本文選題：氯離子通道 + 膜片鉗　； 參考：《吉林大學(xué)》2010年博士論文

【摘要】：研究目的： 青光眼是主要的不可逆性致盲眼病之一,其確切發(fā)病機(jī)制目前仍不十分清楚。房水流出阻力增加導(dǎo)致高眼壓,是青光眼發(fā)生的主要危險(xiǎn)因素。小梁網(wǎng)是房水流出通道及眼壓調(diào)節(jié)的主要部位。房水流出阻力的調(diào)節(jié)是多機(jī)制聯(lián)合的結(jié)果,包括小梁網(wǎng)的收縮性,細(xì)胞骨架的重排,伸展,及細(xì)胞外基質(zhì)的相互作用,基因表達(dá)的變化,和小梁細(xì)胞容積的變化等。小梁細(xì)胞的容積和收縮性決定著組織間隙,因而在房水流出阻力的調(diào)節(jié)方面具有積極重要的作用。小梁細(xì)胞的收縮性和容積的維持可能與不同的離子通道和泵的調(diào)整有關(guān)。 氯離子是生物體內(nèi)含量最豐富的陰離子,通過(guò)跨膜轉(zhuǎn)運(yùn)和離子通道參與機(jī)體多種生物功能。氯通道種類多樣,容積敏感(調(diào)節(jié))性氯通道(volume-activated chloride channel,VACC或Icl,vol)又稱腫脹激活性氯通道(swelling-activated chloride channel,ICl,swell),幾乎表達(dá)于所有真核細(xì)胞,在維持細(xì)胞容積平衡中發(fā)揮重要作用,并參與調(diào)控細(xì)胞的增殖分化和細(xì)胞凋亡。至今Icl, vol的分子基礎(chǔ)仍不清楚,因?yàn)槿狈μ禺愋缘母哂H和力阻斷劑,要確定的Icl, vol蛋白分子結(jié)構(gòu)非常困難,目前主要應(yīng)用膜片鉗技術(shù)對(duì)其進(jìn)行研究。Srinivas等給予培養(yǎng)的牛小梁細(xì)胞低滲刺激,細(xì)胞容積急性增大,膜片鉗顯示激活了特征性氯電流,該電流可被NPPB和fluoxetine抑制,I-的通透性大于Cl-,符合容積敏感性氯電流的特征。Soto等報(bào)導(dǎo)牛小梁細(xì)胞容積調(diào)節(jié)與氯通道和鉀通道有關(guān)。Mitchell等報(bào)導(dǎo)人小梁細(xì)胞容積調(diào)節(jié)與氯通道有關(guān)。電壓門(mén)控性氯通道(voltage-gated chloride channnel, CIC)是氯離子通道中的一個(gè)大家族,廣泛分布于機(jī)體的細(xì)胞膜和細(xì)胞器膜,參與細(xì)胞電活動(dòng)調(diào)節(jié)、容積調(diào)節(jié)、跨上皮物質(zhì)轉(zhuǎn)運(yùn)、細(xì)胞內(nèi)pH調(diào)節(jié),在細(xì)胞免疫應(yīng)答、細(xì)胞遷移、細(xì)胞增生和分化、細(xì)胞凋亡等多種活動(dòng)和功能的調(diào)節(jié)中均發(fā)揮一定的作用。Comes等報(bào)導(dǎo)在培養(yǎng)的人小梁細(xì)胞中ClC-2～ClC-7均有表達(dá),未見(jiàn)ClC-1的表達(dá)。Srinivas等在培養(yǎng)的牛小梁細(xì)胞中存在ClC-2,ClC-5和P糖蛋白的表達(dá),ClC-3沒(méi)有表達(dá)。 目前已經(jīng)可以確認(rèn),容積敏感性氯通道參與小梁細(xì)胞的容積調(diào)節(jié),并且小梁網(wǎng)表達(dá)多種電壓門(mén)控性氯離子通道。但有關(guān)這方面的研究還很少,兩類氯通道在小梁網(wǎng)中的具體作用還不十分清楚,本文研究的目的是進(jìn)一步探討ClC和Icl,vol在小梁中的可能作用,為青光眼發(fā)病機(jī)制的研究提供一定的理論基礎(chǔ)。 研究?jī)?nèi)容： 體外培養(yǎng)永生化人小梁細(xì)胞,利用全細(xì)胞膜片鉗技術(shù),記錄等滲和低滲條件下小梁細(xì)胞膜上的氯通道電流,觀察該電流特點(diǎn)；觀察在浴液中加入氯離子通道阻斷劑NPPB和他莫西芬及ATP后低滲狀態(tài)下電流的變化；觀察在小梁細(xì)胞培養(yǎng)液中加入10-7mo1/L地塞米松后1天、3天、7天時(shí)低滲狀態(tài)下氯電流的變化情況。 建立大鼠慢性高眼壓模型,前房?jī)?nèi)注射玻璃酸鈉,每周1次,注射10周,每次注射前觀察眼壓情況。 利用RT-PCR方法在mRNA水平檢測(cè)正常大鼠小梁網(wǎng)組織電壓門(mén)控性氯離子通道ClC-1、ClC-2、ClC-3、ClC-4、ClC-5、ClC-K1、ClC-K2的表達(dá)情況,利用免疫組化方法從蛋白水平檢測(cè)大鼠小梁網(wǎng)組織中ClC-2和ClC-3的表達(dá)情況。前房?jī)?nèi)注射玻璃酸鈉誘導(dǎo)大鼠慢性高眼壓模型3周、10周后,重復(fù)上述檢查,觀察模型眼與正常眼間表達(dá)量的變化。 研究結(jié)果： 體外培養(yǎng)的人小梁細(xì)胞,在等滲條件下,僅觀察到微弱且穩(wěn)定的背景電流,低滲刺激后,細(xì)胞迅速腫脹,電流較等滲時(shí)明顯增大,呈外向優(yōu)勢(shì)。此電流對(duì)細(xì)胞容積改變敏感,細(xì)胞腫脹時(shí)被激活,細(xì)胞體積逐漸減少,電流逐漸減弱。該電流呈非時(shí)間依從性,電流未見(jiàn)明顯失活,翻轉(zhuǎn)電位接近Cl-離子的平衡電位。在低滲狀態(tài)下的浴液中分別加入NPPB(100μmol·L-1)、他莫昔芬(50μmol·L-1)和ATP(2μumol·L-1)20min后,電流明顯被抑制。在小梁細(xì)胞培養(yǎng)液中加入10-7mol/L地塞米松繼續(xù)培養(yǎng)1天、3天、7天后,發(fā)現(xiàn)隨著地塞米松處理時(shí)間的延長(zhǎng),小梁細(xì)胞低滲刺激后外向和內(nèi)向電流密度值逐漸減低,加入地塞米松1天,外向和內(nèi)向電流密度值均較低滲時(shí)減低,但無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),加入地塞米松3天、7天外向和內(nèi)向電流密度值均較低滲時(shí)明顯減低,差異有顯著性(P0.05)。 采用前房?jī)?nèi)注射玻璃酸鈉誘導(dǎo)大鼠慢性高眼壓模型,術(shù)前及術(shù)后1周、3周、6周、10周模型眼眼壓值分別為11.47±0.90 mmHg；13.20±0.85 mmHg；21.79±1.47 mmHg；22.96±1.04 mmHg；23.27±1.42 mmHg,可見(jiàn)單次注射眼壓增高幅度較小,反復(fù)多次注射后眼壓持續(xù)緩慢升高,形成穩(wěn)定的慢性高眼壓模型。 利用RT-PCR檢測(cè),結(jié)果顯示：正常大鼠小梁組織中ClC-1、ClC-2、ClC-3、ClC-4、ClC-5、ClC-K1、ClC-K2七種電壓門(mén)控性氯離子通道均有表達(dá),免疫組化方法從蛋白水平證實(shí)了大鼠小梁網(wǎng)組織中存在ClC-2和ClC-3的表達(dá)。前房?jī)?nèi)注射玻璃酸鈉誘導(dǎo)大鼠慢性高眼壓模型3周后,發(fā)現(xiàn)模型組大鼠小梁組織中七種ClC氯通道在mRNA轉(zhuǎn)錄水平的表達(dá)及ClC-2、ClC-3在蛋白水平的表達(dá)均較正常大鼠增高,而高眼壓模型10周后則較正常大鼠減少。 研究成果及在研究領(lǐng)域的地位、意義和價(jià)值： 小梁細(xì)胞的容積和收縮性在房水流出阻力的調(diào)節(jié)中具有積極重要的作用。國(guó)外已有學(xué)者證實(shí)容積敏感性氯通道參與小梁細(xì)胞的容積調(diào)節(jié),國(guó)內(nèi)未見(jiàn)相關(guān)報(bào)道,本文首次在國(guó)內(nèi)對(duì)培養(yǎng)的人小梁細(xì)胞的容積敏感性氯電流的生理學(xué)特點(diǎn)進(jìn)行了研究,并觀察了地塞米松對(duì)該電流的影響,認(rèn)為培養(yǎng)的人小梁細(xì)胞低滲刺激后形成的氯電流符合容積敏感性氯電流(Icl,vol)特點(diǎn),該通道參與小梁細(xì)胞的容積調(diào)節(jié),地塞米松可能通過(guò)影響小梁細(xì)胞容積敏感性氯通道參與激素性青光眼的發(fā)生。 國(guó)外已有學(xué)者證實(shí)在培養(yǎng)的人和牛小梁細(xì)胞中存在電壓門(mén)控性氯通道的表達(dá),國(guó)內(nèi)外均未見(jiàn)關(guān)于大鼠小梁網(wǎng)中ClC氯通道的相關(guān)報(bào)道,已知鼠眼的構(gòu)造及生理學(xué)特點(diǎn)在很多方面和人眼類似,如小梁、Schlemn管、睫狀體、視網(wǎng)膜血管、鞏膜上靜脈等,其房水引流途徑也與人很相似。我們首次證實(shí)了大鼠小梁組織中存在電壓門(mén)控性氯通道的表達(dá),并發(fā)現(xiàn)前房?jī)?nèi)注射玻璃酸鈉誘導(dǎo)大鼠慢性高眼壓模型影響其表達(dá),認(rèn)為ClC型氯通道與小梁細(xì)胞功能密切相關(guān)。 本研究為探討I c1.vol和ClC氯通道在青光眼發(fā)病機(jī)制中的可能作用提供了一定的理論依據(jù),并為進(jìn)一步研究氯離子通道在青光眼發(fā)病機(jī)制中作用及抗青光眼藥物的開(kāi)發(fā)提供新的思路。
[Abstract]:The purpose of the study is:
Glaucoma is one of the main irreversible blindness ophthalmopathy, its exact pathogenesis is still not very clear. The increase of water flow resistance and high intraocular pressure is the main risk factor for glaucoma. Trabecular network is the main part of the flow of aqueous humor and the regulation of intraocular pressure. The adjustment of the resistance of aqueous humor is the result of the combination of multi mechanism. It includes the contractility of the trabecular meshwork, the rearrangement of the cytoskeleton, the extension, the interaction of the extracellular matrix, the change of the gene expression and the change of the volume of the trabecular cells. The volume and contractility of the trabecular cells determine the intertissue gap and thus play an active and important role in the regulation of the flow resistance of the aqueous humor. The contractility and tolerance of the trabecular cells The maintenance of product may be related to the adjustment of different ion channels and pumps.
Chloride ion is the most abundant anion in the organism. It participates in a variety of biological functions through transmembrane transport and ion channels. Chloride channels are diverse and volume sensitive (volume-activated chloride channel, VACC or Icl, vol) are also known as swelling activated chlorine channels (swelling-activated chloride channel, ICl, swell). It is almost expressed in all eukaryotic cells, plays an important role in maintaining cell volume balance, and participates in the regulation of cell proliferation and differentiation and cell apoptosis. So far, the molecular basis of Icl, Vol is still unclear, because the lack of specific high affinity blockers, to determine the Icl, Vol protein molecular structure is very difficult, the main application of the membrane at present The forceps technique studied the hypotonic stimulation of Niu Xiaoliang cells, such as.Srinivas and so on. The cell volume was acute. The patch clamp showed that the characteristic chlorine current was activated by the patch clamp. The current could be suppressed by NPPB and fluoxetine. The permeability of I- was greater than that of Cl-. The volume regulation and chlorine of the bovine trabecular cells were reported in accordance with the characteristic.Soto of the volume sensitive chlorine current. The channel and potassium channel related.Mitchell and other reports of human trabecular cell volume regulation are related to the chloride channel. The voltage gated chloride channel (voltage-gated chloride channnel, CIC) is a large family in the chloride channel. It is widely distributed in the cell membrane and organelles membrane of the body. It participates in the regulation of cell electrical activity, volume regulation, and trans epithelial material transfer. Transport, intracellular pH regulation, in cellular immune response, cell migration, cell proliferation and differentiation, cell apoptosis and other activities and functions play a certain role in the regulation of.Comes and other reports in cultured human trabecular cells ClC-2 to ClC-7 are expressed, no ClC-1 expression of.Srinivas in the existence of ClC-2, ClC in the cultured Niu Xiaoliang cells The expression of -5 and P glycoproteins was not expressed in ClC-3.
It is now confirmed that the volume sensitive chloride channel is involved in the volume regulation of trabecular cells, and the trabecular meshwork network expresses a variety of voltage-gated chloride channels. However, there are few studies on this aspect. The specific role of the two types of chlorine channels in small Liang Wangzhong is not very clear. The purpose of this study is to further explore ClC and Icl, Vol. The possible role of trabeculae provides a theoretical basis for the study of the pathogenesis of glaucoma.
Research content:
In vitro culture of immortalized human trabecular cells, a whole cell patch clamp technique was used to record the current of chloride channel on the membrane of the trabecular cells under isotonic and hypotonic conditions. The characteristics of the current were observed. The changes in the current of the chloride channel blocker NPPB and tamoxifen and the hypotonic state after ATP were observed in the bath liquid. The changes of chloride currents in 1 days, 3 days and 7 days after 10-7mo1/L were added to dexamethasone.
A rat model of chronic intraocular hypertension was established. Sodium hyaluronate was injected into the anterior chamber, 1 times a week for 10 weeks, and the intraocular pressure was observed before each injection.
The expression of voltage gated chloride channel ClC-1, ClC-2, ClC-3, ClC-4, ClC-5, ClC-K1, ClC-K2 in normal rat trabecular meshwork tissue was detected by RT-PCR method at mRNA level. The expression of ClC-2 and ClC-3 in the trabecular meshwork tissue of rats was detected by immunohistochemistry. Sodium hyaluronate induced chronic higher rats in the anterior chamber After 3 weeks, 10 weeks later, the above examination was repeated to observe the change of expression between the model eye and normal eye.
The results of the study:
In vitro, the cultured human trabecular cells, under the isosmotic condition, only observed the weak and stable background current. After the hypotonic stimulation, the cells swelled rapidly and the current increased obviously when the current was isosmotic. The current was sensitive to the cell volume change, the cell swelling was activated, the volume of the cells gradually decreased and the current gradually weakened. There was no apparent inactivation of the current, the reversal potential was close to the equilibrium potential of Cl- ions. NPPB (100 u mol. L-1), tamoxifen (50 mol. L-1) and ATP (2 u umol. L-1) 20min were added to the bath fluid in the low permeability state, and the current was obviously suppressed. The addition of 10-7mol/L dexamethasone into the culture solution of trabecular cells continued for 1 days, 3 days, 7. After the treatment of dexamethasone, the extroversion and inward current density of the trabecular cells decreased gradually after the hypotonic stimulation of the trabecular cells. The extrovert and inward current density values were reduced in 1 days after adding dexamethasone, but there was no statistical difference (P0.05). The value of the extroversion and inward current density on the 7 day was lower than that of the lower osmotic density for 3 days. The difference was significant (P0.05).
The rat model of chronic ocular hypertension was induced by sodium hyaluronate injection in the anterior chamber. The intraocular pressure of the model eyes was 11.47 + 0.90 mmHg, 13.20 + 0.85 mmHg, 21.79 + 1.47 mmHg, 22.96 + 1.04 mmHg and 23.27 + mmHg, respectively before and after 1 weeks, 3 weeks, 6 weeks and 10 weeks, respectively. A slow increase, forming a stable chronic high intraocular pressure model.
Using RT-PCR detection, the results showed that seven voltage gated chloride channels were expressed in ClC-1, ClC-2, ClC-3, ClC-4, ClC-5, ClC-K1, ClC-K2 in normal rat trabecular tissue. The expression of ClC-2 and ClC-3 in the trabecular meshwork of rats was confirmed by immunohistochemistry. Sodium hyaluronate induced chronic high eye in rats by injection of sodium hyaluronate in the anterior chamber After 3 weeks of pressure model, the expression of seven ClC chloride channels in the trabecular tissue of the rat model group and the expression of ClC-2 at the mRNA transcriptional level were found. The expression of ClC-3 at the protein level was higher than that of the normal rats, while the high intraocular pressure model was less than that of the normal rats after 10 weeks.
The research achievements and their status, significance and value in the research field:
The volume and contractility of the trabecular cells play an important role in regulating the flow resistance of the aqueous humor. The volume sensitivity of the volume sensitive chloride channel in the volume regulation of the trabecular cells has not been reported in the country. The physiological characteristics of the volume sensitive chlorine current of cultured human trabecular cells are first introduced in this paper. The effect of dexamethasone on the current was observed. The chlorine current formed by the cultured human trabecular cell hypotonic stimulation accords with the volume sensitive chlorine current (Icl, vol), which is involved in the volume regulation of trabecular cells. Dexamethasone may be involved in hormonal green through the effect of the chloride channel on the volume sensitivity of the trabecular cells. The occurrence of the eye.
Some foreign scholars have confirmed that there is a voltage gated chloride channel expression in the cultured human and Niu Xiaoliang cells. There are no reports about the ClC chloride channel in the trabecular meshwork at home and abroad. It is known that the structure and physiological characteristics of the rat eye are similar to those of the human eyes in many aspects, such as trabeculae, Schlemn tubes, ciliary body, retinal vessels, and sscleral. It is the first time that the expression of voltage gated chloride channels in the trabecular tissue of rats was confirmed, and the expression of chronic high intraocular pressure model induced by sodium hyaluronate injection in the anterior chamber was found to affect its expression. It was found that the ClC type chloride channel was closely related to the function of small Liang Xi cell.
This study provides a theoretical basis for exploring the possible role of I c1.vol and ClC chloride channels in the pathogenesis of glaucoma, and provides a new way of thinking for further research on the role of chloride channel in the pathogenesis of glaucoma and the development of anti glaucoma drugs.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R775.9

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1 于希軍;彌散性粘彈劑Viscoat對(duì)眼內(nèi)壓的影響的實(shí)驗(yàn)研究[D];四川大學(xué);2006年

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