本文選題：糖原合成酶激酶3 + AP-1　； 參考：《華中科技大學(xué)》2006年博士論文

【摘要】： 第一部分 糖原合成酶激酶3在吸煙誘導(dǎo)的豬氣道上皮細(xì)胞鱗狀分化中的作用研究 氣道(氣管和支氣管)上皮的鱗狀細(xì)胞化生常見(jiàn)于慢性支氣管炎癥、致癌劑刺激(如吸煙)等,一般被認(rèn)為是對(duì)慢性損傷的一種適應(yīng)性反應(yīng),也是肺鱗癌的癌前病變,其分子機(jī)制尚未完全闡明。糖原合成酶激酶3(glycogen synthase kinase 3,GSK3)是一種多功能的蛋白激酶,有結(jié)構(gòu)和功能相似的兩種亞型GSK3α和GSK3β,在靜息細(xì)胞內(nèi)呈組成性激活,其N(xiāo)末端絲氨酸殘基(Ser21-GSK3α,Ser9-GSK3β)的磷酸化可導(dǎo)致其活性抑制。多年來(lái)大量的研究表明,GSK3具有廣泛的底物,包括代謝酶、翻譯起始因子、轉(zhuǎn)錄因子、細(xì)胞周期相關(guān)蛋白、癌基因產(chǎn)物和細(xì)胞骨架蛋白等,可參與糖代謝、蛋白質(zhì)合成以及細(xì)胞的增殖、分化、凋亡和運(yùn)動(dòng)等多種生命活動(dòng)。近來(lái)研究顯示GSK3及其轉(zhuǎn)錄因子底物AP-1(activator protein-1)可能參與氣道上皮鱗狀分化的發(fā)生。因此,本研究對(duì)GSK3的表達(dá)、GSK3和AP-1信號(hào)在吸煙誘導(dǎo)的氣道上皮細(xì)胞鱗狀分化中的作用進(jìn)行了初步探討。 本實(shí)驗(yàn)首先用免疫組化和免疫細(xì)胞熒光法檢測(cè)GSK3在人和多種實(shí)驗(yàn)動(dòng)物(大鼠、小鼠和豬)的肺組織及培養(yǎng)的豬氣道上皮細(xì)胞中的表達(dá),結(jié)果顯示GSK3α和GSK3β廣泛表達(dá)于人、鼠和豬的肺組織中,定位于胞漿;它們?cè)趲追N動(dòng)物肺組織中的表達(dá)分布大致相似,主要見(jiàn)于各級(jí)支氣管上皮細(xì)胞、肺泡上皮細(xì)胞、粘膜平滑肌細(xì)胞和粘膜下腺體;但GSK3α在軟骨細(xì)胞中表達(dá)明顯強(qiáng)于GSK3p;幾種哺乳動(dòng)物肺組織中均未檢測(cè)到GSK3α/β的磷酸化。在培養(yǎng)的豬支氣管上皮細(xì)胞中有豐富的GSK3α、β的表達(dá),磷酸化的GSK3α/β信號(hào)弱。 其次,細(xì)胞毒性實(shí)驗(yàn)和形態(tài)學(xué)觀(guān)察顯示香煙煙霧提取物和尼古丁處理可抑制豬氣道上皮細(xì)胞的增殖并導(dǎo)致細(xì)胞出現(xiàn)較為伸展和扁平、融合減緩、細(xì)胞間隙增寬等形態(tài)學(xué)改變。Western blot和RT-PCR檢測(cè)發(fā)現(xiàn),香煙煙霧提取物和尼古丁處理后鱗狀分化標(biāo)記物外皮蛋白(involucrin)和小脯氨酸豐富蛋白(small proline-richprotein,SPRP)的表達(dá)增強(qiáng),證實(shí)了吸煙能夠誘導(dǎo)豬氣道上皮細(xì)胞的鱗狀分化。進(jìn)一步的研究顯示,香煙煙霧提取物和尼古丁處理后,抑制性磷酸化GSK3α/β(Ser-21-GSK3α/Ser-9-GSK3β)的水平升高、GSK3p表達(dá)降低,表明香煙成分可抑制GSK3的表達(dá)和活性;而且,用GSK3的抑制劑SB216763處理細(xì)胞后檢測(cè)外皮蛋白的表達(dá)變化,結(jié)果顯示外皮蛋白的表達(dá)升高并呈現(xiàn)濃度和時(shí)間依賴(lài)性。這提示GSK3可能在香煙成分誘導(dǎo)豬氣道上皮的鱗狀分化中起重要作用。 最后,轉(zhuǎn)錄因子活性檢測(cè)顯示香煙煙霧提取物和尼古丁處理可顯著增強(qiáng)轉(zhuǎn)錄因子AP-1與外皮蛋白基因上游調(diào)節(jié)區(qū)域的結(jié)合活性;而用GSK3的抑制劑LiCl和SB216763模擬香煙成分的作用也得到了類(lèi)似的結(jié)果,提示GSK3可能通過(guò)負(fù)向調(diào)節(jié)AP-1的活性從而介導(dǎo)了香煙成分誘導(dǎo)的豬氣道上皮鱗狀分化。 小結(jié) 上述結(jié)果表明:(1)香煙成分可抑制豬氣道上皮細(xì)胞的增殖并誘導(dǎo)其發(fā)生鱗狀分化。(2)GSK3在氣道上皮細(xì)胞中有豐富的表達(dá),可能通過(guò)負(fù)向調(diào)節(jié)AP-1的活性從而介導(dǎo)了香煙成分誘導(dǎo)的豬氣道上皮鱗狀分化。 第二部分吸煙致肺泡Ⅱ型上皮細(xì)胞損傷中GSK3p的活性變化對(duì) β-catenin/TCF信號(hào)途徑的影響 大量研究證明,吸煙能夠從多個(gè)方面損傷肺泡上皮細(xì)胞,阻礙其遷移、增生和分化以修復(fù)受損區(qū)域,因而有助于吸煙相關(guān)疾病(如慢性阻塞性肺疾病和支氣管源性肺癌等)的發(fā)生與發(fā)展。然而,吸煙致肺泡上皮細(xì)胞損傷的分子機(jī)制還遠(yuǎn)未闡明。 β-連環(huán)素(p-catenin)是一種多功能蛋白,既可作為細(xì)胞骨架蛋白與E-鈣粘附素結(jié)合,參與組成中間連接,維持上皮細(xì)胞的極性和組織結(jié)構(gòu)的完整性;又是Wnt信號(hào)途徑的重要成員,可與TCF/LEF(T cell factor/lymphoid enhancer factor)結(jié)合形成轉(zhuǎn)錄因子復(fù)合體,調(diào)節(jié)多種基因的表達(dá),參與細(xì)胞和組織的發(fā)育分化、損傷修復(fù)以及腫瘤的發(fā)生發(fā)展等生物學(xué)過(guò)程。最近研究顯示,尼古丁和香煙致癌物NNK可促進(jìn)糖原合成酶激酶3(glycogen synthase kinase 3,GSK3)的抑制性磷酸化,而GSK3β可磷酸化調(diào)節(jié)p-catenin,在經(jīng)典的Wnt信號(hào)途徑中起關(guān)鍵性抑制作用。我們課題組的早期工作發(fā)現(xiàn),在吸煙導(dǎo)致的氣道上皮損傷修復(fù)過(guò)程中β-catenin的表達(dá)和定位發(fā)生了變化。但未知GSK3β和p-catenin/TCF信號(hào)是否介導(dǎo)了吸煙誘導(dǎo)的肺泡上皮損傷。因此,本實(shí)驗(yàn)利用人肺泡Ⅱ上皮細(xì)胞株(A549)對(duì)吸煙致肺泡上皮損傷中GSK3p的活性變化及其對(duì)p-catenin/TCF信號(hào)的影響進(jìn)行了初步探討。 本實(shí)驗(yàn)首先采用免疫細(xì)胞熒光檢測(cè)發(fā)現(xiàn)GSK3β在肺泡Ⅱ型上皮細(xì)胞(A549細(xì)胞)中高表達(dá)。然后用不同濃度的香煙煙霧提取物(CSE)處理細(xì)胞24h后,進(jìn)行Western blot分析。結(jié)果顯示,GSK3β的表達(dá)降低、抑制性磷酸化GSK3β的水平升高,并呈現(xiàn)濃度依賴(lài)性,表明CSE可抑制肺泡Ⅱ型上皮細(xì)胞中GSK3β的表達(dá)和活性。 其次,為分析CSE對(duì)p-catenin的影響,用不同濃度的CSE處理細(xì)胞24h后提取細(xì)胞總蛋白,進(jìn)行Western blot檢測(cè)發(fā)現(xiàn)CSE促進(jìn)了β-catenin的表達(dá)并呈現(xiàn)濃度依賴(lài)性。為進(jìn)一步闡明表達(dá)增強(qiáng)的p-catenin是否向核內(nèi)轉(zhuǎn)位,用4%CSE處理細(xì)胞24h后分別提取細(xì)胞漿和細(xì)胞核蛋白進(jìn)行檢測(cè),結(jié)果顯示細(xì)胞漿和細(xì)胞核中β-catenin的表達(dá)均增加,表明CSE促進(jìn)了β-catenin向核內(nèi)轉(zhuǎn)位。 接著,為分析CSE處理后β-catenin/TCF信號(hào)的狀態(tài),用含有TCF/LEF結(jié)合序列的熒光素酶報(bào)告基因質(zhì)粒(pGL3-OT,突變型質(zhì)粒pGL3-OT作為對(duì)照)轉(zhuǎn)染細(xì)胞,再行CSE處理后,檢測(cè)熒光素酶活性。結(jié)果顯示,4%CSE處理組的熒光素酶活性明顯高于對(duì)照組,差異有顯著意義(P0.05),提示CSE可激活肺泡Ⅱ型上皮細(xì)胞中β-catenin/TCF信號(hào)。 最后,為進(jìn)一步分析GSK3p是否在其中發(fā)揮了作用,用持續(xù)激活突變型GSK3β(GSK3βS9A,不受GSK3的上游激酶下調(diào)而持續(xù)激活)轉(zhuǎn)染細(xì)胞后檢測(cè)GSK3β和β-catenin的水平,結(jié)果顯示GSK3p的表達(dá)明顯增強(qiáng),而β-catenin的表達(dá)則顯著降低,證實(shí)了肺泡Ⅱ型上皮細(xì)胞中GSK3β的表達(dá)和活性增強(qiáng)可促進(jìn)β-catenin的降解;與此同時(shí),也證明所用質(zhì)粒GSK3βS9A是可靠的。然后,將GSK3βS9A與pGL3-OT共轉(zhuǎn)染后,再加入CSE處理,進(jìn)行熒光報(bào)告基因分析。分析發(fā)現(xiàn),與僅用CSE處理組相比,共轉(zhuǎn)染GSK3βS9A組的熒光素酶活性顯著降低,差異有極顯著意義(P0.01),說(shuō)明細(xì)胞轉(zhuǎn)染GSK3βS9A后表達(dá)的突變型GSK3p不受CSE的抑制而持續(xù)激活,促進(jìn)了β-catenin的降解,因而導(dǎo)致β-catenin/TCF信號(hào)的抑制。上述結(jié)果表明,在肺泡Ⅱ型上皮細(xì)胞中,CSE可通過(guò)抑制GSK3β而增強(qiáng)β-catenin/TCF的轉(zhuǎn)錄活性。 小結(jié) 本實(shí)驗(yàn)表明:(1)CSE可抑制肺泡Ⅱ型上皮細(xì)胞中GSK3β的表達(dá)和活性。(2)CSE可促進(jìn)肺泡Ⅱ型上皮細(xì)胞中β-catenin的表達(dá)和轉(zhuǎn)位從而激活β-catenin/TCF信號(hào)。(3)在肺泡Ⅱ型上皮細(xì)胞中,CSE可通過(guò)抑制GSK3β而增強(qiáng)β-catenin/TCF的轉(zhuǎn)錄活性。 本實(shí)驗(yàn)提示:GSK3β可通過(guò)對(duì)β-catenin/TCF信號(hào)途徑的調(diào)節(jié),參與吸煙致肺泡上皮細(xì)胞的損傷,進(jìn)而可能介導(dǎo)吸煙相關(guān)疾病的發(fā)生與發(fā)展。
[Abstract]:Part one
Role of glycogen synthase kinase 3 in smoking induced squamous cell differentiation of porcine airway epithelial cells
Squamous cell metaplasia of the airway (trachea and bronchus) is common in chronic bronchitis, carcinogenic stimulant (such as smoking), which is generally considered an adaptive response to chronic injury, and is also a precancerous lesion of squamous cell carcinoma of the lung. Its molecular mechanism has not been fully elucidated. Glycogen synthase kinase 3 (glycogen synthase kinase)
3, GSK3) is a multifunctional protein kinase with two subtypes of structure and function similar to GSK3 alpha and GSK3 beta, which are active in resting cells. The phosphorylation of the N terminal serine residue (Ser21-GSK3 alpha, Ser9-GSK3 beta) can lead to the inhibition of its activity. Many studies have shown that GSK3 has a wide range of substrates, including metabolic enzymes, and turns over many years. Translation initiation factors, transcription factors, cell cycle related proteins, oncogene products and cytoskeleton proteins are involved in glycometabolism, protein synthesis, cell proliferation, differentiation, apoptosis and exercise. Recent studies have shown that the GSK3 and its transcription factor substrate AP-1 (activator protein-1) may be involved in the squamous cell differentiation of the airway epithelium. Therefore, the role of GSK3 expression, GSK3 and AP-1 signaling in smoking induced squamous cell differentiation of airway epithelial cells is discussed in this study.
In this experiment, the expression of GSK3 in lung tissues and cultured porcine airway epithelial cells in human and a variety of experimental animals (rats, mice and pigs) was detected by immunohistochemistry and immunofluorescence. The results showed that GSK3 alpha and GSK3 beta were widely expressed in the lung tissues of human, rat and pig, and located in the cytoplasm; their expression in several animal lung tissues was expressed. The distribution is roughly similar, mainly in bronchial epithelial cells at all levels, alveolar epithelial cells, mucous smooth muscle cells and submucosal glands, but the expression of GSK3 alpha in chondrocytes is obviously stronger than that of GSK3p; there are no phosphorylation of GSK3 alpha / beta in several mammalian lung tissues. There are abundant GSK3 alpha, beta in the cultured porcine bronchial epithelial cells. The expression of phosphorylated GSK3 alpha / beta signal is weak.
Secondly, cytotoxicity test and morphological observation showed that cigarette smoke extract and nicotine treatment could inhibit the proliferation of porcine airway epithelial cells and lead to more extensional and flat cells, slow fusion and widening of intercellular space, such as.Western blot and RT-PCR detection, cigarette smoke extract and nicotine treated scales. The expression of involucrin and small proline rich protein (small proline-richprotein, SPRP) was enhanced, which confirmed that smoking can induce the squamous differentiation of porcine airway epithelial cells. Further studies showed that the inhibitory phosphorylation of GSK3 alpha / beta (Ser-21-GSK3 alpha /Ser-9-GS) after cigarette smoke extract and nicotine treatment. The level of K3 beta and the expression of GSK3p decreased, indicating that the cigarette composition could inhibit the expression and activity of GSK3. Moreover, the expression of the outer skin protein was detected with the GSK3 inhibitor SB216763 treated cells. The results showed that the expression of the outer skin protein increased and showed a concentration and time dependence. This suggests that GSK3 may induce the pig airway in the cigarette composition. The scaly differentiation of the skin plays an important role.
Finally, the activity detection of transcription factor showed that cigarette smoke extract and nicotine treatment could significantly enhance the binding activity of the transcription factor AP-1 and the upstream regulation region of the outer skin protein gene, while the effect of GSK3 inhibitor LiCl and SB216763 on the simulation of cigarette composition was similar, suggesting that GSK3 may regulate the activity of AP-1 by negative direction. Sex thus mediated the differentiation of porcine airway epithelium induced by cigarette components.
Summary
The above results show that: (1) cigarette composition can inhibit the proliferation and induce squamous differentiation of porcine airway epithelial cells. (2) GSK3 has a rich expression in the airway epithelial cells, which may mediate the squamous differentiation of porcine airway epithelium induced by cigarette composition by negatively regulating the activity of AP-1.
The second part is about the change of GSK3p activity in alveolar type II epithelial cells induced by smoking.
The effect of beta -catenin/TCF signal pathway
A large number of studies have shown that smoking can damage alveolar epithelial cells from many aspects, obstruct their migration, proliferation and differentiation to repair the damaged areas, thus contributing to the occurrence and development of smoking related diseases such as chronic obstructive pulmonary disease and bronchogenic lung cancer. However, the molecular mechanism of smoking induced alveolar epithelial cell damage is far from being explained. Ming.
Beta catenin (P-catenin) is a multifunctional protein that can be used as a cytoskeleton and E- calcium adhesion element to form intermediate connections to maintain the polar and tissue integrity of epithelial cells. It is also an important member of the Wnt signaling pathway and can be combined with TCF/LEF (T cell factor/lymphoid enhancer factor) to form a transcription factor complex. The recent study shows that nicotine and cigarette carcinogen NNK can promote the inhibitory phosphorylation of glycogen synthase kinase 3 (glycogen synthase kinase 3, GSK3), while GSK3 beta phosphorylation regulates p-caten. In, which plays a key inhibitory role in the classic Wnt signaling pathway, has been found in our early work group that changes in the expression and localization of beta -catenin during the repair of airway epithelial injury caused by smoking. But the unknown GSK3 beta and p-catenin/TCF signals mediate the alveolar epithelial injury induced by smoking. Therefore, this experiment is used in this experiment. The effect of human alveolar epithelial cell line (A549) on GSK3p activity and p-catenin/TCF signaling in cigarette smoke induced alveolar epithelial injury is discussed.
This experiment first used immunofluorescence to detect the high expression of GSK3 beta in the alveolar type II epithelial cells (A549 cells). Then the Western blot analysis was carried out after the treatment of cell 24h with different concentrations of cigarette smoke extract (CSE). The results showed that the expression of GSK3 beta was reduced, the level of GSK3 beta phosphorylated, and the concentration dependence of the inhibitory phosphorylation of GSK3 beta. It indicates that CSE can inhibit the expression and activity of GSK3 beta in alveolar type II epithelial cells.
Secondly, in order to analyze the effect of CSE on P-catenin, the total protein of cell was extracted with 24h of different concentrations of CSE, and Western blot detection found that CSE promoted the expression of beta -catenin and showed a concentration dependence. The results showed that the expression of beta -catenin in cytoplasm and nucleus increased, indicating that CSE promoted the translocation of beta -catenin into the nucleus.
Then, in order to analyze the state of the beta -catenin/TCF signal after CSE treatment, the luciferase activity was detected by using the luciferase reporter gene plasmid containing the TCF/LEF binding sequence (pGL3-OT, the mutant plasmid pGL3-OT as the control). The luciferase activity was detected after CSE treatment. The results showed that the luciferase activity of the 4%CSE treatment group was significantly higher than that of the control group, and the difference was found. Significant (P0.05) indicates that CSE can activate the beta -catenin/TCF signal in alveolar type II epithelial cells.
Finally, in order to further analyze whether GSK3p played a role in it, the level of GSK3 beta and beta -catenin was detected by continuous activation of mutant GSK3 beta (GSK3 beta S9A, not down regulated by the upstream kinase of GSK3). The results showed that the expression of GSK3p was obviously enhanced and the expression of beta -catenin decreased significantly, which confirmed the type II of alveolar type. The expression and activity enhancement of GSK3 beta in epithelial cells can promote the degradation of beta -catenin; at the same time, it is also proved that the plasmid GSK3 beta S9A is reliable. Then, after CO transfection of GSK3 beta S9A and pGL3-OT, then CSE treatment is added to the fluorescence report gene analysis. The analysis found that the luciferase of GSK3 beta S9A group was co transfected to the GSK3 beta S9A group compared with the CSE treatment group. The activity significantly decreased, and the difference was significant (P0.01), indicating that the mutant GSK3p expressed after the transfection of GSK3 beta S9A was not inhibited by CSE, which promoted the degradation of beta -catenin and resulted in the inhibition of beta -catenin/TCF signal. The results showed that CSE could enhance the beta -cate in the alveolar type II epithelial cells by inhibiting the GSK3 beta. The transcriptional activity of nin/TCF.
Summary
This experiment shows: (1) CSE can inhibit the expression and activity of GSK3 beta in the alveolar type II epithelial cells. (2) CSE can promote the expression and transposition of beta -catenin in the alveolar type II epithelial cells and activate the beta -catenin/TCF signal. (3) in the alveolar type II epithelial cells, CSE can enhance the transcriptional activity of beta -catenin/TCF by inhibiting the GSK3 beta.
This experiment suggests that GSK3 beta may be involved in the injury of alveolar epithelial cells induced by smoking by regulating the signaling pathway of beta -catenin/TCF, and may also mediate the occurrence and development of smoking related diseases.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 陳文書(shū),郝天玲,王曦,田丹,吳人亮;豬氣道上皮細(xì)胞的快速分離培養(yǎng)[J];中華病理學(xué)雜志;2005年10期

2 李文俊,吳人亮,李娜萍,周晟,郝春榮,王曦;β連環(huán)素在吸煙小鼠氣道上皮損傷修復(fù)中的作用[J];中華結(jié)核和呼吸雜志;2001年08期

3 陳芳,吳人亮,王曦,郝天玲;香煙煙霧提取物對(duì)豬氣道上皮細(xì)胞β-連環(huán)素及酪氨酸磷酸化的影響[J];中華醫(yī)學(xué)雜志;2001年07期

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本文編號(hào)：1781946