列规则;经高浓度(25ng/ml及50ng/ml)MGF处理后,TSCs形态成不规则形,细胞骨架断裂,排列紊乱,荧光强度变弱;经10μg/ml PQ401处理后,TSCs细胞形态出现明显改变,细胞骨架断裂,呈颗粒样,荧光强度极弱。3.Western Blot结果显示TSCs经5ng/ml及10ng/mlMGF处理72h后,TNC、SCX蛋白表达量均较对照组显著升高,差异有统计学意义(P0.05)。而经1ng/ml及25ng/ml MGF处理后,蛋白表达较对照组无明显差异(P0.05)。第三部分:1.Real-time PCR结果显示:TSCs经0ng/ml、2.5ng/ml以及5.0ng/ml MGF处理后,在4%2Hz机械牵伸条件下牵伸24h,SCX和TNC mRNA相对表达较静态培养组明显升高,差异有统计学意义(P0.05)。RUNX2、DLX5以及AP2、PPARγmRNA相对表达量较静态培养组明显降低,差异有统计学意义(P0.05)。50μM PD98059+5ng/ml MGF组TSCs经4%2Hz机械牵伸24h后,全部基因的mRNA相对表达量较未添加抑制剂的5ng/ml MGF组变化倍数显著降低,差异有统计学意义(P0.05)。2.Western blot结果显示:TSCs经0、2、5ng/ml MGF处理后,在4%2Hz机械牵伸下牵拉24h,TNC蛋白相对表达量较静态培养组升高,差异有统计学意义(P0.05)。RUNX2以及CEBPα蛋白相对表达量较静态培养组显著降低,差异有统计学意义(P0.05)。50μM PD98059+5ng/ml MGF组TSCs经4%2Hz机械牵伸24h后,全部分化相关基因的蛋白相对表达量较未添加抑制剂的5.0ng/mlMGF组变化倍数显著降低,差异有统计学意义(P0.05)。结论第一部分:不同条件的机械牵伸会引起TSCs向不同方向分化。4%2Hz 24h为诱导成肌腱分化的最佳牵伸条件,8%1Hz 24h为诱导成骨分化的最佳牵伸条件,8%2Hz 48h为诱导成脂肪分化的最佳牵伸条件。机械牵伸可上调TSCs中MGF表达水平,且与成肌腱分化相关。第二部分:MGF可增强TSCs迁移及增殖能力,并且可诱导TSCs向肌腱细胞方向分化,其中对增殖和迁移促进作用与MAPK/ERK通路激活相关。第三部分:机械牵伸与MGF协同促进TSCs向肌腱方向分化,抑制成脂及成骨分化。机械牵伸条件下MGF对TSCs分化的诱导调控是通过激活MAPK/ERK通路发挥作用。
[Abstract]:Preface tendons disease (Tendinopathy) is a common cause of a variety of factors caused by tendon dysfunction syndrome, its treatment is diverse, but the overall treatment effect is limited, the reason is that the pathogenesis of tendon disease is not fully elucidated by the [1-3]. tendon stem cell (tendon stem cells, TSCs) as a tendon source of special stem cells in maintenance The normal physiological function of tendon and the pathophysiological process of tendon disease all play an important role [4,5]. how to regulate the normal differentiation of TSCs into tendon cells to repair the tendon micro injury, which is of great significance for the improvement of the prevention and treatment of tendon disease. At present, it has been proved that TSCs can be regulated by many factors, such as mechanical stress [6-11], inflammatory factor [11-15], skin [16], cytokine [14,17-21] and so on. In many regulatory factors, mechanical stress is the starting factor of the pathogenesis of tendon disease, and it is self-evident for the importance of TSCs regulation. Mechano growth factor (MGF), also known as mechanical growth factor, is widely found in bone, skeletal muscle, tendon, brain and other tissues, which are in the machine. The selective splicing isomer of an insulin-like growth factor -1, produced by mechanical stress, electrical stimulation or tissue damage, has attracted many scholars' attention since the discovery of MGF, and its unique E peptide structure and its unique functions have been shown to play an important role in the proliferation, migration and differentiation of various cells, [28-36]., and [28-36].. It was found that the expression of MGF increased significantly after the mechanical stress stimulation, and the reaction time was significantly earlier than that of IGF-1[37-41].. TSCs was the "star cell" in the pathogenesis of tendon disease, and how the expression of MGF was changed after the mechanical drafting stress was applied and whether MGF played a role in the proliferation and differentiation and migration ability of MGF. No related reports have been reported. At the same time, mechanical drafting and MGF play a separate role or a common effect on TSCs differentiation, are they synergistic or antagonistic? This series of problems need to be solved. Therefore, the main purpose of this study is to verify the regulation of TSCs differentiation by mechanical drafting and the expression of MGF. The influence of MGF on the proliferation, differentiation, migration and regulation mechanism of TSCs, and 3. to elucidate the effect and mechanism of MGF on the regulation of TSCs differentiation under the condition of mechanical drafting. Method first part: TSCs isolation and culture identification of rats, and Real-time PCR, Western Blot respectively from the transcriptional and Translation levels to detect different mechanical drafting conditions. Bone formation, mRNA and protein expression levels of lipid and tendon differentiation in TSCs, and the level of MGF mRNA expression in TSCs. The second part: CCK8 and real-time unmarked dynamic cell analysis were used to detect the proliferation of TSCs after different concentrations of MGF, and through Transwell, scratch test and confocal laser confocal The effects of different concentrations of MGF on the mobility and cytoskeleton changes of TSCs were detected by the microscope. The expression of gene protein expression related to the differentiation of the tendon was detected by Western Blot after the treatment of TSCs with different concentrations of MGF and MAPK/ERK pathway inhibitor PD98059. The third part: the mechanical drafting was detected by Real-time PCR and Western Blot, respectively. After the same treatment with TSCs, the relative expression of mRNA and protein in the three lineage differentiation related genes of the tendon, osteogenesis and fat formation. Results the first part: the relative expression of TNC and the osteogenic differentiation index RUNX2 in the 1.4%1Hz group was significantly higher than that in the static culture group (P0.05), and the relative expression of TNC in the 4% 2Hz group was more than that in the static culture. The expression of CEBP alpha was significantly inhibited (P0.05) and the relative expression of RUNX2 was significantly higher than that in the control group (P0.05), while the RUNX2 expression level of 24h in 8%1Hz group was significantly higher than that in the static culture group (P0.05), and the expression of CEBP in 8%1Hz group was significantly higher than that in the static culture group (P0.05). There was no significant change in the expression of the adult tendon differentiation and the expression of the adipose differentiation related genes in the 8%1Hz group than that in the static culture group (P). 0.05); in group 8%2Hz, the expression level of CEBP a was significantly higher than that in the static culture group (P0.05), while the relative expression of RUNX2 was not significantly different than that in the static culture group (P0.05). The relative expression of TNC was significantly lower than that in the static culture group (P0.05). (P0.05) the different conditions of the mechanical drafting would lead to the increase of the relative expression of MGF mRNA in TSCs. The level of MGF mRNA increased significantly (P0.05) for 12 hours in group Z and 8%1Hz, and the expression of MGF mRNA reached the peak at the time of drawing 24h. The difference was statistically significant (P0.05). The second part: 1.CCK-8 results showed that TSCs after 24 hours, the OD value began to rise significantly, and the difference was statistically significant compared with the rest of the other groups. However, there was no significant difference in absorbency between the other groups (P0.05).RTCA results showed that after 1ng/ml and 5ng/ml MGF treatment, the TSCs cell index was higher than the rest of the other groups, and the difference was statistically significant (P0.05), but there was no significant difference between the two groups (P0.05). Compared with the other groups, the cell index of 5ng/mlMGF+50 M PD98059 group decreased significantly, and the difference was statistically significant (P0.05).2. scratch test results showed that after 1ng/ml, 5ng/ml and 10ng/ml MGF treatment, TSCs migration ability was significantly faster than that of the control group. The results showed that after the treatment of TSCs with different concentrations of MGF, the number of TSCs migration increased significantly, and the OD value increased significantly (P0.05), among which the 5ng/ml and 7.5ng/ml groups were the most obvious. In addition, the number of migration in 5ng/ml MGF+50 mu M PD98059 group was significantly reduced and the OD value decreased significantly. The cell morphology of the g/ml PQ401 group changed obviously, the number of cell migration was the least, and the OD value was the lowest. The confocal laser scanning microscope showed that the cytoskeleton was good, the cytoskeleton was good, the fluorescence was strong, the cell became the typical long shuttle shape, the myocutaneous fibrin was arranged regularly, and the high concentration (25ng/ml and 50ng/ml) MGF (25ng/ml and 50ng/ml) MGF were observed by laser confocal microscopy. After treatment, the morphology of TSCs was irregular, the cytoskeleton was broken, the arrangement was disorderly and the fluorescence intensity was weak. After 10 u g/ml PQ401 treatment, the morphology of TSCs cells changed obviously, the cytoskeleton broke, the particle like, the fluorescence intensity was very weak.3.Western Blot results showed that TSCs was treated with 5ng/ ml and 10ng/mlMGF 72h, TNC, the expression of TNC and protein expression were both compared to the control. The difference was statistically significant (P0.05). The protein expression was not significantly different from the control group after 1ng/ml and 25ng/ml MGF treatment (P0.05). The third part: 1.Real-time PCR results showed that TSCs was drafted under the condition of 0ng/ml, 2.5ng/ml and 5.0ng/ml MGF. The difference was statistically significant (P0.05).RUNX2, DLX5 and AP2, and the relative expression of PPAR gamma mRNA was significantly lower than that in the static culture group (P0.05), the difference was statistically significant (P0.05).50 u M PD98059+5ng/ml MGF group after TSCs via mechanical draft, the relative expression of all genes was more than that of the non inhibitor. The difference was statistically significant (P0.05).2.Western blot results showed that after 0,2,5ng/ml MGF treatment, TSCs was pulled 24h under 4%2Hz mechanical drafting, and the relative expression of TNC protein was higher than that in the static culture group, and the difference was statistically significant (P0.05).RUNX2 and the relative expression of CEBP alpha protein was significantly lower than that in the static culture group, and the difference was found. Statistical significance (P0.05).50 mu M PD98059+5ng/ml MGF group TSCs after 4%2Hz mechanically drafted 24h, the relative expression of all the related genes was significantly lower than that of the 5.0ng/mlMGF group without the inhibitor, and the difference was statistically significant (P0.05). Conclusion the first part: the mechanical drafting of different conditions will cause TSCs to different directions. Differentiation.4%2Hz 24h is the best drawing condition for inducing tendon differentiation, 8%1Hz 24h is the best drawing condition for inducing osteogenic differentiation, 8%2Hz 48h is the best drawing condition for inducing adipose differentiation. Mechanical drawing can increase the level of MGF expression in TSCs, and is related to the differentiation of tendon. The second part: MGF can enhance the ability of TSCs migration and proliferation, and It can induce the differentiation of TSCs to tendon cells, and the promotion of proliferation and migration is related to the activation of MAPK/ERK pathway. The third part: the synergistic effect of mechanical drafting and MGF to promote the direction differentiation of TSCs to the tendons and inhibit the formation of lipid and osteogenic differentiation. The induced regulation of MGF to TSCs differentiation under the condition of mechanical drawing is to play the role by activating the MAPK/ERK pathway.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R686.1

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