VKORC1在草酸钙结晶形成中的作用及其机制研究

发布时间：2018-06-30 04:48

本文选题：草酸钙 + 肾结石　；参考：《复旦大学》2014年硕士论文

【摘要】：[目的]：研究不同浓度草酸钙结晶对HK-2细胞凋亡的影响,找到合适的草酸钙结晶浓度,以排除其对后续试验的影响,观察过表达VKORC1蛋白对草酸钙结晶粘附和形成能力的影响。shRNA vkorc1,观察VKORC 1蛋白下调对草酸钙结晶形成能力的影响。推测VKORC 1蛋白在草酸钙结晶形成过程中的作用,为进一步研究肾结石形成机制提供新的方向和思路。[方法]：将HK-2细胞铺在六孔板中,与混有草酸钙结晶的培养基培养48小时,用AnnexinV/propidium iodide双染试剂盒处理后,观察细胞的凋亡情况。HK-2细胞分别转染pFLAG-CMV-7.1-VKORC1, pFLAG-CMV-7.1质粒,通过RT-PCR, Western blotting分别检测两组mRNA和蛋白的表达情况。在共聚焦显微镜下观察并比较两组结晶粘附和形成的差异。HK-2细胞分别转染pGPU6/GFP/Neo-VKORC 1 shRNAs 和 pGPU6/GFP/Neo-shRNA-NC质粒,分别在24,48,72小时行RT-PCR检测RNA干扰的效果,筛选出干扰效果最好的质粒。获得稳定的pGPU6/GFP/Neo-VKORC 1 shRNA和pGPU6/GFP/Neo-shRNA-NC稳定细胞株,通过Western blotting来验证pGPU6/GFP/Neo-VKORC 1 shRNA2和pGPU6/GFP/Neo-VKORC1 shNC组蛋白的表达情况,然后在共聚焦显微镜下观察并比较两组结晶粘附和形成的差异。[结果]：取不同浓度荧光标记草酸钙结晶分别为：50ug/ml, 100ug/ml,400ug /ml,800ug/ml。凋亡细胞占活细胞总数的百分比：50ug/ml组为6.30±0.25；100ug/ml组为：7.0±0.64；400ug/ml组为：10.7±0.44；800ug/ml组为：17.2±0.29；mock组为：5.8±0.4。50ug/ml,100ug/ml相比于mock组没有统计学意义。(P0.05),400ug/ml,800ug/ml与mock组有统计学意义。(P0.05)HK-2细胞分别转染pFLAG-CMV-7.1-VKORC 1和pFLAG-CMV-7.1质粒,分别取24,48小时两个时间点,行RT-PCR实验得知：pFLAG-CMV-7.1-VKORC 1组mRNA的表达量与pFLAG-CMV-7.1相比有显著差异。(P0.001)。Western blotting验证实验组VKORC 1蛋白的表达量高于对照组。将转染的pCMV-7.1-VKORC 1和pFLAG-CMV-7.1质粒的HK-2细胞,与混有草酸钙结晶的培养基共同培养48小时,通过激光共聚焦显微镜观察结晶形成情况,在100倍视野下观察结晶形成数目,pFLAG-CMV-7.1-VKORC 1组为：14±4;pFLAG-CMV-7.1为：26±4。实验组结晶形成能力比对照组相比有统计学意义。(P0.05),分别转染pGPU6/GFP/Neo-VKORC 1 shRNAs和pGPU6/GFP/Neo-shRNA-NC质粒,取三个时间点24,48,72小时来检测mRNA,我们发现pGPU6/GFP/Neo-VKORC 1 shRNA2明显抑制VKORC1 mRNA的表达。筛选稳定细胞株,通过激光共聚焦显微镜观察结晶形成情况,在100倍视野下结晶形成数目,干扰组为：65+11,NC组为：24+8,干扰组比pGPU6/GFP/Neo-shRNA-NC组有统计学意义。(P0.05)。[结论]：低浓度的草酸钙结晶对肾小管细胞的凋亡影响相较于对照组没有明显的差异。高于400ug/ml草酸钙盐结晶才会对肾小管细胞产生损伤。通过过表达和下调VKORC1的表达,发现VKORC1有抑制草酸钙盐结晶粘附和聚集的作用。本研究为了解结晶和上皮细胞的相互作用、结晶的形成机制提供了新的方法和思路。
[Abstract]:[Objective] to study the effect of calcium oxalate crystallization on HK-2 cell apoptosis in different concentrations and find the appropriate concentration of calcium oxalate crystal to exclude the effect on the follow-up test, and observe the effect of VKORC1 protein on the adhesion and formation of calcium oxalate crystallization and the formation ability of.ShRNA VKORC1, and observe the effect of VKORC 1 protein down on the crystallization of calcium oxalate. The effect of VKORC 1 protein in the crystallization of calcium oxalate can provide new directions and ideas for further study of the mechanism of kidney stone formation. [method]: the HK-2 cells were spread in the six pore plate and cultured with calcium oxalate crystals for 48 hours, and the cell withering was observed with the AnnexinV/propidium iodide double staining kit. .HK-2 cells were transfected with pFLAG-CMV-7.1-VKORC1 and pFLAG-CMV-7.1 plasmid respectively. The expression of mRNA and protein in two groups was detected by RT-PCR and Western blotting respectively. The difference of.HK-2 cells from the two groups was observed and compared to pGPU6/GFP/Neo-VKORC 1 shRNAs and pGPU6/GFP/Neo-shR by the confocal microscopy. NA-NC plasmid, the effect of RNA interference was detected by RT-PCR at 24,48,72 hours respectively, and the plasmid with the best interference effect was screened. Stable pGPU6/GFP/Neo-VKORC 1 shRNA and pGPU6/GFP/Neo-shRNA-NC stable cell lines were obtained, and Western blotting was used to verify the expression of pGPU6/GFP/Neo-VKORC 1 shRNA2 and pGPU6/GFP/Neo-VKORC1 histones. And then observed and compared the differences between the two groups of adhesion and formation under confocal microscopy. [results]: the crystallization of calcium oxalate with different concentrations was 50ug/ml, 100ug/ml, 400ug /ml, and the percentage of 800ug/ml. apoptotic cells accounted for the total number of living cells: 50ug/ml group was 6.30 + 0.25; 100ug/ml group was 7 + 0.64; 400ug/ml group. 10.7 + 0.44, group 800ug/ml was 17.2 + 0.29, group mock was 5.8 + 0.4.50ug/ml, 100ug/ml was not statistically significant compared with mock group. (P0.05), 400ug/ml, 800ug/ml and mock group had statistical significance. (P0.05) HK-2 cells were transfected respectively for pFLAG-CMV-7.1-VKORC 1 and plasmid, respectively, for two hours, respectively. The experimental results showed that the expression of mRNA in pFLAG-CMV-7.1-VKORC 1 groups was significantly different from that of pFLAG-CMV-7.1. (P0.001) the expression of VKORC 1 protein in the.Western blotting test group was higher than that of the control group. The transfected pCMV-7.1-VKORC 1 and pFLAG-CMV-7.1 plasmids HK-2 cells were co cultured with the culture medium with calcium oxalate crystallization for 48 hours. The formation of crystallization was observed by laser confocal microscopy, and the number of crystal formation was observed at 100 times of visual field. The pFLAG-CMV-7.1-VKORC 1 groups were 14 + 4, and pFLAG-CMV-7.1 was: the crystallization ability of the 26 + 4. experimental group was statistically significant compared with the control group. (P0.05), pGPU6/GFP/Neo-VKORC 1 shRNAs and pGPU6/GFP/Neo-shRNA-NC plasmids were transfected respectively. Taking three time points for 24,48,72 hours to detect mRNA, we found that pGPU6/GFP/Neo-VKORC 1 shRNA2 significantly inhibited the expression of VKORC1 mRNA. The stable cell lines were screened and the crystallization formation was observed by laser confocal microscopy, and the number of crystals formed under 100 times of visual field. The interference group was 65+11, NC group was 24+8, and the interference group was more than pGPU6/GFP/Neo-sh. RNA-NC group had statistical significance (P0.05). [Conclusion]: the effect of low concentration calcium oxalate crystallization on renal tubular cell apoptosis was not significantly different from that of the control group. Higher than the 400ug/ml calcium oxalate crystallization of 400ug/ml could damage the renal tubule cells. Through overexpression and down regulation of VKORC1 expression, it was found that VKORC1 inhibited calcium oxalate crystallization. This study provides new methods and ideas for understanding the interaction between crystallization and epithelial cells and the formation mechanism of crystallization.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R691.4

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