Study On Molecular And Cellular Pathogenesis Of Differential Biological Behavior Between Transition Zone And Peripheral Zone In Human Prostate

Benign prostatic hyperplasia (BPH) is a common disorder in aging males, which is characterized histologically by the enlargement of glandular nodules in the transition zone(TZ), and never happened in peripheral zone(PZ), central zone(CZ) of human prostate. Little is known why BPH only was located in TZ.The axis of the prostate ductal system may be divided into three regions. TZ correspondes to the proximal region in the prostatic ductal system, while PZ correspondes to the distal region. The morphology and the function of stroma and epithelial cells in the ductal system are different in different sections of the system. Cell death is not evident in the distal region, while in the proximal region, cells are actively undergoing apoptosis. It is established that epidermal growth factor(EGF, growth stimulate factor) is produced by fibroblasts, which are around in distal region , and transforming growth factor (TGF-β1 growth inhibitive factor) produced by stroma smooth muscle cells , which are located in proximal region in ductal system. Under the influence of these growth factors, stroma cells act on epithelial cells.The effect of androgen on the prostate is mediated through stroma cells, this process is called cell-to-cell interaction. In proximal region of ductal system, androgen can stimilate epithelial cells proliferation, while in proximal region androgen can also induce epithelial cells apoptosis by TGF-β₁.we can conclude that androgen is not a necessary stimulative factor for epithelial cells growth , and also not a necessary inhibitive factor for cells apoptosis. In the stage of prostate growth, stroma cells are the key point. If stroma cell phenotype was changed , the role of androgen and the biological behavior of epithelia cell would also changed . This may be the potential etiology of BPH or Pea.We have detected the expression of DHT in TZ, PZ of human normal prostateand BPH tissues in 2003, and found that there were no significant difference in the expression of DHT in these samples. This result was coincidenced to the study reported by other scholars. But EGFR was significantly increased in TZ than that in PZ, this result suggested that atroma cell may have occurred somewhat changes and attributed the alterations of regional zonal distribution of growth factors .But we did not know the specific process of this phenomenon. Under the enlighten of this observation, we further detect stroma cell phenotype and its change in peripheral transition zone of human normal prostate and benign prostatic hyperplasia, and compare their difference in phenotype in these different regions. We also investigate the changes in proliferation and apoptosis of the epithelial cells and regional distribution of growth stimulative/inhibitive factor. This will hope to give a rational interpretation about the reason why BPH only originate from TZ. we also search for differential gene expression between peripheral and transition zone in normal prostate, hope to defy new gene that specifically expressed in peripheral zone or in transition zone. This will attribute to understand the etiology of BPH or Pea, and provide a new target for molecular therapy on BPH and Pea.Objective:To investigate stroma cell phenotype and its change in peripheral transition zone of human normal prostate and benign prostatic hyperplasia, explain the mechanism of role of stroma -epithelial on the point of stroma cellular function . On this background, we further detect cellular alterations that include changes in proliferation and apoptosis in the epithelium ,zonal differential expression of growth stimulate factor and growth inhibitive factor , regional distribution of apoptotic modulation gene bcl-2.we also search for differential gene expression between peripheral and transition zone in normal prostate , clone and sequence differentially expressed sequence-tags(ESTs ), verify these ESTs on the lever of nucleic acid, defy new gene specifically expressed in peripheral zone or in transition zone. This will attribute to understand the etiology of BPH or Pea..Methods:17 normal prostate glands were obtained from adult donors (mean age 28.9) and sampled according to McNeal's zonal anatomy. 20 BPH samples were obtained from patients undergoing retropubic prostatectomy, nodules near the urethral area weredissected for experiment analysis. Transmission electron microscopy was used to observed cell ultrastructure, especial stroma cell in different zones of normal prostate and BPH, and compared cellular phenotype and its difference in these zones . Total RNA was isolated by two-step protocol. Reversal transcript-polymerase chain reaction (RT-PCR) was used to investigate semi-quantitatively the expression of EGF mRNA and TGF-(3i mRNA in different regions of normal prostate and BPH. EGF and TGF-(3i protein expression was also examined with Western blot. Cell proliferation and bcl-2 expression were assessed by immunostaining using PCNA and anti-bcl-2 antibodies, while apoptotic bodies were specifically stained using in situ nick translation, bcl-2 expression was also detected by RT-PCR. Gene expression between peripheral and transition zone of normal prostate was investigated with an improved mRNA differential display approach. Differentially expressed ESTs were inserted into PMD-18 vector and sequenced. Homology analysis with nucleotide BLAST technique was done. Then we designed corresponding primer according to acquired sequence of ESTs , verified reliability of these ESTs expressed in peripheral and transition zone by the technique of Real-time PCR.Results :1. Alterations of stroma cell phenotype were detected in different zones of normal prostate and BPH. Fibroblast served as functional active in transition zone, while degenerated in peripheral zone. Smooth muscle cell were actived in peripheral zone than that in transition zone. Fibroblast moved to ductal proximal regions in BPH was the significant character of BPH tissue. These phenomenon are different from that in normal prostate of ductal system in mouse, and would be attribute to result in proliferate in proximal epithelial cell and apoptosis in distal epithelial cell. The phenotype alteration of stroma cell should be regarded as a dynamic process.2. There were different regional expression of EGF and TGF-p\ between normal and enlarged prostate tissues. In transition zone, levels of EGF mRNA and protein were significantly higher than that in peripheral zone, while TGF-(3i showed no significant difference between these two regions. TGF-f-Siin transition was slightly higher with no statistical significance. In BPH, both EGF and TGF-(3iwere significantly elevated together than in any zone of normal prostate. These findings are coincidenced to the phenomenon of stroma cellular phenotype changes described upon.3. There also existed regional difference in modulation on epithelial cell proliferationand apoptosis. In normal epithelium, the rates of proliferation (13.7 + 4.32%) and apoptosis (14.6±4.34%) were decreased in the transition zone compared with the peripheral zone (20.9 + 6.44%, 25.6±6.35%) . Proliferation was significantly greater in BPH (29.2 + 4.81%) than in normal prostate, in contrast, there were fewer apoptotic cells in BPH(5.1±2.26%). Bcl-2 was expressed by glandular and basal cells in prostate. Bcl-2 was overexpressed in basal cells in transition zone, while in peripheral zone limited to dispersed basal cells. In BPH, bcl-2 was strongly expressed by epithelial cell. There was significantly negatively correlation with bcl-2-positive epithelial cells and the number of apoptotic cells in BPH (rs=0.867, PO.001) . However, bcl-2 was not seen in normal stroma or in BPH stroma. 4. Significant difference was observed between normal peripheral zone and transition zone in gene expression. Some genes only expressed in peripheral zone, some only in transition zone, and some both expressed in these two regions but manifested stronger or weaker. We selected 4 ESTs to clone and sequence, verified these ESTs expression in normal peripheral zone and transition zone through Real-time PCR technique. We got a new EST (513bp, named clone-D) specifically expressed in peripheral zone, shared 100% homology with BI430542 (Human endometrium mRNA), belonged to a function unknown gene. Whole sequence of EST clone-D and functional evaluation expressed in prostate would be further investigated.Conclusion :1. Stromal cellular phenotype were varied between normal peripheral, transition zone and enlarged BPH tissue. Function of fibroblast was active in transition zone, while smooth muscle cell actived in peripheral zone. Fibroblast moved to ductal proximal regions was the significant character of BPH tissue. Alterations in stroma would lead to changes in stromal-epithelial interactions and BPH progression.2. Cellular alterations that include changes in proliferation and apoptosis in the epithelium, zonal differential expression of growth stimulate factor and growth inhibitive factor, regional distribution of apoptotic modulation gene bcl-2 were implicated in normal peripheral transition zone and BPH pathogenesis .These all changes coincidenced to the phenomenon of stroma cellular phenotype changes.3. There are significant differently expressed genes between normal peripheral zone and transition zone. Through the techniques of clone, sequence and Real-time PCR ,we got a new EST (513bp, named clone-D) specifically expressed in peripheral...