ZIP1蛋白對酒精誘導(dǎo)的兔BMSCs成脂基因PPARγ、aP2的影響研究
發(fā)布時(shí)間：2018-09-08 18:19
【摘要】：目的:酒精性股骨頭壞死是因?yàn)殚L期大量酗酒而引起的一種非創(chuàng)傷性的股骨頭壞死,是以中青年男性患者居多的骨科常見疾病。脂質(zhì)紊亂及脂肪分化異常是導(dǎo)致酒精性股骨頭壞死的重要機(jī)制,鋅鐵調(diào)控蛋白1(ZIP1)作為鋅離子的轉(zhuǎn)運(yùn)體,能將鋅離子轉(zhuǎn)運(yùn)到細(xì)胞內(nèi),促進(jìn)骨髓間充質(zhì)干細(xì)胞的成骨分化,但ZIP1蛋白是否能通過影響骨髓間充質(zhì)干細(xì)胞分化,從而抑制脂肪細(xì)胞的形成尚不明確,本實(shí)驗(yàn)將探討ZIP1蛋白對兔骨髓間充質(zhì)干細(xì)胞成脂分化的影響,從而為酒精性股骨頭壞死的防治提供思路。方法:1、取兔骨髓間充質(zhì)干細(xì)胞進(jìn)行復(fù)蘇,再用完全培養(yǎng)基培養(yǎng),傳代培養(yǎng)后獲得狀態(tài)良好的骨髓間充質(zhì)干細(xì)胞,定向誘導(dǎo)骨髓間充質(zhì)干細(xì)胞并進(jìn)行油紅“O”染色,分析骨髓間充質(zhì)干細(xì)胞成脂分化能力;設(shè)計(jì)合成ZIP1siRNA1、2、3三條序列,并兔轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞,RT-PCR檢測骨髓間充質(zhì)干細(xì)胞中ZIP1m RNA表達(dá)量,篩選ZIP1siRNA最佳序列;同時(shí)設(shè)計(jì)合成ZIP1目的片段,構(gòu)建ZIP1表達(dá)載體,予FD Kpn I和FD Eco R限制性內(nèi)切酶進(jìn)行雙切驗(yàn)證和重組載體測序,將成功構(gòu)建的重組載體轉(zhuǎn)染兔骨髓間充質(zhì)干細(xì)胞。2、先將骨髓間充質(zhì)干細(xì)胞分為9組,即正常對照組、模型組(0.03mol/L酒精組、0.09mol/L酒精組、0.15 mol/L酒精組、0.21mol/L酒精組)、實(shí)驗(yàn)組(0.03mol/L酒精+ZIP1siRNA組、0.09mol/L酒精+ZIP1siRNA組、0.15mol/L酒精+ZIP1siRNA組、0.21mol/L酒精+ZIP1siRNA組)。正常對照組不做任何處理正常培養(yǎng),模型組分別用含有0.03mol/L、0.09mol/L、0.15mol/L、0.21mol/L酒精濃度的培養(yǎng)基進(jìn)行培養(yǎng),實(shí)驗(yàn)組予轉(zhuǎn)染ZIP1siRNA序列后分別用含有0.03mol/L、0.09mol/L、0.15mol/L、0.21mol/L酒精濃度的培養(yǎng)基進(jìn)行培養(yǎng),每組均培養(yǎng)6h。采用試劑盒檢測各組細(xì)胞內(nèi)甘油三酯含量,記錄甘油三酯含量變化,Western blotting分別檢測各組骨髓間充質(zhì)干細(xì)胞中PPARγ,aP2蛋白表達(dá)的含量。3、將骨髓間充質(zhì)干細(xì)胞分為3組,正常對照組、ZIP1表達(dá)組、ZIP1siRNA組。正常對照組未經(jīng)任何處理正常培養(yǎng),表達(dá)轉(zhuǎn)染組予ZIP1表達(dá)載體轉(zhuǎn)染后正常培養(yǎng),ZIP1siRNA轉(zhuǎn)染組予ZIP1siRNA序列轉(zhuǎn)染后正常培養(yǎng),每組均培養(yǎng)6h。采用Western blotting方法分別檢測各組骨髓間充質(zhì)干細(xì)胞中PPARγ,aP2蛋白表達(dá)的含量。結(jié)果:1、在濃度為siRNA 25n M情況下ZIP1 siRNA序列1、2、3組與ZIP1 siRNA空轉(zhuǎn)染序列組比較,差異具有統(tǒng)計(jì)學(xué)意義(p㩳0.05),但ZIP1siRNA序列2組ZIP1m RNA表達(dá)含量明顯升高;ZIP1siRNA序列1、3組ZIP1m RNA表達(dá)含量明顯降低,相比較ZIP1siRNA序列3組ZIP1m RNA表達(dá)含量更低。ZIP1表達(dá)載體經(jīng)FD Kpn I和FD Eco RI雙酶切和測序鑒定后,目的片段和重組載體插入片段完全一致重組載體構(gòu)建成功。2、0.03mol/L酒精組、0.09mol/L酒精組、0.15mol/L酒精組、0.21mol/L酒精組中aP2、PPARγ蛋白表達(dá)量明顯升高,與正常對照組比較差異有統(tǒng)計(jì)學(xué)意義(P㩳0.05);甘油三酯含量在0.03mol/L酒精組與正常對照組比較時(shí)無明顯升高,差異無統(tǒng)計(jì)學(xué)意義(p㧐0.05)外,其他組與正常組比較差異均有統(tǒng)計(jì)學(xué)意義(P㩳0.05),其中0.21mol/L酒精組aP2、PPARγ蛋白表達(dá)量及甘油三酯含量最高,與其他組比較差異均有統(tǒng)計(jì)學(xué)意義(P㩳0.05);而在同一濃度為0.21 mol/L酒精培養(yǎng)下,0.21mol/L酒精+ZIP1siRNA組aP2、PPARγ蛋白表達(dá)含量及甘油三酯含量較0.21mol/L酒精組明顯升高,差異均有統(tǒng)計(jì)學(xué)意義(P㩳0.05)。3、ZIP1表達(dá)組aP2、PPARγ蛋白表達(dá)含量明顯降低,與正常對照組比較差異有統(tǒng)計(jì)學(xué)意義(P㩳0.05),ZIP1siRNA組aP2、PPARγ蛋白表達(dá)含量明顯升高,與正常對照組比較差異有統(tǒng)計(jì)學(xué)意義(P㩳0.05)。結(jié)論:1、成功合成并篩選ZIP1siRNA序列3為最佳抑制序列,能夠明顯抑制ZIP1m RNA的表達(dá),成功構(gòu)建了表達(dá)ZIP1基因的載體,經(jīng)酶切和測序鑒定正確。2、酒精能夠誘導(dǎo)骨髓間充質(zhì)干細(xì)胞成脂分化,在一定范圍內(nèi)隨著酒精濃度增加骨髓間充質(zhì)干細(xì)胞成脂分化的能力增強(qiáng),并且在酒精作用下ZIPsiRNA能促進(jìn)骨髓間充質(zhì)干細(xì)胞成脂分化。3、表達(dá)ZIP1基因能夠抑制骨髓間充質(zhì)干細(xì)胞成脂分化,沉默ZIP1基因能夠促進(jìn)骨髓間充質(zhì)干細(xì)胞成脂分化。
[Abstract]:Objective: Alcoholic femoral head necrosis is a non-traumatic osteonecrosis of the femoral head caused by long-term heavy drinking. It is a common orthopaedic disease in young and middle-aged men. Lipid disorders and abnormal adipogenesis are important mechanisms leading to alcoholic head necrosis. Zinc-iron regulatory protein 1 (ZIP1) acts as a transporter of zinc ions. Zinc ions can be transported into cells to promote the osteogenic differentiation of bone marrow mesenchymal stem cells, but whether ZIP1 protein can inhibit the formation of adipocytes by affecting the differentiation of bone marrow mesenchymal stem cells is not clear. This experiment will explore the effect of ZIP1 protein on adipogenic differentiation of bone marrow mesenchymal stem cells in rabbits, so as to alcoholic femoral head. Methods: 1. Rabbit bone marrow mesenchymal stem cells (BMSCs) were resuscitated and cultured in complete medium. After subculture, BMSCs in good condition were obtained. BMSCs were induced to differentiate into adipogenic cells by oil red "O" staining, and ZIP1 was designed and synthesized. The expression of ZIP1m RNA in bone marrow mesenchymal stem cells was detected by RT-PCR, and the optimal sequence of ZIP1siRNA was screened. At the same time, the ZIP1 target fragment was designed and synthesized, and the ZIP1 expression vector was constructed. The recombinant vector was sequenced by restriction endonuclease of FD Kpn I and FD Eco R. Rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with recombinant vector. The BMSCs were divided into 9 groups: normal control group, model group (0.03 mol/L alcohol group, 0.09 mol/L alcohol group, 0.15 mol/L alcohol group, 0.21 mol/L alcohol group), experimental group (0.03 mol/L alcohol + ZIP1 siRNA group, 0.09 mol/L alcohol + ZIP1 siRNA group, 0.15 mol/L alcohol + IP1 RNA group, 0.2 mol/L alcohol + ZIP1 siRNA group). 1 mol/L alcohol + ZIP1 siRNA group). Normal control group was not treated with normal culture. Model group was cultured with medium containing 0.03 mol/L, 0.09 mol/L, 0.15 mol/L, 0.21 mol/L alcohol concentration respectively. Experimental group was transfected with ZIP1 siRNA sequence and then cultured with medium containing 0.03 mol/L, 0.09 mol/L, 0.15 mol/L, 0.21 mol/L alcohol concentration respectively. The content of intracellular triglyceride was detected by kit and the content of triglyceride was recorded. The expression of PPAR-gamma and aP2 protein in bone marrow mesenchymal stem cells was detected by Western blotting. The bone marrow mesenchymal stem cells were divided into three groups: normal control group, ZIP1 expression group and ZIP1 siRNA group. After normal culture, the expression transfection group was transfected with ZIP1 expression vector, and the ZIP1 siRNA transfection group was transfected with ZIP1 siRNA sequence. Each group was cultured for 6 hours. In the case of ZIP1 siRNA sequence 1,2,3 groups and ZIP1 siRNA empty transfection sequence group, the difference was statistically significant (p?0.05), but ZIP1 siRNA expression in two groups of ZIP1 siRNA sequence was significantly higher; ZIP1 siRNA expression in 1,3 groups of ZIP1 siRNA sequence was significantly lower than ZIP1 siRNA expression in three groups of ZIP1 siRNA sequence. After digestion and sequencing of I and FD Eco RI, the recombinant vectors were successfully constructed. 2,0.03 mol/L ethanol group, 0.09 mol/L ethanol group, 0.15 mol/L ethanol group, 0.21 mol/L ethanol group, the expression of aP2 and PPAR gamma protein was significantly higher than the normal control group (P?0.05). The content of triglyceride in 0.03 mol/L alcohol group was not significantly higher than that in the normal control group, and there was no significant difference (p?0.05). There was significant difference between the other groups and the normal group (P?0.05). Among them, the expression of aP2, PPAR-gamma protein and triglyceride content in 0.21 mol/L alcohol group were the highest, and there was significant difference between the other groups (P?0.05). In the same concentration of 0.21 mol/L alcohol culture, 0.21 mol/L alcohol + ZIP1 siRNA group aP2, PPAR gamma protein expression and triglyceride content were significantly higher than 0.21 mol/L alcohol group, the difference was statistically significant (P?0.05). 3, ZIP1 expression group aP2, PPAR gamma protein expression was significantly lower than the normal control group, the difference was statistically significant. Significance (P? 0.05), ZIP1 siRNA group aP2, PPAR gamma protein expression significantly increased, compared with the normal control group was statistically significant (P? 0.05). Conclusion: 1, ZIP1 siRNA sequence 3 was successfully synthesized and screened as the best inhibitory sequence, can significantly inhibit the expression of ZIP1m RNA, successfully constructed the expression vector of ZIP1 gene, identified by enzyme digestion and sequencing. Indeed, alcohol can induce adipogenic differentiation of bone marrow mesenchymal stem cells, and the ability of adipogenic differentiation of bone marrow mesenchymal stem cells increases with the increase of alcohol concentration in a certain range. ZIPsiRNA can promote adipogenic differentiation of bone marrow mesenchymal stem cells under the effect of alcohol. 3. Expression of ZIP1 gene can inhibit adipogenic differentiation of bone marrow mesenchymal stem cells. Silencing ZIP1 gene can promote the adipogenic differentiation of bone marrow mesenchymal stem cells.
【學(xué)位授予單位】：廣西中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R274.9
本文編號(hào)：2231316
[Abstract]:Objective: Alcoholic femoral head necrosis is a non-traumatic osteonecrosis of the femoral head caused by long-term heavy drinking. It is a common orthopaedic disease in young and middle-aged men. Lipid disorders and abnormal adipogenesis are important mechanisms leading to alcoholic head necrosis. Zinc-iron regulatory protein 1 (ZIP1) acts as a transporter of zinc ions. Zinc ions can be transported into cells to promote the osteogenic differentiation of bone marrow mesenchymal stem cells, but whether ZIP1 protein can inhibit the formation of adipocytes by affecting the differentiation of bone marrow mesenchymal stem cells is not clear. This experiment will explore the effect of ZIP1 protein on adipogenic differentiation of bone marrow mesenchymal stem cells in rabbits, so as to alcoholic femoral head. Methods: 1. Rabbit bone marrow mesenchymal stem cells (BMSCs) were resuscitated and cultured in complete medium. After subculture, BMSCs in good condition were obtained. BMSCs were induced to differentiate into adipogenic cells by oil red "O" staining, and ZIP1 was designed and synthesized. The expression of ZIP1m RNA in bone marrow mesenchymal stem cells was detected by RT-PCR, and the optimal sequence of ZIP1siRNA was screened. At the same time, the ZIP1 target fragment was designed and synthesized, and the ZIP1 expression vector was constructed. The recombinant vector was sequenced by restriction endonuclease of FD Kpn I and FD Eco R. Rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with recombinant vector. The BMSCs were divided into 9 groups: normal control group, model group (0.03 mol/L alcohol group, 0.09 mol/L alcohol group, 0.15 mol/L alcohol group, 0.21 mol/L alcohol group), experimental group (0.03 mol/L alcohol + ZIP1 siRNA group, 0.09 mol/L alcohol + ZIP1 siRNA group, 0.15 mol/L alcohol + IP1 RNA group, 0.2 mol/L alcohol + ZIP1 siRNA group). 1 mol/L alcohol + ZIP1 siRNA group). Normal control group was not treated with normal culture. Model group was cultured with medium containing 0.03 mol/L, 0.09 mol/L, 0.15 mol/L, 0.21 mol/L alcohol concentration respectively. Experimental group was transfected with ZIP1 siRNA sequence and then cultured with medium containing 0.03 mol/L, 0.09 mol/L, 0.15 mol/L, 0.21 mol/L alcohol concentration respectively. The content of intracellular triglyceride was detected by kit and the content of triglyceride was recorded. The expression of PPAR-gamma and aP2 protein in bone marrow mesenchymal stem cells was detected by Western blotting. The bone marrow mesenchymal stem cells were divided into three groups: normal control group, ZIP1 expression group and ZIP1 siRNA group. After normal culture, the expression transfection group was transfected with ZIP1 expression vector, and the ZIP1 siRNA transfection group was transfected with ZIP1 siRNA sequence. Each group was cultured for 6 hours. In the case of ZIP1 siRNA sequence 1,2,3 groups and ZIP1 siRNA empty transfection sequence group, the difference was statistically significant (p?0.05), but ZIP1 siRNA expression in two groups of ZIP1 siRNA sequence was significantly higher; ZIP1 siRNA expression in 1,3 groups of ZIP1 siRNA sequence was significantly lower than ZIP1 siRNA expression in three groups of ZIP1 siRNA sequence. After digestion and sequencing of I and FD Eco RI, the recombinant vectors were successfully constructed. 2,0.03 mol/L ethanol group, 0.09 mol/L ethanol group, 0.15 mol/L ethanol group, 0.21 mol/L ethanol group, the expression of aP2 and PPAR gamma protein was significantly higher than the normal control group (P?0.05). The content of triglyceride in 0.03 mol/L alcohol group was not significantly higher than that in the normal control group, and there was no significant difference (p?0.05). There was significant difference between the other groups and the normal group (P?0.05). Among them, the expression of aP2, PPAR-gamma protein and triglyceride content in 0.21 mol/L alcohol group were the highest, and there was significant difference between the other groups (P?0.05). In the same concentration of 0.21 mol/L alcohol culture, 0.21 mol/L alcohol + ZIP1 siRNA group aP2, PPAR gamma protein expression and triglyceride content were significantly higher than 0.21 mol/L alcohol group, the difference was statistically significant (P?0.05). 3, ZIP1 expression group aP2, PPAR gamma protein expression was significantly lower than the normal control group, the difference was statistically significant. Significance (P? 0.05), ZIP1 siRNA group aP2, PPAR gamma protein expression significantly increased, compared with the normal control group was statistically significant (P? 0.05). Conclusion: 1, ZIP1 siRNA sequence 3 was successfully synthesized and screened as the best inhibitory sequence, can significantly inhibit the expression of ZIP1m RNA, successfully constructed the expression vector of ZIP1 gene, identified by enzyme digestion and sequencing. Indeed, alcohol can induce adipogenic differentiation of bone marrow mesenchymal stem cells, and the ability of adipogenic differentiation of bone marrow mesenchymal stem cells increases with the increase of alcohol concentration in a certain range. ZIPsiRNA can promote adipogenic differentiation of bone marrow mesenchymal stem cells under the effect of alcohol. 3. Expression of ZIP1 gene can inhibit adipogenic differentiation of bone marrow mesenchymal stem cells. Silencing ZIP1 gene can promote the adipogenic differentiation of bone marrow mesenchymal stem cells.
【學(xué)位授予單位】：廣西中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R274.9
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