前列腺癌细胞的原代和传代培养的研究

目的探讨前列腺癌细胞的培养方法,以便建立前列腺癌细胞系,为前列腺癌的基础研究提供实验模型。方法前列腺腺癌组织来源于我院两例患者的手术标本,分别采用组织块培养法、消化培养法和裸鼠肿瘤移植动物模型法对癌组织细胞进行体外分离培养。培养细胞按恶性肿瘤连续细胞系建系标准进行各项指标检测。结果组织块培养法有一例已稳定传为13代,消化培养法有一例已稳定传为23代,裸鼠肿瘤移植动物模型法未见癌肿生长。结论组织块培养法和消化培养法均为前列腺癌细胞培养的可行方法。     

羊水细胞原代培养和传代培养方法比较

<正>羊水细胞培养及染色体核型分析是产前确诊染色体疾病诊断的金标准,为妊娠中晚期胎儿临床诊断提供可靠的科学依据。羊膜腔穿刺是损伤性的取样技术,对孕妇和胎儿有造成损伤、感染、流产的潜在危险,流产发生率约0.5%[1]。羊水细胞培养因技术要求高、难度大、周期长、易污染、分裂相少,一旦培养失败,孕妇难接受第二次穿刺取样。因此,提高羊水细胞培养成功率是关键。本研究就羊水细胞的原代培养和传代培养两种方法比较。     

骨髓基质细胞转化为神经细胞的实验研究

背景：研究发现骨髓基质细胞移植后能够游走到全身不同部位，增生转化为相应细胞，能不同程度恢复损伤器官功能。以证实骨髓基质细胞可在体外转化成中胚层和内胚层细胞。目的：探讨骨髓基质细胞体外转化为神经细胞的可能性。设计：设立对照的重复测量设计。地点和对象：哈尔滨医科大学附属第一医院骨科。健康雄性SD大鼠，120∽150g，清洁级，由哈尔滨医科大学附属第一医院动物室提供。干预：骨髓基质细胞原代培养、传代后使用3种不同方法诱导。行大体观察，免疫组织化学及细胞电生理检查和长期培养研究。主要结局观察指标：骨髓基质细胞诱导、细胞免疫组化和细胞电生理检测结果。结果：第1诱导方案加入诱导剂1h50％转化成神经细胞，90min70％∽80％转化为神经细胞。第2诱导方案加入诱导剂24h10％转化成神经细胞。第3诱导方案BMSC40％加入，24h90％转化成神经细胞，胞体小突触短。BMSC 80％时加入，转化的神经细胞呈巢状排列占40％。骨髓基质细胞体外可诱导为突触明确的神经细胞(诱导率&;gt;80％)；诱导后细胞表面有神经特异性抗原NSE，GFAP表达；同时具有早期神经细胞电流特性。结论：骨髓基质细胞可横向转化为早期神经细胞。     

心脏组织工程学研究的进展:成人体外心肌细胞原代与传代培养(英文)

目的:建立成人心肌细胞原代培养和传代培养的方法。方法:用0.2%胰蛋白酶(trypsin)和0.1%胶原酶(collagenase)进行消化,用差速黏附贴壁法纯化心肌细胞,用IMDM(Iscove's改良的Dul-becco'smedium)培养基培养,倒置显微镜下观察,用苔盼蓝(trypan)染色法做心肌细胞质量评价,心肌细胞进行Actin免疫组化、Myoglobin荧光免疫组化和电镜分析。结果:心肌细胞存活率为99%,24h后见有95%细胞贴壁,心肌细胞达95%,心肌细胞为圆形状、梭形状、椭圆状、棒状、星状及分叉状等;Actin免疫组化和Myoglobin荧光免疫组化强阳性,电镜见心肌细胞结构存在;原代培养第30天和传代培养第15天心肌细胞生长良好。结论:本方法可很好的用于成人心肌细胞原代培养和传代培养,为进一步建立心肌病理模型打好基础。     

低密度传代培养人脂肪基质细胞的体外增殖和表面抗原表达

目的：观察低密度传代培养对人脂肪基质细胞的形态、增殖能力和表面抗原表达的影响。 方法：实验于2004-05／2006—01在上海交通大学医学院附属第九人民医院整形外科和组织工程研究中心完成。①吸脂术获得脂肪组织，经胶原酶消化分离。（函培养脂肪基质细胞，原代细胞80％融合后分别以5000／cm^2和1000／cm^2的接种密度传代至第3代。③四唑盐比色法和流式细胞法检测比较不同密度传代培养对细胞形态、增殖能力及表面抗原表达的影响。 结果：①低密度传代培养的细胞形态相对均一、体积较小。②四唑盐比色法结果显示低密度传代细胞增殖活力与高密度传代培养组差异无显著性。③流式细胞学检测结果表明，低密度培养组表达CD105，CD166，Stro-1，Flk-1等干细胞相关表面抗原的细胞比例显著高于高密度培养组。 结论：低密度传代培养有利于脂肪基质细胞的体外增殖，并能显著提高细胞群体中干细胞的比例，有可能发展成为一种简便的脂肪干细胞纯化方法。     

低密度传代培养人脂肪基质细胞的体外增殖和表面抗原表达

目的:观察低密度传代培养对人脂肪基质细胞的形态、增殖能力和表面抗原表达的影响。方法:实验于2004-05/2006-01在上海交通大学医学院附属第九人民医院整形外科和组织工程研究中心完成。①吸脂术获得脂肪组织,经胶原酶消化分离。②培养脂肪基质细胞,原代细胞80%融合后分别以5000/cm2和1000/cm2的接种密度传代至第3代。③四唑盐比色法和流式细胞法检测比较不同密度传代培养对细胞形态、增殖能力及表面抗原表达的影响。结果:①低密度传代培养的细胞形态相对均一、体积较小。②四唑盐比色法结果显示低密度传代细胞增殖活力与高密度传代培养组差异无显著性。③流式细胞学检测结果表明,低密度培养组表达CD105,CD166,Stro-1,Flk-1等干细胞相关表面抗原的细胞比例显著高于高密度培养组。结论:低密度传代培养有利于脂肪基质细胞的体外增殖,并能显著提高细胞群体中干细胞的比例,有可能发展成为一种简便的脂肪干细胞纯化方法。     

染料木黄酮对前列腺癌细胞系PC-3生物学行为影响的意义

目的：观察染料木黄酮(genistein)对前列腺癌细胞PC-3生物学行为的影响，探讨genistein预防前列腺癌的可能性。方法：用MTT法绘制生长曲线，观察0，10，20，40μmol／L的Genistein对PC-3细胞生长能力的影响；PC-3细胞经Genistein处理3d后，流式细胞术观察Genistein对前列腺癌细胞PC-3细胞周期的影响，Transwell小室法重建基底膜侵袭模型研究Genistein处理后细胞侵袭性的改变。结果：经Genistein处理后，PC-3细胞的生长受到抑制，作用3d时的药物半数致死量(ICso)为25μmol／L；细胞周期改变主要为G2／M阻滞，凋亡率(AI)明显增加，0，10，20，40μmol／L组的G2／M的百分比和AI分别为14．9％，27．4％，33．1％，31．9％和0，6．5％，14．2％，25．4％。侵袭能力分别下降到未加药组的31．8％，8．6％和3．96％。结论：Genistein通过引起PC-3细胞G2／M阻滞，诱导PC-3细胞凋亡，从而抑制PC-3细胞的恶性增殖以及降低PC-3细胞的侵袭能力可能是Genistein降低前列腺癌的发病率的一个重要原因。Genistein有可能成为预防前列腺癌的药物之一。     

熊果酸对前列腺癌细胞作用的实验研究

目的 探讨熊果酸对前列腺癌的治疗作用及机制.方法 MTT法检测熊果酸对体外培养的人前列腺癌细胞LNCaP和DUl45生长的影响.结果 熊果酸对不同浓度雄激素下的LNCaP细胞均呈浓度和时间依赖性生长抑制,熊果酸作用后,LNCaP细胞生长的最适雄激素浓度上升了10倍.DU145细胞对雄激素阻断剂氟他胺缺乏反应,熊果酸对DU145细胞具有浓度和时间依赖性抑制效应,熊果酸作用同时再应用氟他胺比单纯熊果酸的作用更明显,对细胞抑制率明显上升.结论 熊果酸能改善前列腺癌细胞对雄激素的反应性,使LNCaP细胞对雄激素的依赖性加强,并诱发了DUl45细胞对雄激素的反应性,延缓或者阻止了雄激素非依赖性的发生.     

骨髓基质细胞转化为神经细胞的实验研究

背景研究发现骨髓基质细胞移植后能够游走到全身不同部位,增生转化为相应细胞,能不同程度恢复损伤器官功能.以证实骨髓基质细胞可在体外转化成中胚层和内胚层细胞.目的探讨骨髓基质细胞体外转化为神经细胞的可能性.设计设立对照的重复测量设计.地点和对象哈尔滨医科大学附属第一医院骨科.健康雄性SD大鼠,120～150 g,清洁级,由哈尔滨医科大学附属第一医院动物室提供.干预骨髓基质细胞原代培养、传代后使用3种不同方法诱导.行大体观察,免疫组织化学及细胞电生理检查和长期培养研究.主要结局观察指标骨髓基质细胞诱导、细胞免疫组化和细胞电生理检测结果.结果第1诱导方案加入诱导剂1 h 50%转化成神经细胞,90 min 70%～80%转化为神经细胞.第2诱导方案加入诱导剂24 h 10%转化成神经细胞.第3诱导方案BMSC 40%加入,24 h 90%转化成神经细胞,胞体小突触短.BMSC 80%时加入,转化的神经细胞呈巢状排列占40%.骨髓基质细胞体外可诱导为突触明确的神经细胞(诱导率＞80%);诱导后细胞表面有神经特异性抗原NSE,GFAP表达;同时具有早期神经细胞电流特性.结论骨髓基质细胞可横向转化为早期神经细胞.     

Fas-mediated killing of primary prostate cancer cells is increased by mitoxantrone and docetaxel

Therapies for prostate cancer based on Fas (CD95) modulation have been under active development at the preclinical stage using immortalized cell lines. To address clinical applicability, the potential of 11 cultures of primary prostate cancer cells to be killed by Fas-mediated apoptosis was investigated. In addition, the effect of the chemotherapeutic agents mitoxantrone and docetaxel on this killing was determined. Apoptosis was induced in patient-derived, primary prostate cancer cells using effector cells engineered by recombinant lentivirus infection to express Fas ligand (FasL) and measured by 51Cr release assays. All cultured prostate cells were found to undergo Fas-mediated killing; cytotoxicity ranged from 12% to 87% after 6 h. These cells were significantly more sensitive to FasL-mediated killing than PC-3 cells. The basal expression of Fas or the expression of five inhibitors of apoptosis (c-FLIP, survivin, cellular inhibitors of apoptosis protein 1 and 2, and bcl-2) was not found to correlate with susceptibility to Fas-mediated killing. Both mitoxantrone and docetaxel were able to induce Fas receptor expression on primary prostate cancer cells, which translated into a 1.5- to 3-fold enhancement of apoptosis mediated by FasL. Whereas mitoxantrone increased the Fas-induced apoptotic response of all cultured prostate cells tested, docetaxel pretreatment was found to preferentially enhance the killing of bcl-2-expressing cells. These findings show that cultured primary prostate cancer cells are sensitive to Fas-mediated apoptosis. Furthermore, the incidence of apoptosis was found to be improved by combining Fas-mediated therapy with standard chemotherapeutic agents. These findings may have significant implications for prostate cancer therapy.     

新生大鼠心肌细胞原代培养方法的改良

目的探讨酶法分离新生大鼠心肌细胞,提高心肌原代细胞存活率及纯度。方法应用0.1%的Ⅱ型胶原酶消化出生第1d乳鼠的心肌组织,收集的细胞用含10%胎牛血清的DMEM培养基中和,加入DNA酶消化一次,用差速贴壁分离法分离。结果在分离培养新生大鼠心肌细胞过程中,胶原酶和DNA酶的组合使用,并改变差速贴壁时间,可得到高存活率和高纯度的心肌细胞,细胞搏动率高,持续时间久。结论本研究应用改良的新生乳鼠心肌细胞培养方法,心肌细胞存活率高,纯度高,且操作简便,重复性好,是一种较为理想的心肌细胞原代培养方法,可满足实验要求。     

Technique for the primary culture of human breast cancer cells and measurement of their prostaglandin secretion.

1. A method is described for the primary culture of human breast tumour cells on feeder layers of the STO mouse embryo fibroblast cell line. 2. The secretion of the prostaglandins E2 and F2 alpha from the cells was measured and the results indicate that the secretion of both prostaglandins was dependent on oestrogen-receptor status, with cells from oestrogen-receptor-positive tumours secreting significantly more prostaglandin than cells from oestrogen-receptor-negative tumours. 3. Prostaglandin E2, but not prostaglandin F2 alpha, secretion was also significantly greater from cells of tumours from postmenopausal women than from cells of tumours from premenopausal women. Small (< 3 cm) tumours secreted significantly more prostaglandin than large (> 3 cm) tumours, and increased levels of prostaglandin were secreted with advancing clinical stage (T1-T4). 4. Additional evidence for increased prostaglandin metabolism in oestrogen-receptor-positive tumours compared with oestrogen-receptor-negative tumours was obtained from studies on the uptake of [14C]arachidonic acid from the cultures. Significantly more labelled arachidonic acid was incorporated into cells from oestrogen-receptor-positive tumours compared with oestrogen-receptor-negative tumours, with the subsequent release of more prostaglandin in response to various stimuli.     

Apoptosis-mediated cytotoxic effects of ibandronic acid on hormone- and drug-refractory prostate cancer cells and human breast cancer cells

Over 80% of patients with advanced breast and prostate cancer ultimately develop bone metastases. Ibandronic acid has proven efficacy for treatment of bone metastasis secondary to breast cancer. This study was designed to investigate the cytotoxic and apoptotic effects of ibandronic acid on hormone- and drug-refractory prostate carcinoma DU-145 and human breast cancer MCF-7 cell lines. Cytotoxicity was evaluated using an XTT cell proliferation kit, and apoptosis was assessed by enzyme-linked immunosorbent assay (histone-DNA fragmentation) and measurement of caspase 3/7 activity. With increasing concentrations of ibandronic acid there was a dose- and time-dependent decrease in cell numbers. MCF-7 cells were more resistant than DU-145 cells (half maximal inhibitory concentrations of 122 and 90 microM, respectively). Ibandronic acid induced apoptosis in both cell lines. The study showed an apoptosis-mediated cytotoxic effect for ibandronic acid (in addition to the already known osteoclast inhibiting effect) in breast cancer patients with bone metastases; which was also observed in prostate cancer cells. Further clinical studies involving breast and prostate cancer patients with bone metastases are warranted to confirm these findings.     

Stem cells in prostate cancer initiation and progression

Peter Nowell and David Hungerford's discovery of the Philadelphia chromosome facilitated many critical studies that have led to a paradigm shift in our understanding of cancer as a disease of stem cells. This Review focuses on the application of these concepts to investigation of the role of stem cells in prostate cancer initiation and progression. Major strides in the development of in vitro and in vivo assays have enabled identification and characterization of prostate stem cells as well as functional evaluation of the tumorigenic effects of prostate cancer-related genetic alterations.     

Celebrex对雄激素非依赖性前列腺癌细胞的抑制作用

目的探讨选择性环氧化酶-2（COX-2）抑制剂Celehrex对雄激素非依赖性前列腺癌PC3细胞株的抑制作用及机制。方法体外培养人雄激素非依赖性前列腺癌细胞PC3,应用MTT法、RT-PCR和ELISA法检测Celebrex对PC3细胞生长的作用及对COX-2mRNA、前列腺素E2（PGE2）和IL-6表达的影响。结果Celebrex在一定时间和剂量范围内可明显抑制PC3细胞的生长,Celebrex无论在时间-效应或剂量-效应关系上均不影响PC3细胞COX-2mRNA的表达,但能明显减少PGE2和IL-6的表达。结论炎症因子的分泌增多可能是雄激素非依赖性前列腺癌细胞生长的动力,Celebrex对COX-2mRNA的表达无明显影响,主要是通过抑制COX-2及其下游炎症因子PGE2和IL-6等的表达而对雄激素非依赖性前列腺癌发挥抑制效应。     

Approach to primary care follow-up of patients with prostate cancer

OBJECTIVE

To review resources available to aid family physicians in their care of prostate cancer patients and develop an algorithm to summarize these findings.

SOURCES OF INFORMATION

MEDLINE, EMBASE, and relevant website search. All relevant guidelines were level III evidence.

MAIN MESSAGE

Improved screening and treatment of patients with prostate cancer is resulting in an increasing number of survivors. These men require ongoing monitoring, a responsibility that is largely falling to family physicians. We review the expected prostate-specific antigen (PSA) responses to different prostate cancer treatment modalities and provide an appropriate schedule of follow-up and monitoring techniques for prostate cancer patients.

CONCLUSION

In light of the paucity of resources for family physicians in their ongoing care of prostate cancer patients, we present an algorithm, primarily based on PSA kinetics, for practical use in the continuing care of these patients.     

Men, culture and hegemonic masculinity: understanding the experience of prostate cancer

Following a diagnosis of, and treatment for prostate cancer, there is an expectation that men will cope with, adjust to and accept the psychosocial impact on their lives and relationships. Yet, there is a limited qualitative world literature investigating the psychosocial experience of prostate cancer, and almost no literature exploring how masculinity mediates in such an experience. This paper will suggest that the experience of prostate cancer, the process by which it is investigated, and the way in which it is understood has been shaped by an essentialist interpretation of gender, exemplified by hegemonic masculinity as the archetypal mechanism of male adaptation. In response to this static and limiting view of masculinity, this paper will offer a reframe of hegemonic masculinity. This reframe, being more aligned with common experience, will portray masculinity as a dynamic and contextual construct, better understood as one of a number of cultural reference points around which each man organises and adopts behaviour. It will be suggested that the extant literature, in being organised around hegemonic masculinity, obfuscates the experience of prostate cancer and acts to render covert any collateral masculinities, public or private, that may also be operating.     

前列腺癌干细胞分离方法的对比及筛选

背景：前列腺癌干细胞是前列腺癌复发侵袭的重要原因,目前研究难点在于前列腺癌干细胞分离技术效率较低。目的：探索高效地从人前列腺癌PC-3及LNCap细胞株分离前列腺癌干细胞的方法。方法：采用含血清贴壁培养法及无血清悬浮培养法培养PC-3及LNCap细胞株,然后利用流式细胞表面标记CD133及CD44检测两种细胞在不同培养条件下可获取前列腺癌干细胞的比例,同时采用诱导分化实验初步鉴定前列腺癌干细胞特性。结果与结论：PC-3及LNCap细胞能在添加生长因子的无血清培养基中形成悬浮细胞球,接种在含血清培养基后可以诱导分化为贴壁细胞;无血清培养组的CD44^＋/CD133^＋细胞比例：PC-3为0.59%,LNCap为1.71%,含血清培养组中的CD44^＋/CD133^＋细胞比例：PC-3为0.32%,LNCap为0.73%,其中LNCap细胞采用两种方法所获的CD44^＋/CD133^＋细胞均高于PC-3所获的的细胞（P〈0.05）,在两种细胞中无血清悬浮培养和含血清贴壁培养差异无显著性意义（P〉0.05）,但无血清悬浮培养周期长,获得细胞数相对较少,直接影响分选后肿瘤干细胞功能测定。因此可以证实含血清贴壁培养LNCap细胞较无血清悬浮培养法更能高效快捷的获取前列腺癌干细胞。