【摘要】：實(shí)驗(yàn)?zāi)康?硫氧還蛋白互作蛋白(thioredoxin-interacting protein,Txnip)發(fā)現(xiàn)于1,25-二羥維生素D3處理的HL-60細(xì)胞,因此早前命名為維生素D3上調(diào)蛋白1(vitamin D3 upregulated protein 1,VDUP1)。Txnip可以與硫氧還蛋白(thioredoxin,Trx)結(jié)合,從而抑制Trx的抗氧化功能,導(dǎo)致活性氧的累積與細(xì)胞壓力。經(jīng)葡萄糖處理后,Txnip在人胰島中表達(dá)高度上調(diào),具有平衡代謝和調(diào)節(jié)細(xì)胞生長(zhǎng)、分化的功能。但Txnip在雄性睪丸中的表達(dá)定位與功能研究尚未見報(bào)道,是否在糖尿病導(dǎo)致的雄性生殖功能受損中扮演某種角色值得研究。基于此,本研究擬通過制備糖尿病小鼠模型,觀察Txnip基因敲除是否具有拮抗睪丸損傷的效應(yīng)及其可能的機(jī)制,為臨床上防治糖尿病生殖損傷提供實(shí)驗(yàn)依據(jù)。實(shí)驗(yàn)方法:1.免疫組化、免疫熒光、PCR和western blot方法檢測(cè)Txnip在小鼠睪丸中表達(dá)情況。2.繁殖Txnip基因敲除小鼠(TALEN技術(shù)制備)與野生型小鼠,用于糖尿病模型制備。首次注射鏈脲佐菌素(streptozotocin,STZ)前禁食12 h,按55 mg/kg劑量腹腔內(nèi)注射STZ,連續(xù)5d。實(shí)驗(yàn)分組:野生型小鼠組(WT)、Txnip基因敲除組(KO)、WT+STZ組和KO+STZ組,每組6只小鼠。6 d后用血糖測(cè)定儀(ACCU CHEK,羅氏)檢測(cè)血糖。野生型小鼠血糖大于16.7 mmol/L視為造模成功。常規(guī)飼養(yǎng)60 d后處死小鼠,取血液與睪丸標(biāo)本,用于進(jìn)一步實(shí)驗(yàn)。3.免疫組化、western blot檢測(cè)Txnip基因敲除對(duì)小鼠睪丸Txnip表達(dá)的影響。睪丸標(biāo)本制成石蠟切片,通過HE染色和TUNEL實(shí)驗(yàn)觀察小鼠睪丸的形態(tài)學(xué)變化及檢測(cè)精細(xì)胞的凋亡情況。并且測(cè)定小鼠睪丸組織中MDA含量與SOD活性。實(shí)驗(yàn)結(jié)果:1.免疫組化、免疫熒光、PCR及western blot實(shí)驗(yàn)結(jié)果顯示Txnip表達(dá)于小鼠睪丸組織中支持細(xì)胞、間質(zhì)細(xì)胞及精細(xì)胞區(qū)域;體外實(shí)驗(yàn)顯示Txnip表達(dá)于睪丸TM4支持細(xì)胞及TM3間質(zhì)細(xì)胞的胞質(zhì)。2.免疫組化與western blot實(shí)驗(yàn)結(jié)果表明在Txnip敲除小鼠睪丸中未檢測(cè)到Txnip表達(dá)。STZ誘導(dǎo)野生型小鼠血糖明顯增加,Txnip基因敲除明顯抑制血糖的增加。3.睪丸組織學(xué)結(jié)果顯示Txnip基因敲除抑制睪丸形態(tài)學(xué)損傷與生精細(xì)胞凋亡,下調(diào)凋亡相關(guān)蛋白Bax/Bcl-2表達(dá)比值。此外,Txnip敲除明顯下調(diào)糖尿病小鼠模型睪丸MDA水平,上調(diào)SOD活性。實(shí)驗(yàn)結(jié)論:Txnip表達(dá)在小鼠睪丸組織中支持細(xì)胞、間質(zhì)細(xì)胞及精細(xì)胞區(qū)域,體外實(shí)驗(yàn)顯示Txnip表達(dá)于TM4支持細(xì)胞及TM3間質(zhì)細(xì)胞的胞質(zhì)。STZ可以誘導(dǎo)血糖上升,但敲除Txnip明顯抑制血糖的增加。通過分析睪丸形態(tài)學(xué),發(fā)現(xiàn)Txnip基因敲除抑制睪丸形態(tài)學(xué)損傷與生精細(xì)胞凋亡,下調(diào)凋亡相關(guān)蛋白Bax/Bcl-2表達(dá)比值。此外,Txnip敲除明顯下調(diào)糖尿病小鼠模型睪丸MDA水平,上調(diào)SOD活性。本研究揭示Txnip基因敲除可以拮抗高血糖引起的睪丸生精細(xì)胞凋亡與氧化壓力增加,從而有效發(fā)揮抗生殖損傷的作用。進(jìn)一步研究其機(jī)理對(duì)防治糖尿病導(dǎo)致的雄性生殖功能異常具有重要意義。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2

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