Txnip基因敲除抑制糖尿病诱导的小鼠睾丸生精细胞凋亡与氧化压力

发布时间：2018-07-15 14:58

【摘要】：实验目的:硫氧还蛋白互作蛋白(thioredoxin-interacting protein,Txnip)发现于1,25-二羟维生素D3处理的HL-60细胞,因此早前命名为维生素D3上调蛋白1(vitamin D3 upregulated protein 1,VDUP1)。Txnip可以与硫氧还蛋白(thioredoxin,Trx)结合,从而抑制Trx的抗氧化功能,导致活性氧的累积与细胞压力。经葡萄糖处理后,Txnip在人胰岛中表达高度上调,具有平衡代谢和调节细胞生长、分化的功能。但Txnip在雄性睾丸中的表达定位与功能研究尚未见报道,是否在糖尿病导致的雄性生殖功能受损中扮演某种角色值得研究。基于此,本研究拟通过制备糖尿病小鼠模型,观察Txnip基因敲除是否具有拮抗睾丸损伤的效应及其可能的机制,为临床上防治糖尿病生殖损伤提供实验依据。实验方法:1.免疫组化、免疫荧光、PCR和western blot方法检测Txnip在小鼠睾丸中表达情况。2.繁殖Txnip基因敲除小鼠(TALEN技术制备)与野生型小鼠,用于糖尿病模型制备。首次注射链脲佐菌素(streptozotocin,STZ)前禁食12 h,按55 mg/kg剂量腹腔内注射STZ,连续5d。实验分组:野生型小鼠组(WT)、Txnip基因敲除组(KO)、WT+STZ组和KO+STZ组,每组6只小鼠。6 d后用血糖测定仪(ACCU CHEK,罗氏)检测血糖。野生型小鼠血糖大于16.7 mmol/L视为造模成功。常规饲养60 d后处死小鼠,取血液与睾丸标本,用于进一步实验。3.免疫组化、western blot检测Txnip基因敲除对小鼠睾丸Txnip表达的影响。睾丸标本制成石蜡切片,通过HE染色和TUNEL实验观察小鼠睾丸的形态学变化及检测精细胞的凋亡情况。并且测定小鼠睾丸组织中MDA含量与SOD活性。实验结果:1.免疫组化、免疫荧光、PCR及western blot实验结果显示Txnip表达于小鼠睾丸组织中支持细胞、间质细胞及精细胞区域;体外实验显示Txnip表达于睾丸TM4支持细胞及TM3间质细胞的胞质。2.免疫组化与western blot实验结果表明在Txnip敲除小鼠睾丸中未检测到Txnip表达。STZ诱导野生型小鼠血糖明显增加,Txnip基因敲除明显抑制血糖的增加。3.睾丸组织学结果显示Txnip基因敲除抑制睾丸形态学损伤与生精细胞凋亡,下调凋亡相关蛋白Bax/Bcl-2表达比值。此外,Txnip敲除明显下调糖尿病小鼠模型睾丸MDA水平,上调SOD活性。实验结论:Txnip表达在小鼠睾丸组织中支持细胞、间质细胞及精细胞区域,体外实验显示Txnip表达于TM4支持细胞及TM3间质细胞的胞质。STZ可以诱导血糖上升,但敲除Txnip明显抑制血糖的增加。通过分析睾丸形态学,发现Txnip基因敲除抑制睾丸形态学损伤与生精细胞凋亡,下调凋亡相关蛋白Bax/Bcl-2表达比值。此外,Txnip敲除明显下调糖尿病小鼠模型睾丸MDA水平,上调SOD活性。本研究揭示Txnip基因敲除可以拮抗高血糖引起的睾丸生精细胞凋亡与氧化压力增加,从而有效发挥抗生殖损伤的作用。进一步研究其机理对防治糖尿病导致的雄性生殖功能异常具有重要意义。
[Abstract]:Objective: thioredoxin-interacting protein (Txnip) was found in HL-60 cells treated by 1,25- dihydroxyvitamin D3, so it was named as vitamin D3 up-regulated protein 1 earlier (vitamin D3 upregulated protein 1, VDUP1) can be combined with thioredoxin to inhibit the antioxidant activity. Function, leading to the accumulation of active oxygen and cell pressure. After glucose treatment, the expression of Txnip in human islets is highly up-regulated and has the function of balancing metabolism and regulating cell growth and differentiation. However, there is no report on the expression and function of Txnip in the male testis, whether it plays a certain role in the damage of male reproductive function caused by diabetes. In this study, the purpose of this study is to investigate the effect of Txnip gene knockout against testicular damage and its possible mechanism to provide experimental basis for the prevention and treatment of diabetic reproductive injury by preparing a diabetic mouse model. Experimental methods: 1. immunofluorescence, immunofluorescence, PCR and Western blot methods for the detection of Txnip in the mice. The expression in mice testis.2. reproduction Txnip gene knockout mice (TALEN Technology) and wild type mice used for diabetes model preparation. The first injection of streptozotocin (streptozotocin, STZ) was fasting 12 h, STZ was injected intraperitoneally at the dose of 55 mg/kg, and continuous 5d. experimental group: wild type mice (WT), Txnip gene knockout group (KO), In group and KO+STZ group, 6 mice in each group were tested with blood glucose meter (ACCU CHEK, Roche) after.6 D. The blood sugar of wild type mice was more than 16.7 mmol/L as a successful model. After 60 d, mice were killed and samples of blood and testis were taken for further experiment of.3. immunization. Western blot test Txnip gene knockout on mice testis Txnip table HE staining and TUNEL test were used to observe the morphological changes of the testis and detect the apoptosis of spermatocyte by HE staining and TUNEL test. The test results were as follows: 1. immunohistochemistry, immunofluorescence, PCR and Western blot experimental results showed that Txnip was expressed in mouse testis. In the pill tissue, cells, stromal cells and spermatocyte areas were supported; in vitro experiments showed that Txnip was expressed in the cytoplasm of TM4 Sertoli cells and TM3 interstitial cells by.2. immunohistochemistry and the results of Western blot experiment showed that no Txnip expression in Txnip knockout mouse testis was significantly increased in.STZ induced wild type mice and Txnip gene knockout. The.3. testicular histological results showed that Txnip knockout inhibited the morphological damage of the testis and the apoptosis of spermatogenic cells, and down regulated the Bax/Bcl-2 expression ratio of the apoptosis related protein. In addition, Txnip knockout significantly lowered the MDA level of the testis and up regulation of SOD activity in the diabetic mice model. The experimental conclusion: Txnip expression in mouse testis tissue In vitro, cells, stromal cells and spermatocyte areas were supported. In vitro experiments showed that Txnip expressed in TM4 support cells and cytoplasmic.STZ of TM3 stromal cells could induce blood glucose increase, but Txnip knockout obviously inhibited the increase of blood sugar. Through the analysis of testicular morphology, it was found that Txnip gene knockout and inhibited the morphological damage of testis and the apoptosis of spermatogenic cells. The expression ratio of apoptosis related protein Bax/Bcl-2. In addition, Txnip knockout obviously down regulate the MDA level of testis in diabetic mice and up regulation of SOD activity. This study reveals that Txnip gene knockout can antagonize the increase of apoptosis and oxidative stress in testicular spermatogenic cells induced by hyperglycemia, and thus effectively play the role of anti reproductive damage. Further study the mechanism of its mechanism. It is important for preventing and treating male reproductive dysfunction caused by diabetes.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2

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