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HSP90α亚型对GC-GR核转位的影响研究

发布时间:2018-04-12 21:05

  本文选题:糖皮质激素受体 + 热休克蛋白90α亚型 ; 参考:《重庆医科大学》2007年硕士论文


【摘要】: 糖皮质激素(glucocorticoid GC)在治疗原发性肾病综合征、支气管哮喘、风湿性疾病、炎症性肠炎、多发性硬化症、淋巴瘤、白血病等疾病时,发挥了很好的作用。但有少数患者对糖皮质激素治疗不敏感,甚至抵抗。造成这种不敏感和抵抗的原因是我们关心的问题。 糖皮质激素广泛而复杂的生物学效应是通过糖皮质激素受体(glucocorticoid receptor GR)介导的,GR存在于胞浆中,结合GC后能形成二聚体,并转移到细胞核中发挥对目标基因的转录调节作用,从而发挥对细胞功能的调节。热休克蛋白90(HSP90)是GC-GR效应全程伴侣蛋白,在GC通过GR介导产生作用的过程中,HSP90起到非常重要的作用(参见图1)。HSP90的质(不同亚型)、量(数量)可能直接影响到GC-GR效应。 HSP90是热休克蛋白家族成员之一,占胞浆蛋白的1%~2%。胞浆中HSP90主要有两种亚型,分别是HSP90α(诱导型,小鼠中称为HSP86)和HSP90β(组成型,小鼠中称为HSP84)。HSP90主要以同源双聚体的形式(αα,证据。两亚型的基因组来源、基因序列、mRNA和氨基酸序列都存在很大差别。在诱导方面也存在差别(HSP90α受热诱导而显著升高,HSP90β受丝裂原诱导可显著升高)。提示HSP90亚型在功能上可能存在明显差异。 另外,如下的一些现象也引起我们关注: 1)HSP90α是诱导性蛋白,当机体受到应激时,HSP90α会很快得到补充和提高;而HSP90β是结构性蛋白,HSP90β(小鼠叫HSP84)的反应和提高则需要一定时间, 6h内存在差异是否说明HSP90α也参与了GC-GR通路; 2)HSP90α,HSP90β来自不同的基因,且在HSP90α,HSP90β的c端和铰链区差异较大(是与GR结合和协助GR入核的区域)是否就是这个差异使得只有其中一个亚型在维持GR结构和功能。我们进一步推测:HSP90的质(不同亚型)可能对GC-GR效应有不同贡献。深入研究HSP90亚型对GC-GR通路的作用将有助于阐明HSP90亚型质和量对GC-GR效应的影响,和解决糖皮质激素抵抗问题,也可能为对糖皮质激素治疗不敏感、甚至抵抗的患者带来新的希望。本研究首先构建HSP90α的RNA干扰的真核表达质粒;然后在降低HSP90α的情况下观察GC-GR核转位的变化.最后得到了以下的结果: 1.将化学合成的三段寡聚核苷酸与线性化的pSUPER-EGFP载体连接获得了可用于沉默HSP90α基因实验的三个质粒,经酶切鉴定、电泳证明,含作用序列的重组质粒pSuper-HSP90α1, pSuper-HSP90α2,及pSuper-HSP90α3构建是成功的,重组质粒pSuper-HSP90α1, pSuper-HSP90α2测序分析完全正确。实验中先后用不同的脂质体和质粒比例转染HUVEC、HEPG2、HEK 293三种细胞,以绿色荧光蛋白作为评价转染效率的指标,确定HEK-293细胞为靶细胞.在HEK-293中优化转染条件,转染效率可以达到80%左右。 2.用半定量PCR的方法,通过与对照质粒转染组和未处理组比较发现: pSuper-HSP90α转染后24小时HEK-293细胞中HSP90αmRNA水平均有下降,pSuper-HSP90α1最明显。 3.用western方法,通过与对照转染组和未处理组比较发现:pSuper-HSP90α转染后48小时HEK-293细胞中HSP90α水平明显下降。 4.用50 nM GC处理2小时后,细胞核中GR与细胞浆中GR数量的比值增大,表明GR转位到细胞核以发挥调节功能。pSuper-HSP90α转染HEK-293细胞会增强这种GR核浆比值增大的趋势。这种效应都是在质粒转染细胞后48小时观察到的,这提示HSP90α可能直接负调控GR核转位;也可能是HSP90α下降,其他亚型反应性增高间接导致GR核转位增强。
[Abstract]:Glucocorticoid (glucocorticoid GC) in the treatment of primary nephrotic syndrome, bronchial asthma, rheumatic diseases, inflammatory bowel disease, multiple sclerosis, lymphoma, leukemia and other diseases, played a very good role. But there are a few cases that are not sensitive to glucocorticoid therapy and even cause resistance. This is not sensitive and resistance is our concern.
The biological effect of glucocorticoids is wide and complex through the glucocorticoid receptor (glucocorticoid receptor GR) mediated, GR exists in the cytoplasm, combined with GC after the formation of two dimers, and transferred to play the transcription of target genes in the nucleus of regulation, which play a role in regulation of cell function. Shock protein 90 (HSP90) is the GC-GR effect throughout the chaperone, GC through GR mediated effect, HSP90 plays a very important role (see Figure 1).HSP90 matter (different subtypes), quantity (quantity) may directly affect the GC-GR effect.
HSP90 is one of the heat shock protein family members, accounting for 1% ~ HSP90 cytoplasmic protein 2%. in cytoplasm mainly has two subtypes, namely HSP90 (alpha inducible mouse, called HSP86) and HSP90 (beta type composition in mice, referred to as HSP84).HSP90 mainly in the homologous dimer form (alpha alpha, evidence. Two subtypes of genomic, gene sequence and amino acid sequence of mRNA, there are great differences. There are differences in the induction (HSP90 alpha heating induced significantly increased, HSP90 significantly increased the mitogen induced by beta) suggest that HSP90 subtype in function may be significantly different.
In addition, some of the following phenomena have aroused our concern:
1) HSP90 alpha is an inducible protein. When HSP90 is stressed, HSP90 alpha will soon be replenished and improved, while HSP90 beta is a structural protein, while HSP90 beta (mouse called HSP84) needs a certain time to react and improve. 6h difference indicates whether HSP90 is also involved in GC-GR pathway.
2) HSP90 alpha, HSP90 beta from different genes, and in the C end and HSP90 alpha, HSP90 beta larger differences in the hinge region (region is combined with GR and GR to assist in nucleus) is the difference that makes only one subtype in maintaining the structure and function of GR. We further speculate: HSP90 quality (subtypes) may have different contribution to the GC-GR effect. Further study on effect of HSP90 subtype of GC-GR pathway will help to elucidate the HSP90 subtype of quality and quantity for the GC-GR effect, and solve the problem of glucocorticoid resistance, may also be less sensitive to glucocorticoid therapy, even resistant patients bring new hope. The eukaryotic expression plasmid of RNA interference in the dissertation, HSP90 alpha; then observe the change of nuclear translocation of GC-GR in reducing the HSP90 alpha case. Finally, the results obtained are as follows:
1. pSUPER-EGFP carrier chemical synthesis of three oligonucleotides with linear connection could be used in the three plasmid HSP90 gene silence experiment, identified by enzyme digestion, electrophoresis demonstrated that the recombinant plasmid pSuper-HSP90 containing sequences of pSuper-HSP90 alpha 1, alpha 2, alpha 3 and pSuper-HSP90 to construct recombinant plasmid is successful. PSuper-HSP90 alpha 1, alpha 2 pSuper-HSP90 sequencing analysis is completely correct. The experiment has used the liposome and plasmid transfection ratio HUVEC, different HEPG2, 293 HEK three cells with green fluorescent protein as the evaluation index to determine the transfection efficiency, HEK-293 cells as target cells. Optimization of transfection conditions in HEK-293, the transfection efficiency can reach 80% left and right.
2. by semi quantitative PCR method, by comparing with control plasmid transfection group and untreated group, it was found that the level of HSP90 alpha mRNA in HEK-293 cells decreased after 24 hours of pSuper-HSP90 alpha transfection, and pSuper-HSP90 alpha 1 was the most obvious.
3. by western method, the level of HSP90 alpha in HEK-293 cells decreased significantly in 48 hours after transfection with the control group and the untreated group.
4. with 50 nM GC after 2 hours of treatment, increasing the ratio of GR and GR in the number of nuclei in the cytoplasm, showed that GR translocates to the nucleus to play a regulatory function of.PSuper-HSP90 alpha transfected HEK-293 cells will enhance the karyoplasmic ratio of GR increased trend. This effect is in the transfected cells after 48 hours observed, suggesting that HSP90 may direct the negative regulation of GR nuclear translocation; it may be HSP90 a drop, the other subtypes hyperergy indirectly lead to nuclear translocation of GR increased.

【学位授予单位】:重庆医科大学
【学位级别】:硕士
【学位授予年份】:2007
【分类号】:R341

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