| Background:Chronic prostatitis(CP) is a multifactorial problem, which affects men of all ages and in all demographics, and it is the main reason for the patients under 50 years old to go to hospital. Chronic prostatitis symptoms usually include pain, voiding and sexual dysfunction, even some patients usually experience psycological problems. Men with prostatitis tend to experience serious impairments in health-related quality of life (QOL). The QOL of patients with prostatitis is comparable with that of a patient suffering from recent myocardial infarction, unstable angina, or Crohn's disease. The morbidity of CP is about 10-14%, and more than 50% of the whole male suffered this disease in their life. A native population-based cross-sectional study reported by Liang-Zhaozhao indicated that 8.4% of them in china had symptoms of CP,4.5% of them were diagnosed as CP by NIH-CPSI and CP symptomatology. The classification of prostatitis is very complicated, and it was classify into 4 kinds by the traditional classification, they were (1) Acute bacterial prostatitis (ABP);(2) Chronic bacterial prostatitis (CBP);(3) Non-bacterial prostatitis (NBP); and (4) Prostatodynia (PD). This classification is based on the duration of the symptoms of CP and the serious of the symptoms of it. Though it was never validated but it was used by the urological doctors for some generations. In 1999, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)) proposed a new classification:(1) Acute bacterial prostatitis is an acute infection of the prostate(ABP); (2) Chronic bacterial prostatitis is a recurrent infection of the prostate(CBP); (3) Chronic nonbacterial prostatitis/chronic pelvic pain syndrome (CP/CPPS), where there is no demonstrable infection. Subgroup of this class are:(3.1) Inflammatory CPPS, where white cells are found in the semen, EPS, or VB3. (3.2) Non-inflammatory CPPS, where white cells are not found in the semen, EPS, or VB3. (4) Asymptomatic inflammatory prostatitis (AIP), where there are no subjective symptoms, but white blood cells are found in prostate secretions or in prostate tissue during an evaluation for other disorders.The constituent ratio of CP/CPPS is the highest one in the 4 kinds of prostatitis, about 90% percent of the whole patients with prostatitis belong to CP/CPPS, different from other 3 kinds, there is not evidence which can prove that there is bacteria in the prostatic tissue or EPS in CPPS patients by the traditional bacterial technology, and pain is the most common symptom of this kind of prostatitis. The early classification is based on this assumption that 1) the finding of bacteria in EPS, semen or the urine after prostate message is the reason of the symptoms of prostatitis and the abnormity of semen; 2)the exist of white blood cells in semen or EPS is the marker of the inflammation of prostate; 3)the inflammation or infection can be excluded if the white blood cells or bacteria can not be found in EPS or semen. But all above were not validated up to now, so the term "nonbacterial chronic prostatitis" and "Prostatodynia" were taken the place by the term "CPPS". The reason is whether there is inflammation in prostate tissue or not, the symptoms of the prostatitis were not influenced, and the prognosis and the curative effects of it were not influenced too. On the other hand, the new concept indicate that we know little about the etiology of chronic prostatitis, and maybe the symptoms of chronic prostatitis were involved more than one organ. The etiology of CPPS is not clear up to now, and many factors were thought to be related to its oncoming, include infection, injury, nerves factors, autoimmune, urine follow-metry, and psychological problems,et al.The rats or mice were injected with The purified prostate tissue protein combined with complete Freund's adjuvant(CFA) and diphtheria pertussis tetanus (DPT) vaccine at 0 and 30 days, The model of animal chronic prostatitis was made successfully in rodent animals. And the adoptive transfer of the lymphocytes suspended from the immuned mice spleens can also induce the prostate inflammation of mice and the obvious infiltration of inflammatory cells in the prostate can be found. It was proved that the main kind of lymphocytes infiltrate in the prostate was Th1 lymphocyte, and the mice immuned with this method can be found the similar symptoms of clinical prostatitis. Applying the rats model of autoimmune prostatitis to study the influence on the reproduction by this disease, the quality of semen of the rats was injured obviously. According to the types of cytokines secreted by the CD4+ T helper lymphocytes, it was divided into two kinds, include Thl and Th2 helper T cells. The main cytokines secreted by the former cells include IFNy and IL12, and the function of it was related with IFNy and IL12 to regulates the cellular immunity, wiping out the pathogen in the cells; the main cytokines secreted by the later T cells were IL4,IL5,IL13 et al, and the function of it was related with IL4, regulating the humoral immunity to wipe out the parasite. In recent years a new kind of assistant T lymphocyte was found which secret IL17 mainly, and it has many to do with lots of autoimmune diseases such as rheumatoid arthritis(RA), multiple sclerosis, systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis et al. IL17 is belong to pro-inflammatory cytokines and it can induce the secretion of IL6, NO, PGE2 so as to amplify the local inflammation; it can induce the recruiting response of neutrophile granulocyte by stimulating the secretion of colony stimulating factor and IL8; IL17 can promote the secretion of Tumor necrosis factor a(TNFa), Interleukin 1B(IL-1B), and they have synergistic reaction and thereby strengthen the inflammation reaction. IL23 is one kind of pro-inflammatory cytokine secreted by DCs and other antigen presenting cells, it was made of two subunits include p19 and p40 which is shared with Interleukin 12(IL12), and it is a important factor regulates the production of IL17. IL23 can promote the expression of Th17 cell, and has important funtion in the amplification of Th17 cells.We use the method introduced by papers to make the C57BL/6 mice model of experimental autoimmune prostatitis, and measure the expression of Th17 related cytokines include IL17, IL23p19 and IL23p40 and study the infiltration of Th17 lymphocytes in the prostate tissue by real time RT-PCR and immunol histochemistry respectively as well.Part one The making of experimental autoimmune prostatitis(EAP) with C57BL/6 mouseMethods:40 C57BL/6 mice were used to make the model of autoimmune prostatitis. They were divided into 2 groups randomly, each group had 20 mice. One group was as EAP group and the other was as control group. The prostates of Wistar rats were used to make purified extract, which would be used as antigen. The concentration of the extract was diluted to 2mg/ml. after the mouse were anesthetized by chloral hydrate by intraperitoneal injection,0.5ml prostate extract was injected subcutaneously as the same time each mouse was injected with CFA intra-peritonealy (0.5ml) combined with DPT vaccine(0.1ml),30 days after the first injection, the second injection was performed.60 days after the first injection, the mice were operated by open surgery to harvest the prostates. And the EAP group and the control group was divided in to two groups individually randomly, in the other words, the EAP group was divided into 2 groups and as the same time the control group was divided into 2 groups too, one group has 8 mice and the other group has 12 mice. The group of 12 mice was used to do the study of the CD4+,CD8+ and IL17+ lymphocytes infiltration in the prostate tissue, and the other group of 8 mice was used to study the expression of Thl7cell related cytokines in the prostate tissue.The mice were anesthetized by chloral hydrate, the abdominal wall of the mice were opened and the prostate were harvest. The prostate which would be used to check the cytokines were divided in to two parts, one part was about 1/4 volume of the whole prostate was fixed into 4% paraformldehyde for about 24 hours for the use to observe the infiltration of inflammatory corpuscle in the prostate tissue, and the other part was snapped in to the liquid nitrogen and then shift to-80℃low temperature refrigerator so as to be used to do the RT-PCR.The prostates of the other 12 mice whose prostates would be used to do immunol histochemistry were fixed into paraformldehyde for about 24 hours and then shift to the-80℃low temperature refrigerator for the use of immuno histochemistry later.Results:1 The observation of the gross appearance of the prostate60 days after the first immune injection, the prostates of the C57BL/6 mice were harvest. In the operation we could find a serious adhension between the prostate and the adjacent tissues and organs, include the pelvic wall and the rectum in the EAP group, the prostate was congestion with blood and has obvious oedema, and the operation was difficult to isolate the prostate.but in the control group there was not adhenssion and it was easily to isolate the prostate.The weight of the mice body of the control group was 22.7～26.1g, and the mean weight of it was 25.0±2.2g; the weight of the mice body of the EAP group was 21.7～28.3g, and the mean weight was 23.7±1.9g, t=1.6, p>0.05, and the differentce between the two groups has not statistics significance.The weight of the prostate of the control group was 23.2～33.5mg, and the mean weight was 28.5±3.9 mg; the weight of the prostate of the EAP group was 26.1～42.3mg, and the mean weight was 32.7±5.4mg, and the difference between the two group has not statistics too(t=-1.5, p>0.05).The index of the prostate of the two groups were 0.0011±0.0002(0.0008～0.0014)and 0.0014±0.0001 (0.0012～0.0018) respectively and the difference was statistics (t=-2.6, p<0.05).2 The histopathologyThe frozen section was made with 5μm. And the histo pathology was observed by microscope. In the EAP group the inflammation was founded and there were a lot of infiltration of inflammation cells in the proastate, the cells infiltrate in the prostate was lymphocytes, granulocyte cells and plasmacytoid lymphocyte.The most seriously places infiltrated with inflammation cells was in the interstitial, and the surrounding of the vessils. It could be found oedema, dillation of the gland, and the destruction in the gland.On the other hand, there was not infiltration of inflammation cells in the control group.Part 2 The study of the infiltration of Thl7 cells in the prostate tissueMethods:The prostate stored in the low temperature ice box was made frozen section in 5μm, mounted on the slide glass, dry by airing, fixed in 4% paraformaldehyde for about 30 minutes, according to the instruction of SABC kit to do the immuno histochemistry test. The diagnosis of the result was according to the stain of the cells. if the membrane and the plasm was stained brown madder it was looked as possive.Five high power field was selected and count the number of possive cells in each field, then make the average number as the result of each cells.Results:The result of immuno histochemistry test:The number of CD4 possive cells was 1.0～4.4 in each high field and the average was 2.4±1.1 in the control group; The number of CD4 possive cells was 5.6～12.7 in each high field and the average was 9.1±2.1 in the EAP group; there is statistic significance between the two groups (t=9.7,p<0.05).The number of CD8 possive cells in the control group was 0.2-1.4 each high field and the average was 0.79±0.32; The number of CD8 possive cells was 2.7～6.8 in each high field and average was 4.82±1.31 in the EAP group; there is statistic significance between the two groups (t=10.35,p<0.05).The number of IL17 possive cells in the control group was 0.3～5.1 in each high field and the average was 2.28±1.77 in the control group; The number of IL17 possive cells was 3.2～8.2 in each high field and the average number of IL17 positive cells was 5.67±1.76 in the EAP group; there is statistic significance between the two groups (t=4.70,p<0.05).Part 3 The study of the expression of the Th17 cell related cytokines in the prostate tissueMethods:About 20mg tissue of the prostate was used to extract RNA. The method was according to the illustrate of the kit.The concentration of the RNA was 118ng/μl, and the rate of purity was 1.95, it was fit for the RT-PCR.One step real time RT-PCR was used to check the mRNA expression of Th17 lymphocytes related cytokines including IL17 and IL23 (p19/p40) in the protate tissue of EAP mice, and compared the difference of the cytokines between the EAP and control group.2"-CT was used to express the content of mRNA of each cytokine.Results: The mRNA relative amount of IL17, IL23p19 and IL23p40 in the mice prostate tissue of control and EAP group were 0.0036±0.0029 vs 0.0530±0.0265 (t=-4.5,p<0.05), 0.0051±0.0025 vs 0.0177±0.0065 (t=-4.5,p<0.05),0.0041±0.0028 vs 0.0289±0.0283 (t=-2.1,p<0.05) respectively, and the difference between the two groups were statistically.Conclussion:The EAP model was made successfully by immuned with prostate extract.1) The expression of Th17 cell related cytokines raised and the infiltration of CD4+,CD8+ and IL17 positive cells was increased, we can conclude that the Th17 cell was related with the ongoing of the EAP prostatitis.2)The therapeutic method related with Th17 cells would benefit to the treatment of CPPS.
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