本文選題：痛風(fēng)　切入點：尿酸鹽晶體　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:觀察尿酸鹽晶體(MSU)對成骨前體細胞(3T3-E1)生長、分化過程的影響,并探討其與內(nèi)質(zhì)網(wǎng)應(yīng)激的關(guān)系。方法:1.配置尿酸鹽晶體(MSU),并在顯微鏡下觀察尿酸鹽晶體(MSU)的形態(tài)。2.取培養(yǎng)至對數(shù)期的成骨前體細胞3T3-E1,設(shè)置對照組(不加入MSU),實驗組分別加入0.02、0.06、0.10、0.30、0.50mg/ml的MSU刺激培養(yǎng)48h后,用MTT法觀察尿酸鹽晶體MSU對成骨前體細胞增殖情況的影響。3.取對數(shù)期的成骨前體細胞3T3-E1,分別加入成骨誘導(dǎo)劑,設(shè)置對照組(不加入MSU),實驗組分別加入0.02、0.10、0.30mg/ml的尿酸鹽晶體(MSU),誘導(dǎo)7天后,行堿性磷酸酶染色、茜素紅染色觀察尿酸鹽晶體(MSU)對成骨前體細胞(3T3-E1)分化為成骨細胞的情況,并測定吸光度值。4.取對數(shù)期的成骨前體細胞3T3-E1,分別加入成骨誘導(dǎo)劑,設(shè)置對照組(不加入MSU),實驗組分別加入0.02、0.10mg/ml的尿酸鹽晶體(MSU),分別誘導(dǎo)3天、7天、14天提取RNA樣品,行RT-PCR測定基因Runx2、Osterix、Osteocalcin、Collal I的表達。5.取對數(shù)期的成骨前體細胞3T3-E1,加入成骨誘導(dǎo)劑,設(shè)置對照組(不加入MSU),實驗組分別加入0.02、0.04、0.06、0.08、0.10mg/ml的尿酸鹽晶體(MSU),分別誘導(dǎo)7天,提取RNA、蛋白,觀察尿酸鹽晶體(MSU)刺激成骨前體細胞分化為成骨細胞過程中的分泌情況及其內(nèi)質(zhì)網(wǎng)應(yīng)激的關(guān)系。結(jié)果:1.顯微鏡下,尿酸鹽晶體(MSU)有兩種形態(tài):一種呈柱狀;一種呈針狀,該形狀與臨床病理中觀察的尿酸鹽晶體形態(tài)極其相似。2.不同濃度的尿酸鹽晶體(MSU)均能抑制成骨前體細胞的增殖,且隨著尿酸鹽濃度的增加抑制作用愈明顯,且不同濃度間的抑制作用均具有明顯的差異(p0.01)。3.堿性磷酸酶、茜素紅染色顯示:不同濃度的尿酸鹽晶體(MSU)均能抑制成骨前體細胞(3T3-E1)向成骨細胞的分化。4.在成骨誘導(dǎo)下分別培養(yǎng)3天、7天、14天,不同濃度的尿酸鹽晶體(MSU)均抑制Runx2、Osterix、Osteocalcin、Collal I的表達,且隨著尿酸鹽(MSU)濃度的增加作用愈加明顯。5.在成骨誘導(dǎo)的第7天,在m RNA水平,尿酸鹽晶體(MSU)能夠促進Bip、CHOP、ATF4其表達;在蛋白水平,隨著尿酸鹽晶體(MSU)濃度的增加,也能促進Bip、CHOP、ATF4、e IF2α蛋白的表達。即尿酸鹽晶體(MSU)抑制成骨前體細胞的增殖、分化、及分泌功能可能是由內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的。結(jié)論:1.配置尿酸鹽晶體(MSU),形態(tài)與痛風(fēng)石臨床病理圖片極其相似。2.尿酸鹽晶體(MSU)可抑制成骨前體細胞(3T3-E1)的增殖。3.尿酸鹽晶體(MSU)可抑制成骨前體細胞(3T3-E1)向成骨細胞的分化。4.尿酸鹽晶體(MSU)可抑制成骨前體細胞(3T3-E1)向成骨細胞分化過程中Runx2、Osterix的表達及骨鈣素、I型膠原蛋白的分泌。5.該過程能夠激活內(nèi)質(zhì)網(wǎng)應(yīng)激,在m RNA和蛋白水平,促進Bip、CHOP的表達、降低ATF4、e IF2α的表達。即尿酸鹽晶體(MSU)抑制成骨前體細胞(3T3-E1)的增殖、分化及分泌功能可能是通過內(nèi)質(zhì)網(wǎng)應(yīng)激ATF4-e IF2α信號通路實現(xiàn)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 偽 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 偽 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 偽 signaling pathway.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R589.7

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