促甲状腺激素对小鼠原代软骨细胞增殖和分化的影响

发布时间：2019-02-15 08:59

【摘要】：目的亚临床甲状腺功能减退症(subclinicalhypothyroidism,SCH),简称亚甲减,是临床上常见的内分泌代谢性疾病。亚甲减患者虽然甲状腺激素水平在正常范围内,但是血清中促甲状腺激素(thyroid stimulating hormone,TSH)水平升高,偏离正常参考范围。已有研究证实,高TSH与甲状腺功能减退的小鼠骨骼发育异常有关,然而其细胞机制仍不清楚。本研究旨在探讨:TSH能否不依赖甲状腺激素独立作用于软骨细胞,即TSH刺激对软骨细胞增殖和分化的直接影响以及自噬在该过程中的可能作用。方法1.小鼠原代软骨细胞(PMCs)的培养及处理:取小鼠膝关节,去除周围结缔组织,将软骨组织与深层软骨下骨仔细分离,缓冲液冲洗,剪碎软骨组织后用0.1%胶原酶消化18.5 h,离心去上清,依次经100μm和40μm滤网过滤。洗涤后用含10%胎牛血清(FBS)、1%双抗和100μM维生素C的培养液分散培养。隔天换液,待细胞融合度达到70%-80%时,换成含1%FBS的培养基分别以不同处理培养48h,尽可能减少血清中脂蛋白及各种激素对实验的干扰作用。2.用10 mU/ml bTSH处理PMCs特定的时间,设置对照组。用CCK8和EdU试剂盒检测细胞增殖活性。3.用JC-1检测细胞线粒体膜电位的改变来观察细胞凋亡情况。应用Western blot检测促凋亡因子Bax和Bcl-2的表达水平。4.用Western blot和免疫荧光染色检测自噬相关标志物如LC3、Beclin-1和p62,Western blot mTOR、p-AMPK和AMPK,用透射电镜检测PMCs的自噬情况。5.采用Alcian blue染色检测亚甲减小鼠和正常小鼠软骨蛋白聚糖的表达水平。通过免疫荧光染色检测collagen ll和collagen l的表达情况。6.通过RT-PCR检测不同整合素亚基的表达情况。结果1.CCK-8细胞增值活性检测发现,细胞活性在高浓度TSH(10 mU/ml)刺激24h后下降明显,低浓度组(1 mU/ml)在TSH刺激72h后,细胞增殖活性也比对照组产生显著降低(P0.05),并持续下降。EdU细胞增殖检测结果也表明,10mU/mlTSH刺激48h显著降低PMCs增殖能力(P0.01),对照组有增值活性的细胞百分比为TSH刺激组的6倍。2.流式结果显示,TSH刺激增加线粒体膜电位的去极化,细胞发生凋亡。通过Western blot实验进一步验证,TSH刺激使促凋亡因子BAX蛋白表达增加(P0.01),Bcl-2表达水平降低(P0.01),Bcl-2与BAX蛋白表达水平的比值降低(P0.05)。3.TSH刺激降低软骨细胞自噬水平。Western blot检测结果表明,LC3 11(P0.05)和Beclin-1(P0.01)的表达水平降低,且在免疫荧光试验的结果中得到了验证。透射电镜也得出相同结论。4.Western blot和免疫荧光检测得出TSH刺激使p62表达水平明显增加的结论。免疫共聚焦结果显示,高表达p62的细胞伴随有凋亡的细胞核,表明TSH刺激抑制自噬,可能进一步诱导凋亡的发生。5.Western blot检测发现mTOR/AMPK通路在TSH刺激后产生损伤,提示TSH可能通过损伤自噬上游的mTOR/AMPK通路,从而抑制自噬水平。6.TSH抑制2型胶原蛋白和蛋白聚糖的合成,同时伴随整合素亚基表达形式的变化。结论1.TSH刺激抑制软骨细胞增殖,增加Bcl-2信号介导的细胞凋亡。2.TSH刺激自噬标志物beclin-1和LC3II的表达减少,伴随p62累积而抑制自噬潮。3.TSH抑制collagen 11和蛋白聚糖的合成,改变整合素亚基的表达,加速软骨细胞分化过程。
[Abstract]:Objective To study the subclinical hypothyroid function (SCH), which is a common endocrine and metabolic disorder in the clinic. Although the thyroid hormone level is in the normal range, the level of the thyroid stimulating hormone (TSH) in the serum is raised and the normal reference range is deviated. It has been demonstrated that high TSH is associated with an abnormal bone development in a mouse with hypothyroid function, but its cellular mechanism remains unclear. The purpose of this study is to investigate whether TSH does not depend on the independent effect of thyroid hormone on the chondrocytes, that is, the direct effect of TSH stimulation on the proliferation and differentiation of the chondrocytes and the possible effects of autophagy in the process. Method 1. Culture and treatment of mouse primary chondrocyte (PMCs): taking the knee of the mouse, removing the surrounding connective tissue, carefully separating the cartilage tissue from the subchondral bone, washing the buffer solution, and digesting the cartilage tissue with 0.1% collagenase for 18-5 h, and centrifuging to remove supernatant. The filter is filtered through a filter screen of 100. m u.m and 40. m After washing, the culture was dispersed with a culture solution containing 10% fetal bovine serum (FBS), 1% double anti-and 100.mu. M vitamin C. when the cell fusion degree reaches 70-80%, the culture medium with 1% FBS is used for culturing for 48hr in different treatment, so as to minimize the interference effect of the lipoprotein and various hormones on the experiment in the serum. The specific time of the PMCs was treated with 10 mU/ ml of bTSH and the control group was set. Cell proliferation activity was tested with the CCK8 and the EdU kit. The changes of mitochondrial membrane potential of the cells were detected by JC-1 to observe the cell apoptosis. The expression level of the pro-apoptotic factor Bax and Bcl-2 was detected by Western blot. The autophagy of PMCs was detected by Western blot and immunofluorescence staining, such as LC3, Beclin-1 and p62, Western blot mTOR, p-AMPK and AMPK. The expression level of subungual and normal mouse cartilage proteoglycans was detected by Alcian blue staining. The expression of the collection 11 and the collection l was detected by immunofluorescence staining. The expression of different integrin subunits was detected by RT-PCR. Results 1. The value-added activity of CCK-8 cells showed that the cell activity decreased after the stimulation of high-concentration TSH (10 mU/ ml) for 24h, and the cell proliferation activity was significantly lower in the low-concentration group (1 mU/ ml) than in the control group (P0.05). The results of the proliferation of EU cells also showed that the proliferation ability of PMCs was significantly reduced by the stimulation of 10mU/ ml of TSH (P 0.01), and the percentage of cells with value-added activity in the control group was 6times that of the TSH stimulation group. The results showed that the stimulation of TSH increased the depolarization of the mitochondrial membrane potential and the apoptosis of the cells. The results of Western blot showed that the expression of the pro-apoptotic factor BAX protein was increased (P0.01), the level of Bcl-2 expression was decreased (P0.01), and the ratio of Bcl-2 to BAX protein expression was decreased (P0.05). The results of Western blot showed that the expression level of LC3 11 (P0.05) and Beclin-1 (P0.01) was reduced, and the results of the immunofluorescence test were verified. The same conclusion was also obtained by transmission electron microscope. The results showed that the cells with high expression of p62 were accompanied by apoptotic nuclei, indicating that TSH stimulation inhibited autophagy and could further induce apoptosis. 5. Western blot showed that the mTOR/ AMPK pathway was damaged after the stimulation of TSH, suggesting that the TSH may pass through the mTOR/ AMPK pathway upstream of the autophagy. 6. TSH inhibits the synthesis of type 2 collagen and proteoglycan, and is accompanied by the change of the expression of integrin subunit. Conclusion 1. TSH stimulates the proliferation of chondrocytes and increases the apoptosis of Bcl-2 signaling.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R684.3

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