AQP1基因表达下调对晶体上皮细胞活性和晶体蛋白含量影响的实验性研究

发布时间：2018-05-26 06:39

本文选题：水通道蛋白-1 + 晶状体上皮细胞　；参考：《福建医科大学》2014年博士论文

【摘要】：目的:研究水通道蛋白1(Aquaporin1 AQP1)对晶体上皮细胞(Lens Epithelial Cells,LECs)活力和晶体蛋白含量的影响,探讨AQP1在白内障发生发展中的作用及可能机制。方法:实验分三个部分进行:(1)将设计好的针对AQP1基因的si RNA通过慢病毒感染人晶体上皮细胞,对其AQP1基因进行RNA干扰,抑制AQP1基因在LECs上的表达;并通过实时荧光定量PCR和Western Blot检测AQP1基因表达下调情况。(2)AQP1基因表达下调后,分别通过透射电镜、CCK-8试剂盒和流式细胞仪观察人晶体上皮细胞形态改变、细胞增殖能力和凋亡情况。(3)AQP1基因表达下调后,利用激光共聚焦显微镜和荧光钙离子探针检测晶体细胞内游离钙离子浓度;并通Western Blot检测钙蛋白酶2和α、β、γ三种晶体蛋白的表达变化情况。结果:1、经过慢病毒感染针对AQP1基因进行RNA干扰的干扰组1和干扰组2细胞与正常细胞对照组相比,其AQP1基因的m RNA水平有显著下调(P0.05);同时其AQP1基因的蛋白表达有明显下调(P0.05)。而针对基因库中所有人源基因均无同源性的非特异干扰组与正常对照组相比其AQP1基因的m RNA水平和蛋白表达无差别(P0.05)。2、与正常对照组相比,在透射电镜下观察干扰组1和干扰组2凋亡细胞较为多见,凋亡细胞表面微绒毛减少,胞质浓缩、胞体变小,细胞核体积缩小,核内染色质凝聚,但核膜完整,并出现凋亡小体。CCK-8试剂盒检测发现干扰组1、干扰组2与正常对照组,非特异干扰组相比,其在450nm波长处的OD值明显低于正常组(P0.05)。流式细胞仪检测发现干扰组1、干扰组2的细胞凋亡率明显高于正常对照组和非特异干扰组(P0.05)。3、通过激光扫描共聚焦显微和荧光钙离子探针检测发现干扰组1、干扰组2与正常对照组、非特异干扰组相比,其细胞内游离钙离子荧光强度明显增强(P0.05)。同时通过Western Blot法发现干扰组1、干扰组2细胞内的钙蛋白酶2表达含量增加,而α、β、γ三种晶体蛋白的表达含量减少,与对照组相比有统计学意义(P0.05)。结论:1、应用携带设计好的si RNA序列质粒的慢病毒感染人晶体上皮,对其AQP1基因进行RNA干扰,可明显下调AQP1基因的m RNA和蛋白的表达量,达到沉默人晶状体上皮细胞AQP1基因的目的。2、AQP1是晶状体上皮细胞主要的膜蛋白,在维持晶体上皮细胞的正常生理功能中起重要作用,AQP1的减少将导致细胞内钙离子浓度增高,打破细胞内环境的稳定,从而触发细胞凋亡。而晶体上皮细胞是人透明晶状体营养代谢不可或缺的关键环节,因此AQP1的下调,可通过晶体上皮细胞,在白内障发生发展过程中产生作用。3、AQP1的减少,可导致细胞内钙离子浓度的升高,继而激发钙蛋白酶2表达含量增高,最终不同程度降解α、β、γ三种晶体蛋白;而α、β、γ晶体蛋白是维护人晶状体透明性的重要蛋白质,并且在白内障的形成中都会有所改变,因此AQP1的减少与白内障形成之间存在着某种联系。
[Abstract]:Aim: to study the effect of aquaporin 1(Aquaporin1 AQP1 on the activity and the content of crystal protein in lens epithelial cells (Lens Epithelial cells), and to explore the role and possible mechanism of AQP1 in the development of cataract. Methods: the designed si RNA for AQP1 gene was infected with human lens epithelial cells by lentivirus. The AQP1 gene was interfered by RNA to inhibit the expression of AQP1 gene on LECs. After the down-regulation of AQP1 gene expression was detected by real-time fluorescence quantitative PCR and Western Blot, the morphological changes of human lens epithelial cells were observed by transmission electron microscope (TEM) CCK-8 kit and flow cytometry, respectively. After the expression of AQP1 gene was down-regulated, the intracellular free calcium concentration was detected by laser confocal microscopy and fluorescence calcium probe. The expression of calpain 2 and 伪, 尾, 纬 was detected by Western Blot. Results compared with normal cells, the m RNA level of AQP1 gene and the protein expression of AQP1 gene were significantly down-regulated in RNA interference group 1 and control group 2, which were infected with lentivirus and infected with lentivirus, respectively. However, there was no difference in m RNA level and protein expression of AQP1 gene between nonspecific interference group and normal control group, and there was no significant difference in m RNA level and protein expression between non-specific interference group and normal control group. Under transmission electron microscope, apoptotic cells were observed more frequently in interference group 1 and interference group 2. The surface microvilli of apoptotic cells were decreased, cytoplasm was concentrated, cell body became smaller, nuclear volume was reduced, chromatin condensed in nucleus, but nuclear membrane was intact. The detection of apoptotic corpuscles. CCK-8 kit showed that the OD value of interference group 2 was significantly lower than that of normal group at the wavelength of 450nm compared with normal control group and non-specific interference group (P 0.05). The apoptosis rate of interference group 2 was significantly higher than that of normal control group and non-specific interference group (P0.05. 3). The results of laser scanning confocal microscopy and fluorescence calcium probe test showed that interference group 1 and interference group 1. (2) compared with the normal control group, Compared with the nonspecific interference group, the fluorescence intensity of intracellular free calcium ion increased significantly (P 0.05). At the same time, it was found by Western Blot method that the expression of calpain 2 in interference group 1 was increased, while the expression of 伪, 尾 and 纬 crystal proteins was decreased, which was significantly higher than that in control group (P 0.05). Conclusion: the expression of m RNA and protein in AQP1 gene can be significantly down-regulated by RNA interference of AQP1 gene in human lens epithelium infected with lentivirus carrying the designed si RNA sequence plasmid. The aim of silencing the AQP1 gene of human lens epithelial cells. 2AAQP1 is the main membrane protein of lens epithelial cells. The decrease of AQP1 plays an important role in maintaining the normal physiological function of lens epithelial cells. The decrease of AQP1 will lead to the increase of intracellular calcium concentration. Break the stability of the cell environment, thus triggering cell apoptosis. The lens epithelium is an indispensable key link in the nutritional metabolism of human transparent lens. Therefore, the down-regulation of AQP1 may play a role in the reduction of AQP1 during the development of cataract through lens epithelial cells. It can lead to the increase of intracellular calcium ion concentration, and then stimulate the increase of the expression of calpain 2, and eventually degrade 伪, 尾, 纬 to varying degrees, and 伪, 尾, 纬 crystal protein is an important protein to maintain the transparency of human lens. And the formation of cataract will change, so there is a relationship between the decrease of AQP1 and cataract formation.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R776.1

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