尿酸盐晶体通过内质网应激对成骨前体细胞生长及其分化的影响

发布时间：2018-03-14 21:08

本文选题：痛风　切入点：尿酸盐晶体　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:观察尿酸盐晶体(MSU)对成骨前体细胞(3T3-E1)生长、分化过程的影响,并探讨其与内质网应激的关系。方法:1.配置尿酸盐晶体(MSU),并在显微镜下观察尿酸盐晶体(MSU)的形态。2.取培养至对数期的成骨前体细胞3T3-E1,设置对照组(不加入MSU),实验组分别加入0.02、0.06、0.10、0.30、0.50mg/ml的MSU刺激培养48h后,用MTT法观察尿酸盐晶体MSU对成骨前体细胞增殖情况的影响。3.取对数期的成骨前体细胞3T3-E1,分别加入成骨诱导剂,设置对照组(不加入MSU),实验组分别加入0.02、0.10、0.30mg/ml的尿酸盐晶体(MSU),诱导7天后,行碱性磷酸酶染色、茜素红染色观察尿酸盐晶体(MSU)对成骨前体细胞(3T3-E1)分化为成骨细胞的情况,并测定吸光度值。4.取对数期的成骨前体细胞3T3-E1,分别加入成骨诱导剂,设置对照组(不加入MSU),实验组分别加入0.02、0.10mg/ml的尿酸盐晶体(MSU),分别诱导3天、7天、14天提取RNA样品,行RT-PCR测定基因Runx2、Osterix、Osteocalcin、Collal I的表达。5.取对数期的成骨前体细胞3T3-E1,加入成骨诱导剂,设置对照组(不加入MSU),实验组分别加入0.02、0.04、0.06、0.08、0.10mg/ml的尿酸盐晶体(MSU),分别诱导7天,提取RNA、蛋白,观察尿酸盐晶体(MSU)刺激成骨前体细胞分化为成骨细胞过程中的分泌情况及其内质网应激的关系。结果:1.显微镜下,尿酸盐晶体(MSU)有两种形态:一种呈柱状;一种呈针状,该形状与临床病理中观察的尿酸盐晶体形态极其相似。2.不同浓度的尿酸盐晶体(MSU)均能抑制成骨前体细胞的增殖,且随着尿酸盐浓度的增加抑制作用愈明显,且不同浓度间的抑制作用均具有明显的差异(p0.01)。3.碱性磷酸酶、茜素红染色显示:不同浓度的尿酸盐晶体(MSU)均能抑制成骨前体细胞(3T3-E1)向成骨细胞的分化。4.在成骨诱导下分别培养3天、7天、14天,不同浓度的尿酸盐晶体(MSU)均抑制Runx2、Osterix、Osteocalcin、Collal I的表达,且随着尿酸盐(MSU)浓度的增加作用愈加明显。5.在成骨诱导的第7天,在m RNA水平,尿酸盐晶体(MSU)能够促进Bip、CHOP、ATF4其表达;在蛋白水平,随着尿酸盐晶体(MSU)浓度的增加,也能促进Bip、CHOP、ATF4、e IF2α蛋白的表达。即尿酸盐晶体(MSU)抑制成骨前体细胞的增殖、分化、及分泌功能可能是由内质网应激介导的。结论:1.配置尿酸盐晶体(MSU),形态与痛风石临床病理图片极其相似。2.尿酸盐晶体(MSU)可抑制成骨前体细胞(3T3-E1)的增殖。3.尿酸盐晶体(MSU)可抑制成骨前体细胞(3T3-E1)向成骨细胞的分化。4.尿酸盐晶体(MSU)可抑制成骨前体细胞(3T3-E1)向成骨细胞分化过程中Runx2、Osterix的表达及骨钙素、I型胶原蛋白的分泌。5.该过程能够激活内质网应激,在m RNA和蛋白水平,促进Bip、CHOP的表达、降低ATF4、e IF2α的表达。即尿酸盐晶体(MSU)抑制成骨前体细胞(3T3-E1)的增殖、分化及分泌功能可能是通过内质网应激ATF4-e IF2α信号通路实现的。
[Abstract]:Objective: to observe the effect of uric acid crystal (MSU) on the growth and differentiation of osteoblast precursor cells (3T3-E1). To study the relationship between Urogen and endoplasmic reticulum stress. Methods: 1. Configure the uric acid crystal and observe the morphology of MSU under microscope. Take 3T3-E1 from osteoblast precursor cells cultured to logarithmic phase and set up control group (not adding MSUU). Group A was incubated with 0.02mg / ml of MSU for 48h, 0.10g / ml 0.10g / ml, 0.30mg / ml MSU, 0.30mg / ml MSU for 48h, respectively. The effect of uric acid crystal MSU on the proliferation of osteoblast precursor cells was observed by MTT. 3T3-E1 was taken from the osteoblast precursor cells in logarithmic phase, and the osteoblast inducer was added respectively. After 7 days of induction, alkaline phosphatase staining and alizarin red staining were used to observe the differentiation of osteoblasts into osteoblasts. The absorbance value was determined. 3T3-E1 was taken from the osteoblast precursor cells in logarithmic phase, and the osteogenic inducer was added respectively. The control group was set up. The control group was treated with 0.02mg / ml of uric acid crystal 0.10mg / ml, respectively, and the RNA samples were extracted from the cells for 3 days, 7 days and 14 days, respectively. RT-PCR was used to determine the expression of the gene Runx2OsterixOsterixOsteocalcinia Collal I. 5. 3T3-E1 was taken from the osteoblast precursor cells in logarithmic phase, and the osteoblast inducer was added to the control group. The control group was set up as control group (without MSUU, the experimental group was treated with 0.02OF0.04 + 0.080.10 mg / ml of uric acid crystal) for 7 days, the RNAs, proteins and proteins were extracted, respectively. To observe the secretion of osteoblast precursor cells and the relationship between the endoplasmic reticulum stress and the process of stimulating osteoblast precursor cells to differentiate into osteoblasts. Results: under the microscope, there are two forms of uric acid crystals: one is columnar, the other is needle-like. The shapes of these crystals were similar to those observed in clinicopathology. (2) different concentrations of uric acid crystals (MSU) could inhibit the proliferation of osteoblast precursor cells, and the inhibitory effect was more obvious with the increase of uric acid concentration. The inhibitory effects of different concentrations of alkaline phosphatase were significantly different from those of P0.01U. 3. alkaline phosphatase, alkaline phosphatase (ALP), alkaline phosphatase (ALP), Alizarin red staining showed that different concentrations of uric acid crystals (MSU) could inhibit the differentiation of osteoblasts from osteoblasts 3T3-E1) into osteoblasts. 4. Cultured for 3 days, 7 days and 14 days, different concentrations of uric acid crystals (MSU) inhibited the expression of Runx2Osterix Osteocalcinia Collal I. On the 7th day after osteogenesis induction, at m RNA level, uric acid crystals could promote the expression of Bip-CHOP-ATF4, and at the protein level, with the increase of the concentration of uric acid crystals, the expression of Bip-CHOP-ATF4 was enhanced at the level of m RNA, and at the protein level, it increased with the increase of the concentration of uric acid crystals, and the expression of Bip-CHOPP-ATF4 was increased at the level of m RNA. It can also promote the expression of Bip-CHOPOP-ATF4- IF2 伪 protein, that is, uric acid crystal can inhibit the proliferation and differentiation of osteoblast precursor cells. The secretory function and secretion function may be mediated by endoplasmic reticulum stress. Conclusion 1.Conclusion 1. The configuration of uric acid crystal is very similar to that of gout clinicopathologic picture. (MSU) can inhibit the proliferation of osteoblast precursor cell (3T3-E1) .3.The crystal of uric acid salt can inhibit the proliferation of osteoblast precursor cell (3T3-E1). MSU) can inhibit the differentiation of osteoblasts from osteoblasts 3T3-E1) .4. uric acid crystals can inhibit the expression of Runx2Osterix and the secretion of osteocalcin type I collagen during the differentiation of osteoblast cells into osteoblasts. This process can inhibit the expression of Osterix and the secretion of osteocalcin type I collagen in the process of osteoblast differentiation. Activate endoplasmic reticulum stress, At the level of m RNA and protein, the expression of Bipnchop was promoted, and the expression of ATF4 IF2 伪 was decreased. The inhibition of proliferation, differentiation and secretory function of osteoblast precursor cells (3T3-E1) may be realized by endoplasmic reticulum stress ATF4-e IF2 伪 signaling pathway.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.7

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