Proliferation and differentiation of canine prostatic epithelial cells in culture

When cultured in monolayers, non-secretory epithelial cells from canine prostates actively synthesize DNA, RNA and proteins; subsequently, mitotic figures and an increase in cell number are observed. During this culture period, cell size and acid phosphatase activity remain constant. As the culture proceeds, these cells mature into secretory cells as evidenced by a gradual shift in their density in Percoll gradients to the density of secretory cells and by an increase in the cellular content of acid phosphatase. During maturation, the size of the non-secretory cells increases and their morphology changes and becomes similar to that of the secretory cells. When a homogeneous population of secretory cells is cultured, DNA synthesis is minimal and few mitotic figures may be observed while cell number and cell density remain constant. Early in the culture period, their size increases and by 2 weeks their acid phosphatase activity is 2--3-fold higher than that of the non-secretory cells. Thus, upon culture, the non-secretory epithelial cells enter and proceed through the cell cycle with evidence of DNA synthesis and mitosis. Those cells leaving the cycle undergo maturation into secretory cells which further differentiate with the concomitant appearance of acid phosphatase activity. This model will be useful to study prostatic hyperplasia and hypertrophy and the control mechanisms involved in these phenomena.     

Reciprocal epithelial: fibroblast interactions in the control of fetal and adult rat lung cells in culture

Intercellular communication between epithelial cells and fibroblasts of the alveolar wall has been postulated from studies of lung development and repair. We examined the epithelial cell-fibroblast interactions with respect to growth control and epithelial cell function using cultured fetal and adult lung cells. The role of diffusing factor(s) as compared to direct cell-to-cell contact was studied by culturing epithelial cells either on a permeable culture well insert over fibroblasts or in co-culture with fibroblasts. The results show that the normal low proliferative rate of epithelial cells in culture is increased when exposed to fibroblast supernatants. In contrast, epithelial cells (particularly from adult lung) secrete a factor that suppresses fibroblast growth when cultured with a filter between the cell types. However, when cell-cell contact occurs in co-culture, the growth rate of fibroblasts is greatly increased. Synthesis of disaturated phosphatidylcholine by epithelial cells is increased under serum-free conditions and further rises when fetal epithelial cells are exposed to steroid-treated fibroblasts, when the cell types are separated, and when cells are in contact. This indicates that a fibroblast-derived factor stimulates epithelial differentiation, and morphologic evidence relating the appearance of lamellar bodies to the areas of direct epithelial cell-fibroblast contact was found. The results indicate the complex interdependence of these two types of cell where a secretory product of one cell or direct cell-cell contact may alter when regulatory control of the other cell type. These interactions are likely to be important in orderly development and in the reparative response of the lung to injury.     

Suppressor role of androgen receptor in proliferation of prostate basal epithelial and progenitor cells

Early studies have reported the differential roles of androgen receptor (AR) in different types (luminal, basal intermediate, and stromal) of prostate cancer cells. In vivo mouse model tumor studies using the total prostate epithelial knockout mice (pes-ARKO) also revealed that AR played a suppressive role in proliferation of the CK5(+)/CK8(+) progenitor/intermediate cells but a positive role in the CK5(-)/CK8(+) luminal epithelial cells. Using three different resources (one human basal epithelial cell line, one mouse basal epithelial originated progenitor cell line, and a basal epithelium-specific ARKO mouse model), we here demonstrated that the AR in basal epithelial cells of normal prostate plays a suppressive role in their proliferation but a positive role in differentiation into luminal epithelial cells. These results led us to conclude that ARs may play a negative role to suppress CK5(+) basal epithelial and progenitor cell proliferation, yet play an essential role to drive basal epithelial cells into more differentiated states. These results may explain why differential AR expression in different cell types within normal prostate is needed and suggest that ARs in prostate basal epithelial cells, although expressed at a very low level, are necessary to maintain the balance between progenitor cells and differentiated luminal epithelial cells.     

Androgen-induced cell growth and c-myc expression in human non-transformed epithelial prostatic cells in primary culture.

We assessed androgen-induced cell growth and c-myc expression in human non-transformed epithelial prostatic (HNTEP) cells in primary culture. Prostatic tissue was obtained from 48 retropubic prostatectomy patients (age: 61-77years) with benign prostatic hyperplasia (malignant tumors excluded). HNTEP cells were treated with testosterone or DHT, alone or in association with hydroxyflutamide. DHT action on c-myc mRNA was examined using Northern blots and RT-PCR. RT-PCR also was used to verify if HNTEP cells expressed the androgen receptor gene. Cell proliferation was assessed on days 3 and 6. Testosterone (2 x 10(-11) M) and DHT (10(-13)M) caused a significant increase (P < 0.05) in cell proliferation on both days. Addition of hydroxyflutamide (10(-6) M) to DHT abolished cell proliferation. HNTEP cells expressed androgen receptor (AR) gene and the treatment with DHT increased AR mRNA levels. C-myc expression was maximal at 30 min and 1 h with DHT (10(-13) M). C-myc seems to play a key role in the control of hormone responsiveness and cell proliferation in epithelial prostatic cells. The detection of androgen receptor gene expression and the increase in this expression with the addition of androgen shows that the HTNEP cells maintain functional characteristics and hormone dependence, and that they are a fruitful in vitro model for studying steroid hormone action mechanisms.     

Interaction between normal and leukaemic human cells in agar culture.

The influence of leukaemic cells from 12 patients with acute leukaemia on normal granulopoiesis in agar culture was investigated using leukeamic cell feeder layers. Leukaemic feeder cells from 7 of the 12 patients elicited no colony growth, while cells from the remaining 5 stimulated normal colony growth. In 3 of the 7 non-stimulatory patients release of inhibitory factors from the leukaemic cells seemed responsible for the effect on normal granulopoiesis, while inappropriate colony stimulating factor (CSF) production by the feeder cells could not be ruled out in the remaining 4 patients. When the leukaemic cells were cultured with, as well as without, conditioned medium, cells from 5 of the patients formed clusters. Growth in these cultures did not correlate to the effect found in the feeder layer experiments.     

A link between lung androgen metabolism and the emergence of mature epithelial type II cells

Lung maturation is delayed in male fetuses compared with female fetuses, which has been attributed to higher levels of androgens in the male lung. Our previous studies demonstrated that the genes encoding for the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) type 5 (androstenedione --> testosterone) and type 2 (the opposite reaction) are expressed in human epithelial type II (PTII)-like A549 cells and in human lung fibroblasts, respectively. Here, we aim to explain the physiological relevance of androgen synthesis by PTII cells. We showed that both 17 beta-HSD type 2 and type 5 genes are upregulated in correlation with the emergence of mature PTII cells in both male and female developing lungs of the mouse. In contrast, the androgen receptor gene is expressed equally in both sexes with no temporal regulation. We conclude that the expression profile of the 17 beta-HSD type 5 gene does not explain the presence of higher levels of androgen in the male fetal lung, but that androgen synthesis must be a normal feature of mature PTII cells for both sexes. The production of androgens after the emergence of mature PTII cells should negatively regulate PTII cell maturation and, thus, a role for androgens in cell reprogramming is suggested.     

Effect of ionizing radiation on acinar morphogenesis of human prostatic epithelial cells under three-dimensional culture conditions

Homeostasis is maintained by the interplay of multiple factors that directly or indirectly regulate cell proliferation and cell death. Complex multiple interactions between cells and the extracellular matrix occur during acinar morphogenesis and changes in these might indicate carcinogenesis of cells from a normal to a malignant, invasive phenotype. In this study, the human prostatic epithelial cell line RWPE-1 was cultured under three-dimensional (3-D) culture conditions, and the effect of ionizing radiation on acinar morphogenesis and its association with autophagy were discussed. The results illustrated that formation of specific spheroid (acinar) structures was detectable under 3-D culture conditions. Radiation induced the disruption of acini in different cell models using either gene overexpression (Akt) or gene knock-down (Beclin 1 and ATG7). Introduction of Akt not only accelerated the growth of cells (i.e., caused the cells to manifest elongating and microspike-like structures that are obviously different from structures seen in wild-type RWPE-1 cells under two-dimensional conditions), but also changed their morphological characteristics under 3-D culture conditions. Knock-down of autophagy-related genes (Beclin 1 and ATG7) increased the radiosensitivity of cells under 3-D culture conditions, and cells died of non-apoptotic death after radiation. The results suggested that ionizing radiation may change the cell phenotype and the formation of acini. Additionally even the autophagy mechanism may play a role in these processes.     

Antiinflammatory effect of androgen receptor activation in human benign prostatic hyperplasia cells

Progression of benign prostatic hyperplasia (BPH) involves chronic inflammation and immune dysregulation. Preclinical studies have demonstrated that prostate inflammation and tissue remodeling are exacerbated by hypogonadism and prevented by testosterone supplementation. We now investigated whether, in humans, hypogonadism was associated with more severe BPH inflammation and the in vitro effect of the selective androgen receptor agonist dihydrotestosterone (DHT) on cultures of stromal cells derived from BPH patients (hBPH). Histological analysis of inflammatory infiltrates in prostatectomy specimens from a cohort of BPH patients and correlation with serum testosterone level was performed. Even after adjusting for confounding factors, hypogonadism was associated with a fivefold increased risk of intraprostatic inflammation, which was also more severe than that observed in eugonadal BPH patients. Triggering hBPH cells by inflammatory stimuli (tumor necrosis factor α, lipopolysaccharide, or CD4(+)T cells) induced abundant secretion of inflammatory/growth factors (interleukin 6 (IL6), IL8, and basic fibroblast growth factor (bFGF)). Co-culture of CD4(+)T cells with hBPH cells induced secretion of Th1 inducer (IL12), Th1-recruiting chemokine (interferon γ inducible protein 10, IP10), and Th2 (IL9)- and Th17 (IL17)-specific cytokines. Pretreatment with DHT inhibited NF-κB activation and suppressed secretion of several inflammatory/growth factors, with the most pronounced effects on IL8, IL6, and bFGF. Reduced inflammatory cytokine production by T-cells, an increase in IL10, and a significant reduction of T cells proliferation suggested that DHT exerted a broad anti inflammatory effect on testosterone cells [corrected]. In conclusion, our data demonstrate that DHT exerts an immune regulatory role on human prostatic stromal cells, inhibiting their potential to actively induce and/or sustain autoimmune and inflammatory responses.     

Isolation and culture of biliary epithelial cells.

At one time it was thought that biliary epithelial cells simply formed the lining to the tubular conduits which constitute the biliary tract. Development of in vitro systems for culturing biliary epithelial cells has enabled functional studies which increasingly show that this is far from true, and that biliary epithelial cells do have important functional roles. Disruption of these functions may be involved in the generation of pathology. Most functional studies to date have utilised cells isolated from rat liver. Increasingly, variations are being found between human and animal cells both in terms of function and phenotype. The relevance of animal cells in the study of human disease therefore remains obscure. Human biliary tract disease has to date been studied almost exclusively by examination of histological sections. The development of improved methods for isolating highly pure biliary epithelial cells from human liver provides a new technology with which to investigate directly the dynamics of human biliary epithelial cell biology and pathobiology. It is predicted that further progress will now be made in dissecting the biology and physiology of human biliary epithelium.     

巨噬细胞与间充质干细胞相互作用及其对缺血性心脏病的影响

心肌梗死发生后,缺血及周边区域发生的炎症反应贯穿了心肌组织修复及纤维化的始终。巨噬细胞作为炎症反应的重要组分,在心肌损伤后的修复过程中至关重要。不同亚型的巨噬细胞参与了心肌梗死的各个阶段,并发挥着吞噬凋亡细胞、促进血管新生、促进瘢痕形成等多种作用。移植间充质干细胞也因具有免疫调控、旁分泌心肌保护等多种效应,被广泛用于缺血性心衰的研究中。近年来研究发现,巨噬细胞和间充质干细胞在治疗缺血性心脏病中有着密切联系,间充质干细胞可减少促炎型巨噬细胞在炎症部位的募集,并诱导巨噬细胞由促炎型向抗炎型转化,从而促进心肌修复。而巨噬细胞可影响间充质干细胞的生存、增殖、迁移及免疫调控等功能,从而改变其心肌修复效果。本文就巨噬细胞与间充质干细胞相互影响及其在缺血性心脏病中的作用与应用前景进行综述。     

Primary culture of human gallbladder epithelial cells

Growth requirements of epithelial cells isolated from the human gallbladder were examined. The growth of gallbladder epithelial cells (GBEC) was stimulated by medium conditioned with gallbladder fibroblasts (CM-GBF) in a dose-dependent manner. The conditioned medium derived from human embryo lung fibroblasts also showed similar growth stimulating activity for GBEC, suggesting that fibroblasts secrete a factor or factors which induce GBEC growthin vitro. CM-GBF in the presence of 10% fetal bovine serum increased GBEC growth up to 10 times the growth in the absence of CM-GBF supplement. GBEC cultured with CM-GBF showed hexagonal shape under a phase-contrast microscope, and also expressed cytokeratin and mucopolysaccharide in the cytoplasm, both of which are specific for GBEC. Electron microscopy revealed desmosomes and tight junctions between the cells and microvilli on the apical plasma membrane, suggesting that they regained morphological polarity in the medium containing CM-GBF. These results shows that CM-GBF is essential for the growth and the differentiation of GBEC in culture.     

Growth and differentiation of hamster tracheal epithelial cells in culture

The purpose of these studies was to define culture conditions that support growth and differentiation of normal epithelial cells obtained from hamster tracheas. Epithelial cells from tracheas of adult hamsters were collected using enzymatic procedures and cultured under various conditions. The medium used consisted of a 1:1 mixture of medium 199 and Dulbecco's modified Eagle's medium with 2% fetal bovine serum, which was conditioned by mouse 3T3 cells before use. Insulin, transferrin, hydrocortisone, epidermal growth factor, and an extract from bovine hypothalamus were used as supplements. When seeded on uncoated or collagen-coated tissue culture dishes, the hamster cells grew only poorly. When the cells were seeded on collagen gels, however, rapid and prolonged growth ensued. The cultures had a population doubling time of 20 hr and a colony-forming efficiency of 7-10%, and they could be grown for up to three passages. Growth was dependent on the presence of transferrin, insulin, epidermal growth factor, and 3T3 conditioning factors in the medium. The latter could be omitted if the concentration of serum was increased. Less important for growth was the presence of hydrocortisone and bovine hypothalamus extract. In contrast to results with tracheal epithelial cells from adult rabbits, rats, and mice, differentiation into ciliated cells regularly occurred in cultures of cells derived from hamster tracheas. The appearance of ciliated cells in the cultures was dependent on the presence of collagen gel as a substratum and of 3T3 conditioning factors in the medium. In addition, there were numerous cells that contained electron-dense cytoplasmic granules. The granules were not stained by dialyzed iron, which stains acidic glycoproteins, but were stained positively by periodic acid-Schiff reagents and the periodic acid-thiocarbohydrazide-silver proteinate method, suggesting the presence of secretory granules containing neutral glycoproteins. A similar staining pattern was observed for the secretory granules of intact hamster tracheas. The culture system described supports growth and cellular differentiation of normal tracheal epithelial cells of hamsters. We believe therefore that it will be a useful model for studying the regulation of tracheal cell function on the cellular and biochemical level.     

Ion transport by tracheal epithelial cells in culture

In culture, cells from the dog tracheal epithelium form confluent cell sheets that have ion transport processes similar to those of the original tissue. Cell sheets grown from "normal" human tracheal mucosa resemble those of the dog in the presence of active chloride secretion, which can be stimulated by endogenous mediators. Tracheal cells cultured from patients with cystic fibrosis lack chloride secretion because their apical membranes are impermeable to this anion.     

Induction of acid phosphatase synthesis in canine prostatic epithelial cells in vitro

The increase in acid phosphatase (AP) activity in cultured canine prostatic epithelial cells was investigated as a biochemical marker of in vitro cellular differentiation. The enzyme was studied in secretory and non-secretory epithelial cell populations obtained from control and cycloheximide-treated cultures over a period of 3 weeks and compared to the AP present in tissue and cellular extracts from normal canine prostates. The progressive increase in AP activity with the duration of culture was strongly inhibited by cycloheximide in both cell populations. The degree of inhibition was more pronounced late in the culture when AP activity increased at a faster rate in secretory cells. Cycloheximide inhibited protein biosynthesis by 70-80% as evidenced by a reduction in the incorporation of amino acids into acid-insoluble material. However, the specific activities of AP in the cellular extracts were similar in control and cycloheximide-treated cultures and increased sharply by 3-4-fold in the secretory cells after 12 days of culture. When extracts derived from control and cycloheximide-treated cells of various duration were submitted to electrophoresis in polyacrylamide gels (PAGE), a unique pattern of three bands of AP activity with Rf values of 0.18, 0.27 and 0.38 was obtained. In controls the AP activity in the band with an Rf of 0.18 increased preferentially during the culture period and was more important quantitatively in secretory cells. In cycloheximide-treated cultures the increase of AP activity associated with the band with an Rf of 0.18 was more strongly inhibited. The addition of tartrate to the staining mixture inhibited all three bands of AP activity. Similar results were obtained when extracts derived from freshly dispersed cells as well as from normal canine prostatic tissue were submitted to PAGE; the AP activity was resolved into 3 bands with Rf values of 0.15-0.18, 0.23-0.27 and 0.33-0.38; all three bands were inhibited by the addition of tartrate and the first band was predominant. Thus, the increase in AP activity in prostatic epithelial cells in a culture medium supplemented with serum and deprived of sex steroids is due to the de novo synthesis of a major form of the enzyme by the secretory cells.     

良性前列腺增生和前列腺癌组织中雄激素受体的表达

目的　探讨雄激素受体 (AR)与良性前列腺增生 (BPH)和前列腺癌 (PCa)的关系。　方法　应用免疫组织化学技术检测 4 0例BPH、4 0例PCa患者及 2 0例正常前列腺组织 (正常对照组 )AR的表达。　结果　BPH组、PCa组、正常对照组AR表达的阳性率分别为 98%、5 3%、75 % ,相互比较差异均有显著性 (P <0 0 5 )。高分化癌、中分化癌、低分化癌的AR表达的阳性率分别为 6 7%、5 6 %、30 % ,高、中分化癌的AR表达的阳性率比低分化癌高 (P <0 0 5 )。早期前列腺癌的AR表达阳性率 (6 9% )比晚期前列腺癌 (4 2 % )高 (P <0 0 5 )。　结论　BPH组织中AR的表达高于正常前列腺组织 ,AR在BPH的发生、发展中起着重要作用 ;PCa组织中AR的表达低于正常前列腺组织。AR的表达与肿瘤分级、分期相关。     

Interaction of an artificial surfactant in human pulmonary epithelial cells

Surfaxin (lucinactant), a peptide-based surfactant consisting of dipalmitoylphosphatidylcholine (DPPC) plus KL(4) (sinapultide) (a synthetic peptide modeled after human surfactant protein-B), is effective in treating respiratory distress syndrome in preterm infants. Our goal was to determine the uptake and effects of Surfaxin on human pulmonary type II cells isolated from fetal tissue and other lung cell types. Based on previous published reports, we hypothesized that this exogenous synthetic surfactant would have little effect on type II cell surfactant-related physiological features. Human type II cells and A549 and NCI-H441 adenocarcinoma cells incorporated (3)H-KL(4) and (14)C-DPPC components in Surfaxin, but with different kinetics. Fractionation of NCI-H441 and A549 cellular components indicated that the highest specific activity of (3)H-KL(4) was present in the 18,000g cellular fraction (which contains vesicles and lysosomes). The number of lamellar bodies (LBs) appears to increase in human type II cells incubated in the presence of Surfaxin when visualized by light microscopy, while LB structure (determined by electron microscopy) was not altered. Expression of endogenous surfactant protein (SP-A, SP-B, and SP-C) mRNA levels in human type II cells was not altered by the presence of Surfaxin. We conclude that while human type II cells and other lung cell types can incorporate the components of Surfaxin, the surfactant-related physiological functions of these cells are not altered.     

A monolayer culture of human gastric epithelial cells

Our aim was to develop a fibroblast-free monolayer culture of human gastric mucosal cells, using the specimens obtained by routine endoscopic biopsy. Human gastric mucosa obtained from normal volunteers by endoscopic biopsy was dissociated from collagenase and hyaluronidase. Dissociated cells were cultured in supplemented Coon's modified Ham's F-12 medium. Within 24 hr of inoculation, the cells were attached to the culture dishes. This was followed by cellular outgrowth. On phase-contrast microscopy, all cells had epithelial characteristics and fibroblasts were not observed. Ninety percent of cells contained periodic acid Schiff reaction-positive mucous granules after diastase digestion consistent with mucous epithelial cells. Two percent of the cells gave a strong reaction for succinic dehydrogenase activity (parietal cells). Immunohistochemical staining for pepsinogen in cultured cells was negative. On EM, microvilli-like projections, junctional complexes, Golgi apparatus, and mucous granules were apparent in the majority of cells. Mitotic figures were observed by day 3 with Giemsa staining. Autoradiographically, these cells were able to incorporate [3H]TdR into the nuclei. Cells were capable of synthesizing DNA, and this function was inhibited by cycloheximide. Cells could be cultured for up to two weeks without fibroblast contamination. A method of primary monolayer culture of human gastric mucosa obtained by a routine endoscopic biopsy has been successfully developed.