不同年龄段人前列腺外周带基质细胞表型特征的研究

目的:探讨体外培养的不同年龄段人前列腺外周带基质细胞的表型特征和生长特性差异,分析前列腺基质细胞微环境对老年男性前列腺癌促发的可能原因。方法:原代培养不同年龄段前列腺外周带基质细胞,利用免疫细胞化学技术分析细胞表型特征、透射电镜观察细胞超微结构、琥珀酸脱氢酶法观察细胞体外增殖以及流式细胞术检测细胞凋亡情况。结果:不同年龄段前列腺基质细胞均表达成纤维细胞标记物prolyl-4-hydroxy-lase,而平滑肌肌动蛋白(α-SMA)、肌间线蛋白(desmin)的表达随年龄逐渐增加,即α-SMA的阳性表达:年轻组vs老年组=(2.56±1.81)%vs(38.89±11.22)%,(P<0.01);而desmin阳性表达:年轻组vs老年组=(0.89±0.93)%vs(14.89±5.97)%,(P<0.01)。并且α-SMA和/或desmin阳性细胞形态相对宽大、扁平、多形性。超微结构观察发现,前列腺基质细胞内蛋白合成有关的细胞器随着年龄的增加不断增多,主要为粗面内质网和高尔基复合体。老年组前列腺基质细胞的增殖率低于年轻组(P<0.01);而两组基质细胞凋亡率均在1%～3%之间,组间差异无统计学意义。结论:前列腺外周带基质细胞中具有活性的肌成纤维细胞随年龄不断增加可能是老年男性前列腺癌高发和恶性进展的原因之一。     

Pygeum africanum extract inhibits proliferation of human cultured prostatic fibroblasts and myofibroblasts

OBJECTIVE: To investigate the effect of Pygeum africanum (PA) extract on the proliferation of cultured human prostatic myofibroblasts and fibroblasts; this extract is used for treating urinary disorders associated with benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: Primary cultures of prostatic stromal cells were obtained from histologically confirmed human BPH by enzymatic digestion. Cell proliferation was measured by 5-bromo2'-deoxy-uridine (BrdU) incorporation assays, and cytotoxicity by luminescent quantification of adenylate kinase activity. RESULTS: Cultured cells were labelled by an anti-vimentin antibody, and most of them by an alpha-smooth-muscle-actin antibody, revealing the presence of fibroblasts and myofibroblasts. BrdU incorporation tests showed that proliferation of cultured human stromal cells, stimulated by fetal calf serum, by basic fibroblast growth factor and by epidermal growth factor, was dose-dependently inhibited by PA extract (5-100 microg/mL). Except at 100 microg/mL, no acute cytotoxicity of the extract was detected after 24 h of culture. Similarly, the extract dose-dependently inhibited the proliferation of Madin-Darby canine kidney epithelial cells, but to a lesser extent; whatever the dose of extract, no acute toxicity was evident on this cell line. CONCLUSION: PA extract inhibits the proliferation of cultured human prostatic myofibroblasts and fibroblasts. We propose that cultured human prostatic cells offer a reliable model for preclinical screening of therapeutic agents, and to study the mechanisms underlying the inhibition of proliferation.     

正常前列腺不同区带及癌组织来源原代基质细胞的生物学特性及其对前列腺癌细胞株C4-2B的作用

目的 比较正常前列腺外周带来源原代基质细胞(NPPF),移行带来源原代基质细胞(NPTF)及前列腺癌组织来源原代基质细胞(CAF)的生物学特性差异,以及它们对前列腺癌细胞株C4-2B的不同影响.方法 苏木素-伊红(HE)染色鉴定前列腺不同区带及癌组织的组织学特征差异;原代培养NPPF、NPTF、CAF,免疫细胞化学染色观察其波形蛋白(Vimentin)、平滑肌动蛋白(SMA)及前列腺特异性抗原(PSA)的表达,生长曲线比较其增殖能力,流式细胞仪检测比较其凋亡率,透射电镜观察比较其超微结构;建立不同来源原代基质细胞与C4-2B细胞株共培养系统,比较不同来源基质细胞对C4-2B细胞增殖(MTT)和凋亡(FCM)方面的影响.结果 NPPF、NPTF、CAF的生长、凋亡、超微结构及对C4-2B细胞的生物学影响存在明显差异,其生长速度依次递增,凋亡率分别为(9.25±2.24)%、(5.98±0.74)%、(2.63±0.96)%,透射电镜提示CAF蛋白质合成最旺盛;C4-2B细胞与基质细胞体外共培养后出现增殖加速,凋亡减少,MTT结果显示共培养2 d后吸光度分别0.540、0.471、0.632,共培养4 d后吸光度分别为0.554、0.488、0.670,P均<0.05;FCM结果显示与对照组(10.32±0.43)%比较,CAF对C4-2B细胞凋亡的抑制能力最强(3.36±0.17)%,其次是NPPF(5.97±0.70)%和NPTF(8.01±0.22)%,P<0.05.结论 NPPF、NPTF、CAF的生物学特性存在明显差异,这种差异可能是导致前列腺增生或前列腺癌发生发展的重要原因.     

Transrectal hyperthermia-induced histological and ultrastructural changes of human benign prostatic hyperplasia tissue.

This prospective study evaluated the tissutal, cellular and intracellular effects of transrectal microwave hyperthermia on human benign prostatic hyperplasia. Forty-eight patients with benign prostatic hyperplasia underwent ten 60-min-long sessions of transrectal hyperthermia with an intraprostatic calculated temperature of 42 +/- 0.5 degrees C. Ultrasound-guided transperineal biopsies of the prostate were taken before and 1 month after completion of treatment. Postoperatively, morphometric analysis of bioptic specimens showed a statistically significant (p < 0.01) increase in the number of intraprostatic arterioles and capillary-like vessels. Diffused inflammatory infiltrates were also noted. Postoperative integrity of intracellular organelles and cellular membranes was evidenced by transmission electron microscopy. Our regimen of transrectal prostatic hyperthermia did not cause any irreversible histological or ultrastructural damage to the prostatic tissue. Hyperthermia-induced increase in blood flow could enhance drug delivery to the prostate gland.     

Effects of cyclic stretch on prostatic cells in culture

PURPOSE: The fundamental process in the development of benign prostatic hyperplasia (BPH) is a loss of homeostasis between cell proliferation and apoptosis. Prostatic smooth muscle cells contract under adrenergic control. The response of a cell to stretch may have a role in the pathogenesis of BPH. MATERIALS AND METHODS: Monolayer cultures of human prostatic stromal and epithelial cell lines were exposed to cyclic stretch for 48 hours. RESULTS: Cyclic stretch conferred resistance to etoposide induced apoptosis. Underlying this apoptotic resistance was increased expression of the anti-apoptotic Bcl-2 family of proteins. As measured by thymidine incorporation, the rate of proliferation also increased in benign epithelial cells under cyclic stretch conditions. Furthermore, an increase in the production of platelet-derived growth factor by stromal cells and transforming growth factor-beta by epithelial cells occurred under such conditions. CONCLUSIONS: The observed changes in proliferation and apoptosis may contribute to the understanding of BPH, ultimately leading to therapeutic and preventive applications.     

Prostatic stromal cells derived from benign prostatic hyperplasia specimens possess stem cell like property

INTRODUCTION: The hyper-proliferative activity of stromal smooth muscle (SM) cells is believed to be responsible for the pathogenesis of benign prostatic hyperplasia (BPH). We have observed that those stromal cells can differentiate into unrelated specialized cells. We thus hypothesize that stromal cells derived from adults prostate specimens may contain adult stem cells. To test this hypothesis, human prostate stromal primary cultures were established and used for characterization of their stem cell properties. METHODS: Immunoblotting, immunohistochemistry, RT-PCR, and tissue culture techniques were used to characterize the primary cultured human prostate-derived stromal cells for their stem cell and differentiation properties. The plasticity of these stromal cells was analyzed using cell culture and histology techniques. RESULTS: Primary cultured prostate stromal cells from BPH patient possess polygonal and elongated fibroblast/myofibroblast cellular morphology. They are positive in CD30, CD34, CD44, NSE, CD133, Flt-1, stem cell factor (SCF), and neuron-specific enolase (NSE), but negative in C-Kit, stem cell antigen (SCA), SH2, CD11b. Expression of SM myogenic markers in these cells may be induced by sodium butyrate (NaBu) treatment. Induction to osteogenic and adipogenic differentiation in these cells is also evident. CONCLUSIONS: Our study on primary stromal cells from BPH patients have yielded many interesting findings that these prostate stroma cells possess: (1) mesenchymal stem cell (MSC) markers; (2) strong proliferative potential; and (3) ability to differentiate or transdifferentiate to myogenic, adipogenic, and osteogenic lineages. These cell preparations may serve as a potential tool for studies in prostate adult stem cell research and the regulation of benign prostatic hyperplasia.     

Secretion of prostatic specific antigen, proliferative activity and androgen response in epithelial-stromal co-cultures from human prostate carcinoma

We investigate the proliferative activity, prostatic specific antigen (PSA) secretion, morphology and androgen response of human prostate tumour epithelial cells co-cultured with stromal cells in a bicameral system. Stromal and epithelial cells were isolated from prostate adenocarcinoma by enzyme digestion and cultured in defined media. Immunocytochemistry for prostate carcinoma tumour antigen (PCTA-1) was performed for culture purity evaluation. Also, the morphology of the epithelial cells in co-culture was evaluated by electron microscopy. PSA was determined by microparticle enzyme immunoassay (MEIA) automatized protocol and the proliferation was evaluated by a commercial spectrophotometric kit, based on formazan salt formation. Both cell cultures showed more than 90% of purity. The epithelial cell co-cultures showed marked membrane processes and cell interdigitations. The proliferative activity of the epithelial cells was increased in presence of stromal cells. Also, PSA secretion was significantly increased and maintained for at least 14 days, whereas the androgen response for PSA secretion was evidenced only in co-culture condition. Primary co-cultures of epithelial and stromal cells from human prostate carcinoma are able to maintain, for a prolonged time, proliferative and secretory properties as well hormone response, and represent a valuable tool for cellular and molecular studies on prostate cancer.     

Role of endogenous transforming growth factor beta (TGFbeta)1 in prostatic stromal cells

BACKGROUND: In this study, defined culture conditions were used to examine the effects of recombinant TGFbeta1 on prostatic stromal cells and to determine the role of endogenous TGFbeta produced by these cells. METHODS: Cells were grown +/- recombinant TGFbeta1 and cell population sizes in replicate cultures determined. In other experiments, TGFbeta1 production by prostatic stromal cells was examined and the effects of neutralization of this activity on cell population sizes and apoptosis evaluated. RESULTS: At > 1 ng/ml, TGFbeta1 reduced cell population sizes while at 0.01 ng/ml cell numbers were increased cf. controls. Stromal cells produced up to 10 ng/ml/48 hr of latent TGFbeta1 of which < 0.2% was biologically active. When cells were treated with anti-TGFbeta1 antibodies, cell numbers decreased cf. controls and the proportion of apoptotic cells increased. CONCLUSIONS: These observations suggest that TGFbeta1 is an autocrine factor made by prostatic stromal cells in which it inhibits apoptosis at the activity levels produced.     

Apoptotic activity of doxazosin on prostate stroma in vitro is mediated through an autocrine expression of TGF-beta1

BACKGROUND: Doxazosin, an alpha-adrenergic antagonist, has been shown to induce apoptosis in prostatic stromal cells. The mechanism of this apoptotic action by Doxazosin remains undefined. The present study was carried out to demonstrate that the effect of Doxazosin on apoptosis of prostate stromal cells is mediated through an autocrine action of TGF-beta1. METHODS: Primary cultures of human prostate cells were treated with varying concentrations of Doxazosin (0, 0.1, 1, 10, and 100 microM) for a period up to 3 days. At the end of the 3-day culture, cell numbers were counted. Apoptosis was assessed by a colorimetric terminal deoxyribonucleotide transferase labeling technique. TGF-beta1 was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared to control cultures, cell numbers were significantly decreased as much as 68.4% in cultures treated with 10 microM of Doxazosin after 3 days incubation, while apoptosis increased by 64.7% in cultures treated with the same concentration of Doxazosin after 24 h. This decrease in cell number was reversed when antibody to TGF-beta1 was added to these cultures. Addition of TGF-beta1 (0, 1.0, and 10 ng/mL) to the cultures also decreased the cell numbers. Quantitation of TGF-beta1 in lysates of cells by ELISA revealed that the cells treated with Doxazosin (10 microM) produced as much as 62.5% more TGF-beta1 than in that of untreated cells. CONCLUSIONS: These results demonstrate that the apoptotic effect of Doxazosin on human prostatic stromal cells is mediated through an autocrine production of TGF-beta1.     

癃必消胶囊对体外培养的人前列腺增生间质细胞TGF-β1和Smoothelin基因表达的影响

目的:研究中药癃必消胶囊对体外培养的人前列腺增生间质细胞TGF-β1和Smoothelin基因表达的影响。方法:把含癃必消胶囊的药物血清加入到体外培养的人前列腺增生间质细胞中,采用实时定量RT-PCR检测TGF-β1和Smoothelin在体外培养的人前列腺增生间质细胞中的表达。结果:高、低中药浓度血清对TGF-β1的相对表达CT值分别为0.158±0.020、0.169±0.020,较对照组显著降低(P<0.01);高、低中药浓度血清对Smoothelin的相对表达值分别为0.035±0.007、0.036±0.007,较对照组显著降低(P<0.01)。结论:癃必消胶囊可能通过抑制前列腺间质细胞TGF-β1和Smoothelin基因的表达达到治疗前列腺增生的目的。     

Interleukin-8 expression is increased in senescent prostatic epithelial cells and promotes the development of benign prostatic hyperplasia

BACKGROUND: Benign prostatic hyperplasia (BPH) is an extremely common disease of older men characterized by increased growth of prostatic epithelial and stromal cells. Previously we showed that senescent epithelial cells accumulate in the prostate of aging men and secrete interleukin-1 alpha (IL-1 alpha). IL-8 is also present at increased levels in BPH tissues and induces expression of FGF2, a potent stromal growth factor. Therefore, we sought to determine if IL-8 is also expressed at increased levels by senescent epithelial cells and if this secreted IL-8 plays a role in the pathogenesis of BPH. METHODS: Expression of IL-8 in human BPH tissue and primary cultures of prostatic epithelial cells was analyzed using an enzyme-linked immunoabsorption assay (ELISA). Tissue senescence was assessed by a quantitative assay for senescence-associated beta galactosidase (SA-beta gal). Proliferation of primary and immortalized prostatic epithelial cells in response to IL-8 was determined by counting of cells at intervals after addition of IL-8. RESULTS: Expression of IL-8 is significantly increased in vitro when cultured prostatic epithelial cells undergo senescence. Quantitative assay of BPH tissue extracts revealed that tissue IL-8 levels are correlated with both SA-beta gal activity and prostate weight. IL-8 promotes proliferation of primary and immortalized prostatic epithelial cells in culture. CONCLUSIONS: Senescence of prostatic epithelial cells results in increased expression of IL-8, which can promote proliferation of non-senescent epithelial and stromal cells by direct and indirect mechanisms, and in this manner contributes to the increased tissue growth seen in BPH.     

Human prostatic smooth muscle cells in culture: Estradiol enhances expression of smooth muscle cell-specific markers

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB-131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL-5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 μM of estradiol to the growth medium dramatically increased expression of these SMC-specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle α-actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs. Prostate 30:117–129, 1997. © 1997 Wiley-Liss, Inc.     

bFGF和TGF-β1对原代培养的前列腺间质细胞的作用

目的:探讨碱性成纤维细胞生长因子(bFGF)和转化生长因子β1(TGF-β1)在良性前列腺增生(BPH)中的作用。方法:培养了人BPH间质细胞,采用MTT法检测无血清培养的间质细胞的增殖,用免疫组化方法检测平滑肌细胞表型变化,观察不同浓度bFGF和TGF-β1对培养的人BPH间质细胞的影响。结果:bFGF促进间质细胞增殖(P<0.05、P<0.01),较高浓度时(10μg/L)降低平滑肌细胞表型表达;TGF-β1(>0.1μg/L)抑制间质细胞增殖并增加平滑肌细胞表型表达(P<0.05、P<0.01);5μg/L的bFGF与0.001μg/L和0.01μg/L TGF-β1作用间质细胞,促进细胞增殖(P<0.01),与0.1μg/L,1μg/L及10μg/L TGF-β1作用间质细胞,抑制细胞增殖,0.1μg/L时对细胞的抑制作用轻微(P>0.05),1μg/L及10μg/L时出现明显的抑制(P<0.01),同时TGF-β1在较高浓度时(>1μg/L),平滑肌细胞表型表达明显增加(P<0.01)。结论:bFGF以时间和浓度依赖的方式促进培养的增生前列腺间质细胞的增殖,并减少平滑肌细胞表型表达;TGF-β1抑制间质细胞的生长并诱导间质细胞向平滑肌细胞分化,两者共同在BPH的形成机制中发挥着重要作用。     

Androgen and prostatic stroma

Aim: To investigate the effect of androgen on the proliferation, differentiation and regression of canine prostatic stromal cells in vivo and human stromal cells in vitro. Methods: Twenty-two dogs, including 15 normal prostate dogs and 7 prostatic hyperplasia dogs, had their serum concentration of testosterone and estrodiol determined by radioimmunoassay before and after castration. The expression of androgen receptor (AR) and estrogen receptor (ER) in the prostate were analysed by immunohistochemistry and semi-quantitative RT-PCR before and after castration. Light microscopy, transmission electron microscopy and TUNEL assay were carried out successively before and after castration to evaluate the prostatic histomorphology. In vitro serum-free cell cultures from human prostatic stroma were established and exposed to dihydrotestosterone (DHT). The proliferation of the cell culture was detected by MTT assay. The expression of TGFβ, bFGF, AR, and smooth muscle cell (SMC) specific proteins (myosin and/or sm     

Identification of apoptotic and antiangiogenic activities of terazosin in human prostate cancer and endothelial cells

PURPOSE: It has been suggested that terazosin has an inhibitory effect on prostate tumor growth. We determined if terazosin action contributes to direct suppression of the angiogenic effect. MATERIALS AND METHODS: PC-3 cells and primary cultures of human benign prostatic cells were used in this study. The cytotoxic effect was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and lactate dehydrogenase release reaction. The in vivo angiogenic effect was determined in nude mice models, followed by histological examination and quantification by the hemoglobin detection assay. In vitro determination of cell migration, proliferation and tube formation was performed in cultured human umbilical vein endothelial cells.RESULTS Terazosin induced cytotoxicity in PC-3 and human benign prostatic cells with an IC50 of more than 100 microM. The positive terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling and lactate dehydrogenase release reaction was associated with terazosin induced cytotoxicity, indicating apoptotic and necrotic cell death. Furthermore, cytotoxicity due to terazosin action was not a common characteristic of a quinazoline based structure. Terazosin significantly inhibited vascular endothelial growth factor induced angiogenesis in nude mice with an IC50 of 7.9 microM., showing that it had a more potent anti-angiogenic than cytotoxic effect. Terazosin also effectively inhibited vascular endothelial growth factor induced proliferation and tube formation in cultured human umbilical vein endothelial cells (IC50 9.9 and 6.8 microM., respectively). CONCLUSIONS: Together our data suggest that terazosin shows direct anti-angiogenic activity through the inhibition of proliferation and tube formation in endothelial cells. This action may partly explain the in vivo antitumor potential of terazosin.