【作者】 潘恩山；

【导师】 郑泽棠；

【作者基本信息】 广州中医药大学 ， 中医外科， 2008， 博士

【摘要】 一、研究目的良性前列腺增生(benign prostatic hyperplasia,BPH)是老年男性的常见病、多发病。随着老龄人口日趋增多,良性前列腺增生的发病率不断攀升,严重影响着老年男性的身心健康和生存质量。BPH的发病机理目前尚未完全明了,研究资料显示BPH是因为尿道周围前列腺基质和腺体增生所致。从细胞凋亡的角度来看,由于细胞过度增殖或者凋亡障碍(程序性细胞死亡),导致细胞净增加有关,因此细胞凋亡学说越来越受到重视,并认为凋亡减少和细胞增殖参与了BPH的发生,所以维持前列腺组织内环境的稳定及干预BPH的进展是药物治疗的一个重要目标。本论文分别在临床以及动物实验两方面,通过免疫组织化学的方法,探讨中成药益肾通胶囊对良性前列腺增生的作用机制与环节,以及其对人以及大鼠前列腺组织细胞增殖与细胞凋亡状态的影响,为中医中药治疗良性前列腺增生提供科学的依据。二、研究内容(一)临床研究1.资料与方法按随机、对照的原则,将符合纳入标准的受试对象按顺序编号,按1:1的比例,根据治疗用药的情况分为益肾通胶囊治疗组、非那雄胺对照组。治疗组给予口服益肾通胶囊共3个疗程;对照组给予口服非那雄胺共3个疗程。3个疗程后,具手术指征的患者接受手术治疗,术后取得患者的增生的前列腺病理组织。为对照需要,加入正常前列腺组。应用免疫组化方法检测益肾通胶囊治疗组、非那雄胺对照组以及正常前列腺组,通过观察其中的Ki-67/cD34、bcl-2/bax这些指标表达的强弱,来判断前列腺组织细胞凋亡的情况,探讨益肾通胶囊对人增生前列腺组织细胞凋亡的影响。2.结果(1)增生前列腺组织的bcl-2蛋白表达阳性率显著高于正常前列腺组织(P＜0.01),提示bcl-2蛋白的过表达而导致细胞凋亡的减少在BPH的发生过程中起着重要作用。bcl-2在BPH中表达较正常前列腺增强,而细胞凋亡正好相反,这与以前报道一致,因此提示在前列腺中bcl-2蛋白表达与细胞凋亡呈负相关。(2)正常前列腺组织上皮细胞和间质中均有bax蛋白表达,以上皮细胞表达明显。虽然增生前列腺组织的bax蛋白表达阳性率要比正常前列腺组织低,但无统计学意义(P＞0.05),提示bax在BPH的发生、发展过程中所起作用并不显著。(3)bcl-2蛋白在治疗组、对照组两组表达阳性率无显著差异(P＞0.05),bax蛋白在治疗组、对照组两组表达阳性率无显著性差异(P＞0.05)。提示益肾通胶囊治疗良性前列腺增生,机理是促进增生前列腺细胞凋亡,从而达到减小前列腺体积,缓解前列腺增生的压迫以及刺激症状等治疗效果,能够达到与非那雄胺相似的治疗效果。(4)益肾通胶囊和非那雄胺均可使Ki-67指数减少,CD34表达含量减少的作用,提示临床上可通过抑制前列腺组织新生血管产生而达到治疗BPH的目的,而益肾通胶囊和非那雄胺的作用机制均可能与此有关。(二)动物实验研究1.资料与方法将75只雄性SD实验大鼠随机分为正常对照组、增生组、益肾通胶囊高剂量组、益肾通胶囊低剂量组、非那雄胺对照组5组。成功建立前列腺增生大鼠模型后,除正常对照组,给予其他不同组大鼠分别以生理盐水、益肾通胶囊高剂量、益肾通胶囊低剂量以及非那雄胺灌胃。完成灌胃后,处死所有的大鼠并取得其前列腺组织,通过免疫组化的方法测定bcl-2/bax、Ki-67表达的强弱,评价益肾通胶囊高、低剂量对大鼠前列腺细胞凋亡的影响。2.结果(1)与增生组比较,益肾通胶囊高、低剂量组及非那雄胺组的前列腺湿重、体积及指数明显低于增生组(P＜0.05或P＜0.01);与非那雄胺对照组相比较,益肾通胶囊高、低剂量组的前列腺湿重、体积及指数虽然高于非那雄胺组,但差别无显著性(P＞0.05)。结果证明益肾通胶囊高、低剂量的效果与非那雄胺相似,能够降低实验性大鼠的前列腺湿重、体积、指数。(2)通过对各组大鼠前列腺组织的HE染色的观察,结果证明益肾通胶囊高、低剂量均能够改善大鼠前列腺组织病理增生改变。(3)与正常组比较,益肾通胶囊高、低剂量组、增生组bcl-2蛋白表达明显高于正常组,差别有显著性差异(P＜0.01);与增生组比较,益肾通胶囊高、低剂量组及非那雄胺组的bcl-2蛋白表达均低于增生组,比较有明显差异性(P＜0.05);与非那雄胺组相比较,益肾通胶囊高、低剂量组的bcl-2蛋白表达与非那雄胺组无显著性差异。而bax蛋白在正常组、增生组、益肾通高、益肾通低组、非那雄胺组各组阳性表达率无显著性差异,(均P＞0.05)。提示益肾通胶囊能够调节大鼠前列腺组织bcl-2及bax表达的平衡,促进前列腺细胞凋亡。(4)正常对照组前列腺组织中Ki-67无表达。与正常组比较,增生组、益肾通胶囊高低组、非那雄胺组的Ki-67表达显著高于正常组(P＜0.01),差异有显著性。与增生组比较,益肾通胶囊高、低组和非那雄胺组Ki-67表达显著下降(P＜0.05或P＜0.01);与非那雄胺组比较,益肾通胶囊高、低剂量组Ki-67表达均高于非那雄胺组,其中益肾通胶囊低剂量组Ki-67表达升高有统计学意义(P＜0.05),益肾通胶囊高剂量组Ki-67升高无统计学意义(P＞0.05);益肾通胶囊高剂量组与益肾通胶囊低剂量相比较无显著性差异(P＞0.05)。证明益肾通胶囊高、低剂量均能够降低大鼠前列腺组织Ki-67蛋白的表达,抑制前列腺细胞增殖、促进细胞凋亡。三结论1.本病虚实夹杂,本虚而标实,肾气虚衰是其根本原因,而血瘀是其主要的病理环节,并贯穿BPH发展的始终,故补益肾气,活血化瘀类中药在药物,在治疗日渐增多的BPH上,愈来愈显示了其重要作用。2.根据临床研究及以往试验研究结果,本课题的机理研究从细胞凋亡着手。益肾通胶囊对BPH的治疗作用是多环节、多靶点的,其能够降低前列腺湿重、体积、指数;能够改善前列腺组织病理增生性改变;能够调节前列腺组织bcl-2及bax表达平衡,促进前列腺细胞凋亡;能够降低前列腺组织Ki-67蛋白的表达,抑制前列腺细胞增殖。3.益肾通胶囊能够促进增生前列腺细胞凋亡,减少组织新生血管产生,降低细胞增殖活性,从而防止前列腺增生的发生,其作用机理可能是综合性的,是一种安全有效的中药品种,具有较大的应用前景。以上研究只是部分和初步的,有待今后进一步探讨。更多还原

【Abstract】 Objective:Benign prostatic hyperplasia(BPH)is the common,frequently occurring disease of aging male.While the aging population increasing gradually,the incidence rate of BPH is ascending too,BPH has already affect their health and quality of life.It is uncertain about the etiopathogenesis of BPH,according to the data,it indicates that the reason results in hyperplasy of prostate interstitial substance around the urethra and glandular organ.As far as apoptosis concerned,because of over-hyperplasy or apoptosis disorder(programmed cell death),leads to the hyperplasy of cell,so,the theory of apoptosis has been paid more and more attention,it is considered that the apoptosis reduction and the cell multiplication participate the generative course of BPH,so how to maintain the stabilization of prostatic internal environment and interfere the progression of BPH,becomes the important aim of drug treatment.The research includes both the clinic and the experiment research,through the method of immunohistochemistry,probes the mechanism and the connection of traditional Chinese medicine YinShenTong capsule(YST capsule)in curing BPH,as well as the effection in the cell multiplication and apotosis of human and rat,so as to provide the data for treating the BPH with traditional Chinese method.Content1.Clinical resarch(1)Material and methodFollowed the principle of randomization,comparison,according to 1:1 proportion,numbers the internalized standard target by order and divided them into the YiSTtreatment capsule group(The YST capsule group)and the finasteride control group(the finasteride group).The YST capsule group was taking orally with YST capsules for 3 courses of treatment,and the the finasteride group was taking orally with finasteride tablet for 3 courses of treatment.After 3 courses of treatmet,operation of BPH treatment were accepted and the patients prostate tissue taken out.for the sake of comparison,an extra normal group was added up just for comparison.By means of immunhistochmisty,determine the expression of bcl-2/bax,Ki-67/CD34 in the 3 groups.Through the expression,observed the information of prostatic apoptosis,and evaluated the influence of YST capsule to human prostatic apoptosis.(2)Result①The bcl-2 protein expression of prostate hyperplasia tissue is obvious higher than the normal prostate tissue(P＜0.01),It indexed that the over-expression of bcl-2 protein induced the apoptosis to decrease,and played an important part during the developing process of BPH.bcl-2 protein expression of prostatic hyperplasia tissue is obvious higher than the normal prostate tissue,but the apoptosis is on the contrary,the result is accord to the report before,so there are inverse correlation of the bcl-2 protein expression and the apoptosis②The bax protein expression appeared in both protstate endothelial cell and interstitium,especially expressed obviously in endothelial cell.Though the bax protein expression was lower in hyperplasia prostate tissue than in the normal prostate tissue,but there was no statistical significance(P＞0.05), it meant that bax protein expression played an unimportant part in the generative、developing procession of BPH.③Bcl-2 protein expression was no obvious difference in both treatment group and control group(P＞0.05),the positive expression rate of bax protein makes no obviously different in both 2 groups(P＞0.05),It proved that the mechanism of curing BPH with YST capsule was promoting prostate hyperplasia apoptosis, so as to diminish the volume of prostate,relieve the oppressive and irritative symptom of BPH,the therapeutic efficacy was similar to finasteride tablet.④Both the YST capsule and the finasteride tablet could lower the expression of Ki-67 and CD34,it proved to be restraining the new vessel of prostate as to cure BPH,and believe that was the mechanism of YST capsule and the finasteride tablet. 2.experiment research(1)Material and method75 Sprague-Dawley male rats were randomly divided into 5 groups.Included normal group,hyperplasia group,YST capsule large-dosage group,YST capsule low-dosage group,finasteride group.When experiment models were finished, except the normal group,the rest of groups were given intragastric administration with different drugs.The rats were killed and prostate were removed after intragastric administration for 30 days.By means of immunhistochmisty,determine the expression of bcl-2/bax、Ki-67,to evaluate the influence of YST capsule to rat prostatic apoptosis.(2)Results①Compared with hyperplasia group,the weight、volume and index of prostate in YST capsule large-dosage group,YST capsule low-dosage group,finasteride group were obviously less(P＜0.05 or P＜0.01);Compared with finasteride group, although the weight、volume and index of prostate in YST capsule large-dosage group,YST capsule low-dosage group werehigher,but there were no obviously difference.The research proved to that the effect of YST capsule large-dosage, YST capsule low-dosage is similar to finasteride group,also could lower the weigh、volume、index of rats prostates.②Through the observation of rats prostate tissue HE corlored,it proved that the YST capsule large-dosage,YST capsule low-dosage could improve the tissue pathohyperplasia of rats prostates.③Compared with normal group,the bacl-2 protein expression of YST capsule large-dosage group and YST capsule low-dosage group are much higher,there are significant different(P＜0.01).Compared with hyperplasia group,the bacl-2 protein expression of YST capsule large-dosage group、YST capsule low-dosage group and finasteride group is lower,it make obviously different (P＜0.05);the bacl-2 protein expression makes no obviously different(P＞0.05) of the YST capsule large-dosage group and YST capsule low-dosage group to be compared with finasteride group;the positive expression rate of bax protein makes no significant different among the 5 groups.It proved that the YST capsule can balance the expression of bcl-2 and bax protein and promoted the prostatic apoptosis.④Ki-67 protein did not express in the prostate tissue of normal group. Compared with normal group,the expression of Ki-67 protein in the hyperplasia group、the YST capsule large-dosage group、YST capsule low-dosage group and the finasteride group was higher(P＜0.01),there was significant difference. Compared with the hyperplasia group,the expression of Ki-67 protein in YST capsule large-dosage group、YST capsule low-dosage group was lower obviously (P＜0.05 or P＜0.01).Compared with the finasteride group,the expression of Ki-67 protein in YST capsule large-dosage group and YST capsule low-dosage group was high,among them,there was statistical significance that the expression of Ki-67 protein increased in YST capsule low-dosage group(P＜0.05). there was no statistical significance that the expression of Ki-67 protein increased in YST capsule large-dosage group(P＞0.05).it was no significant difference between YST capsule large-dosage and YST capsule low-dosage,it was proved that both of them can lower the expression of Ki-67 of rat prostate tissue,restrain the prostate cell multiplication、promote prostatic apoptosis.Conclution①the pathogenesis of this diseases is intermingled deficiency and excess. root cause is deficient and the manifestation is excess,deficiency of kidney qi is the main reason,and the blood stasis is the main part of Pathological Mechanism,and survives all through the process of BPH,so,the Chinese medicine which invigorate kidney qi and promote blood circulation for removing blood stasis,is more and more used in curing BPH,and show their important effect.②The research are engaged in the method of apoptosis according the former clinic and experience research result.It is poly-link and multitarget for the YST capsule to cure BPH,it can lower the weigh,volume、index of rats prostates;it can improve the tissue pathohyperplasia of rats prostates; It can balance the expression of bcl-2 and bax protein;it can lower the expression of Ki-67 of rat prostate tissue,restrain the prostate cell multiplication,promote prostatic apoptosis.③It proves that the mechanism of curing BPH with YST capsule is promoting prostate hyperplasia apoptosis,restraining the new vessel of prostate, lowering the cell activity of cell multiplication,so as to prevent the BPH from happening,and its mechanism of action is synthetic.It is a variety of safe and effective Chinese medicine,and get a great application prospect. Butall the research above is partly and preliminary,it is expected to be deeply approached in the future.更多还原