DLX1联合BMP9促进人骨肉瘤细胞成骨分化的研究

发布时间：2018-04-28 18:23

本文选题：DLX1 + 骨肉瘤　；参考：《重庆医科大学》2017年硕士论文

【摘要】：第一部分DLX1过表达对人骨肉瘤细胞恶性生物学行为及成骨分化的影响目的探讨DLX1过表达对人骨肉瘤细胞MG63恶性生物学行为及成骨分化的影响。方法采用构建有DLX1基因的重组腺病毒(adenovirus,Ad)Ad DLX1和空载腺病毒Ad RFP,分别感染人骨肉瘤细胞MG63,分为DLX1实验组(Ad DLX1感染组)和RFP对照组(Ad RFP感染组),观察细胞荧光表达情况并以RT-PCR和Western blot验证DLX1的表达情况;Transwell实验检测MG63细胞迁移和侵袭;DAPI染色和流式细胞术检测MG63细胞凋亡;CCK-8检测MG63细胞增殖;碱性磷酸酶(alkaline phosphatase,ALP)染色和ALP读数分析MG63细胞早期成骨能力,Western blot检测骨桥蛋白(osteopontin,OPN)的蛋白表达,茜素红染色检测细胞晚期成骨能力;RT-PCR初筛相关信号通路,Western blot验证初筛阳性信号分子的蛋白表达情况。结果1.Ad DLX1和Ad RFP分别感染MG63细胞后,与RFP对照组相比,Ad DLX1能显著增加骨肉瘤细胞MG63中DLX1的表达。2.MG63细胞中DLX1过表达后,与RFP对照组相比,细胞迁移能力和侵袭能力下降。3.MG63细胞中DLX1过表达后,与RFP对照组相比,过表达DLX1对细胞增殖、凋亡无明显影响。4.MG63细胞中DLX1过表达后,与RFP对照组相比,过表达DLX1对细胞成骨分化无明显影响。5.MG63细胞中DLX1过表达后,与RFP对照组相比,DLX1过表达可上调β-catenin m RNA和蛋白的表达。结论1.MG63细胞中DLX1过表达后,对MG63细胞的恶性生物学行为有不同程度的影响:可抑制MG63细胞的迁移和侵袭,对MG63细胞的增殖和凋亡无显著影响;对MG63细胞成骨分化无明显促进作用。2.DLX1在MG63细胞中过表达后,Wnt/β-catenin信号通路参与DLX1对MG63细胞生物学行为的调控。第二部分DLX1联合BMP9对人骨肉瘤细胞恶性生物学行为及成骨分化的影响目的探讨DLX1联合BMP9对人骨肉瘤细胞MG63恶性生物学行为及成骨分化的影响。方法采用构建有DLX1和BMP9基因的重组腺病毒Ad DLX1和Ad BMP9,单独或联合感染人骨肉瘤MG63细胞,分为RFP组(Ad RFP感染组)、BMP9组(Ad BMP9+Ad RFP感染组)、DLX1+BMP9组(Ad DLX1+Ad BMP9感染组)、DLX1组(Ad DLX1+Ad GFP感染组),观察细胞荧光表达情况并以RT-PCR和Western blot验证DLX1、BMP9m RNA和蛋白表达情况;Transwell实验检测MG63细胞迁移和侵袭;DAPI染色和流式细胞术检测MG63细胞凋亡;CCK-8检测MG63细胞增殖;ALP染色和ALP读数分析细胞早期成骨能力,Western blot检测骨桥蛋白(osteopontin,OPN)的蛋白表达,茜素红染色检测细胞晚期成骨能力;采用RT-PCR初筛相关信号通路,Western blot验证初筛阳性信号分子的蛋白表达情况。结果1.Ad DLX1单独感染MG63细胞后,细胞中DLX1的m RNA和蛋白表达上调;Ad BMP9单独感染MG63细胞后,细胞中BMP9的m RNA和蛋白表达上调;Ad DLX1和Ad BMP9联合感染MG63细胞后,细胞中DLX1、BMP9的m RNA和蛋白表达均上调。2.与RFP组相比,BMP9组、DLX1+BMP9组、DLX1组中MG63细胞迁移和侵袭能力均下降,且DLX1+BMP9组对迁移和侵袭能力的抑制较BMP9组和DLX1组有所增加。3.与RFP组相比,BMP9组、DLX1+BMP9组中MG63细胞增殖能力均下降,且两组MG63细胞受到的增殖抑制无明显差异;DLX1组细胞增殖较RFP组无明显差异。4.与RFP组相比,BMP9组、DLX1+BMP9组、DLX1组中MG63细胞凋亡无明显差异。5.与RFP组相比,BMP9组中MG63细胞ALP活性、OPN蛋白表达和钙盐沉积有所增加,但增加程度有限;DLX1+BMP9组中MG63细胞ALP活性增强、OPN蛋白表达上调、钙盐沉积明显增加;DLX1组中MG63细胞ALP活性、OPN蛋白表达和钙盐沉积无明显增加。结论1.DLX1单独作用于人骨肉瘤细胞MG63时,可抑制MG63细胞迁移和侵袭,对MG63细胞凋亡和增殖无明显影响;不能促进MG63细胞成骨分化。2.BMP9单独作用于人骨肉瘤细胞MG63时,可抑制MG63细胞迁移、侵袭和增殖,对MG63细胞凋亡无明显影响;对MG63细胞成骨分化的促进作用较弱。3.DLX1与BMP9联合作用后,可抑制MG63细胞迁移、侵袭和增殖,对MG63细胞凋亡无明显影响;可促进MG63细胞成骨分化。4.骨肉瘤是一种分化异常导致的恶性增生物,DLX1联合BMP9作用后可降低人骨肉瘤细胞迁移、侵袭和增殖的恶性生物学行为,促进人骨肉瘤细胞成骨分化。
[Abstract]:The effect of overexpression of DLX1 on the malignant biological behavior and osteogenic differentiation of human osteosarcoma cells in order to explore the effect of overexpression of DLX1 on the malignant biological behavior and osteogenic differentiation of human osteosarcoma cell MG63. Methods the recombinant adenovirus (adenovirus, Ad) Ad DLX1 and Ad RFP of the empty adenovirus were used to infect people respectively. Osteosarcoma cell MG63 was divided into DLX1 experimental group (Ad DLX1 infection group) and RFP control group (Ad RFP infection group). The expression of cell fluorescence was observed and the expression of DLX1 was tested with RT-PCR and Western blot. Alkaline phosphatase (alkaline phosphatase, ALP) staining and ALP reading were used to analyze the early osteogenesis of MG63 cells. Western blot was used to detect the protein expression of osteopontin (osteopontin, OPN). Alizarin red staining was used to detect the late osteogenesis of the cells; RT-PCR initial screening related signal pathways, Western blot to verify the protein expression of the positive signal molecules of the initial screening. Results after 1.Ad DLX1 and Ad RFP infected MG63 cells respectively, compared with the RFP control group, Ad DLX1 significantly increased the expression of DLX1 in the MG63 cells of osteosarcoma cells after the DLX1 over expression, and compared with the control group, the cell migration ability and invasion ability decreased. After cell proliferation, apoptosis had no obvious effect on the overexpression of DLX1 in.4.MG63 cells. Compared with the RFP control group, overexpression of DLX1 had no significant effect on DLX1 over expression in.5.MG63 cells. Compared with the RFP control group, the over expression of DLX1 could up regulate the beta -catenin m RNA and the expression of protein. The malignant biological behavior has different effects: it can inhibit the migration and invasion of MG63 cells, have no significant effect on the proliferation and apoptosis of MG63 cells; there is no obvious promoting effect on the osteogenic differentiation of MG63 cells. After the overexpression of.2.DLX1 in MG63 cells, Wnt/ beta -catenin signaling pathway and DLX1 regulate the biological behavior of MG63 cells by DLX1. Second parts. The effect of DLX1 combined with BMP9 on the malignant biological behavior and osteogenic differentiation of human osteosarcoma cells in order to explore the effect of DLX1 combined with BMP9 on the malignant biological behavior and osteogenic differentiation of human osteosarcoma cells MG63. Methods the recombinant adenovirus Ad DLX1 and Ad BMP9, which had DLX1 and BMP9 genes, were used to infect human osteosarcoma MG63 cells alone or in combination. They were divided into group RFP (Ad RFP infection group), group BMP9 (Ad BMP9+Ad RFP infection group), DLX1+BMP9 group (Ad DLX1+Ad BMP9 infection group), DLX1 group (Ad DLX1+Ad). MG63 cell apoptosis was detected by cytometer, MG63 cell proliferation was detected by CCK-8, ALP staining and ALP reading were used to analyze the early osteogenic ability of cells, Western blot was used to detect the protein expression of osteopontin (osteopontin, OPN), and alizarin red staining was used to detect the late osteogenesis of the cells; RT-PCR initial screening signal pathway was used, and Western blot was used to verify the positive signal of initial screening. Results when 1.Ad DLX1 infecting MG63 cells alone, the expression of M RNA and protein expression of DLX1 in the cells was up-regulated, and Ad BMP9 was infected with MG63 cells alone, and m RNA and protein expression of BMP9 in the cells were up regulated. The migration and invasion ability of MG63 cells in group BMP9, group DLX1+BMP9 and group DLX1 decreased, and the inhibition of migration and invasion in group DLX1+BMP9 was higher than that in group BMP9 and DLX1 group, and the proliferation ability of MG63 cells in BMP9 group and DLX1+BMP9 group decreased, and there was no significant difference in proliferation inhibition between the two groups. There was no significant difference between group RFP and group RFP. Compared with group RFP, there was no significant difference in the apoptosis of MG63 cells in group BMP9, DLX1+BMP9 and DLX1. The MG63 cell ALP activity, the increase of protein expression and calcium salt deposition in the BMP9 group increased, but the increase degree was limited. The ALP activity of MG63 cells, OPN protein expression and calcium salt deposition in DLX1 group did not increase obviously. Conclusion 1.DLX1 can inhibit the migration and invasion of MG63 cells in human osteosarcoma cells alone, and have no obvious effect on the apoptosis and proliferation of MG63 cells, and can not promote the MG63 cell osteogenic differentiation.2.BMP9 alone to act on the human osteosarcoma cell MG63. It could inhibit the migration, invasion and proliferation of MG63 cells, and had no obvious effect on the apoptosis of MG63 cells. The combination of.3.DLX1 and BMP9 could inhibit the migration, invasion and proliferation of MG63 cells, and had no obvious effect on the apoptosis of MG63 cells, and could promote the differentiation of.4. osteosarcoma of MG63 cells to be a kind of abnormal differentiation. DLX1 combined with BMP9 can reduce the malignant biological behavior of human osteosarcoma cell migration, invasion and proliferation, and promote osteosarcoma cells osteogenic differentiation.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738

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