IGFBP-3在糖尿病ED大鼠陰莖海綿體組織中的表達(dá)及其意義
發(fā)布時(shí)間：2019-06-12 12:26
【摘要】：研究背景：勃起功能障礙(erectile dysfunction, ED)嚴(yán)重影響著患者和伴侶的生活質(zhì)量,治療ED不僅是患者的需要,而且有重要的社會(huì)意義。目前估計(jì)全球ED患者超過(guò)1億,糖尿病ED (diabetes mellitus, DM ED)是ED的一種重要類(lèi)型。調(diào)查顯示約50%的糖尿病患者有ED,但具體機(jī)制不清楚。糖尿病能引起包括陰莖小動(dòng)脈在內(nèi)的全身小血管結(jié)構(gòu)改變,如內(nèi)皮損傷,基膜增厚,血粘度增高,紅細(xì)胞聚集,血小板粘附和聚集,最后導(dǎo)致微血栓形成和或微血管閉塞等,是組織缺血缺氧的重要原因。陰莖勃起的分子機(jī)制主要是在各種性刺激作用下陰莖海綿體神經(jīng)末梢中非腎上腺素能非膽堿能(NENA)神經(jīng)的神經(jīng)源性(nNOS)及內(nèi)皮源性(eNOS)釋放,eNOS分解左旋精氨酸生成NO, NO可自由穿過(guò)細(xì)胞膜刺激海綿體平滑肌細(xì)胞(CCSMC)內(nèi)的可溶性鳥(niǎo)苷酸環(huán)化酶(sGC),使三磷酸鳥(niǎo)苷(GTP)轉(zhuǎn)化為第二信使cGMP,從而激活依賴(lài)于cGMP的蛋白激酶(cGMP dependent protein kinase I, cGKI), CCSMC內(nèi)Ca2+濃度降低,引起海綿體平滑肌舒張。在勃起過(guò)程中,陰莖海綿體平滑肌舒張的基礎(chǔ)上使血流不斷流人陰莖海綿竇間隙內(nèi),使其擴(kuò)大,陰莖腫脹使白膜受壓,導(dǎo)致溝通海綿竇與陰莖背靜脈的溝通靜脈受壓,限制了陰莖海綿體內(nèi)的血流流出,使血管內(nèi)回流阻力上升,以至于靜脈血不能外流,陰莖內(nèi)血壓上升,產(chǎn)生了堅(jiān)硬的勃起。大量的研究說(shuō)明：IGF-1即能促進(jìn)新生血管的形成,又能在血管損傷修復(fù)中起重要作用,同時(shí)能夠促進(jìn)細(xì)胞的增值、遷移以及抑制凋亡而保持細(xì)胞的完整性,IGF-1并且還參與NO/sGC/cGMP信號(hào)傳導(dǎo)通路,導(dǎo)致血管擴(kuò)張,從而改善勃起功能障礙。而IGF-1在血循環(huán)中半衰期短,主要與IGFBP-3相結(jié)合而存在；IGFBP-3既是IGF-1的載體蛋白,又可延長(zhǎng)IGF-1的半衰期,IGFBP-3攜帶了絕大部分的IGF-1, IGFBP-3通過(guò)競(jìng)爭(zhēng)性抑制IGF-1與胰島素樣生長(zhǎng)因子受體(insulin like growth factor receptor, IGFR)的結(jié)合限制IGF-1進(jìn)入靶細(xì)胞而調(diào)節(jié)IGF-1的有效性。高水平表達(dá)的IGFBP-3能夠減少I(mǎi)GF-1在陰莖海綿體組織中的作用。 目的：本課題主要通過(guò)建立糖尿病性ED大鼠、糖尿病性非ED大鼠及正常大鼠的動(dòng)物模型,了解其DM組大鼠生活習(xí)性及勃起功能的改變。研究糖尿病性勃起功能障礙大鼠陰莖海綿體組織中IGF-BP3的表達(dá),觀察其對(duì)陰莖勃起功能的影響,然后進(jìn)行NO-cGMP通路方面的檢測(cè),探討IGFBP-3在糖尿病性勃起功能障礙發(fā)病過(guò)程中的作用,為臨床開(kāi)發(fā)新的ED治療藥物提供前期的研究基礎(chǔ)。 方法：選取2月齡的健康雄性Wistar大鼠30只,經(jīng)阿樸嗎啡(Apomorphine,APO)功能試驗(yàn)提示均有正常的勃起功能,隨機(jī)分為2組：①正常對(duì)照組：9只,給予普通飲食喂養(yǎng)；②糖尿病模型組：21只,腹腔注射60mg/kg STZ誘導(dǎo)糖尿病大鼠模型,于2周后采用割尾法采血測(cè)大鼠的隨機(jī)血糖值,以隨機(jī)血糖水平16.7mmol/L為糖尿病動(dòng)物模型建立成功標(biāo)準(zhǔn)。建模成功的糖尿病大鼠8周后,行阿樸嗎啡APO功能試驗(yàn),以陰莖體增長(zhǎng)、龜頭充血、露出為陰莖勃起,記錄30min內(nèi)大鼠陰莖勃起次數(shù),其中30min內(nèi)有1次勃起的稱(chēng)勃起功能正常,從而篩選出糖尿病非ED模型組、糖尿病ED模型組。對(duì)糖尿病模型組及正常對(duì)照組造模前后的體重及血糖進(jìn)行比較,再分別對(duì)正常對(duì)照組、糖尿病非ED模型組、糖尿病ED模型組行：大鼠陰莖海綿體內(nèi)壓(ICP)/平均動(dòng)脈壓(MAP)和total ICP的檢測(cè)、RT-PCI測(cè)定IGF-BP3mRNA的表達(dá)、Western Blot檢鋇IGF-BP3蛋白的表達(dá)、ELISA法檢測(cè)cGMP的含量。 結(jié)果：(1)糖尿病造模組與正常組造模前后大鼠體重及血糖測(cè)定結(jié)果：正常對(duì)照組大鼠體重隨周齡的增加而增長(zhǎng)；DM組大鼠較正常大鼠每日飲水量及尿量均顯著增加,明顯消瘦且毛色暗淡,體重較對(duì)照組顯著減輕(p0.001),血糖水平較對(duì)照組顯著升高(p0.001)。(2)阿樸嗎啡(AP0)篩選實(shí)驗(yàn)結(jié)果：9只正常組大鼠陰莖勃起功能均正常；19只成模的糖尿病大鼠有11只未見(jiàn)陰莖勃起為糖尿病ED組,有8只糖尿病大鼠可見(jiàn)陰莖勃起為糖尿病非ED組。(3)糖尿病性ED大鼠模型組大鼠陰莖海綿體內(nèi)壓(ICP)/平均動(dòng)脈壓(MAP)和total ICP值均明顯低于正常對(duì)照組及糖尿病非ED組(P0.01),糖尿病非ED組與正常對(duì)照組相比無(wú)明顯統(tǒng)計(jì)學(xué)意義(P0.05)。(4) RT-PCR測(cè)定IGF-BP3mRNA的表達(dá)結(jié)果顯示：糖尿病ED組陰莖海綿體IGFBP-3mRNA表達(dá)水平顯著高于正常對(duì)照組及糖尿病非ED組,糖尿病非ED組IGFBP-3mRNA表達(dá)水平明顯高于正常對(duì)照組。(5) Western Blot檢測(cè)IGF-BP3蛋白的表達(dá)結(jié)果顯示：糖尿病ED組IGF-BP3蛋白的表達(dá)顯著高于正常對(duì)照組及糖尿病非ED組,糖尿病非ED組IGF-BP3蛋白的表達(dá)明顯高于正常對(duì)照組。(6) ELISA法檢測(cè)cGMP的含量結(jié)果顯示：糖尿病ED組cGMP的含量顯著低于正常對(duì)照組及糖尿病非ED組(P0.01),糖尿病非ED組cGMP的含量與正常對(duì)照組相比無(wú)明顯統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：通過(guò)本課題的研究,可以得出以下結(jié)論：1、IGFBP-3在糖尿病大鼠陰莖海綿體組織中的表達(dá)水平升高；2、在糖尿病ED大鼠陰莖海綿體組織中IGFBP-3的表達(dá)水平明顯升高；3、IGFBP-3mRNA及IGF-BP3蛋白的表達(dá)水平在糖尿病ED大鼠陰莖海綿體組織中與cGMP的含量呈負(fù)相關(guān)；4、糖尿病可引起IGFBP-3的表達(dá)增加,IGFBP-3的表達(dá)量增加到一定程度,從而影響陰莖海綿體組織局部cGMP的含量降低,可能是導(dǎo)致勃起功能障礙的重要原因之一。
[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
【學(xué)位授予單位】：長(zhǎng)江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R587.2;R698
本文編號(hào)：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
【學(xué)位授予單位】：長(zhǎng)江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R587.2;R698
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