雌、雄激素对去卵巢雌鼠泪液分泌及泪腺中Bax，bcl-2表达的影响和泪腺中TGF-β_1、MMP-2及其抑制剂TIMP-2表达的实验研究
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摘要
眼表(角膜、结膜、副泪腺和睑板腺)、主泪腺和它们之间的神经放射环由于其密切的解剖和功能联系构成一个整体功能单位,称为“眼表泪腺反馈系统”。它们共同发挥对泪液分泌和泪膜形成的调控作用,维护眼表健康,任一环节的损害均可导致泪膜完整性和功能的破坏,从而导致干眼。干眼的发病机制比较复杂,目前认为主要与炎症、细胞凋亡、神经调节机制异常和性激素水平失调等因素相关。干眼常发生于老年女性,性激素被认为是其发病机制之一。在绝经后的妇女,内分泌变化是干眼发生的重要因素,然而是由于雌激素过剩还是不足、雄激素的缺乏和/或雌、雄激素之间的不平衡影响眼表功能尚有争议,哪些因素起主要作用仍不清楚。
细胞凋亡(apoptosis)是一种特殊的细胞死亡类型,指细胞在一定的生理或者病理条件下,遵循自身固有程序结束生命的现象。细胞凋亡是一种正常的生理现象,通过凋亡,机体清除感染和损伤,维持机体正常生理功能。然而,在一定条件下,细胞凋亡又是一种病理现象。细胞凋亡受多种信号的协同调控,其中最重要的调节细胞凋亡的因子是bcl-2和它相关的基因。细胞凋亡在免疫性疾病中起到十分重要的作用。干眼泪腺和眼表上皮细胞的凋亡异常增加,而局部组织中的淋巴细胞的凋亡却被抑制。
基质金属蛋白酶(matrix metalloproteinases,MMPs)的表达受细胞因子、生长因子、性激素、神经激素、细胞形态改变,细胞间交互作用和细胞转化等多种因素的调节。已证实性激素在许多组织中调节MMP-2和MMP-9的合成,全身性和非全身性干燥综合症(Sj?gren syndrome,SS)病人泪液和唾液中MMP-2和MMP-9均增加,在不同的SS鼠模型中也是如此,泪腺炎症诱导的兔干眼模型泪腺和角膜中MMP-2和MMP-9也同样升高,认为MMPs可能是本病的发病机理。转化生长因子-β1(transforming growth factor-β1, TGF-β1)是一类具有多种生物活性的细胞因子,参与调节细胞生长、分化等多种功能,是免疫系统的主要抑制性分子。TGF-β1刺激细胞外基质(ECM)如胶原蛋白、纤维连接蛋白、层粘蛋白和蛋白多糖的合成,抑制基质蛋白酶的产生,促进调节各种细胞连接蛋白受体的表达及ECM成分与这些受体的结合。然而关于性激素调节泪腺中MMP-2及其抑制剂TIMP-2表达,以及与TGF-β1的相互关系却不清楚。以往研究表明:17β-雌二醇促进人角膜上皮细胞中促炎细胞因子和MMPs的表达,而MMPs与干眼症又有非常密切的关系。近些年,性激素在干眼中的作用逐渐引起人们的重视,成为研究的热点。
为此本实验设计了通过对雌性性成熟Wistar大鼠行双侧卵巢切除术(Ovariectomy,OVX),模拟人工绝经状态,给予大鼠雌、雄激素全身及局部治疗,观察血清雌、雄激素水平、泪液分泌量、泪膜稳定性、大鼠泪腺和眼表组织学的变化,检测泪腺中凋亡基因Bax、bcl-2、TGF-β1因子表达及MMP-2及其抑制剂TIMP-2的表达,探讨雌、雄激素对去卵巢雌鼠泪液分泌及泪腺功能的影响及其作用机制,为临床医生应用雌、雄激素治疗绝经期妇女干眼提供实验依据和理论基础。本实验分为三部分。
第一部分雌、雄激素对去卵巢雌鼠泪液分泌及眼表和泪腺组织病理学和超微结构改变的影响
目的:探讨雌、雄激素对去卵巢雌鼠泪液分泌及眼表和泪腺组织病理和超微结构改变的影响。方法:雌性性成熟Wistar大鼠64只,随机分为正常对照组8只、假手术组8只、实验组48只。假手术组仅打开腹腔,切除部分脂肪,实验组行双侧OVX。术后5个月将实验组随机分为实验1组、实验2组、实验3组、实验4组、实验5组、实验6组,每组8只大鼠。自术后第5个月起,实验1组不予任何干预,实验2组给予粟米油100ul皮下注射每3天一次,实验3组给予苯甲酸雌二醇200ug/kg皮下注射每3天一次,实验4组给予苯甲酸雌二醇点眼,每日4次,实验5组给予3.75mg/kg丙酸睾丸酮肌肉注射每3天一次,实验6组给予2.5%丙酸睾丸酮点眼,每日4次。分别于术前、术后第1、2、3、4、5个月及给药后6周,测大鼠泪液分泌量(SchirmerⅠtest,SⅠt)、泪膜破裂时间(tear film break-up time,BUT)及角膜荧光素钠染色情况。给药6周后处死动物,立刻取所有大鼠泪腺、角膜和结膜,行眼表和泪腺组织病理学和透射电镜检查。分别于术前、术后第5个月及给药后6周测大鼠血清雌二醇和睾酮水平。结果:1实验组动物在OVX术后1个月BUT较术前缩短(P<0.01);实验3组给药6周后BUT结果较给药前缩短(P<0.01);实验5组给药6周后BUT结果较给药前延长(P<0.05)。其余组治疗前后BUT结果无显著性差异(P>0.05)。2实验组动物在OVX术后3个月SⅠt结果较术前下降50%,且与术前比较有非常显著性差异(P<0.01)。实验3组给药6周后SⅠt结果较给药前缩短(P<0.05)。实验5组给药6周后SⅠt结果较给药前延长(P<0.05)。其余组治疗前后SⅠt结果无显著性差异(P>0.05)。3实验组动物在OVX术后4个月角膜染色评分较术前增加(P<0.05);实验3组给药6周后角膜染色评分较给药前增加(P<0.05);实验5组给药6周后角膜染色评分较给药前减少(P<0.05);其余组治疗前后角膜染色评分无显著性差异(P>0.05)。4眼表及泪腺组织病理学及泪腺超微结构:实验3组组织病理学及超微结构病变均较实验1组加重;实验5组组织病理学及超微结构病变均较实验1组有明显改善。5血清性激素水平:OVX术后5个月大鼠血清雌二醇水平明显低于术前(P<0.01)。实验3组给药6周后血清雌激素水平上升。OVX术后5个月大鼠血清睾酮水平低于术前(P<0.05)。实验5组给药6周后血清睾酮水平上升。结论:1通过OVX模拟人工绝经状态,去卵巢雌鼠血清中雌、雄激素水平均较术前降低,泪液分泌减少,泪膜稳定性下降。全身补充雄激素使泪液分泌量增加,泪膜稳定性提高;全身补充雌激素使泪液分泌量减少,泪膜稳定性下降。推测OVX引起的泪液分泌减少与血清雄激素水平下降有关。2全身补充雄激素能够改善由于OVX术后出现的泪腺萎缩,缓解眼表损害,促进泪液分泌,改善泪膜稳定性。3全身补充雌激素加重泪腺萎缩及眼表损害,减少泪液分泌,破坏泪膜稳定性。提醒临床医生应慎用激素替代疗法。
第二部分雌、雄激素对去卵巢雌鼠泪腺中凋亡基因Bax,bcl-2表达的影响
目的:探讨雌、雄激素对去卵巢雌鼠泪腺中凋亡基因Bax、bcl-2表达的影响。方法:实验动物及分组同第一部分。给药6周后处死动物,立刻摘取大鼠泪腺,采用免疫组化(SP法)观察泪腺上皮细胞凋亡率及凋亡基因Bax和bcl-2的表达。结果:1泪腺上皮凋亡相关基因Bax和bcl-2的表达:实验1组泪腺上皮中Bax的平均光密度高于A组(P<0.01);实验3组给药6周后泪腺上皮中Bax的平均光密度高于实验1组(P<0.01)。实验5组给药6周后泪腺上皮中Bax的平均光密度低于实验1组(P<0.05)。实验1组泪腺上皮中bcl-2的平均光密度低于A组(P<0.01)。实验3组给药6周后泪腺上皮中bcl-2的平均光密度低于实验1组(P<0.01)。实验5组给药6周后泪腺上皮中bcl-2的平均光密度高于实验1组(P<0.01)。2泪腺上皮细胞凋亡率:实验1组泪腺上皮细胞凋亡率高于A组(P<0.01);实验3组给药6周后泪腺上皮细胞凋亡率高于实验1组(P<0.01);实验5组给药6周后泪腺上皮细胞凋亡率低于实验1组(P<0.01)。结论:正常大鼠泪腺上皮中存在Bax的表达,OVX术后泪腺上皮细胞中Bax表达增加,bcl-2表达减少,泪腺上皮细胞凋亡可能是去卵巢雌鼠泪液分泌减少的作用机制之一。全身应用雌激素加重去卵巢大鼠泪腺上皮细胞凋亡,全身应用雄激素减轻去卵巢大鼠泪腺上皮细胞凋亡。提示雄激素通过改善泪腺上皮细胞凋亡,使泪液分泌量增加,泪膜稳定性提高,眼表损害减轻;雌激素与之作用相反。
第三部分雌、雄激素对去卵巢雌鼠泪腺中TGF-β1、MMP-2及其抑制剂TIMP-2表达及其相关性的实验研究
目的:探讨雌、雄激素对去卵巢雌鼠泪腺中MMP-2及其抑制剂TIMP-2的表达的影响及TGF-β1的表达变化,并对TGF-β1、MMP-2及TIMP-2进行相关性分析,进一步探讨MMP-2及TIMP-2在干眼中的作用机制。方法:实验动物及分组同第一部分。给药6周后处死动物,立刻摘取大鼠泪腺,应用RT-PCR技术检测MMP-2mRNA和TIMP-2mRNA水平,采用Western-blot方法检测泪腺中MMP-2蛋白含量的变化,应用酶联免疫吸附试验(ELISA)检测泪腺中TGF-β1的质量浓度。结果:1 RT-PCR法检测去卵巢雌鼠泪腺中MMP-2mRNA水平结果:各组用药6周后泪腺中MMP-2mRNA水平比较有非常显著性差异(P<0.01)。实验1组泪腺中MMP-2mRNA与A组比较表达增加(P<0.05);实验3组泪腺中MMP-2mRNA与实验1组比较表达增加(P<0.05);实验5组泪腺中MMP-2mRNA与实验1组比较表达减少(P<0.01)。2 RT-PCR法检测去卵巢雌鼠泪腺中TIMP-2mRNA水平结果:各组用药6周后泪腺中TIMP-2mRNA水平比较有非常显著性差异(P<0.01)。实验1组泪腺中TIMP-2mRNA与A组比较表达增加(P<0.05);实验3组泪腺中TIMP-2mRNA与实验1组比较表达增加(P<0.05);实验5组泪腺中TIMP-2mRNA与实验1组比较表达减少(P<0.01)。3 Western Blot法检测雌、雄激素对去卵巢雌鼠泪腺中MMP-2蛋白的表达:各组用药6周后泪腺中MMP-2蛋白的表达比较有非常显著性差异(P<0.01)。实验1组泪腺中MMP-2蛋白的表达与A组比较表达增加(P<0.05);实验3组泪腺中MMP-2蛋白的表达与实验1组比较表达增加(P<0.05);实验5组泪腺中MMP-2蛋白的表达与实验1组比较表达减少(P<0.01)。4泪腺中TGF-β1因子的表达:各组用药6周后泪腺匀浆上清液中TGF-β1质量浓度比较有非常显著性差异(P<0.01),实验5组与实验1组间有非常显著性差异(P<0.01)。5 TGF-β1、MMP-2mRNA及TIMP-2mRNA相关性分析结果:TGF-β1与MMP-2mRNA间无相关关系(r=0.089,P=0.587);TGF-β1与TIMP-2mRNA间无相关关系(r=0.106,P=0.515);MMP-2mRNA与TIMP-2mRNA间有相关关系(r=0.445,P=0.004)。结论:1 OVX术后泪腺中MMP-2mRNA和TIMP-2mRNA水平较正常组增加;雌激素上调泪腺中MMP-2mRNA和TIMP-2mRNA水平;雄激素下调泪腺中MMP-2mRNA和TIMP-2mRNA水平;且MMP-2与TIMP-2间有相关关系。2 OVX术后泪腺中MMP-2蛋白的表达较正常组增加;雌激素上调泪腺中MMP-2蛋白的表达;雄激素下调泪腺中MMP-2蛋白的表达。3雄激素促进去卵巢雌鼠泪腺中TGF-β1的表达。4推测雌激素通过上调泪腺中MMP-2的水平导致泪液分泌减少,泪膜稳定性下降,雄激素与之作用相反,一方面通过增加TGF-β1的含量,抑制泪腺的免疫反应,另一方面,通过抑制MMP-2的表达,改善干眼的泪液分泌功能和减缓泪腺损害。
Ocular surface(cornea、conjunctiva、accessory lacrimal gland and meibomian gland)、lachrymal gland and their nerve conjunction setting up a function unit for their intimate anatomy and function relationship, is called“ocular surface-lachrymal gland feedback system”.They play an important role in regulating lacrimal secretion and tear-film formation together and maintaining the health of ocular surface. The damage of every element will induce to destroy the integrality and function of tear film and lead to dry eye. It is multiplicity about the pathogenesy of dry eye.Now it is sugested that dry eye is related to inflammation, apoptosis and the abnormality of neuromodulation and the imbalance of sex hormone. Dry eye is most often in old women. It is sugested that sex hormone is one of the pathogenesies of dry eye. The change of internal hormone is the important factor in post-menopause women.It remains to be ascertained, however, whether dry eye relates to estrogen excess or deficiency, androgen deficiency and/or estrogen/androgen imbalance.
Apoptosis,a special cell death, is a phenomenon that in definite physiology and pathology condition cells end the life following their own program. Apoptosis is a normal physiology phenomenon,through which the body clear infect and damage and retain the body normal physiological functions.However, in certain conditions, apoptosis is a pathology phenomenon.Apoptosis coordinated regulation through many signals. The most important regulation factor is bcl-2 and its related gene. Apoptosis plays most important role in immune diseases.Apoptosis in lacrimal gland and ocular surface epithelium increase abnormally,but in local lymphocytic cell is inhibitted in dry eye .
The expression of matrix metalloproteinase(MMPs) is regulated by many factors such as cytokine, growth factor, sex hormone, neurohormone,the change of cell appearance,the interaction between cells and cell transformation.Elevated levels of both MMP-2 and MMP-9, which have been found in the tears and saliva of patients with systemic dry eye disease and non-systemic dry eye disease, as well as in the lacrimal gland and cornea of a rabbit model with lacrimal gland inflammation-induced dry eye, suggest that both MMP-2 and MMP-9 may be implicated in the pathogenesis of this disease. Transforming growth factor-β1 is a kind of bioactive cytokine. It participates in regulating cell growth and differentiation. It is major rejection capability molecule in immune system. TGF-β1 stimulates the synthesis of extracellular matrix such as collagen protein, fibrous joint protein, laminin and proteoglycan and inhibits the production of MMPs. It promotes the expression of connexin receptor and the combination of ECM with these receptors. However, it is not clear that whether sex hormone regulates the expression of MMP-2 and it’s inhibitor TIMP-2 in lachrymal gland and that the relationship among MMPs,sex hormone and TGF-β1.The research indicated that 17β-estradiol up-regulation proinflammatory cytokine and MMPs in human corneal epithelial cells.There is a close relationship between MMPs and dry eye.Investigators have paid more attention to dry eye and the role of sex hormone recently.
Therefore,this investigation as follows: sexual mature rats had been done ovariectomy(OVX), modelling artificial menopause state, administrated systemic and local eyedrop application with estrogen and androgen. The estrogen and androgen levels in serum and lacrimal secretion, tear film break-up time and corneal fluorescence staining were measured.Lachrymal gland was obtained for pathohistological observation. The expressions of Bax, bcl-2, TGF-β1, MMP-2 and it’s inhibitor TIMP-2 in lachrymal gland and relationship were studied. The effect of estrogen and androgen on lacrimal secretion and lachrymal gland function in ovariectomized rats and mechanism were investigated.It provided experiment evidence and rationale for the treatment of dry eye with estrogen and androgen to clinician. This experiment devided into 3 parts.
Part 1 Effect of estrogen and androgen on lacrimal secretion and the histological and ultramicrostructure change in lacrimal gland and ocular surface in ovariectomized rats
Objective:To investigate the effect of estrogen and androgen on lacrimal secretion and the histological and ultramicrostructure change of lacrimal gland and ocular surface in ovariectomized rat. Methods: sixty-four female Wistar rats were devided into contol group, sham operation group and experiment group randomly. In sham operation group, rats were only cut little fat in abdominal cavity.In experiment group, rats had been done ovariectomy(OVX). Lacrimal secretion (SⅠt), tear film break-up time (BUT test) and corneal fluorescence staining were measured in all groups before the operation and 1,2,3,4, 5 months after the operation.Five months later, rats of OVX experiment group were treated with systemic application and eyedrop application with corn oil, estrogen and androgen and devided into experiment group 1, experiment group 2, experiment group 3, experiment group 4, experiment group 5, experiment group 6.There was no any intervention in experiment group 1.The rats in experiment group 2 were given hypodermical injection of oil (100ul )every 3 days. The rats in experiment group 3 were given hypodermical injection of estradiol benzoate (200ug/kg)every 3 days. The rats in experiment group 4 were given eyedrop of estradiol benzoate 4 times every day. The rats in experiment group 5 were given intramuscular injection of testosterone (3.75mg/kg)every 3 days. The rats in experiment group 6 were given eyedrop of testosterone 4 times every day.Six weeks later, SIt and BUT test and corneal fluorescence staining were measured.Then all the rats were sacrificed. The lachrymal gland, cornea and conjunctiva were obtained for pathohistological ultramicrostructure observation. The serum estrogen and androgen were measured before and 5 months after the operation,as well as before death. Results: 1 The result of BUT in the experiment group 1 month after OVX was shortter than that before OVX (P<0.01). The result of BUT in the experiment group 3 with estradiol enzoate injected hypodermically for 6 weeks was shortter than that before injection (P<0.01).While the BUT of the experiment group 3 with testosterone intramuscular injected for 6 weeks was longer than that before injection (P<0.01). There was no difference in other groups (P>0.05).2 The result of SⅠt 3 months after OVX decreased to 50% of the result before OVX (P<0.01). The result of SⅠt in experiment group 3 was shorter than that before injection(P<0.05). The result of SⅠt in experiment group 5 was longer than that before injection(P<0.05). There was no difference in other groups (P>0.05). 3 The score of corneal fluorescence staining 4 months after OVX increased compared with the score before OVX(P<0.05). The score of corneal fluorescence staining in experiment group 3 increased compared with the score before injection(P<0.05). The score of corneal fluorescence staining in experiment group 5 decreased compared with the score before injection(P<0.05). There was no difference in other groups(P>0.05).4 The histological change and ultrastructure of lacrimal gland and ocular surface:There had been obvious improvement in experiment group 5 than experiment group 1. There had been more serious in experiment group 3 than experiment group 1. 5 The estrogen and androgen levels in serum decreased after OVX,compared with those before OVX. After estrogen or androgen injected, both the serum estrogen level in experiment group 3 and the serum androgen level in experiment group 5 increased.Conclusion:1 The sexual mature rat had been done ovariectomy(OVX), modelling artificial menopause state , the level of estrogen and androgen in serum decreased, lacrimal secretion decreased, the stability of tear-film decreased.In systemic androgen treatment group, lacrimal secretion increased, the stability of tear-film increased. In systemic estrogen treatment group, lacrimal secretion decreased, the stability of tear-film decreased. It indicated that the decrease of lacrimal secretion and stability of tear-film is related to the decrease of the level of androgen in serum. 2 Systemic androgen treatment can improve atrophy in the lacrimal gland caused by OVX It relieve the damage of ocular surface and increase lacrimal secretion and the stability of tear-film. 3 Systemic estrogen treatment can aggravate the atrophy in the lacrimal gland and the damage of ocular surface and decrease lacrimal secretion and the stability of tear-film. We remind clinician that it must be cautious when they applicate hormone replacement therapy.
Part 2 Effect of estrogen and androgen on the expression of apoptosis in lachrymal gland in ovariectomized rats
Objective: To investigate the effect of estrogen and androgen on the expression in apoptosis gene in lachrymal gland in ovariectomized rats. Methods: The rats handled as same as part one. Six weeks after treatment, all rats were sacrificed, and the lachrymal glands were obtained. The expression of Bax and bcl-2 were detected by immunohistochemical staining in different groups. Results: 1 The optical density(OD) of Bax in lacrimal epithelium cell :The OD in experiment group 1 rats was higher than A group (P<0.01), The OD in experiment group 3 rats was higher than experiment group 1 (P<0.01).The OD in experiment group 5 rats was lower than experiment group 1 (P<0.05).The OD of bcl-2 in lacrimal epithelium cell: The OD in experiment group 1 rats was lower than A group (P<0.01), The OD in experiment group 3 rats was lower than experiment group 1(P<0.01).The OD in experiment group 5 rats was higher than experiment group 1(P<0.01).2 Apoptosis index of lacrimal epithelium:The percent of positive cell in experiment group 1 was higher than A group (P<0.01),The percent of positive cell in experiment group 3 higher than experiment group 1(P<0.01). The percent of positive cell in experiment group 5 was lower than experiment group 1 (P<0.01). Conclusions: There was expression on apoptosis of Bax in normal lacrimal epithelium. After OVX, the expression of Bax increased and the expression of bcl-2 decreased in lacrimal epithelium.The apoptosis in lachrymal gland epithelium may be one of mechanisms of action. Apoptosis in lachrymal gland epithelium became more serious in systemic estrogen group and relieved in systemic androgen group. It suggested that androgen treatment made lacrimal secretion and the stability of tear-film increase through relieving apoptosis. However, the role of estrogen is opposite.
Part 3: The experiment study of estrogen and androgen on expression of TGF-β1、MMP-2、it’s inhibitor TIMP-2 and relationship to TGF-β1 in lachrymal gland in ovariectomized rats.
Objectives: To investigate the effect of estrogen and androgen on the expression of MMP-2mRNA、TIMP-2mRNA and MMP-2 protein in lachrymal gland in ovariectomized rats.To make correlation analysis of TGF-β1,MMP-2 and TIMP-2.To explore the function of MMP-2 and TIMP-2 in dry eye.Methods: The rats handled as same as part one. Six weeks after treatment, all rats were sacrificed, and the lachrymal gland was obtained.The expression of MMP-2mRNA and TIMP-2mRNA was detected by RT-PCR.The expression of MMP-2 protein was detected by Western-blot. The level of TGF-β1was detected by ELISA.Results:1 The expression of MMP-2mRNA in lachrymal gland:Six weeks after treatment, there was significant differences on the expression of MMP-2mRNA in lachrymal gland in different groups(P<0.01).Experiment group 1 was hihger than A group(P<0.05).Experiment group 3 was hihger than experiment group 1(P<0.05).Experiment group 5 was lower than experiment group 1(P<0.01).2 The expression of TIMP-2 mRNA in lachrymal gland: Six weeks after treatment, there was significant differences of the expression of TIMP-2 mRNA in lachrymal gland in different groups(P<0.01). Experiment group 1 was higher than A group (P<0.05).Eexperiment group 3 was hihger than experiment group 1(P<0.05).Experiment group 5 was lower than experiment group 1(P<0.01).3 The expression of MMP-2 protein in lachrymal gland: Six weeks after treatment, there were significant difference of the expression of MMP-2 protein in lachrymal gland in different groups(P<0.01). Experiment group 1 was higher than A group(P<0.05).Experiment group 3 was higher than experiment group 1(P<0.05).Experiment group 5 was lower than experiment group 1(P<0.01). 4 The expression of TGF-β1 in lachrymal gland: There was significant difference in different groups after treatment for 6 weeks about mass concentration of TGF-β1 in supernatant of lachrymal gland homogenate(P<0.01). There was significant difference between experiment group 5 and experiment group 1 after treatment for 6 weeks about mass concentration of TGF-β1 in supernatant of lachrymal gland homogenate(P<0.01).5 The correlation analysis among TGF-β1、MMP-2mRNA and TIMP-2mRNA:There was no correlation between TGF-β1 and MMP-2mRNA(r=0.089,P=0.587).There was no correlation between TGF-β1 and TIMP-2 mRNA(r=0.106,P=0.515).There was correlation between MMP-2 mRNA and TIMP-2 mRNA(r=0.445,P = 0.004). Conclusion:1 After OVX the level of MMP-2 mRNA and TIMP-2 mRNA in lachrymal gland is higher than normal group.Estrogen up-regulate the expression of MMP-2mRNA and TIMP-2mRNA in lachrymal gland. Androgen down-regulate the expression of MMP-2mRNAand TIMP-2mRNA in lachrymal gland. There was correlation between MMP-2 mRNA and TIMP-2 mRNA.2 After OVX the level of MMP-2 protein in lachrymal gland is higher than normal group.Estrogen up-regulate the expression of MMP-2 protein in lachrymal gland. Androgen down-regulate the expression of MMP-2 protein in lachrymal gland. Androgen can promote lacrimal secretion and the expression of TGF-β1 in lacrimal gland in OVX rats.It suggested that estrogen induced the decrease of lacrimal secretion and the stability of tear-film through up-regulation of MMP-2. The role of androgen is opposite.In one hand,it increase the level of TGF-β1 and inhibit immune reaction in lachrymal gland. In another hand, it inhibit the expression of MMP-2.
细胞凋亡(apoptosis)是一种特殊的细胞死亡类型,指细胞在一定的生理或者病理条件下,遵循自身固有程序结束生命的现象。细胞凋亡是一种正常的生理现象,通过凋亡,机体清除感染和损伤,维持机体正常生理功能。然而,在一定条件下,细胞凋亡又是一种病理现象。细胞凋亡受多种信号的协同调控,其中最重要的调节细胞凋亡的因子是bcl-2和它相关的基因。细胞凋亡在免疫性疾病中起到十分重要的作用。干眼泪腺和眼表上皮细胞的凋亡异常增加,而局部组织中的淋巴细胞的凋亡却被抑制。
基质金属蛋白酶(matrix metalloproteinases,MMPs)的表达受细胞因子、生长因子、性激素、神经激素、细胞形态改变,细胞间交互作用和细胞转化等多种因素的调节。已证实性激素在许多组织中调节MMP-2和MMP-9的合成,全身性和非全身性干燥综合症(Sj?gren syndrome,SS)病人泪液和唾液中MMP-2和MMP-9均增加,在不同的SS鼠模型中也是如此,泪腺炎症诱导的兔干眼模型泪腺和角膜中MMP-2和MMP-9也同样升高,认为MMPs可能是本病的发病机理。转化生长因子-β1(transforming growth factor-β1, TGF-β1)是一类具有多种生物活性的细胞因子,参与调节细胞生长、分化等多种功能,是免疫系统的主要抑制性分子。TGF-β1刺激细胞外基质(ECM)如胶原蛋白、纤维连接蛋白、层粘蛋白和蛋白多糖的合成,抑制基质蛋白酶的产生,促进调节各种细胞连接蛋白受体的表达及ECM成分与这些受体的结合。然而关于性激素调节泪腺中MMP-2及其抑制剂TIMP-2表达,以及与TGF-β1的相互关系却不清楚。以往研究表明:17β-雌二醇促进人角膜上皮细胞中促炎细胞因子和MMPs的表达,而MMPs与干眼症又有非常密切的关系。近些年,性激素在干眼中的作用逐渐引起人们的重视,成为研究的热点。
为此本实验设计了通过对雌性性成熟Wistar大鼠行双侧卵巢切除术(Ovariectomy,OVX),模拟人工绝经状态,给予大鼠雌、雄激素全身及局部治疗,观察血清雌、雄激素水平、泪液分泌量、泪膜稳定性、大鼠泪腺和眼表组织学的变化,检测泪腺中凋亡基因Bax、bcl-2、TGF-β1因子表达及MMP-2及其抑制剂TIMP-2的表达,探讨雌、雄激素对去卵巢雌鼠泪液分泌及泪腺功能的影响及其作用机制,为临床医生应用雌、雄激素治疗绝经期妇女干眼提供实验依据和理论基础。本实验分为三部分。
第一部分雌、雄激素对去卵巢雌鼠泪液分泌及眼表和泪腺组织病理学和超微结构改变的影响
目的:探讨雌、雄激素对去卵巢雌鼠泪液分泌及眼表和泪腺组织病理和超微结构改变的影响。方法:雌性性成熟Wistar大鼠64只,随机分为正常对照组8只、假手术组8只、实验组48只。假手术组仅打开腹腔,切除部分脂肪,实验组行双侧OVX。术后5个月将实验组随机分为实验1组、实验2组、实验3组、实验4组、实验5组、实验6组,每组8只大鼠。自术后第5个月起,实验1组不予任何干预,实验2组给予粟米油100ul皮下注射每3天一次,实验3组给予苯甲酸雌二醇200ug/kg皮下注射每3天一次,实验4组给予苯甲酸雌二醇点眼,每日4次,实验5组给予3.75mg/kg丙酸睾丸酮肌肉注射每3天一次,实验6组给予2.5%丙酸睾丸酮点眼,每日4次。分别于术前、术后第1、2、3、4、5个月及给药后6周,测大鼠泪液分泌量(SchirmerⅠtest,SⅠt)、泪膜破裂时间(tear film break-up time,BUT)及角膜荧光素钠染色情况。给药6周后处死动物,立刻取所有大鼠泪腺、角膜和结膜,行眼表和泪腺组织病理学和透射电镜检查。分别于术前、术后第5个月及给药后6周测大鼠血清雌二醇和睾酮水平。结果:1实验组动物在OVX术后1个月BUT较术前缩短(P<0.01);实验3组给药6周后BUT结果较给药前缩短(P<0.01);实验5组给药6周后BUT结果较给药前延长(P<0.05)。其余组治疗前后BUT结果无显著性差异(P>0.05)。2实验组动物在OVX术后3个月SⅠt结果较术前下降50%,且与术前比较有非常显著性差异(P<0.01)。实验3组给药6周后SⅠt结果较给药前缩短(P<0.05)。实验5组给药6周后SⅠt结果较给药前延长(P<0.05)。其余组治疗前后SⅠt结果无显著性差异(P>0.05)。3实验组动物在OVX术后4个月角膜染色评分较术前增加(P<0.05);实验3组给药6周后角膜染色评分较给药前增加(P<0.05);实验5组给药6周后角膜染色评分较给药前减少(P<0.05);其余组治疗前后角膜染色评分无显著性差异(P>0.05)。4眼表及泪腺组织病理学及泪腺超微结构:实验3组组织病理学及超微结构病变均较实验1组加重;实验5组组织病理学及超微结构病变均较实验1组有明显改善。5血清性激素水平:OVX术后5个月大鼠血清雌二醇水平明显低于术前(P<0.01)。实验3组给药6周后血清雌激素水平上升。OVX术后5个月大鼠血清睾酮水平低于术前(P<0.05)。实验5组给药6周后血清睾酮水平上升。结论:1通过OVX模拟人工绝经状态,去卵巢雌鼠血清中雌、雄激素水平均较术前降低,泪液分泌减少,泪膜稳定性下降。全身补充雄激素使泪液分泌量增加,泪膜稳定性提高;全身补充雌激素使泪液分泌量减少,泪膜稳定性下降。推测OVX引起的泪液分泌减少与血清雄激素水平下降有关。2全身补充雄激素能够改善由于OVX术后出现的泪腺萎缩,缓解眼表损害,促进泪液分泌,改善泪膜稳定性。3全身补充雌激素加重泪腺萎缩及眼表损害,减少泪液分泌,破坏泪膜稳定性。提醒临床医生应慎用激素替代疗法。
第二部分雌、雄激素对去卵巢雌鼠泪腺中凋亡基因Bax,bcl-2表达的影响
目的:探讨雌、雄激素对去卵巢雌鼠泪腺中凋亡基因Bax、bcl-2表达的影响。方法:实验动物及分组同第一部分。给药6周后处死动物,立刻摘取大鼠泪腺,采用免疫组化(SP法)观察泪腺上皮细胞凋亡率及凋亡基因Bax和bcl-2的表达。结果:1泪腺上皮凋亡相关基因Bax和bcl-2的表达:实验1组泪腺上皮中Bax的平均光密度高于A组(P<0.01);实验3组给药6周后泪腺上皮中Bax的平均光密度高于实验1组(P<0.01)。实验5组给药6周后泪腺上皮中Bax的平均光密度低于实验1组(P<0.05)。实验1组泪腺上皮中bcl-2的平均光密度低于A组(P<0.01)。实验3组给药6周后泪腺上皮中bcl-2的平均光密度低于实验1组(P<0.01)。实验5组给药6周后泪腺上皮中bcl-2的平均光密度高于实验1组(P<0.01)。2泪腺上皮细胞凋亡率:实验1组泪腺上皮细胞凋亡率高于A组(P<0.01);实验3组给药6周后泪腺上皮细胞凋亡率高于实验1组(P<0.01);实验5组给药6周后泪腺上皮细胞凋亡率低于实验1组(P<0.01)。结论:正常大鼠泪腺上皮中存在Bax的表达,OVX术后泪腺上皮细胞中Bax表达增加,bcl-2表达减少,泪腺上皮细胞凋亡可能是去卵巢雌鼠泪液分泌减少的作用机制之一。全身应用雌激素加重去卵巢大鼠泪腺上皮细胞凋亡,全身应用雄激素减轻去卵巢大鼠泪腺上皮细胞凋亡。提示雄激素通过改善泪腺上皮细胞凋亡,使泪液分泌量增加,泪膜稳定性提高,眼表损害减轻;雌激素与之作用相反。
第三部分雌、雄激素对去卵巢雌鼠泪腺中TGF-β1、MMP-2及其抑制剂TIMP-2表达及其相关性的实验研究
目的:探讨雌、雄激素对去卵巢雌鼠泪腺中MMP-2及其抑制剂TIMP-2的表达的影响及TGF-β1的表达变化,并对TGF-β1、MMP-2及TIMP-2进行相关性分析,进一步探讨MMP-2及TIMP-2在干眼中的作用机制。方法:实验动物及分组同第一部分。给药6周后处死动物,立刻摘取大鼠泪腺,应用RT-PCR技术检测MMP-2mRNA和TIMP-2mRNA水平,采用Western-blot方法检测泪腺中MMP-2蛋白含量的变化,应用酶联免疫吸附试验(ELISA)检测泪腺中TGF-β1的质量浓度。结果:1 RT-PCR法检测去卵巢雌鼠泪腺中MMP-2mRNA水平结果:各组用药6周后泪腺中MMP-2mRNA水平比较有非常显著性差异(P<0.01)。实验1组泪腺中MMP-2mRNA与A组比较表达增加(P<0.05);实验3组泪腺中MMP-2mRNA与实验1组比较表达增加(P<0.05);实验5组泪腺中MMP-2mRNA与实验1组比较表达减少(P<0.01)。2 RT-PCR法检测去卵巢雌鼠泪腺中TIMP-2mRNA水平结果:各组用药6周后泪腺中TIMP-2mRNA水平比较有非常显著性差异(P<0.01)。实验1组泪腺中TIMP-2mRNA与A组比较表达增加(P<0.05);实验3组泪腺中TIMP-2mRNA与实验1组比较表达增加(P<0.05);实验5组泪腺中TIMP-2mRNA与实验1组比较表达减少(P<0.01)。3 Western Blot法检测雌、雄激素对去卵巢雌鼠泪腺中MMP-2蛋白的表达:各组用药6周后泪腺中MMP-2蛋白的表达比较有非常显著性差异(P<0.01)。实验1组泪腺中MMP-2蛋白的表达与A组比较表达增加(P<0.05);实验3组泪腺中MMP-2蛋白的表达与实验1组比较表达增加(P<0.05);实验5组泪腺中MMP-2蛋白的表达与实验1组比较表达减少(P<0.01)。4泪腺中TGF-β1因子的表达:各组用药6周后泪腺匀浆上清液中TGF-β1质量浓度比较有非常显著性差异(P<0.01),实验5组与实验1组间有非常显著性差异(P<0.01)。5 TGF-β1、MMP-2mRNA及TIMP-2mRNA相关性分析结果:TGF-β1与MMP-2mRNA间无相关关系(r=0.089,P=0.587);TGF-β1与TIMP-2mRNA间无相关关系(r=0.106,P=0.515);MMP-2mRNA与TIMP-2mRNA间有相关关系(r=0.445,P=0.004)。结论:1 OVX术后泪腺中MMP-2mRNA和TIMP-2mRNA水平较正常组增加;雌激素上调泪腺中MMP-2mRNA和TIMP-2mRNA水平;雄激素下调泪腺中MMP-2mRNA和TIMP-2mRNA水平;且MMP-2与TIMP-2间有相关关系。2 OVX术后泪腺中MMP-2蛋白的表达较正常组增加;雌激素上调泪腺中MMP-2蛋白的表达;雄激素下调泪腺中MMP-2蛋白的表达。3雄激素促进去卵巢雌鼠泪腺中TGF-β1的表达。4推测雌激素通过上调泪腺中MMP-2的水平导致泪液分泌减少,泪膜稳定性下降,雄激素与之作用相反,一方面通过增加TGF-β1的含量,抑制泪腺的免疫反应,另一方面,通过抑制MMP-2的表达,改善干眼的泪液分泌功能和减缓泪腺损害。
Ocular surface(cornea、conjunctiva、accessory lacrimal gland and meibomian gland)、lachrymal gland and their nerve conjunction setting up a function unit for their intimate anatomy and function relationship, is called“ocular surface-lachrymal gland feedback system”.They play an important role in regulating lacrimal secretion and tear-film formation together and maintaining the health of ocular surface. The damage of every element will induce to destroy the integrality and function of tear film and lead to dry eye. It is multiplicity about the pathogenesy of dry eye.Now it is sugested that dry eye is related to inflammation, apoptosis and the abnormality of neuromodulation and the imbalance of sex hormone. Dry eye is most often in old women. It is sugested that sex hormone is one of the pathogenesies of dry eye. The change of internal hormone is the important factor in post-menopause women.It remains to be ascertained, however, whether dry eye relates to estrogen excess or deficiency, androgen deficiency and/or estrogen/androgen imbalance.
Apoptosis,a special cell death, is a phenomenon that in definite physiology and pathology condition cells end the life following their own program. Apoptosis is a normal physiology phenomenon,through which the body clear infect and damage and retain the body normal physiological functions.However, in certain conditions, apoptosis is a pathology phenomenon.Apoptosis coordinated regulation through many signals. The most important regulation factor is bcl-2 and its related gene. Apoptosis plays most important role in immune diseases.Apoptosis in lacrimal gland and ocular surface epithelium increase abnormally,but in local lymphocytic cell is inhibitted in dry eye .
The expression of matrix metalloproteinase(MMPs) is regulated by many factors such as cytokine, growth factor, sex hormone, neurohormone,the change of cell appearance,the interaction between cells and cell transformation.Elevated levels of both MMP-2 and MMP-9, which have been found in the tears and saliva of patients with systemic dry eye disease and non-systemic dry eye disease, as well as in the lacrimal gland and cornea of a rabbit model with lacrimal gland inflammation-induced dry eye, suggest that both MMP-2 and MMP-9 may be implicated in the pathogenesis of this disease. Transforming growth factor-β1 is a kind of bioactive cytokine. It participates in regulating cell growth and differentiation. It is major rejection capability molecule in immune system. TGF-β1 stimulates the synthesis of extracellular matrix such as collagen protein, fibrous joint protein, laminin and proteoglycan and inhibits the production of MMPs. It promotes the expression of connexin receptor and the combination of ECM with these receptors. However, it is not clear that whether sex hormone regulates the expression of MMP-2 and it’s inhibitor TIMP-2 in lachrymal gland and that the relationship among MMPs,sex hormone and TGF-β1.The research indicated that 17β-estradiol up-regulation proinflammatory cytokine and MMPs in human corneal epithelial cells.There is a close relationship between MMPs and dry eye.Investigators have paid more attention to dry eye and the role of sex hormone recently.
Therefore,this investigation as follows: sexual mature rats had been done ovariectomy(OVX), modelling artificial menopause state, administrated systemic and local eyedrop application with estrogen and androgen. The estrogen and androgen levels in serum and lacrimal secretion, tear film break-up time and corneal fluorescence staining were measured.Lachrymal gland was obtained for pathohistological observation. The expressions of Bax, bcl-2, TGF-β1, MMP-2 and it’s inhibitor TIMP-2 in lachrymal gland and relationship were studied. The effect of estrogen and androgen on lacrimal secretion and lachrymal gland function in ovariectomized rats and mechanism were investigated.It provided experiment evidence and rationale for the treatment of dry eye with estrogen and androgen to clinician. This experiment devided into 3 parts.
Part 1 Effect of estrogen and androgen on lacrimal secretion and the histological and ultramicrostructure change in lacrimal gland and ocular surface in ovariectomized rats
Objective:To investigate the effect of estrogen and androgen on lacrimal secretion and the histological and ultramicrostructure change of lacrimal gland and ocular surface in ovariectomized rat. Methods: sixty-four female Wistar rats were devided into contol group, sham operation group and experiment group randomly. In sham operation group, rats were only cut little fat in abdominal cavity.In experiment group, rats had been done ovariectomy(OVX). Lacrimal secretion (SⅠt), tear film break-up time (BUT test) and corneal fluorescence staining were measured in all groups before the operation and 1,2,3,4, 5 months after the operation.Five months later, rats of OVX experiment group were treated with systemic application and eyedrop application with corn oil, estrogen and androgen and devided into experiment group 1, experiment group 2, experiment group 3, experiment group 4, experiment group 5, experiment group 6.There was no any intervention in experiment group 1.The rats in experiment group 2 were given hypodermical injection of oil (100ul )every 3 days. The rats in experiment group 3 were given hypodermical injection of estradiol benzoate (200ug/kg)every 3 days. The rats in experiment group 4 were given eyedrop of estradiol benzoate 4 times every day. The rats in experiment group 5 were given intramuscular injection of testosterone (3.75mg/kg)every 3 days. The rats in experiment group 6 were given eyedrop of testosterone 4 times every day.Six weeks later, SIt and BUT test and corneal fluorescence staining were measured.Then all the rats were sacrificed. The lachrymal gland, cornea and conjunctiva were obtained for pathohistological ultramicrostructure observation. The serum estrogen and androgen were measured before and 5 months after the operation,as well as before death. Results: 1 The result of BUT in the experiment group 1 month after OVX was shortter than that before OVX (P<0.01). The result of BUT in the experiment group 3 with estradiol enzoate injected hypodermically for 6 weeks was shortter than that before injection (P<0.01).While the BUT of the experiment group 3 with testosterone intramuscular injected for 6 weeks was longer than that before injection (P<0.01). There was no difference in other groups (P>0.05).2 The result of SⅠt 3 months after OVX decreased to 50% of the result before OVX (P<0.01). The result of SⅠt in experiment group 3 was shorter than that before injection(P<0.05). The result of SⅠt in experiment group 5 was longer than that before injection(P<0.05). There was no difference in other groups (P>0.05). 3 The score of corneal fluorescence staining 4 months after OVX increased compared with the score before OVX(P<0.05). The score of corneal fluorescence staining in experiment group 3 increased compared with the score before injection(P<0.05). The score of corneal fluorescence staining in experiment group 5 decreased compared with the score before injection(P<0.05). There was no difference in other groups(P>0.05).4 The histological change and ultrastructure of lacrimal gland and ocular surface:There had been obvious improvement in experiment group 5 than experiment group 1. There had been more serious in experiment group 3 than experiment group 1. 5 The estrogen and androgen levels in serum decreased after OVX,compared with those before OVX. After estrogen or androgen injected, both the serum estrogen level in experiment group 3 and the serum androgen level in experiment group 5 increased.Conclusion:1 The sexual mature rat had been done ovariectomy(OVX), modelling artificial menopause state , the level of estrogen and androgen in serum decreased, lacrimal secretion decreased, the stability of tear-film decreased.In systemic androgen treatment group, lacrimal secretion increased, the stability of tear-film increased. In systemic estrogen treatment group, lacrimal secretion decreased, the stability of tear-film decreased. It indicated that the decrease of lacrimal secretion and stability of tear-film is related to the decrease of the level of androgen in serum. 2 Systemic androgen treatment can improve atrophy in the lacrimal gland caused by OVX It relieve the damage of ocular surface and increase lacrimal secretion and the stability of tear-film. 3 Systemic estrogen treatment can aggravate the atrophy in the lacrimal gland and the damage of ocular surface and decrease lacrimal secretion and the stability of tear-film. We remind clinician that it must be cautious when they applicate hormone replacement therapy.
Part 2 Effect of estrogen and androgen on the expression of apoptosis in lachrymal gland in ovariectomized rats
Objective: To investigate the effect of estrogen and androgen on the expression in apoptosis gene in lachrymal gland in ovariectomized rats. Methods: The rats handled as same as part one. Six weeks after treatment, all rats were sacrificed, and the lachrymal glands were obtained. The expression of Bax and bcl-2 were detected by immunohistochemical staining in different groups. Results: 1 The optical density(OD) of Bax in lacrimal epithelium cell :The OD in experiment group 1 rats was higher than A group (P<0.01), The OD in experiment group 3 rats was higher than experiment group 1 (P<0.01).The OD in experiment group 5 rats was lower than experiment group 1 (P<0.05).The OD of bcl-2 in lacrimal epithelium cell: The OD in experiment group 1 rats was lower than A group (P<0.01), The OD in experiment group 3 rats was lower than experiment group 1(P<0.01).The OD in experiment group 5 rats was higher than experiment group 1(P<0.01).2 Apoptosis index of lacrimal epithelium:The percent of positive cell in experiment group 1 was higher than A group (P<0.01),The percent of positive cell in experiment group 3 higher than experiment group 1(P<0.01). The percent of positive cell in experiment group 5 was lower than experiment group 1 (P<0.01). Conclusions: There was expression on apoptosis of Bax in normal lacrimal epithelium. After OVX, the expression of Bax increased and the expression of bcl-2 decreased in lacrimal epithelium.The apoptosis in lachrymal gland epithelium may be one of mechanisms of action. Apoptosis in lachrymal gland epithelium became more serious in systemic estrogen group and relieved in systemic androgen group. It suggested that androgen treatment made lacrimal secretion and the stability of tear-film increase through relieving apoptosis. However, the role of estrogen is opposite.
Part 3: The experiment study of estrogen and androgen on expression of TGF-β1、MMP-2、it’s inhibitor TIMP-2 and relationship to TGF-β1 in lachrymal gland in ovariectomized rats.
Objectives: To investigate the effect of estrogen and androgen on the expression of MMP-2mRNA、TIMP-2mRNA and MMP-2 protein in lachrymal gland in ovariectomized rats.To make correlation analysis of TGF-β1,MMP-2 and TIMP-2.To explore the function of MMP-2 and TIMP-2 in dry eye.Methods: The rats handled as same as part one. Six weeks after treatment, all rats were sacrificed, and the lachrymal gland was obtained.The expression of MMP-2mRNA and TIMP-2mRNA was detected by RT-PCR.The expression of MMP-2 protein was detected by Western-blot. The level of TGF-β1was detected by ELISA.Results:1 The expression of MMP-2mRNA in lachrymal gland:Six weeks after treatment, there was significant differences on the expression of MMP-2mRNA in lachrymal gland in different groups(P<0.01).Experiment group 1 was hihger than A group(P<0.05).Experiment group 3 was hihger than experiment group 1(P<0.05).Experiment group 5 was lower than experiment group 1(P<0.01).2 The expression of TIMP-2 mRNA in lachrymal gland: Six weeks after treatment, there was significant differences of the expression of TIMP-2 mRNA in lachrymal gland in different groups(P<0.01). Experiment group 1 was higher than A group (P<0.05).Eexperiment group 3 was hihger than experiment group 1(P<0.05).Experiment group 5 was lower than experiment group 1(P<0.01).3 The expression of MMP-2 protein in lachrymal gland: Six weeks after treatment, there were significant difference of the expression of MMP-2 protein in lachrymal gland in different groups(P<0.01). Experiment group 1 was higher than A group(P<0.05).Experiment group 3 was higher than experiment group 1(P<0.05).Experiment group 5 was lower than experiment group 1(P<0.01). 4 The expression of TGF-β1 in lachrymal gland: There was significant difference in different groups after treatment for 6 weeks about mass concentration of TGF-β1 in supernatant of lachrymal gland homogenate(P<0.01). There was significant difference between experiment group 5 and experiment group 1 after treatment for 6 weeks about mass concentration of TGF-β1 in supernatant of lachrymal gland homogenate(P<0.01).5 The correlation analysis among TGF-β1、MMP-2mRNA and TIMP-2mRNA:There was no correlation between TGF-β1 and MMP-2mRNA(r=0.089,P=0.587).There was no correlation between TGF-β1 and TIMP-2 mRNA(r=0.106,P=0.515).There was correlation between MMP-2 mRNA and TIMP-2 mRNA(r=0.445,P = 0.004). Conclusion:1 After OVX the level of MMP-2 mRNA and TIMP-2 mRNA in lachrymal gland is higher than normal group.Estrogen up-regulate the expression of MMP-2mRNA and TIMP-2mRNA in lachrymal gland. Androgen down-regulate the expression of MMP-2mRNAand TIMP-2mRNA in lachrymal gland. There was correlation between MMP-2 mRNA and TIMP-2 mRNA.2 After OVX the level of MMP-2 protein in lachrymal gland is higher than normal group.Estrogen up-regulate the expression of MMP-2 protein in lachrymal gland. Androgen down-regulate the expression of MMP-2 protein in lachrymal gland. Androgen can promote lacrimal secretion and the expression of TGF-β1 in lacrimal gland in OVX rats.It suggested that estrogen induced the decrease of lacrimal secretion and the stability of tear-film through up-regulation of MMP-2. The role of androgen is opposite.In one hand,it increase the level of TGF-β1 and inhibit immune reaction in lachrymal gland. In another hand, it inhibit the expression of MMP-2.
引文
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