IGFBP-3在糖尿病ED大鼠阴茎海绵体组织中的表达及其意义
发布时间：2019-06-12 12:26
【摘要】：研究背景：勃起功能障碍(erectile dysfunction, ED)严重影响着患者和伴侣的生活质量,治疗ED不仅是患者的需要,而且有重要的社会意义。目前估计全球ED患者超过1亿,糖尿病ED (diabetes mellitus, DM ED)是ED的一种重要类型。调查显示约50%的糖尿病患者有ED,但具体机制不清楚。糖尿病能引起包括阴茎小动脉在内的全身小血管结构改变,如内皮损伤,基膜增厚,血粘度增高,红细胞聚集,血小板粘附和聚集,最后导致微血栓形成和或微血管闭塞等,是组织缺血缺氧的重要原因。阴茎勃起的分子机制主要是在各种性刺激作用下阴茎海绵体神经末梢中非肾上腺素能非胆碱能(NENA)神经的神经源性(nNOS)及内皮源性(eNOS)释放,eNOS分解左旋精氨酸生成NO, NO可自由穿过细胞膜刺激海绵体平滑肌细胞(CCSMC)内的可溶性鸟苷酸环化酶(sGC),使三磷酸鸟苷(GTP)转化为第二信使cGMP,从而激活依赖于cGMP的蛋白激酶(cGMP dependent protein kinase I, cGKI), CCSMC内Ca2+浓度降低,引起海绵体平滑肌舒张。在勃起过程中,阴茎海绵体平滑肌舒张的基础上使血流不断流人阴茎海绵窦间隙内,使其扩大,阴茎肿胀使白膜受压,导致沟通海绵窦与阴茎背静脉的沟通静脉受压,限制了阴茎海绵体内的血流流出,使血管内回流阻力上升,以至于静脉血不能外流,阴茎内血压上升,产生了坚硬的勃起。大量的研究说明：IGF-1即能促进新生血管的形成,又能在血管损伤修复中起重要作用,同时能够促进细胞的增值、迁移以及抑制凋亡而保持细胞的完整性,IGF-1并且还参与NO/sGC/cGMP信号传导通路,导致血管扩张,从而改善勃起功能障碍。而IGF-1在血循环中半衰期短,主要与IGFBP-3相结合而存在；IGFBP-3既是IGF-1的载体蛋白,又可延长IGF-1的半衰期,IGFBP-3携带了绝大部分的IGF-1, IGFBP-3通过竞争性抑制IGF-1与胰岛素样生长因子受体(insulin like growth factor receptor, IGFR)的结合限制IGF-1进入靶细胞而调节IGF-1的有效性。高水平表达的IGFBP-3能够减少IGF-1在阴茎海绵体组织中的作用。 目的：本课题主要通过建立糖尿病性ED大鼠、糖尿病性非ED大鼠及正常大鼠的动物模型,了解其DM组大鼠生活习性及勃起功能的改变。研究糖尿病性勃起功能障碍大鼠阴茎海绵体组织中IGF-BP3的表达,观察其对阴茎勃起功能的影响,然后进行NO-cGMP通路方面的检测,探讨IGFBP-3在糖尿病性勃起功能障碍发病过程中的作用,为临床开发新的ED治疗药物提供前期的研究基础。 方法：选取2月龄的健康雄性Wistar大鼠30只,经阿朴吗啡(Apomorphine,APO)功能试验提示均有正常的勃起功能,随机分为2组：①正常对照组：9只,给予普通饮食喂养；②糖尿病模型组：21只,腹腔注射60mg/kg STZ诱导糖尿病大鼠模型,于2周后采用割尾法采血测大鼠的随机血糖值,以随机血糖水平16.7mmol/L为糖尿病动物模型建立成功标准。建模成功的糖尿病大鼠8周后,行阿朴吗啡APO功能试验,以阴茎体增长、龟头充血、露出为阴茎勃起,记录30min内大鼠阴茎勃起次数,其中30min内有1次勃起的称勃起功能正常,从而筛选出糖尿病非ED模型组、糖尿病ED模型组。对糖尿病模型组及正常对照组造模前后的体重及血糖进行比较,再分别对正常对照组、糖尿病非ED模型组、糖尿病ED模型组行：大鼠阴茎海绵体内压(ICP)/平均动脉压(MAP)和total ICP的检测、RT-PCI测定IGF-BP3mRNA的表达、Western Blot检钡IGF-BP3蛋白的表达、ELISA法检测cGMP的含量。 结果：(1)糖尿病造模组与正常组造模前后大鼠体重及血糖测定结果：正常对照组大鼠体重随周龄的增加而增长；DM组大鼠较正常大鼠每日饮水量及尿量均显著增加,明显消瘦且毛色暗淡,体重较对照组显著减轻(p0.001),血糖水平较对照组显著升高(p0.001)。(2)阿朴吗啡(AP0)筛选实验结果：9只正常组大鼠阴茎勃起功能均正常；19只成模的糖尿病大鼠有11只未见阴茎勃起为糖尿病ED组,有8只糖尿病大鼠可见阴茎勃起为糖尿病非ED组。(3)糖尿病性ED大鼠模型组大鼠阴茎海绵体内压(ICP)/平均动脉压(MAP)和total ICP值均明显低于正常对照组及糖尿病非ED组(P0.01),糖尿病非ED组与正常对照组相比无明显统计学意义(P0.05)。(4) RT-PCR测定IGF-BP3mRNA的表达结果显示：糖尿病ED组阴茎海绵体IGFBP-3mRNA表达水平显著高于正常对照组及糖尿病非ED组,糖尿病非ED组IGFBP-3mRNA表达水平明显高于正常对照组。(5) Western Blot检测IGF-BP3蛋白的表达结果显示：糖尿病ED组IGF-BP3蛋白的表达显著高于正常对照组及糖尿病非ED组,糖尿病非ED组IGF-BP3蛋白的表达明显高于正常对照组。(6) ELISA法检测cGMP的含量结果显示：糖尿病ED组cGMP的含量显著低于正常对照组及糖尿病非ED组(P0.01),糖尿病非ED组cGMP的含量与正常对照组相比无明显统计学意义(P0.05)。 结论：通过本课题的研究,可以得出以下结论：1、IGFBP-3在糖尿病大鼠阴茎海绵体组织中的表达水平升高；2、在糖尿病ED大鼠阴茎海绵体组织中IGFBP-3的表达水平明显升高；3、IGFBP-3mRNA及IGF-BP3蛋白的表达水平在糖尿病ED大鼠阴茎海绵体组织中与cGMP的含量呈负相关；4、糖尿病可引起IGFBP-3的表达增加,IGFBP-3的表达量增加到一定程度,从而影响阴茎海绵体组织局部cGMP的含量降低,可能是导致勃起功能障碍的重要原因之一。
[Abstract]:Background: Erectile dysfunction (ED) seriously affects the quality of life of patients and partners, and the treatment of ED is not only the patient's needs, but also of important social significance. At present, it is estimated that the number of ED patients in the world is more than 100 million, and the diabetic ED (DM ED) is an important type of ED. The survey showed that about 50% of the patients with diabetes had ED, but the specific mechanism was not clear. Diabetes can cause a change of the whole body of the whole body, including the arterioles of the penis, such as the endothelial injury, the thickening of the basal membrane, the increase of the blood viscosity, the aggregation of the red blood cells, the adhesion and the aggregation of the platelets, and finally the formation of the microthrombus and the microvessel occlusion, and is an important cause of the ischemia and hypoxia of the tissue. The molecular mechanism of the erection of the penis is mainly the neurogenic (nNOS) and endogenic (eNOS) release of the non-adrenergic non-cholinergic (NEA) nerve in the nerve terminal of the corpus cavernosum of the penis under various sexual stimulation, and the eNOS is decomposed into L-arginine to generate NO, NO can freely pass through the cell membrane to stimulate the soluble ornithine acid cyclase (sGC) in the corpus cavernosum smooth muscle cell (CCSMC), so that the triphosphate (GTP) is converted into a second messenger cGMP, so that the cGMP dependent protein kinase I (cGKI) is activated, and the concentration of the Ca2 + in the CCSMC is reduced, causing the smooth muscle of the corpus cavernosum to relax in that erection proces, the blood flow is continuously flow through the gap of the penis sponge of the penis, so that the blood flow is expanded, the penis is swollen, the white film is pressed, the communication vein between the communication sponge and the back vein of the penis is compressed, blood flow in the corpus cavernosum of the penis is limited, The flow resistance in the blood vessel is increased so that the venous blood can not flow out, the blood pressure in the penis rises, and a hard erection is produced. A large number of studies show that IGF-1 can promote the formation of new blood vessels and play an important role in the repair of vascular injury, and can promote the value-added, migration and apoptosis of the cells, maintain the integrity of the cells, IGF-1 and also participate in the NO/ sGC/ cGMP signaling pathway, leading to the expansion of the blood vessel, So as to improve the erectile dysfunction. IGFBP-3 is not only a carrier protein of IGF-1, but also can prolong the half-life of IGF-1. IGFBP-3 carries most of IGF-1 and IGFBP-3 by competitive inhibition of IGF-1 and insulin-like growth factor receptor (IGF-1). The combination of IGFR limits the effectiveness of IGF-1 by limiting the entry of IGF-1 into the target cell. The high level of IGFBP-3 can reduce the role of IGF-1 in the tissue of the corpus cavernosum. Objective: To study the life habits and erectile function of diabetic ED rats, diabetic non-ED rats and normal rats by establishing animal models of diabetic ED rats, diabetic non-ED rats and normal rats. To study the expression of IGF-BP3 in the corpus cavernosum tissue of diabetic erectile dysfunction rats, to observe the effect of IGFBP-3 on the function of erectile dysfunction, and to explore the role of IGFBP-3 in the pathogenesis of diabetic erectile dysfunction. To provide a preliminary study group for the clinical development of new ED therapeutic drugs Methods:30 healthy male Wistar rats were randomly divided into two groups: normal control group (n = 9), normal diet (n = 9), and diabetic model group (n = 9). The model of diabetic rats was induced by injection of 60 mg/ kg STZ in the abdominal cavity. The random blood glucose level of the rats was measured by the method of cutting the tail after two weeks, and the random blood glucose level of 16.7 mmol/ L was established as the animal model of the diabetes. After 8 weeks of successful modeling of diabetic rats, the functional test of apomorphine APO was performed to increase the number of erection of the penis in 30 minutes with the increase of the penis body and the hyperemia of the glans and the erection of the penis. The erection function of the rats was normal in 30 minutes, and the non-ED type of diabetes was selected. model group, diabetic ED The body weight and blood sugar before and after the model group and the normal control group were compared, and the normal control group, the diabetic non-ED model group and the diabetic ED model group were divided into the normal control group, the diabetic non-ED model group and the diabetic ED model group. The expression of IGF-BP3 mRNA was determined by RT-PCI, and the expression of IGF-BP3 protein was detected by Western Blot. The results were as follows: (1) The body weight and blood glucose of the rats in the normal control group increased with the increase of the age of week before and after the model group and the normal group, and the daily water consumption and the urine volume of the DM group in the normal rats increased significantly, and the rats were obviously emaciated. And the blood sugar level was significantly higher than that of the control group (p0.001), and the blood sugar level was significantly higher than that in the control group (p0.001). (1). (2) The experimental results of apomorphine (AP0):9 normal groups and 9 normal rats were normal in the function of the erection of the penis.11 of the 19 diabetic rats were not found to be in the ED group. (3) The internal pressure of the corpus cavernosum (ICP)/ mean arterial pressure (MAP) and total ICP of the model group of the diabetic ED rats were significantly lower than those in the control group and the non-ED group (P0.01), and the diabetic non-ED group had no significant statistical significance as compared with the normal control group (P (4) The expression of IGFBP-3 mRNA in the diabetic ED group was significantly higher than that of the normal control group and the diabetic non-ED group, and the expression level of IGFBP-3 mRNA in the non-ED group of the diabetic ED group was significantly higher than that of the normal control group and the diabetic non-ED group. The expression of IGF-BP3 protein in the diabetic ED group was significantly higher than that of the normal control group and the diabetic non-ED group, and the expression of the IGF-BP3 protein in the diabetic ED group was significantly higher than that of the normal control group and the diabetic non-ED group. (6) The content of cGMP in the diabetic ED group was significantly lower than that in the control group and the non-ED group (P0.01), and the content of cGMP in the diabetic ED group was not significantly higher than that of the normal control group (P The results were as follows:1. The expression level of IGFBP-3 in the corpus cavernosum tissue of diabetic rats increased;2. The expression of IGFBP-3 in the corpus cavernosum tissue of the diabetic ED rats The levels of IGFBP-3 mRNA and IGF-BP3 protein were negatively correlated with the content of cGMP in diabetic ED rats.4. The expression of IGFBP-3 increased and the expression of IGFBP-3 increased to a certain extent. The reduction in the content of MP may result in erectile dysfunction
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R698
本文编号：2498018
[Abstract]:Background: Erectile dysfunction (ED) seriously affects the quality of life of patients and partners, and the treatment of ED is not only the patient's needs, but also of important social significance. At present, it is estimated that the number of ED patients in the world is more than 100 million, and the diabetic ED (DM ED) is an important type of ED. The survey showed that about 50% of the patients with diabetes had ED, but the specific mechanism was not clear. Diabetes can cause a change of the whole body of the whole body, including the arterioles of the penis, such as the endothelial injury, the thickening of the basal membrane, the increase of the blood viscosity, the aggregation of the red blood cells, the adhesion and the aggregation of the platelets, and finally the formation of the microthrombus and the microvessel occlusion, and is an important cause of the ischemia and hypoxia of the tissue. The molecular mechanism of the erection of the penis is mainly the neurogenic (nNOS) and endogenic (eNOS) release of the non-adrenergic non-cholinergic (NEA) nerve in the nerve terminal of the corpus cavernosum of the penis under various sexual stimulation, and the eNOS is decomposed into L-arginine to generate NO, NO can freely pass through the cell membrane to stimulate the soluble ornithine acid cyclase (sGC) in the corpus cavernosum smooth muscle cell (CCSMC), so that the triphosphate (GTP) is converted into a second messenger cGMP, so that the cGMP dependent protein kinase I (cGKI) is activated, and the concentration of the Ca2 + in the CCSMC is reduced, causing the smooth muscle of the corpus cavernosum to relax in that erection proces, the blood flow is continuously flow through the gap of the penis sponge of the penis, so that the blood flow is expanded, the penis is swollen, the white film is pressed, the communication vein between the communication sponge and the back vein of the penis is compressed, blood flow in the corpus cavernosum of the penis is limited, The flow resistance in the blood vessel is increased so that the venous blood can not flow out, the blood pressure in the penis rises, and a hard erection is produced. A large number of studies show that IGF-1 can promote the formation of new blood vessels and play an important role in the repair of vascular injury, and can promote the value-added, migration and apoptosis of the cells, maintain the integrity of the cells, IGF-1 and also participate in the NO/ sGC/ cGMP signaling pathway, leading to the expansion of the blood vessel, So as to improve the erectile dysfunction. IGFBP-3 is not only a carrier protein of IGF-1, but also can prolong the half-life of IGF-1. IGFBP-3 carries most of IGF-1 and IGFBP-3 by competitive inhibition of IGF-1 and insulin-like growth factor receptor (IGF-1). The combination of IGFR limits the effectiveness of IGF-1 by limiting the entry of IGF-1 into the target cell. The high level of IGFBP-3 can reduce the role of IGF-1 in the tissue of the corpus cavernosum. Objective: To study the life habits and erectile function of diabetic ED rats, diabetic non-ED rats and normal rats by establishing animal models of diabetic ED rats, diabetic non-ED rats and normal rats. To study the expression of IGF-BP3 in the corpus cavernosum tissue of diabetic erectile dysfunction rats, to observe the effect of IGFBP-3 on the function of erectile dysfunction, and to explore the role of IGFBP-3 in the pathogenesis of diabetic erectile dysfunction. To provide a preliminary study group for the clinical development of new ED therapeutic drugs Methods:30 healthy male Wistar rats were randomly divided into two groups: normal control group (n = 9), normal diet (n = 9), and diabetic model group (n = 9). The model of diabetic rats was induced by injection of 60 mg/ kg STZ in the abdominal cavity. The random blood glucose level of the rats was measured by the method of cutting the tail after two weeks, and the random blood glucose level of 16.7 mmol/ L was established as the animal model of the diabetes. After 8 weeks of successful modeling of diabetic rats, the functional test of apomorphine APO was performed to increase the number of erection of the penis in 30 minutes with the increase of the penis body and the hyperemia of the glans and the erection of the penis. The erection function of the rats was normal in 30 minutes, and the non-ED type of diabetes was selected. model group, diabetic ED The body weight and blood sugar before and after the model group and the normal control group were compared, and the normal control group, the diabetic non-ED model group and the diabetic ED model group were divided into the normal control group, the diabetic non-ED model group and the diabetic ED model group. The expression of IGF-BP3 mRNA was determined by RT-PCI, and the expression of IGF-BP3 protein was detected by Western Blot. The results were as follows: (1) The body weight and blood glucose of the rats in the normal control group increased with the increase of the age of week before and after the model group and the normal group, and the daily water consumption and the urine volume of the DM group in the normal rats increased significantly, and the rats were obviously emaciated. And the blood sugar level was significantly higher than that of the control group (p0.001), and the blood sugar level was significantly higher than that in the control group (p0.001). (1). (2) The experimental results of apomorphine (AP0):9 normal groups and 9 normal rats were normal in the function of the erection of the penis.11 of the 19 diabetic rats were not found to be in the ED group. (3) The internal pressure of the corpus cavernosum (ICP)/ mean arterial pressure (MAP) and total ICP of the model group of the diabetic ED rats were significantly lower than those in the control group and the non-ED group (P0.01), and the diabetic non-ED group had no significant statistical significance as compared with the normal control group (P (4) The expression of IGFBP-3 mRNA in the diabetic ED group was significantly higher than that of the normal control group and the diabetic non-ED group, and the expression level of IGFBP-3 mRNA in the non-ED group of the diabetic ED group was significantly higher than that of the normal control group and the diabetic non-ED group. The expression of IGF-BP3 protein in the diabetic ED group was significantly higher than that of the normal control group and the diabetic non-ED group, and the expression of the IGF-BP3 protein in the diabetic ED group was significantly higher than that of the normal control group and the diabetic non-ED group. (6) The content of cGMP in the diabetic ED group was significantly lower than that in the control group and the non-ED group (P0.01), and the content of cGMP in the diabetic ED group was not significantly higher than that of the normal control group (P The results were as follows:1. The expression level of IGFBP-3 in the corpus cavernosum tissue of diabetic rats increased;2. The expression of IGFBP-3 in the corpus cavernosum tissue of the diabetic ED rats The levels of IGFBP-3 mRNA and IGF-BP3 protein were negatively correlated with the content of cGMP in diabetic ED rats.4. The expression of IGFBP-3 increased and the expression of IGFBP-3 increased to a certain extent. The reduction in the content of MP may result in erectile dysfunction
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R698
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