大鼠视网膜兴奋损伤中HSP70与TNF-α表达的相关性研究

发布时间：2018-06-18 05:10

本文选题：硫酸锌 + 热休克蛋白70　；参考：《青岛大学》2012年硕士论文

【摘要】：目的探讨大鼠视网膜兴奋损伤中HSP70与TNF-α表达的相关变化。方法健康成年清洁级Wistar大鼠50只随机分为三组,正常对照组(5只)、阳性对照组即NMDA玻璃体腔注射组(20只)、实验组即NMDA玻璃体腔注射组+硫酸锌腹腔注射组(25只)。实验组腹腔注射硫酸锌(24.6mg/Kg),10h后随即处死5只。14h后实验组和阳性对照组均做单眼的玻璃体腔注射NMDA (2μl 80mmol/l),并做好标记。NMDA注射后2h,8h,16h,24h,实验组和阳性组各组均随机处死5只大鼠。正常对照组不做任何处理,可以于任何时间点处死。HE染色光镜下观察各组视网膜组织的形态学变化,实时定量RT-PCR方法观察视网膜TNF-αmRNA和HSP70mRNA表达的变化。结果1.HE染色：正常对照组大鼠视网膜组织结构层次清楚,染色均匀,细胞形态规则。阳性对照组造模后2h,神经节细胞开始水肿；造模后8h,神经节细胞水肿最严重,其内可见空泡,视网膜增厚；造模后16h,神经节细胞水肿较前减轻,排列疏松,形态不规则,可见核固缩,数量减少；造模后24h,神经节细胞数目减少排列紊乱,核固缩明显,神经纤维层、神经节细胞层变薄。各时间点实验组与阳性对照组相比,视网膜的厚度较厚,存活的视网膜神经节细胞的数量较多,损伤均轻于阳性对照组。2.荧光定量RT-PCR:正常对照组大鼠视网膜未见TNF-αmRNA表达,可见HSP70微量表达。阳性对照组中NMDA注射后2h, TNF-αmRNA水平较正常对照组上调(P0.05),注射后8h, TNF-αmRNA水平达高峰(P0.05),注射后16h表达量降低,但仍高于对照组(P0.05),注射后24h, TNF-αmRNA表达量与16h无差别(p0.05),较正常对照组增多(P0.05); NMDA注射后2h, HSP70 mRNA水平较正常对照组上调(P0.05),注射后8h, HSP70 mRNA水平明显上调(P0.05),注射后16h表达量达高峰(P0.05),注射后24h, HSP70 mRNA表达量降低,较正常对照组增多(P0.05)。实验组中硫酸锌注射后10h TNF-αmRNA未见表达,NMDA注射后2h,TNF-αmRNA水平较正常对照组上调(P0.05),较阳性对照组下降(P0.05),注射后8h,TNF-αmRNA水平达高峰(P0.05),较阳性对照组下降(P0.05),注射后16h表达量降低,但仍高于对照组(P0.05),较阳性对照组下降(P0.05),注射后24h,TNF-αmRNA表达量与16h无差别(p0.05),较阳性对照组下降(P0.05),较正常对照组增多(P0.05)；硫酸锌注射后10h HSP70 mRNA水平较正常对照组上调(P0.05),NMDA注射后2h,HSP70 mRNA水平明显上调(P0.05),较阳性对照组增加(P0.05),注射后8h, HSP70 mRNA水平达高峰,与2h的无差别(p0.05),较阳性对照组增加(P0.05),注射后16h,HSP70 mRNA水平与8h的无差别(p0.05),较阳性对照组增加(P＜0.05),注射后24h,HSP70 mRNA水平下降,较阳性对照组增加(P0.05)。结论在视网膜兴奋损伤中,硫酸锌可增加HSP70的表达量,降低TNF-α的表达量,随着HSP70的表达量的增加,TNF-α表达量下降,表明HSP70对TNF-α的表达有抑制作用。
[Abstract]:Objective to investigate the correlation between HSP70 and TNF- 伪 expression in rat retina excitatory injury. Methods Fifty healthy adult clean grade Wistar rats were randomly divided into three groups: normal control group (n = 5), NMDA vitreous injection group (n = 20) and zinc sulfate injection group (n = 25). After 10 hours of intraperitoneal injection of zinc sulfate 24.6 mg 路kg ~ (-1) 路L ~ (-1) 路L ~ (-1), 5 rats in the experimental group and the positive control group were killed immediately after 10 h. All the experimental group and the positive control group were given intravitreal injection of NMDA 2 渭 l 80 mmol 路L ~ (-1), and then 2 h, 8 h and 16 h ~ 24 h after injection of .NMDA, 5 rats were randomly killed in the experimental group and the positive group. The normal control group, without any treatment, could be killed at any time. The morphological changes of retinal tissue were observed under light microscope with HE staining, and the expression of TNF- 伪 mRNA and HSP70 mRNA in retina were observed by real-time quantitative RT-PCR. Results 1. HE staining: the retinal tissue structure of normal control group was clear, stained evenly, and the cell morphology was regular. 2. In the positive control group, the ganglion cells began to edema at 2 h after modeling, the ganglion cells showed the most severe edema at 8 h after modeling, with vacuoles and thickened retina, 16 h after modeling, the edema of ganglion cells decreased, the arrangement of ganglion cells was loose, and the shape was irregular. The number of ganglion cells decreased and the number of ganglion cells decreased. The thickness of retina was thicker and the number of surviving retinal ganglion cells was more in the experimental group than in the positive control group at each time point, and the damage was lighter than that in the positive control group. Fluorescence quantitative RT-PCR: no expression of TNF- 伪 mRNA was found in the retina of normal control group, but HSP70 mRNA was detected in the retina. The level of TNF- 伪 mRNA was up-regulated at 2 h after NMDA injection in the positive control group compared with that in the normal control group. The level of TNF- 伪 mRNA reached the peak at 8 h after injection and decreased at 16 h after injection. However, the expression of TNF- 伪 mRNA was still higher than that of the control group at 24 h after injection, and there was no difference between the expression of TNF- 伪 mRNA and that of the control group at 16 h, which was higher than that in the normal control group at 2 h after NMDA injection, and the level of HSP70 mRNA was up-regulated at 2 h after NMDA injection as compared with that in the control group. At 8 h after injection, the mRNA level of HSP70 increased significantly, and the expression of HSP70 mRNA reached to P0.05% at 16 h after injection. The expression of HSP70 mRNA decreased 24 hours after injection. Compared with the normal control group, the number of P0.05 was increased. There was no expression of TNF- 伪 mRNA in the experimental group at 10 h after injection of zinc sulfate. The expression of TNF- 伪 mRNA was higher than that of the normal control group at 2 h after injection, and decreased significantly than that in the positive control group. The level of TNF- 伪 mRNA reached the peak level at 8 h after injection and decreased at 16 h after injection, and decreased at 16 h after injection. However, the expression of TNF- 伪 mRNA was still higher than that of the control group (P 0.05) and decreased than that of the positive control group. The expression of TNF- 伪 mRNA at 24 h after injection was not significantly different from that of the control group at 16 h (P 0.05), but it was lower than that in the positive control group (P 0.05) and increased than that in the normal control group (P 0.05). HSP70 mRNA level was significantly up-regulated at 10 h after injection of zinc sulfate compared with that in normal control group. HSP70 mRNA level in HSP70 was significantly up-regulated at 2 h after injection of P0.05N MDA, and increased significantly than that in positive control group. The HSP70 mRNA level reached its peak at 8 h after injection. The level of HSP70 mRNA was increased at 16 h after injection and the level of HSP70 mRNA at 8 h was similar to that in the positive control group (P < 0.05). The level of HSP70 mRNA decreased 24 hours after injection and increased P0.05 compared with the positive control group at 24 h after injection. Conclusion Zinc sulfate can increase the expression of HSP70 and decrease the expression of TNF- 伪 in retina excitatory injury. With the increase of HSP70, the expression of TNF- 伪 decreases, indicating that HSP70 can inhibit the expression of TNF- 伪.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1

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