| Background and ObjectivesType?prostatitis (CPPS) is the most common Chronic prostatitis,with uncertain etiological factor and extremely difficult treatment. Surveys and numerous hypothesis on the cause of the infection haven been proposed, yet the clinical characteristics of the disease could not be thoroughly elucidated. The existence of calcification foci and the significant therapeutic effect of antibiotic therapy indicate the possible existence of infective factor that could not be detected through routine examination methods.Calculus could be usually found in the prostate of patients with CPPS,and such prostatic calculus was a kind of true calculus formed in gland alveolus and glandular tube of prostate with uncertain factors for its formation including chronic inflammation of prostate gland alveolus and excretory duct, functions of repressive or promoting nucleating factor, concentration of calcium ion. It is worth exploring whether some unknown infective etiological factor was conductive to the formation of prostatic calculus. The etiological factor of CPPS as well as prostatic calculus could be surveyed by a brand new means with the discovery of nanobacteria and its etiological characteristic of the bacteria.NB is a new micro-organisms and it can not grow in the general culture conditions, but grow in cell culture. Conventional methods can not detect it. With slow growth, insensitivity to the drug and resistance to strong acid and high temperature and other characteristics, it can infect a variety of cells of the human body and induce to tissue inflammation through the release of toxins, while led the infected cells to pathological calcification. Studies have demonstrated the occurrence of human diseases concerned with nanobacterial infection. Some scholars have found that CPPS patients NB detection in blood higher than normal men and patients with benign prostatic hyperplasia by ELISA; the patients with refractory CPPS after anti-NB treatment, the patients pain relief and inflammation was reduced. Therefore, we infer that the incidence of NB may be closely related with CPPS. To confirm our hypothesis, this study intends to isolation, cultivation NB from the prostatic calculi and EPS of the patients with CPPS, which further confirmed the existence of NB and infections; followed by NB infected animals using cultured prostate, in which the pathology observed changes, verify NB occurred in CPPS and may be the role of the etiology; Finally, the research on the pathogenesis of NB with CPPS in the level of genes will provide a new testing approach for the clinical diagnosis of CPPS relevant to nanobacteria and lay a foundation to studying the genetic nanobacteria background and pathogenesis.Methods1. In this study, we isolate NB from the EPS and prostate stones of the CPPS patients and identify cultures by indirect immunofluorescence, electron microscopy and calcium staining methods.2. Selected the adult male SD rats as experimental subjects to establish the animal models. In model group, the SD rats were injected NB at the department of the prostate urethra through retrograde urethral, resulting in rat prostate infection. The control group were injected with saline. At 1 week, 2 weeks, 4 weeks and 8 weeks SD rats in control group and model group were killed, observed the pathological changes of prostate tissue. At the same time, detected the number of leukocytes and related cytokines (IL-1?, TNF-?, IL-6) changes. At the same time, the prostate tissue of all the rats were re-isolated NB to observe whether the NB growth.3. By two-dimensional electrophoresis to look at the differences between cultures NB protein, to obtain the amino acid sequence homology, according to homologous protein sequences to design PCR primers for the amplification of DNA fragments of nanobacteria and sequenced, the sequencing results with the NCBI database for comparison, the initial identification of the target gene. At the same time, specific gene amplification for the clinical diagnosis of various types prostatitis patients. And the results of genetic diagnosis were compared to the results of culture and identification of nanobacteria to further validate the accuracy of gene diagnosis.Results 1.The results of culture and identification of nanobacteria?EPS from the CPPS patients and cultured stone samples isolated after 3-4 weeks, observed that part of the culture tube wall close to the bottom of the growth of white precipitate.?Indirect immunofluorescence antibody identification of NB, CPPS group of 48 patients with EPS was detected in 26 cases of infection exist in NB, the positive rate was 54.2%; 40 cases of normal control group, only 2 cases of EPS specimens exist NB infection The positive rate of 5%. There was significant difference between the two groups(P<0.05). Stone group were 20 cases in 12 cases there were found to have NB infection, the positive rate was 60%.?Transmission electron microscope, we can see NB sizes, between about 200 ~ 500nm, the cell is surrounded by a layer of dark material surrounding, and its thickness is about 10nm or so, the distribution around hydroxyapatite.?Scanning electron microscope, nanobacteria in irregular shape, most of the approximately round or oval diameter between about 150 ~ 600nm; spectrum analysis, the mineralization of nanobacteria shell containing mainly calcium, phosphorus and other elements, the calcium Phosphorus ratio of 1.54, the results of spectrum analysis similar to the results of prostate stones.?Alizarin red staining results: the oil microscope appear orange-red stained calcium particles, most of the orange-red granules densely clustered, individual extremely small, covered the entire field of vision.2. The results of production of nanobacteria induced CPPS model?At the 1 week, 2 weeks, 4 weeks and 8 weeks after NB infection, the number of tissue leukocytes and related cytokines (TNF-?, IL-1?, IL-6) of the model group were significantly different compared with the control group.?Between 1 to 2 weeks after NB infection, the model group showed acute inflammatory changes in rat prostate, tissue congestion, edema, light microscope, interstitial edema, vasodilatation, inflammatory cell infiltration; Between 4 weeks to 8 weeks, SD rat prostate was chronic inflammation and glandular atrophy hardens, the surface points nodular gray, light microscope, luminal narrowing or even closure, interstitial fibrosis was significantly, a large number of lymphocyte infiltration. ?In model rats mitochondria and lysosomes were swollen.Electron microscopy revealed many globoid or racket-shaped particles in cytoplasm. Particle cross-sections were round or oval, suggesting that the particles were nanobacteria.There were transparent, vacuolar changesaround the particles. Under scanning electron microscopy particulate, globoid nanobacteria were observed on the prostate epithelial cell surface and there were micro-calculi in the cells. Particulate or globoid nanobacteria clustered on cross-section.?Immunohistochemistry of prostate tissue: NB with the specific antibody can be seen in model group after the prostate epithelial cells around the glandular lumen and the stromal cells around the brown particles distribution. Control group, no such performance.?Cultured the prostate tissue of model group animal and found 35 cases of prostate NB culture positive specimens, but no one case in control group, there were significant differences between the two groups.3. The results of the role of nanobacteria in the pathogenesis of CPPS at the gene level?By two-dimensional electrophoresis and mass spectrometry analysis revealed a difference of nanobacteria protein, while access to its homologous amino acid sequence.?Obtained by PCR amplification of a new gene sequence, nucleic acid sequences with known sequences in the NCBI database were compared and found no similar results.?We extract EPS template DNA samples from the normal male,?,?,?patients with prostatitis, using specific primers for PCR amplification, from the results, we can find that only type?patients with prostatitis EPS template DNA samples amplified specific Fragment.?Samples of nanobacterial-positive and PCR amplified specific fragment are the same specimen.Conclusion and Prospect1. NB were present in EPS and prostatic calculi of CPPS Patients, and a higher positive rate.2. NB infection can cause inflammatory changes in rat prostate tissue.After 1-2 weeks, the model group were acute inflammatory changes and after 4 weeks showed a chronic inflammatory changes.3. NB rat prostate infection can lead to increases of the number of white blood cells, while the TNF-?, IL-1?, IL-6 levels and other inflammatory mediators. 4. Cultured the prostate tissue of model group animals and found NB culture positive, which confirmed the establish of animal models consistent with Koch's principles.5. Obtained a new gene sequences through PCR amplification, and only EPS samples of CPPS patients were amplified specific fragment of DNA template, which confirmed the specific gene amplification can be used for clinical diagnosis of CPPS caused by NB.6. Samples of nanobacteria-positive and PCR amplified specific fragment are the same specimen, which further confirmed the accuracy of the PCR amplified the specific gene fragment to diagnosis of CPPS caused by NB.7. NB infection and the occurrence and development of CPPS may exist a close relationship.8. Gene level results provide a new means to detection the the clinical diagnosis of CPPS caused by NB, as well as lay a foundation to study the genetic background of nanobacteria and pathogenic mechanism. |
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