人食管上皮细胞原代培养方法的研究

目的探讨适用于组织工程食管研究的食管上皮细胞培养方法.方法对比食管上皮细胞在无血清培养基K-SFM及含血清培养基DMEM中的生长情况.结果食管上皮细胞在无血清培养基K-SFM中生长快,细胞形态好,不易被成纤维细胞污染.结论应用无血清培养基K-SFM培养食管上皮细胞,方法简便,适合推广应用.     

Caffeine stimulates growth hormone secretion by cultured rat pituitary cells

To study the effect of caffeine on growth hormone secretion a culture system of dispersed rat anterior pituitary cells was employed. The cells were incubated overnight in medium 199 containing 10(-5) to 10(-1) M caffeine. The medium was then collected and assayed for rat growth hormone content. A dose dependent stimulatory effect of caffeine on growth hormone secretion into the culture medium was observed. It is concluded that caffeine, like other xanthine phosphodiesterase inhibitors stimulates growth hormone secretion by a direct effect on pituitary cells.     

Three-dimensional perfused human in vitro model of nonalcoholic fatty liver disease

AIM To develop a human in vitro model of non-alcoholic fatty liver disease(NAFLD), utilising primary hepatocytes cultured in a three-dimensional(3D) perfused platform. METHODS Fat and lean culture media were developed to directly investigate the effects of fat loading on primary hepatocytes cultured in a 3D perfused culture system. Oil Red O staining was used to measure fat loading in the hepatocytes and the consumption of free fatty acids(FFA) from culture medium was monitored. Hepatic functions, gene expression profiles and adipokine release were compared for cells cultured in fat and lean conditions. To determine if fat loading in the system could be modulated hepatocytes were treated with known anti-steatotic compounds. RESULTS Hepatocytes cultured in fat medium were found to accumulate three times more fat than lean cells and fat uptake was continuous over a 14-d culture. Fat loading of hepatocytes did not cause any hepatotoxicity and significantly increased albumin production. Numerous adipokines were expressed by fatty cells and genes associated with NAFLD and liver disease were upregulated including: Insulin-like growth factorbinding protein 1, fatty acid-binding protein 3 and CYP7A1. The metabolic activity of hepatocytes cultured in fatty conditions was found to be impaired and the activities of CYP3A4 and CYP2C9 were significantlyreduced, similar to observations made in NAFLD patients. The utility of the model for drug screening was demonstrated by measuring the effects of known antisteatotic compounds. Hepatocytes, cultured under fatty conditions and treated with metformin, had a reduced cellular fat content compared to untreated controls and consumed less FFA from cell culture medium.CONCLUSION The 3D in vitro NAFLD model recapitulates many features of clinical NAFLD and is an ideal tool for analysing the efficacy of anti-steatotic compounds.     

Primary culture of porcine pancreatic acinar cells

INTRODUCTION: The methodology of acinar cell culture has become of primary importance in the research of pancreatic physiology and pharmacology. AIM: To develop a method for primary culture of porcine pancreatic acinar cells. METHODOLOGY: Dispersed pancreatic acinar cells were made by RPMI-1640 medium containing collagenase III. After purification, the isolated acinar cells were cultured in RPMI-1640 medium with 2.5% fetal bovine serum. The morphologic characteristics of acinar cells were described. (3)H-thymidine incorporation of acinar cells and activity of amylase or lipase were determined during the culture. RESULTS: There were no remarkable morphologic changes in the pancreatic acinar cells during the 20-day culture. The acini showed the tendency of gathering but did not attach to the walls of the culture disks. Incorporation of (3)H-thymidine in acinar cells in the primary culture was well kept. The secretion of amylase or lipase from acini decreased with the time of culture. CONCLUSIONS: In the primary culture of acinar cells from porcine pancreas developed in this study, the acinar cells retained normal morphology and ability of growth but not secretion of amylase or lipase. The method would be beneficial for further experiments on acini of porcine pancreas.     

撤除生长因子对间充质干细胞增殖和胞外基质表达的影响

目的:研究撤除表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)和血管内皮细胞生长因子(VEGF)对人脐带间充质干细胞(hUCMSCs)的增殖和胞外基质(ECM)表达的影响。方法:将第5代hUCMSCs分3组:对照组(保持上述生长因子浓度不变),半量撤除组(使生长因子浓度减半),完全撤除组(不添加生长因子)。培养30h后瑞特-姬姆萨法染色观察细胞形态,MTT法细胞计数。培养15h后,定量PCR检测BCL-2、BAX及胞外基质基因的表达情况。结果:撤除生长因子后细胞增殖减慢,形态变大,核质比减小,BCL-2/BAX比率增高,胞外基质表达多数下调。半量撤除组细胞的形态、增殖能力、胞外基质表达的改变相对较小,BCL-2/BAX比率增高更明显。结论:撤除生长因子会影响细胞增殖和胞外基质的表达,保留半量生长因子可以改善这种情况。     

A technique for the culture of Barrett's oesophageal cells

Establishment of cells in tissue culture from Barrett's columnar epithelium has been difficult. The aim of this study was to develop a successful tissue culture method employing a serumfree medium for cultivation of Barrett's epithelial cells. Fragments of Barrett's mucosal tissue were explanted in a 3:1 mixture of Dulbecco's modification of Eagle's medium and Ham's F12, to initiate the outgrowth of epithelial cells. Subsequently, a commercial serum-free medium (formulated for the growth of keratinocytes) was used for the propagation of Barrett's oesophagus cells without fibroblast growth. Cells established in culture retained their epithelial morphology, stained positive for cytokeratin, and contained Alcian blue (pH 2.5) and periodic acid-Schiff reagent-positive/diastase-resistant vacuoles, confirming their origin from Barrett's epithelium. Electron microscopy showed tonofilaments, microvilli and desmosomes. Coating the surface of culture vessels was not required and four cell strains could be passaged up to 20 times with no fibroblast growth, in the keratinocyte serumfree medium.     

The mitosis of fibroblasts in cell culture is enhanced by binding GP IIb-IIIa of activated platelets on fibrinogen

A fibroblast cell culture model enables us to measure the mitogenic ability mediated by growth factors released from stimulated platelets under different conditions. Simultaneously the growth factors secreted in the culture medium were determined. Cell mitotic rate was measured by incorporation of 3H-thymidine on days 3, 5 and 7 of culture. PDGF, TGF-beta, EGF and IGF-I were determined by Western blot. When fibroblasts were grown on surfaces precoated with a mixture of fibrinogen and thrombin-stimulated platelets, the 3H-thymidine uptake (196,645 +/- 56,864 cpm/ml) was increased, in comparison to fibroblasts grown on uncoated surfaces, in medium supplemented with FBS (28,855 +/- 7329 cpm/ml). Neither thrombin-stimulated platelets without fibrinogen nor fibrinogen alone had positive effects on the mitogenic activity of fibroblast. Growth factors were identified only in a culture medium in which the cells were grown on surfaces precoated with fibrinogen and thrombin-stimulated platelets. Blocking the platelet integrin GP IIb-IIIa inhibited the release of growth factors from thrombin-stimulated platelets and consecutively the stimulation of mitosis by fibrinogen and activated platelets was absent. Antibodies against the growth factors added to the medium suppressed the stimulation of cell mitosis. These results show that delivery of growth factors from platelets' secretory granules is dependent on binding of fibrinogen to GP IIb-IIIa.     

The mitosis of fibroblasts in cell culture is enhanced by binding GP IIb-IIIa of activated platelets on fibrinogen

A fibroblast cell culture model enables us to measure the mitogenic ability mediated by growth factors released from stimulated platelets under different conditions. Simultaneously the growth factors secreted in the culture medium were determined. Cell mitotic rate was measured by incorporation of 3 H-thymidine on days 3, 5 and 7 of culture. PDGF, TGF- g , EGF and IGF-I were determined by Western blot. When fibroblasts were grown on surfaces precoated with a mixture of fibrinogen and thrombin-stimulated platelets, the 3 H-thymidine uptake (196645 - 56864 cpm/ml) was increased, in comparison to fibroblasts grown on uncoated surfaces, in medium supplemented with FBS (28855 - 7329 cpm/ml). Neither thrombin-stimulated platelets without fibrinogen nor fibrinogen alone had positive effects on the mitogenic activity of fibroblast. Growth factors were identified only in a culture medium in which the cells were grown on surfaces precoated with fibrinogen and thrombinstimulated platelets. Blocking the platelet integrin GP IIb-IIIa inhibited the release of growth factors from thrombin-stimulated platelets and consecutively the stimulation of mitosis by fibrinogen and activated platelets was absent. Antibodies against the growth factors added to the medium suppressed the stimulation of cell mitosis. These results show that delivery of growth factors from platelets' secretory granules is dependent on binding of fibrinogen to GP IIb-IIIa.     

大鼠胸主动脉平滑肌细胞的培养与鉴定

目的探讨平滑肌细胞培养方法,了解生长特性。方法用组织贴块法培养大鼠胸主动脉平滑肌细胞,用相差显微镜观察其生长情况,用免疫荧光染色分析其抗原的表达。结果培养3日可见组织块周边有细胞长出,2周达亚融合,传代后细胞“谷峰”生长明显。SM-α-肌动蛋白和Calponin抗体免疫荧光染色呈阳性。结论组织贴块法是简单、经济、高效的平滑肌细胞培养方法,为心血管疾病发病机制的研究提供了理想的细胞模型。     

Myofibroblast-derived PDGF-BB promotes Hedgehog survival signaling in cholangiocarcinoma cells

Cholangiocarcinoma (CCA) cells paradoxically express the death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and, therefore, are dependent upon potent survival signals to circumvent TRAIL cytotoxicity. CCAs are also highly desmoplastic cancers with a tumor microenvironment rich in myofibroblasts (MFBs). Herein, we examine a role for MFB-derived CCA survival signals. We employed human KMCH-1, KMBC, HuCCT-1, TFK-1, and Mz-ChA-1 CCA cells, as well as human primary hepatic stellate and myofibroblastic LX-2 cells, for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. Coculturing CCA cells with myofibroblastic human primary hepatic stellate cells or LX-2 cells significantly decreased TRAIL-induced apoptosis in CCA cells, a cytoprotective effect abrogated by neutralizing platelet-derived growth factor (PDGF)-BB antiserum. Cytoprotection by PDGF-BB was dependent upon Hedgehog (Hh) signaling, because it was abolished by the smoothened (SMO; the transducer of Hh signaling) inhibitor, cyclopamine. PDGF-BB induced cyclic adenosine monophosphate-dependent protein kinase-dependent trafficking of SMO to the plasma membrane, resulting in glioma-associated oncogene (GLI)2 nuclear translocation and activation of a consensus GLI reporter gene-based luciferase assay. A genome-wide messenger RNA expression analysis identified 67 target genes to be commonly up- (50 genes) or down-regulated (17 genes) by both Sonic hedgehog and PDGF-BB in a cyclopamine-dependent manner in CCA cells. Finally, in a rodent CCA in vivo model, cyclopamine administration increased apoptosis in CCA cells, resulting in tumor suppression. Conclusions: MFB-derived PDGF-BB protects CCA cells from TRAIL cytotoxicity by a Hh-signaling-dependent process. These results have therapeutical implications for the treatment of human CCA.     

Effect of hepatoma H22 on lymphatic endothelium in vitro

AIM: To determine the effect of metastatic hepatoma cells on lymphangioma-derived endothelium, and to establish in vitro model systems for assessing metastasis-related response of lymphatic endothelium.METHODS: Benign lymphangioma, induced by intraperitoneal injection of the incomplete Freund‘s adjuvant in BALB/c mice, was embedded in fibrin gel or digested and then cultured in the conditioned medium derived from hepatoma H22. Ught and electron microscopy, and the b-answell migration assay were used to determine the effect of H22 on tissue or cell culture. Expressions of Fit-4, c-Fos, proliferating cell nuclear antigen (PCNA), and inducible nitric oxide synthase (iNOS) in cultured cells, and content of nitric oxide in culture medium were also examined.RESULTS: The embedded lymphangioma pieces gave rise to array of capillaries, while separated cells from lymphangioma grew to a cobblestone-like monolayer. H22 activated growth and migration of the capillaries and cells, induced expressions of Flt-4, c-Fos, PCNA and iNOS in cultured cells, and significantly increased the content of NO in the culture medium.CONCLUSION: Lymphangioma-derived cells keep the differentiated phenotypes of lymphatic endothelium, and the models established in this study are feasible for in vitro study of metastasis-related response of lymphatic endothelium.     

乙型肝炎患者骨髓间充质干细胞培养方法的优化和生物学特性

目的比较乙型肝炎患者骨髓间充质干细胞（MSCs）多种体外培养方法的效果，确定乙型肝炎患者MSCs的最佳体外培养方案；并对其生物学特性进行初步研究。方法比较2种接种方案和5种不同成分培养液的乙型肝炎患者MSCs体外培养成功率。比较5种不同成分培养液和4种换液方案的乙型肝炎患者MSCs生长曲线。进行乙型肝炎患者MSCs的形态观察和表面分子鉴定，传代培养以初步了解其生长特性。结果密度梯度离心接种法的体外培养成功率（88％）高于全骨髓接种法（76％），但差异无统计学意义。5种培养液的体外培养成功率差异有统计学意义（P〈0．01），其中患者自体血清培养液的培养成功率最高（100％），3种FBS培养液的培养成功率次之，健康成人血清培养液的最低（56％）。患者自体血清培养液的MSCs生长曲线最高，健康成人血清培养液的生长曲线最低，3种FBS培养液的MSCs生长曲线集中位于上述两者之间。以2d或者3d一次全量换液的MSCs生长曲线较高。乙型肝炎患者MSCs与正常人MSCs形态相同，表面分子表达一致，生长特性近似。结论乙型肝炎患者MSCs的体外培养方案以密度梯度离心法分离后，自体血清培养液进行培养，2～3d全量换液为佳，可以较快获得纯度较高的MSCs。乙型肝炎患者MSCs的生物学特性和正常人的近似。     

Dissociation between microalbuminuria and common carotid thickness in essential hypertensive men

BACKGROUND: The reasons why microalbuminuria (albuminuria > or = 15 microg/min), an expression of a renal microcirculatory abnormality, predicts cardiovascular disease in essential hypertension are unsettled. To test the hypothesis that microalbuminuria represents a marker of subclinical atherosclerosis, we evaluated its association with common carotid artery (CCA) intima media thickness (IMT), a measure of preclinical atherosclerosis and an independent predictor of cardiac and cerebrovascular events, in uncomplicated essential hypertensive individuals. MATERIALS AND METHODS: Albuminuria, ultrasonographic CCA IMT (the mean of six bilateral far wall measurements within 1.5 cm proximally to the flow divider), brachial blood pressure (BP), smoking habits and lipids were evaluated in 136 stage 1-3 untreated essential hypertensive men free of cardiovascular disease. RESULTS: CCA IMT did not differ between normo- (n = 99) and microalbuminuric (n = 37) patients. The correlation between CCA IMT and albuminuria was not significant, and the prevalence of microalbuminuria across IMT quartiles was not different. Microalbuminuric patients showed higher systolic BP and that parameter was the only independent correlate in a multivariate logistic regression model including also age, CCA IMT, diastolic BP, lipids and smoking habits as independent variables and microalbuminuria as the dependent one. CONCLUSION: This cross-sectional study in hypertensive subjects free of cardiovascular disease has shown a dissociation between microalbuminuria and CCA IMT, a surrogate measure of subclinical atherosclerosis, and a parameter linearly related to cardiovascular events. The data do not support the theory of microalbuminuria as a surrogate measure of subclinical atherosclerosis, while confirming the importance of systolic BP levels as an independent correlate of increased albuminuria in essential hypertension. Journal of Human Hypertension (2000) 14, 831-835     

Annexin A1: A new immunohistological marker of cholangiocarcinoma

AIM: To evaluate a new immunohistological marker, annexin A1 (ANXA1), in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC). METHODS: Expression of ANXA1 protein was investigated in liver tissues from patients with CCA and HCC by immunohistochemistry. Its expression on differences stages of tumor development was investigated in hamster CCA tissues induced by Opisthorchis viverrini and N -nitrosodimethylamine. Moreover, mRNA expression of ANXA1 was assessed in CCA cell lines by quantitative real-time polymerase chain reaction and silencing of ANXA1 gene expression using small interfering RNA. RESULTS: In human CCA tissue arrays, immunohistochemical analysis revealed that the positive expression of ANXA1 was 94.1% (64/68 cases) consisting of a high expression (66.2%, 45/68 cases) and a low expression (33.8%, 23/68 cases). However, expression of ANXA1 protein was negative in all histologic patterns for HCC (46/46 cases) and healthy individuals (6/6 cases). In hamster with opisthorchiasis-associated CCA, the expression of ANXA1 was observed in the cytoplasm of inflammatory cells, bile duct epithelia and tumor cells. Grading scores of ANXA1 expression were significantly increased with tumor progression. In addition, mRNA expression of ANXA1 significantly increased in all of the various CCA cell lines tested compared to an immortalized human cholangiocyte cell line (MMNK1). Suppressing the ANXA1 gene significantly reduced the matrix metalloproteinase (MMP) 2 and MMP9, and transforming growth factor-β genes, but increased nuclear factor-kB gene expression. CONCLUSION: ANXA1 is highly expressed in CCA, but low in HCC, suggesting it may serve as a new immunohistochemical marker of CCA. ANXA1 may play a role in opisthorchiasis-associated cholangiocarcinogenesis.     

大鼠海马神经干细胞的原代培养方法

目的探索总结一种简单、高效的大鼠海马神经干细胞原代培养方法。方法选取新生24 h的SD大鼠,剥离出双侧海马,用机械吹打法制成细胞悬液,加入培养液,接种于培养皿中,3 d后半量换液,5~7 d后可传代。每天在荧光倒置显微镜下观察细胞的生长状况,采用免疫荧光法对第2代神经干细胞进行鉴定。结果本方法能准确分离出海马组织,培养出的神经干细胞中无其他混杂的细胞生长,经鉴定呈兔抗巢蛋白(Nestin)阳性和5-嗅-2-脱氧尿苷(BrdU)阳性。结论运用本方法获得的海马神经干细胞纯度高、细胞活力高、增殖能力强。     

Identification of parameters required for efficient lentiviral vector transduction and engraftment of human cord blood CD34(+) NOD/SCID-repopulating cells

OBJECTIVE: Human cord blood (CB) is a potential source of hematopoietic stem cells (HSC) for gene therapy to treat patients with hematopoietic disorders. However, limited numbers of CB CD34(+) cells, low transduction efficiency with lentiviral vectors (LVs), and low engraftment efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRC), a measure of HSC, are blocks to this procedure. To optimize culture and transduction conditions, we compared various lengths of time for prestimulation before transduction, transduction duration, and posttransduction cell culture. MATERIALS AND METHODS: We used a LV to transduce human CB CD34(+) cells followed by engraftment into NOD/SCID mice. We evaluated the effects of prestimulation and transduction time and optimized ex vivo cell culture duration before transplantation. RESULTS: We were able to achieve up to 40% transduction efficiency and up to 50% engraftment efficiency of SRC in CB CD34(+) cells when CB CD34(+) cells were either not prestimulated or prestimulated in 1% fetal bovine serum medium for 1 hour, followed by 5 hours transduction and 3 days culture in a cocktail of growth factors after transduction. No apparent functional changes of CB CD34(+) cells were noted under these conditions. CONCLUSION: This gene-transduction/cell-expansion protocol is the first systematic study to optimize prestimulation time, transduction time, and, very importantly, ex vivo culture time after transduction, and may be of use for LV gene transduction in a gene therapy setting.     

益气活血软坚解毒含药血清诱导人肝癌细胞系Bel-7402细胞的凋亡

目的:研究益气活血软坚解毒(YHRJ)含药血清对人肝癌细胞系Bel-7402生长抑制及诱导凋亡作用.方法:将YHRJ含药血清作用于人肝癌细胞系Bel-7402细胞,应用MTT检测对肝癌细胞生长抑制作用,倒置显微镜、荧光显微镜、激光共聚焦扫描显微镜等影像学方法观察细胞形态学变化,以及PI染色单染、AnnexinV-PI双染后,流式细胞术检测对细胞周期影响及诱导细胞凋亡程度.结果:MTT法检测结果显示:YHRJ含药血清具有抑制Bel-7402肿瘤细胞生长作用(其中20%YHRJ等效剂量抑制率49.1%,与NS比,P<0.01);荧光显微镜及激光共聚焦显微镜可观察到典型的凋亡形态学变化.流式细胞术检测结果,细胞周期出现G0/G1期阻滞,并出现典型的凋亡峰,AnnexinV-PI双染法检测到早期及中晚期细胞凋亡.结论:YHRJ含药血清有抑制人肝癌细胞系Bel-7402细胞生长并有诱导细胞凋亡作用.     

细粒棘球蚴生发细胞体外培养的实验观察

目的 :为培育人源细粒棘球蚴细胞系 (株 ) ,以供用于免疫预防研究。方法 :用外科方法从临床确诊的细粒棘球蚴患者的肝脏中取出包囊 ,以生发层和原头节为培养材料 ,采用RPMI1640、199和改良 DMEM培养液 ,用鼠尾胶原蛋白包被和不包被的培养瓶 ,对其进行初培养 ,并进行对比观察。结果 :以胶原蛋白作为支撑材料 ,应用改良 DMEM培养液培养的人源细粒棘球蚴生发层和原头节效果优于其他 ,已成功地培育了人源细粒棘球蚴细胞系 ,并传至第 2 0代。原代培养时间需 2 8d- 4 5d,3代以内细胞为多形态性。结论 :建立了适合人源细粒棘球蚴细胞体外培养的方法。     

Study on biological characters of SGC7901 gastric cancer cell-dendritic cell fusion vaccines

AIM: To detect the biological characters of the SGC7901 gastric cancer cell-dendritic cell fusion vaccines. METHODS: The suspending living SGC7901 gastric cancer cells and dendritic cells were induced to be fusioned by polyethylene glycol. Pure fusion cells were obtained by selective culture with the HAT/HT culture systems. The fusion cells were counted at different time points of culture and their growth curves were drawn to reflect their proliferative activities. The fusion cells were also cultured in culture medium to investigate whether they could grow into cell clones. MTT method was used to test the stimulating abilities of the fusion cells on T lymphocytes' proliferations. Moreover, the fusion cells were planted into nude mice to observe whether they could grow into new planted tumors in this kind of immunodeficiency animals. RESULTS: The fusion cells had weaker proliferative activity and clone abilities than their parental cells. When they were cultured, the counts of cells did not increase remarkably, nor could they grow into cell clones in culture medium. The fusion cells could not grow into new planted tumors after planted into nude mice. The stimulating abilities of the fusion cells on T lymphocytes' proliferations were remarkably increased than their parental dendritic cells. CONCLUSION: The SGC7901 gastric cancer cell-dendritic cell fusion vaccines have much weaker proliferative abilities than their parental cells, but they keep strong abilities to irritate the T lymphocytes and have no abilities to grow into new planted tumors in immunodeficiency animals. These are the biological basis for their anti-tumor biotherapies.     

Primary culture of purified Leydig cells isolated from adult rat testes

Methods for isolating highly purified Leydig cells permit the study of acute responses and biochemical properties of Leydig cells independent of other testicular cell types. The present study describes the development of a primary culture system for purified Leydig cells from adult rats in which the cells retain their ability to secrete testosterone for at least 72 h in culture. When Leydig cells were cultured in tissue culture medium 199--0.1% BSA (M199-BSA), basal testosterone secretion declined by 72 h, whereas hCGB-stimulated testosterone secretion was reduced by 48 h. Changing the culture medium twice daily or adding 0.5% fetal calf serum (fcs) enhanced basal and gonadotropin-stimulated testosterone secretion at 72 h in culture, although responsiveness to hCG was reduced to 57% of that in freshly isolated cells. Incubation of Leydig cells in the defined culture medium Dulbecco's Modified Eagles-Ham's F-12 (1:1, vol/vol) supplemented with 15 mM Hepes buffer, transferrin, insulin, and epidermal growth factor (DHG:F12 + Hepes + TIE) in either the presence or absence of 0.5% fcs yielded functional Leydig cells for longer intervals in culture. Furthermore, testosterone secretion was greater in DHG:F12 + Hepes + TIE than in M199-BSA at all time intervals tested. In DHG:F12 + Hepes + TIE, basal and gonadotropin-stimulated testosterone production by Leydig cells were maintained for 72 h in culture. Degenerative changes in morphology were apparent in some cells at 72 h, but not at earlier times in culture. This primary culture system for isolated Leydig cells provides a valuable tool to examine the temporally regulated events in Leydig cell function.