氯离子通道在小梁网表达及相关功能的实验研究

发布时间：2018-04-21 04:24

本文选题：氯离子通道 + 膜片钳　；参考：《吉林大学》2010年博士论文

【摘要】：研究目的：青光眼是主要的不可逆性致盲眼病之一,其确切发病机制目前仍不十分清楚。房水流出阻力增加导致高眼压,是青光眼发生的主要危险因素。小梁网是房水流出通道及眼压调节的主要部位。房水流出阻力的调节是多机制联合的结果,包括小梁网的收缩性,细胞骨架的重排,伸展,及细胞外基质的相互作用,基因表达的变化,和小梁细胞容积的变化等。小梁细胞的容积和收缩性决定着组织间隙,因而在房水流出阻力的调节方面具有积极重要的作用。小梁细胞的收缩性和容积的维持可能与不同的离子通道和泵的调整有关。氯离子是生物体内含量最丰富的阴离子,通过跨膜转运和离子通道参与机体多种生物功能。氯通道种类多样,容积敏感(调节)性氯通道(volume-activated chloride channel,VACC或Icl,vol)又称肿胀激活性氯通道(swelling-activated chloride channel,ICl,swell),几乎表达于所有真核细胞,在维持细胞容积平衡中发挥重要作用,并参与调控细胞的增殖分化和细胞凋亡。至今Icl, vol的分子基础仍不清楚,因为缺乏特异性的高亲和力阻断剂,要确定的Icl, vol蛋白分子结构非常困难,目前主要应用膜片钳技术对其进行研究。Srinivas等给予培养的牛小梁细胞低渗刺激,细胞容积急性增大,膜片钳显示激活了特征性氯电流,该电流可被NPPB和fluoxetine抑制,I-的通透性大于Cl-,符合容积敏感性氯电流的特征。Soto等报导牛小梁细胞容积调节与氯通道和钾通道有关。Mitchell等报导人小梁细胞容积调节与氯通道有关。电压门控性氯通道(voltage-gated chloride channnel, CIC)是氯离子通道中的一个大家族,广泛分布于机体的细胞膜和细胞器膜,参与细胞电活动调节、容积调节、跨上皮物质转运、细胞内pH调节,在细胞免疫应答、细胞迁移、细胞增生和分化、细胞凋亡等多种活动和功能的调节中均发挥一定的作用。Comes等报导在培养的人小梁细胞中ClC-2～ClC-7均有表达,未见ClC-1的表达。Srinivas等在培养的牛小梁细胞中存在ClC-2,ClC-5和P糖蛋白的表达,ClC-3没有表达。目前已经可以确认,容积敏感性氯通道参与小梁细胞的容积调节,并且小梁网表达多种电压门控性氯离子通道。但有关这方面的研究还很少,两类氯通道在小梁网中的具体作用还不十分清楚,本文研究的目的是进一步探讨ClC和Icl,vol在小梁中的可能作用,为青光眼发病机制的研究提供一定的理论基础。研究内容：体外培养永生化人小梁细胞,利用全细胞膜片钳技术,记录等渗和低渗条件下小梁细胞膜上的氯通道电流,观察该电流特点；观察在浴液中加入氯离子通道阻断剂NPPB和他莫西芬及ATP后低渗状态下电流的变化；观察在小梁细胞培养液中加入10-7mo1/L地塞米松后1天、3天、7天时低渗状态下氯电流的变化情况。建立大鼠慢性高眼压模型,前房内注射玻璃酸钠,每周1次,注射10周,每次注射前观察眼压情况。利用RT-PCR方法在mRNA水平检测正常大鼠小梁网组织电压门控性氯离子通道ClC-1、ClC-2、ClC-3、ClC-4、ClC-5、ClC-K1、ClC-K2的表达情况,利用免疫组化方法从蛋白水平检测大鼠小梁网组织中ClC-2和ClC-3的表达情况。前房内注射玻璃酸钠诱导大鼠慢性高眼压模型3周、10周后,重复上述检查,观察模型眼与正常眼间表达量的变化。研究结果：体外培养的人小梁细胞,在等渗条件下,仅观察到微弱且稳定的背景电流,低渗刺激后,细胞迅速肿胀,电流较等渗时明显增大,呈外向优势。此电流对细胞容积改变敏感,细胞肿胀时被激活,细胞体积逐渐减少,电流逐渐减弱。该电流呈非时间依从性,电流未见明显失活,翻转电位接近Cl-离子的平衡电位。在低渗状态下的浴液中分别加入NPPB(100μmol·L-1)、他莫昔芬(50μmol·L-1)和ATP(2μumol·L-1)20min后,电流明显被抑制。在小梁细胞培养液中加入10-7mol/L地塞米松继续培养1天、3天、7天后,发现随着地塞米松处理时间的延长,小梁细胞低渗刺激后外向和内向电流密度值逐渐减低,加入地塞米松1天,外向和内向电流密度值均较低渗时减低,但无统计学差异(P0.05),加入地塞米松3天、7天外向和内向电流密度值均较低渗时明显减低,差异有显著性(P0.05)。采用前房内注射玻璃酸钠诱导大鼠慢性高眼压模型,术前及术后1周、3周、6周、10周模型眼眼压值分别为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,可见单次注射眼压增高幅度较小,反复多次注射后眼压持续缓慢升高,形成稳定的慢性高眼压模型。利用RT-PCR检测,结果显示：正常大鼠小梁组织中ClC-1、ClC-2、ClC-3、ClC-4、ClC-5、ClC-K1、ClC-K2七种电压门控性氯离子通道均有表达,免疫组化方法从蛋白水平证实了大鼠小梁网组织中存在ClC-2和ClC-3的表达。前房内注射玻璃酸钠诱导大鼠慢性高眼压模型3周后,发现模型组大鼠小梁组织中七种ClC氯通道在mRNA转录水平的表达及ClC-2、ClC-3在蛋白水平的表达均较正常大鼠增高,而高眼压模型10周后则较正常大鼠减少。研究成果及在研究领域的地位、意义和价值：小梁细胞的容积和收缩性在房水流出阻力的调节中具有积极重要的作用。国外已有学者证实容积敏感性氯通道参与小梁细胞的容积调节,国内未见相关报道,本文首次在国内对培养的人小梁细胞的容积敏感性氯电流的生理学特点进行了研究,并观察了地塞米松对该电流的影响,认为培养的人小梁细胞低渗刺激后形成的氯电流符合容积敏感性氯电流(Icl,vol)特点,该通道参与小梁细胞的容积调节,地塞米松可能通过影响小梁细胞容积敏感性氯通道参与激素性青光眼的发生。国外已有学者证实在培养的人和牛小梁细胞中存在电压门控性氯通道的表达,国内外均未见关于大鼠小梁网中ClC氯通道的相关报道,已知鼠眼的构造及生理学特点在很多方面和人眼类似,如小梁、Schlemn管、睫状体、视网膜血管、巩膜上静脉等,其房水引流途径也与人很相似。我们首次证实了大鼠小梁组织中存在电压门控性氯通道的表达,并发现前房内注射玻璃酸钠诱导大鼠慢性高眼压模型影响其表达,认为ClC型氯通道与小梁细胞功能密切相关。本研究为探讨I c1.vol和ClC氯通道在青光眼发病机制中的可能作用提供了一定的理论依据,并为进一步研究氯离子通道在青光眼发病机制中作用及抗青光眼药物的开发提供新的思路。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R775.9

【参考文献】