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1.
目的 探讨内质网应激对糖尿病小鼠晶状体上皮细胞发生上皮间质转分化(epithelial-mesenchymaltransition,EMT)的作用。方法 42只雄性C57BL/6J小鼠随机分为正常对照组、糖尿病模型组和糖尿病+4-苯基丁酸(4-PBA)干预模型组。糖尿病模型小鼠由腹腔注射STZ建立,糖尿病+4-PBA干预模型组给予4-PBA溶液灌胃。干预过程中监测各组小鼠体质量及随机血糖水平变化。干预开始后每4周散瞳后于裂隙灯显微镜下观察晶状体混浊情况并照相记录,第12周摘取眼球于体式显微镜下观察晶状体混浊情况。在干预的第1、4、8和12周,采用Westernblot方法检测晶状体上皮中葡萄糖调节蛋白78(glucosereg-ulatedprotein,GRP78)、α-平滑肌肌动蛋白(α-smoothmuscleactin,α-SMA)及E-钙黏蛋白(E-cadherin)表达水平。结果 正常对照组小鼠体质量增加明显,观察期结束时体质量为(30.21±1.47)g,糖尿病模型组及糖尿病+4-PBA干预模型组小鼠体质量增长较正常对照组小鼠缓慢,12周末时分别为(25.32±0.73)g及(25.07±0.67)g。随机血糖监测结果显示糖尿病模型组小鼠各时间点血糖均高于正常对照组(t=13.52、19.45、18.86、21.18、21.56,均为P<0.05),糖尿病+4-PBA干预模型组血糖各时间点均高于正常对照组(t=15.23、18.78、13.04、15.06、13.90,均为P<0.05),第4周起低于糖尿病模型组(t=5.819、6.120、7.664,均为P<0.05)。裂隙灯显微镜下检查结果显示,正常对照组小鼠晶状体维持透明,糖尿病模型组小鼠第8周起晶状体出现浅层皮质点状混浊,糖尿病+4-PBA干预模型组小鼠8周时晶状体出现轻度混浊,但程度较糖尿病模型组轻。离体观察正常对照组晶状体保持透明,糖尿病模型组及糖尿病+4-PBA干预模型组晶状体均出现点状混浊,糖尿病+4-PBA干预模型组混浊程度较轻。Westernblot结果显示,与正常对照组相比,糖尿病模型组GRP78蛋白表达量在第12周时明显增高(t=7.153,P<0.05);8周及12周时α-SMA表达量明显升高(t=3.559、4.084,均为P<0.05),各时间点E-cadherin表达量均明显降低(t=4.350、6.139、7.770、3.486,均为P<0.05);糖尿病+4-PBA干预模型组GRP78蛋白表达量低于糖尿病模型组,且在第8周和12周差异均有统计学意义(t=8.600,11.53,均为P<0.05);α-SMA相对表达量在第12周时明显下降(t=4.357,P<0.05),E-cadherin相对表达量在增高且达到正常水平,各时间点差异均有统计学意义(t=4.360、7.633、8.450、5.853,均为P<0.05)。糖尿病+4-PBA干预模型组不同时间点比较,GRP78表达量在8周时达到最低。结论 利用分子伴侣4-PBA抑制糖尿病小鼠内质网应激水平能够抑制糖尿病引起的晶状体上皮细胞发生EMT。 相似文献
2.
晶状体上皮细胞转分化对老年性白内障形成的影响研究 总被引:6,自引:0,他引:6
目的 :为研究晶状体上皮细胞转分化对老年性白内障形成的影响。方法 :对 14例老年性白内障晶状体前囊上皮细胞进行角质蛋白 -8、波形纤维蛋白和纤维粘连蛋白表达的免疫组化研究。结果 :皮质性白内障晶状体上皮的波形纤维蛋白表达明显强于核性白内障 (P <0 0 5 ) ;角质蛋白 -8在皮质性白内障晶状体上皮的表达比核性白内障弱 ((P <0 0 1) ;纤维粘连蛋白在皮质性白内障晶状体上皮的表达明显强于核性白内障 ,而在正常晶状体上皮不表达。结论 :晶状体上皮细胞具有转分化为成纤维样细胞的双向化潜能。晶状体上皮获得转分化能力后而失去原来的细胞学特性。在老年皮质性白内障中 ,晶状体上皮细胞转分化成为成纤维样细胞 ,伴随波形纤维蛋白的过度表达和角质蛋白表达下降。同时合成包括纤维粘连蛋白在内的细胞外基质增多 ,而纤维粘连蛋白能促进晶状体上皮的增殖、迁移及粘附 ,因而晶状体上皮细胞转分化对老年性白内障形成及后囊混浊的形成发挥重要作用。 相似文献
3.
目的建立微创性白内障动物模型,探讨晶状体上皮向平滑肌样细胞的分化。方法4~6周C57BL/6j小鼠24只,随机分为实验组和对照组。实验组采用微创穿破晶状体后囊膜,注入10μLPBS;对照组仅玻璃体腔内注射等量的PBS,术后行眼部检查及裂隙灯照相。术后第3、7、14、28d取眼球,行组织病理学观察及平滑肌肌动蛋白(SMA)、波形纤维蛋白(Vim)免疫组织化学染色。结果术后1~3d,实验组11只眼晶状体相继发生不同程度的白色混浊。混浊眼球术后3d,组织病理学观察示晶状体赤道区上皮开始沿创口处爬行;7d后囊下梭形细胞层增多3~6层,细胞体积增大。14~28d梭形细胞层数基本保持不变。免疫组织化学染色显示增生的梭形细胞SMA强阳性,Vim阴性。对照组均无上述变化。结论微创性损伤晶状体出现白内障,能诱发晶状体上皮向平滑肌样细胞分化。 相似文献
4.
晶体体上皮细胞转分化对老年性白内障形成的影响研究 总被引:1,自引:0,他引:1
目的:为研究晶状体上皮细胞转化对老年性白内障形成的影响。方法:对14例老年性白内障晶状前囊上皮细胞进行角质蛋白-8、波形纤维蛋白和纤维粘连蛋白表达的免疫组化研究,结果:皮质性白内障晶状体上皮的波形纤维蛋白表达明显强于核性白内障(P<0.05),角质蛋白-8在皮质性白内障晶状体上皮的表达比核性白内障弱(P<0.01);纤维粘连蛋白在皮质性白内障晶状体上皮的表达明显强于核性白内障,而在正常晶状体上皮不表达,结论:晶状体上皮细胞具有转分化为成纤维样细胞的双向化潜能,晶状体上皮获转分化能力后而失去原来的细胞学特性,在老年皮质性白内障中,晶状体上皮细胞转化成为纤维样细胞,伴随波形纤维蛋白的过度表达和角质蛋白表达下降,同时合成包括纤维粘连蛋白在内的细胞外基质增多,而纤维粘连蛋白能促进晶状体上皮的增殖,迁移及粘附,因而晶状体上皮细胞转化分对老年性白内障表成及后囊混浊的形成发挥重要作用。 相似文献
5.
波形纤维蛋白在老年性白内障晶状体上皮细胞的表达 总被引:2,自引:0,他引:2
目的观察波形纤维蛋白在老年性白内障晶状体上皮细胞的变化.方法用卵白素-生物素过氧化物酶法对22例老年性白内障患者晶状体前囊膜上皮细胞进行波形纤维蛋白染色,采用包埋前免疫酶电镜技术处理6个晶状体前囊膜标本,并观察其超微结构;利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳及Westemnblot法分析4个晶状体表层组织(前囊膜、上皮细胞和表层皮质)中的波形纤维蛋白.结果老年性白内障晶状体上皮细胞的波形纤维蛋白表达减弱,与对照组比较差异有显著性(t=2.0948,P< 相似文献
6.
目的:研究晶状体中肌节同源盒基因同系物2(Msx2)条件性基因敲除与先天性白内障发生的关系。方法:实验研究。选取条件性基因敲除小鼠Msx2CKO(Msx2fl/fl/Le-Cre+)为实验组,野生型小鼠Msx2WT(Msx2fl/fl)为对照组。取胚胎17.5 d(E17.5)Msx2WT小鼠胚胎头部组织作冰冻切片,采用RNA原位分子杂交方法检测Msx2在眼组织内的正常表达。取2 组小鼠E17.5 和生后8 d(P8)眼球组织石蜡切片HE染色观察晶状体组织形态学变化。比较2 月龄Msx2CKO和Msx2WT小鼠晶状体质量和直径,组间比较采用独立样本t检验。结果:本研究观察到超过50%的2 月龄Msx2CKO小鼠眼部出现角膜轻微混浊,晶状体变小(质量和直径),晶状体混浊及小眼球畸形。石蜡切片HE染色观察到Msx2CKO E17.5及P8小鼠晶状体内分化的纤维细胞排列紊乱,赤道部晶状体上皮细胞及邻近的纤维细胞中有空泡,排列明显紊乱。2月龄Msx2CKO组小鼠的直径小于Msx2WT组小鼠(t=4.80,P < 0.05),重量小于后者(t=14.29,P < 0.05)。结论:Msx2基因对小鼠晶状体发育起重要的调控作用,晶状体条件性敲除该基因可引起先天性白内障发生。 相似文献
7.
目的:探究年龄对水合氯醛诱导的小鼠急性可逆性晶状体混浊及Na+-K+-ATP酶表达的影响。
方法:青年(3月龄)和老年(24月龄)C57BL/6小鼠各12只,4%水合氯醛(400mg/kg)腹腔注射诱导急性可逆性晶状体混浊。在注射后10、20、30、45、60、90、120、150min分别应用裂隙灯观察晶状体混浊程度,根据晶状体混浊评价系统记录混浊程度分级情况并拍照。苏木素-伊红(HE)染色观察晶状体病理改变,免疫组织化学染色检测晶状体Na+-K+-ATP酶表达。
结果:水合氯醛注射后两组小鼠晶状体混浊和消退过程相似,但青年组小鼠晶状体混浊出现早、持续时间略长,混浊厚重,呈乳白色; 老年组小鼠晶状体混浊出现晚,持续时间略短,混浊轻薄,呈薄雾状。HE染色显示水合氯醛注射后晶状体上皮细胞(LECs)下皮质聚集大量水泡,浅层晶状体纤维细胞结构紊乱。免疫组织化学染色显示LECs及纤维Na+-K+-ATP酶的表达呈现阳性,水合氯醛注射前青年组和老年组小鼠LECs中Na+-K+-ATP酶表达较弱,注射后45min LECs中Na+-K+-ATP酶的表达上调,且老年组小鼠LECs的Na+-K+-ATP酶上调更为显著。
结论:年龄影响水合氯醛诱导小鼠急性可逆性晶状体混浊,Na+-K+-ATP酶参与水合氯醛诱导的晶状体混浊。 相似文献
8.
目的 探讨半乳糖性白内障大鼠晶状体上皮细胞的改变。方法 24只SD雌性大鼠,分为正常对照组和白内障组,每组12只。白内障组大鼠用半乳糖饲料喂养,正常对照组大鼠用普通颗粒饲料喂养。裂隙灯显微镜下观察大鼠晶状体混浊程度变化,观察至30d处死大鼠后取晶状体,在光镜和电镜下观察晶状体病理组织和超微结构改变。结果 观察至30d时,正常对照组大鼠晶状体保持透明,白内障组大鼠9眼(37.5%)出现均一的皮质性混浊,15眼(62.5%)出现核混浊。白内障组光镜下可见晶状体皮质和核部大量纤维细胞水肿、崩解,前囊膜下及后囊膜下出现纤维细胞样的有核细胞堆积;透射电镜下可见晶状体上皮细胞变性、增生并突破晶状体上皮层向浅层皮质移行。结论 半乳糖性白内障不仅有晶状体纤维细胞水肿及结构破坏,还存在晶状体上皮细胞的异常增生、分化和移行。 相似文献
9.
HSP70在STZ-糖性白内障发病机制中的研究 总被引:1,自引:1,他引:0
目的:研究HSP70在链脲佐菌素—糖性白内障发生发展中的作用。方法:将66只SD大鼠随机分为2组,正常对照组与白内障组。用链脲佐菌素(STZ)诱发糖性白内障,每周观察晶状体的变化,在实验开始后2,4,8周末,分别摘取眼球,采用免疫组化技术检测热休克蛋白-70(HSP70)在晶状体上皮细胞(LECs)中的表达。结果:对照组晶状体一直保持透明,白内障组晶状体在2周末出现空泡,8周末全部混浊。HSP70在对照组中未见表达,在白内障组中表达明显,并随着白内障的发展而表达增加。结论:HSP70可能通过调节晶状体上皮细胞的生长在糖性白内障的发生、发展中起重要作用。 相似文献
10.
背景实验性糖尿病动物模型的制作是对糖尿病性眼病进行实验研究的关键环节,目前国内普遍应用的方法是链脲佐菌素和四氧嘧啶(ALX)注射,但前者价格昂贵,后者造模过程中动物的死亡率较高。目的探讨ALX注射制作糖尿病兔晶状体后囊膜混浊(PCO)模型并降低死亡率的方法,并观察高血糖对晶状体PCO形成的早期影响。方法将清洁级健康新西兰雄性大白兔40只随机分为2组;其中20只兔经耳缘静脉一次性注射ALX90mg/kg建立糖尿病模型作为高血糖组,另20只兔以同样的方法注射等量生理盐水作为正常血糖组。药物注射后2周时高血糖组兔血糖升高到12.0mmol/L以上可判断为建模成功,2组分别行兔右眼透明晶状体囊外摘出术并对晶状体PCO进行分级。于术后第6、10、14天取眼球,应用免疫组织化学法观察增生细胞核抗原(PCNA)在后囊膜晶状体上皮细胞(LECs)中的表达情况。结果ALX注射后糖尿病兔的成模率为70%。术后第6、10、14天时,高血糖组兔体质量均明显低于正常血糖组,但血糖明显高于正常血糖组,差异均有统计学意义(P〈0.05)。高血糖组术后14d时观察的3只兔中,2只兔出现后囊膜2级混浊,另1只兔出现1级混浊。正常血糖组术后14d时观察的3只兔后囊膜均出现1级混浊。免疫组织化学染色显示,术后第10天高血糖组可见PCNA在LECs的细胞核中表达,但正常血糖组未见PCNA的表达。术后第14天时高血糖组PCNA增生指数为0.86±0.04,明显高于正常血糖组的0.25±0.03,差异有统计学意义(t=-16.171,P=0.000)。结论90mg/kg的ALX静脉注射能形成稳定的糖尿病兔PCO模型;高血糖是促进PCO发生发展的重要因素之一。 相似文献
11.
Background and Purpose: The cataract in Sprague-Dawley rats injected with selenite is a dense nuclear opacity that appears by 4 or 5 days after selenite injection and becomes irreversible by 7 days. Injection of Wistar rats with selenite resulted in a similar nuclear opacity by 4 or 5 days that began to recover transparency by 7 days. In this report, the cytoplasmic proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in lenses from Sprague-Dawley and Wistar rats at 4 and 7 days after injection.Results: In the opaque lens cells, degradation of the 31 kDa protein and cytoskeletal proteins (vimentin, spectrin, and actin) was observed during cataract development using SDS-PAGE and Western blot analysis. During recovery from opacity, the decreased 31 kDa protein and the vimentin increased.Conclusion: The results suggest that the 31 kDa protein and the vimentin may be important for recovery of transparency in a reversible model of cataract formation. 相似文献
12.
Purpose: To investigate the role of vimentin gene in cataractogenesis. Methods: The 12. 7kb chicken vimentin genes were microinjected into the male pronuclei of 918 fertilized mice eggs. 841 injected embryos were transferred into oviducts of pseudopregnant recipient females, of which 12 pregnant mice gave birth to 49 offsping mice. The integration and expression of exogenous gene in the offsping were analysed by Southern and Northern blot hybridizations. In the human senile cataract, the lens vimentin gene was analyzed with the chicken vimentin gene probe.Results: It showed that four of F1 offspring were transgenic mice in which the chicken vimentin gene was integrated in their genomes. The transgenic band was 12kb, similar to the 12. 7kb chicken vimentin fragment injected. One 2kb vimentin mRNA was visualized on E2 mouse lens blot, which revealed that the chicken vimentin gene was efficiently expressed in this transgenic mouse. In the human senile cataract lens, 12kb BamHI-restricted vimentin fragments 相似文献
13.
BACKGROUND AND PURPOSE: The cataract in Sprague-Dawley rats injected with selenite is a dense nuclear opacity that appears by 4 or 5 days after selenite injection and becomes irreversible by 7 days. Injection of Wistar rats with selenite resulted in a similar nuclear opacity by 4 or 5 days that began to recover transparency by 7 days. In this report, the cytoplasmic proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in lenses from Sprague-Dawley and Wistar rats at 4 and 7 days after injection. RESULTS: In the opaque lens cells, degradation of the 31 kDa protein and cytoskeletal proteins (vimentin, spectrin, and actin) was observed during cataract development using SDS-PAGE and western blot analysis. During recovery from opacity, the decreased 31 kDa protein and the vimentin increased. CONCLUSION: The results suggest that the 31 kDa protein and the vimentin may be important for recovery of transparency in a reversible model of cataract formation. 相似文献
14.
目的:通过建立硫醇转移酶(TTase)基因敲除小鼠模型,观察其晶状体形态和生化方面随年龄的改变,探讨TTase在晶状体氧化还原系统中的重要作用及参与年龄相关性白内障形成的机制。方法:建立TTase基因敲除小鼠模型并进行基因型鉴定。裂隙灯观察TTase基因敲除型和野生型小鼠白内障随年龄的发生情况。检测小鼠晶状体中谷胱甘肽(GSH)含量。Western blot检测晶状体中氧化产物蛋白质二硫化物(PSSG)的变化。免疫共沉淀法鉴定形成PSSG的蛋白质。观察纯化的重组人晶状体TTase(RHLT)对PSSG的脱硫醇作用。结果:TTase基因敲除型小鼠和野生型小鼠白内障的发生均随年龄而增加,且主要表现为核性白内障。在TTase基因敲除型小鼠中,白内障最早从4月龄开始发生,9月龄时最为显著;野生型小鼠白内障最早从9月龄开始发生,12月龄时最为显著。两种基因型小鼠晶状体中GSH含量均随年龄增加而下降,9月龄TTase基因敲除型小鼠晶状体中GSH下降更为显著,且PSSG的表达明显高于野生型,主要表现为高分子聚合物。免疫共沉淀反应证实形成PSSG的蛋白质包含肌动蛋白(actin)和三磷酸甘油醛脱氢酶(GAPDH),这种积聚的PSSG可被GSH还原,且与纯化的RHLT反应更有效。结论:TTase基因敲除可以加速小鼠年龄相关性白内障的发生,这与晶状体中PSSG的积聚相关,且形成的PSSG可被TTase脱硫醇,证实TTase在防止年龄相关性白内障的发生中发挥重要作用。 相似文献
15.
Lyu J Kim JA Chung SK Kim KS Joo CK 《Investigative ophthalmology & visual science》2003,44(5):2034-2040
PURPOSE: A side effect associated with long-term treatment of various diseases with steroids is a high incidence of posterior subcapsular cataracts (PSC). To understand the mechanism underlying steroid-induced cataract, the cultured lens model was developed, and the expression of potential candidate proteins during opacity formation was examined. METHOD: Rat lenses were carefully dissected from the surrounding ocular tissue and incubated in medium 199. Dexamethasone was then added to the medium. The lenses were cultured for 7 days and photographed daily to record the development of opacity. Differential expression of candidate proteins was examined by Western blot analysis. RESULT: Various degrees of opacity were observed on the posterior subcapsular region as early as 5 days after incubation with dexamethasone. The expression of E-cadherin and N-cadherin decreased in the cultured rat lenses during the development of opacity. CONCLUSIONS: The pattern of opacity that developed in cultured rat lenses closely resembled that observed in patients with PSC. The results suggest that the decrease in E-cadherin plays a role in the formation of steroid-induced cataract. 相似文献
16.
Purpose: Approximately 50% of congenital and childhood cataracts seen in the clinic is of undetermined origin. Biochemical analysis of the cataracts is rare. This study analyzes lens proteins to determine the mechanism of congenital and childhood cataracts. Method: We analyzed the lens proteins from 10 young patients after cataract operations, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), densitometry analysis, and Western immunoblotting.Results: Densitometry of separated proteins showed a decrease in the high molecular protein bands of posterior subcapsular cataract (PSC). Specifically, spectrin (235 kDa), filensin (100 kDa), and vimentin (57 kDa) were absent from the SDS-PAGE of PSC. Increases in filensin and vimentin were observed in a Christmas tree cataract and lamellar cataracts. Western immunoblots confirmed the densitometry of SDS-PAGE.Conclusion: These results suggest that changes in cytoskeletal proteins may contribute to congenital and childhood cataracts. 相似文献
17.
PURPOSE: Approximately 50% of congenital and childhood cataract seen in the clinic is of undetermined origin. Biochemical analysis of the cataracts is rare. This study analyzes lens proteins to determine the mechanism of congenital and childhood cataracts. METHOD: We analyzed the lens proteins from 10 young patients after cataract operations, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), densitometry analysis, and western immunoblotting. RESULTS: Densitometry of separated proteins showed a decrease in the high molecular protein bands of posterior subcapsular cataract (PSC). Specifically, spectrin (235 kDa), filensin (100 kDa), and vimentin (57 kDa) were absent from the SDS-PAGE of PSC. Increases in filensin and vimentin were observed in a Christmas tree cataract and lamellar cataracts. Western immunoblots confirmed the densitometry of SDS-PAGE. CONCLUSION: These results suggest that changes in cytoskeletal proteins may contribute to congenital and childhood cataract. 相似文献
18.
本文使用S.D.S—聚丙烯酰胺凝胶电泳对人胚胎、青年、成年透明晶体及老年性白内障晶体囊上皮、皮质和核三部分的总蛋白质可溶性和脲溶性蛋白质进行了测定,发现在囊上皮部分自胎儿到老年性白内障晶体均有一条43KD多歇.而自14岁开始在晶体皮质及核的可溶性蛋白质中这条43KD多肽带明显加宽.在晶体皮质及核的碌溶性蛋白中这条谱带随年龄增长变得模糊.在老年性白内障晶体中则几乎消失. 相似文献
19.
Nuclear cataract resulting from an overdose of selenite was characterized by a five-fold increase in nuclear urea-soluble protein. The origin of this urea-soluble protein was examined by two-dimensional electrophoresis, immunoblotting with monospecific antisera against rat lens crystallins, and tryptic mapping. Cataractous urea-soluble protein was primarily composed of insolubilized beta- and gamma-crystallin polypeptides. Polypeptides from cataractous urea-soluble protein, and normal beta L-crystallin aggregates were compared by tryptic mapping. Approximately 19% of the urea-soluble protein from opaque nuclei was composed of 24.7 and 24.0 K polypeptides derived by limited proteolysis of 26.5 K beta L-crystallin polypeptide. Incubation of 26.5 K beta-crystallin polypeptide with purified rat lens calpain II in vitro caused production of fragments with similar molecular weights to polypeptides found in cataractous lenses. These results support the hypothesis that proteolysis may contribute to formation of urea-soluble protein in selenite cataract. 相似文献