高脂和糖尿病SD大鼠辜丸組織中Hsp60的表達(dá)變化研究
發(fā)布時(shí)間：2018-11-14 09:14
【摘要】：目的:復(fù)制雄性Sprague-Dawlay大鼠血脂異常以及2型糖尿病模型,并研究以上兩種疾病對(duì)中年SD大鼠生殖系統(tǒng)的影響,通過對(duì)睪丸內(nèi)生精小管微結(jié)構(gòu)變化和熱休克蛋白60(HSP60)表達(dá)差異,以及曲細(xì)精管中生精細(xì)胞凋亡數(shù)量的改變,研究熱休克蛋白60在大鼠睪丸中的表達(dá)情況與大鼠生精細(xì)胞凋亡數(shù)量變化間的聯(lián)系以及其可能的影響機(jī)制。方法:挑選60只無特定病原體(SPF級(jí))健康雄性Sprague-Dawlay大鼠,每只通過統(tǒng)計(jì)軟件編號(hào)后隨機(jī)劃編成三個(gè)實(shí)驗(yàn)組:對(duì)照組(n=15)、高脂組(n=15)、糖尿病組(n=30)。對(duì)照組全程使用基礎(chǔ)飼料喂養(yǎng);高脂組前期食用高脂飼料,24 W后在原有飼料基礎(chǔ)上添加丙硫氧嘧啶(PTU),加重脂代謝異常;糖尿病組前期進(jìn)食高脂飼料,24 W末腹腔注射鏈脲佐菌素進(jìn)行糖尿病造模(對(duì)照組和高脂組采用檸檬酸注射液代替),完成注射后糖尿病大鼠更換為普通飼料。36周末大鼠尾靜脈采血檢測(cè)大鼠隨機(jī)血糖、血脂四項(xiàng)等指標(biāo),并剔除未成模大鼠。確定造模成功后處死大鼠,迅速分離大鼠一側(cè)睪丸組織,放入Bouin固定液中室溫下浸泡48小時(shí)后送病理實(shí)驗(yàn)室對(duì)標(biāo)本包埋、切片;另一側(cè)睪丸組織離體后立即塞入凍存管后置于液氮罐中暫時(shí)保存,取材結(jié)束后統(tǒng)一移至負(fù)80度冰柜中長(zhǎng)時(shí)間存放。通過免疫組化(SABC)檢測(cè)熱休克蛋白60蛋白表達(dá)情況及所在位置;光鏡分析HE染色切片,觀察大鼠睪丸曲細(xì)精管構(gòu)造變化;蛋白質(zhì)免疫印跡法(Western Blot)分析大鼠睪丸組織中HSP60蛋白表達(dá)程度;通過TUNEL染色了解凋亡細(xì)胞分布情況以及睪丸生精組織細(xì)胞凋亡水平。一切實(shí)驗(yàn)數(shù)據(jù)搜集整理后經(jīng)過spss16.0統(tǒng)計(jì)分析軟件進(jìn)行數(shù)據(jù)處理,以P0.05作為判別數(shù)據(jù)間具有顯著統(tǒng)計(jì)學(xué)差異的標(biāo)準(zhǔn)。結(jié)果:通過隨機(jī)血糖以及血脂四項(xiàng)的檢測(cè)結(jié)果提示高脂和糖尿病SD大鼠模型成功建立。HE染色在鏡下觀可見對(duì)照組生精細(xì)胞按發(fā)育成熟順序由管壁向管腔逐層排列,數(shù)量較多。高脂及糖尿病大鼠睪丸曲細(xì)精管中細(xì)胞排布較對(duì)照組松散、混亂,且各級(jí)精母細(xì)胞數(shù)量出現(xiàn)下降,成熟精子細(xì)胞在管腔中央的分布數(shù)量顯著下降,部分睪丸組織切片中可呈現(xiàn)生精細(xì)胞團(tuán)塊與生精上皮分離;免疫組化顯示高脂組和糖尿病組大鼠睪丸HSP60表達(dá)高于對(duì)照組;Western Blot實(shí)驗(yàn)發(fā)現(xiàn)高脂組和糖尿病組大鼠睪丸組織HSP60表達(dá)水平都出現(xiàn)顯著升高,對(duì)照組的目的蛋白表達(dá)水平表達(dá)則遠(yuǎn)低于其他兩組。Tunel檢測(cè)可觀察到高脂組和糖尿病組睪丸組織凋亡細(xì)胞與對(duì)照組相比數(shù)量較多,糖尿病組細(xì)胞凋亡多于高脂組。結(jié)論:糖尿病和高脂血癥可以使睪丸生精細(xì)胞凋亡數(shù)目上升,精子發(fā)育成熟數(shù)量減少,對(duì)睪丸生精結(jié)構(gòu)產(chǎn)生影響。HSP60蛋白在高脂血癥及糖尿病大鼠睪丸中明顯增加。糖尿病與高脂血癥所致生精細(xì)胞凋亡可能與睪丸組織中的HSP 60蛋白表達(dá)改變以及相關(guān)的凋亡因子表達(dá)改變有關(guān)。
[Abstract]:Objective: to establish a model of dyslipidemia and type 2 diabetes mellitus in male Sprague-Dawlay rats and to study the effects of these two diseases on the reproductive system of middle-aged SD rats. The changes of the microstructures of testicular seminiferous tubules, the expression of heat shock protein 60 (HSP60) and the number of spermatogenic cells apoptosis in seminiferous tubules were studied. To study the relationship between the expression of heat shock protein 60 (HSP60) and the apoptosis of rat spermatogenic cells and its possible mechanism. Methods: sixty healthy male Sprague-Dawlay rats without specific pathogen (SPF grade) were randomly divided into three experimental groups: control group (n = 15), hyperlipidemia group (n = 15) and diabetes group (n = 30). The control group was fed with basic diet for the whole course, and the high-fat group was fed with high-fat diet at the beginning of the experiment. After 24 weeks, the abnormal lipid metabolism was aggravated by the addition of prothiouracil (PTU),) on the basis of the original diet. In the diabetic group, the diabetic model was established by intraperitoneal injection of streptozotocin at the end of 24 weeks after eating high fat diet (citric acid injection was used instead of citric acid injection in the control group and hyperlipidemia group). At the end of the 36th week, the blood samples were collected from the tail vein of the rats to detect the random blood glucose, blood lipid and other indexes, and to eliminate the non-model rats. The rats were killed after the establishment of the model, and the testis of the rats were quickly separated. The rats were immersed in the Bouin fixative solution at room temperature for 48 hours and then sent to the pathology laboratory to bury and slice the specimens. The testicular tissue on the other side of the testis was immediately inserted into a cryopreservation tube and stored in a liquid nitrogen tank for a short time, and then transferred to a negative 80 degree freezer for a long time. The expression and location of heat shock protein 60 (HSP60) were detected by immunohistochemical (SABC), and the changes of testicular seminiferous tubules were observed by HE staining. Western blot (Western Blot) was used to detect the expression of HSP60 protein in rat testis and the distribution of apoptotic cells and the apoptosis level of testicular spermatogenic tissue were studied by TUNEL staining. After all the experimental data were collected and sorted, the data were processed by spss16.0 statistical analysis software, and P05 was taken as the criterion of significant statistical difference among the discriminant data. Results: the results of random blood glucose and blood lipids showed that the SD rat model of hyperlipidemia and diabetes mellitus was successfully established. HE staining showed that the spermatogenic cells in the control group were arranged from the wall to the lumen in the order of development and maturation, and the number of spermatogenic cells in the control group was more than that in the control group. In hyperlipidemic and diabetic rats, the distribution of cells in the seminiferous tubule of testis was looser and confused, and the number of spermatocytes decreased, and the number of mature sperm cells in the center of the tubules decreased significantly. Spermatogenic cell mass and spermatogenic epithelium could be separated in some testicular sections. Immunohistochemistry showed that the expression of HSP60 in testis of hyperlipidemia group and diabetic group was higher than that of control group. Western Blot assay showed that the expression of HSP60 in testis of both hyperlipidemia group and diabetic group was significantly increased. The expression of target protein in the control group was much lower than that in the other two groups. Tunel assay showed that the number of apoptotic cells in testis of hyperlipidemia group and diabetic group was more than that of control group, and that of diabetic group was more than that of hyperlipidemic group. Conclusion: diabetes and hyperlipidemia can increase the number of testicular spermatogenic cells apoptosis and decrease the number of spermatozoa maturation. HSP60 protein in the testis of hyperlipidemia and diabetic rats is obviously increased. The apoptosis of spermatogenic cells induced by diabetes mellitus and hyperlipidemia may be related to the expression of HSP 60 protein and related apoptosis factors in testis.
【學(xué)位授予單位】：廣西醫(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)】：R58;R698.2
本文編號(hào)：2330758
[Abstract]:Objective: to establish a model of dyslipidemia and type 2 diabetes mellitus in male Sprague-Dawlay rats and to study the effects of these two diseases on the reproductive system of middle-aged SD rats. The changes of the microstructures of testicular seminiferous tubules, the expression of heat shock protein 60 (HSP60) and the number of spermatogenic cells apoptosis in seminiferous tubules were studied. To study the relationship between the expression of heat shock protein 60 (HSP60) and the apoptosis of rat spermatogenic cells and its possible mechanism. Methods: sixty healthy male Sprague-Dawlay rats without specific pathogen (SPF grade) were randomly divided into three experimental groups: control group (n = 15), hyperlipidemia group (n = 15) and diabetes group (n = 30). The control group was fed with basic diet for the whole course, and the high-fat group was fed with high-fat diet at the beginning of the experiment. After 24 weeks, the abnormal lipid metabolism was aggravated by the addition of prothiouracil (PTU),) on the basis of the original diet. In the diabetic group, the diabetic model was established by intraperitoneal injection of streptozotocin at the end of 24 weeks after eating high fat diet (citric acid injection was used instead of citric acid injection in the control group and hyperlipidemia group). At the end of the 36th week, the blood samples were collected from the tail vein of the rats to detect the random blood glucose, blood lipid and other indexes, and to eliminate the non-model rats. The rats were killed after the establishment of the model, and the testis of the rats were quickly separated. The rats were immersed in the Bouin fixative solution at room temperature for 48 hours and then sent to the pathology laboratory to bury and slice the specimens. The testicular tissue on the other side of the testis was immediately inserted into a cryopreservation tube and stored in a liquid nitrogen tank for a short time, and then transferred to a negative 80 degree freezer for a long time. The expression and location of heat shock protein 60 (HSP60) were detected by immunohistochemical (SABC), and the changes of testicular seminiferous tubules were observed by HE staining. Western blot (Western Blot) was used to detect the expression of HSP60 protein in rat testis and the distribution of apoptotic cells and the apoptosis level of testicular spermatogenic tissue were studied by TUNEL staining. After all the experimental data were collected and sorted, the data were processed by spss16.0 statistical analysis software, and P05 was taken as the criterion of significant statistical difference among the discriminant data. Results: the results of random blood glucose and blood lipids showed that the SD rat model of hyperlipidemia and diabetes mellitus was successfully established. HE staining showed that the spermatogenic cells in the control group were arranged from the wall to the lumen in the order of development and maturation, and the number of spermatogenic cells in the control group was more than that in the control group. In hyperlipidemic and diabetic rats, the distribution of cells in the seminiferous tubule of testis was looser and confused, and the number of spermatocytes decreased, and the number of mature sperm cells in the center of the tubules decreased significantly. Spermatogenic cell mass and spermatogenic epithelium could be separated in some testicular sections. Immunohistochemistry showed that the expression of HSP60 in testis of hyperlipidemia group and diabetic group was higher than that of control group. Western Blot assay showed that the expression of HSP60 in testis of both hyperlipidemia group and diabetic group was significantly increased. The expression of target protein in the control group was much lower than that in the other two groups. Tunel assay showed that the number of apoptotic cells in testis of hyperlipidemia group and diabetic group was more than that of control group, and that of diabetic group was more than that of hyperlipidemic group. Conclusion: diabetes and hyperlipidemia can increase the number of testicular spermatogenic cells apoptosis and decrease the number of spermatozoa maturation. HSP60 protein in the testis of hyperlipidemia and diabetic rats is obviously increased. The apoptosis of spermatogenic cells induced by diabetes mellitus and hyperlipidemia may be related to the expression of HSP 60 protein and related apoptosis factors in testis.
【學(xué)位授予單位】：廣西醫(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)】：R58;R698.2
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