本文選題：糖皮質(zhì)激素受體 + 熱休克蛋白90α亞型　； 參考：《重慶醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 糖皮質(zhì)激素(glucocorticoid GC)在治療原發(fā)性腎病綜合征、支氣管哮喘、風(fēng)濕性疾病、炎癥性腸炎、多發(fā)性硬化癥、淋巴瘤、白血病等疾病時(shí),發(fā)揮了很好的作用。但有少數(shù)患者對(duì)糖皮質(zhì)激素治療不敏感,甚至抵抗。造成這種不敏感和抵抗的原因是我們關(guān)心的問(wèn)題。 糖皮質(zhì)激素廣泛而復(fù)雜的生物學(xué)效應(yīng)是通過(guò)糖皮質(zhì)激素受體(glucocorticoid receptor GR)介導(dǎo)的,GR存在于胞漿中,結(jié)合GC后能形成二聚體,并轉(zhuǎn)移到細(xì)胞核中發(fā)揮對(duì)目標(biāo)基因的轉(zhuǎn)錄調(diào)節(jié)作用,從而發(fā)揮對(duì)細(xì)胞功能的調(diào)節(jié)。熱休克蛋白90(HSP90)是GC-GR效應(yīng)全程伴侶蛋白,在GC通過(guò)GR介導(dǎo)產(chǎn)生作用的過(guò)程中,HSP90起到非常重要的作用(參見(jiàn)圖1)。HSP90的質(zhì)(不同亞型)、量(數(shù)量)可能直接影響到GC-GR效應(yīng)。 HSP90是熱休克蛋白家族成員之一,占胞漿蛋白的1%～2%。胞漿中HSP90主要有兩種亞型,分別是HSP90α(誘導(dǎo)型,小鼠中稱(chēng)為HSP86)和HSP90β(組成型,小鼠中稱(chēng)為HSP84)。HSP90主要以同源雙聚體的形式(αα,證據(jù)。兩亞型的基因組來(lái)源、基因序列、mRNA和氨基酸序列都存在很大差別。在誘導(dǎo)方面也存在差別(HSP90α受熱誘導(dǎo)而顯著升高,HSP90β受絲裂原誘導(dǎo)可顯著升高)。提示HSP90亞型在功能上可能存在明顯差異。 另外,如下的一些現(xiàn)象也引起我們關(guān)注: 1)HSP90α是誘導(dǎo)性蛋白,當(dāng)機(jī)體受到應(yīng)激時(shí),HSP90α?xí)芸斓玫窖a(bǔ)充和提高;而HSP90β是結(jié)構(gòu)性蛋白,HSP90β(小鼠叫HSP84)的反應(yīng)和提高則需要一定時(shí)間, 6h內(nèi)存在差異是否說(shuō)明HSP90α也參與了GC-GR通路; 2)HSP90α,HSP90β來(lái)自不同的基因,且在HSP90α,HSP90β的c端和鉸鏈區(qū)差異較大(是與GR結(jié)合和協(xié)助GR入核的區(qū)域)是否就是這個(gè)差異使得只有其中一個(gè)亞型在維持GR結(jié)構(gòu)和功能。我們進(jìn)一步推測(cè):HSP90的質(zhì)(不同亞型)可能對(duì)GC-GR效應(yīng)有不同貢獻(xiàn)。深入研究HSP90亞型對(duì)GC-GR通路的作用將有助于闡明HSP90亞型質(zhì)和量對(duì)GC-GR效應(yīng)的影響,和解決糖皮質(zhì)激素抵抗問(wèn)題,也可能為對(duì)糖皮質(zhì)激素治療不敏感、甚至抵抗的患者帶來(lái)新的希望。本研究首先構(gòu)建HSP90α的RNA干擾的真核表達(dá)質(zhì)粒;然后在降低HSP90α的情況下觀察GC-GR核轉(zhuǎn)位的變化.最后得到了以下的結(jié)果: 1.將化學(xué)合成的三段寡聚核苷酸與線性化的pSUPER-EGFP載體連接獲得了可用于沉默HSP90α基因?qū)嶒?yàn)的三個(gè)質(zhì)粒,經(jīng)酶切鑒定、電泳證明,含作用序列的重組質(zhì)粒pSuper-HSP90α1, pSuper-HSP90α2,及pSuper-HSP90α3構(gòu)建是成功的,重組質(zhì)粒pSuper-HSP90α1, pSuper-HSP90α2測(cè)序分析完全正確。實(shí)驗(yàn)中先后用不同的脂質(zhì)體和質(zhì)粒比例轉(zhuǎn)染HUVEC、HEPG2、HEK 293三種細(xì)胞,以綠色熒光蛋白作為評(píng)價(jià)轉(zhuǎn)染效率的指標(biāo),確定HEK-293細(xì)胞為靶細(xì)胞.在HEK-293中優(yōu)化轉(zhuǎn)染條件,轉(zhuǎn)染效率可以達(dá)到80%左右。 2.用半定量PCR的方法,通過(guò)與對(duì)照質(zhì)粒轉(zhuǎn)染組和未處理組比較發(fā)現(xiàn): pSuper-HSP90α轉(zhuǎn)染后24小時(shí)HEK-293細(xì)胞中HSP90αmRNA水平均有下降,pSuper-HSP90α1最明顯。 3.用western方法,通過(guò)與對(duì)照轉(zhuǎn)染組和未處理組比較發(fā)現(xiàn):pSuper-HSP90α轉(zhuǎn)染后48小時(shí)HEK-293細(xì)胞中HSP90α水平明顯下降。 4.用50 nM GC處理2小時(shí)后,細(xì)胞核中GR與細(xì)胞漿中GR數(shù)量的比值增大,表明GR轉(zhuǎn)位到細(xì)胞核以發(fā)揮調(diào)節(jié)功能。pSuper-HSP90α轉(zhuǎn)染HEK-293細(xì)胞會(huì)增強(qiáng)這種GR核漿比值增大的趨勢(shì)。這種效應(yīng)都是在質(zhì)粒轉(zhuǎn)染細(xì)胞后48小時(shí)觀察到的,這提示HSP90α可能直接負(fù)調(diào)控GR核轉(zhuǎn)位;也可能是HSP90α下降,其他亞型反應(yīng)性增高間接導(dǎo)致GR核轉(zhuǎn)位增強(qiáng)。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 劉繼峰,李久宏,翟寧,耿龍,宋芳吉;多發(fā)性肌炎/皮肌炎患者外周血單一核細(xì)胞中糖皮質(zhì)激素受體及熱休克蛋白90 mRNA的表達(dá)[J];中華皮膚科雜志;2004年01期

2 錢(qián)小順,朱元玨,許文兵,林耀廣,李龍蕓;熱休克蛋白90和糖皮質(zhì)激素受體比例失衡對(duì)哮喘患者T淋巴細(xì)胞凋亡的影響[J];中華醫(yī)學(xué)雜志;2001年24期

相關(guān)博士學(xué)位論文 前1條

1 申海鷹;Hsp90功能域多態(tài)性的鑒定及其對(duì)小鼠應(yīng)激反應(yīng)的影響[D];第三軍醫(yī)大學(xué);2002年

相關(guān)碩士學(xué)位論文 前1條

1 呂金利;C57BL/6和BALB/c小鼠急性胰腺炎預(yù)后差異及其機(jī)制研究[D];第三軍醫(yī)大學(xué);2003年

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本文編號(hào)：1741425