这项研究是由美国国立癌症研究所（R01-94160 R01-155086），IU西蒙癌症中心和富国儿科研究中心支持。

IACUC委员会已批准在美国印第安纳大学医学院的这项研究。 1。生成正确的基因型的小鼠<ol><li>建立基底细胞癌的小鼠模型。的队友K14-CREER的小鼠（杰克逊实验室股票号005107）YFP的小鼠R26-SmoM2（杰克逊实验室股票号005130）（18）产生K14-CREER的+和R26-SmoM2的YFP +小鼠。 </li><li>进行基因分型。沉浸尾标本1 mg / ml的蛋白酶K在directPCR尾裂解液（0.5厘米长），在55℃振荡过夜。第二天，通过离心分离除去组织碎片在微型离心机中以最快的速度（保持上清液用于基因分型）。每个PCR反应中的引物和由供应商推荐的条件下，使用1微升上层清液。选择正确的基因型（年龄为3-6周，丁丙诺啡在0.05-0.1毫克/公斤，将被应用于通过SQ每8-12小时后2-3天尾剪断）FO小鼠步骤2ŗ。 </li></ol> 2。诱导表达SmoM2皮肤角质通过喂食针灌胃（弯曲20计）连续5天口服他莫昔芬（5微克/公斤体重每次喂奶50毫升的植物油）角质形成细胞诱导SmoM2的表达。 3。准备皮肤标本在一般情况下，表型在GFP小鼠K14creER/R26SmoM2只耳朵和尾巴上更严重。为了准备细胞分析，我们首先收获小鼠皮肤动物牺牲后与机构的批准过程（CO 2，加上颈椎脱位）。 4。隔离表皮<ol><li>经过牺牲小鼠批准的程序（见第3步），除去毛皮使用一个微调。在分散酶溶液（5毫克/毫升的含10％FBS的DMEM）在37℃下1-2小时（1小时少于8周龄和2小时的小鼠的皮肤浸入试样老鼠老大于8周）。 </li><li>中性蛋白酶处理后，表皮钳真皮分开。 </li></ol> 5。产生单细胞<ol><li>要获得单细胞群体，肉表皮用剪刀，然后沉浸在胶原酶溶液（1毫克/毫升含10％FBS）为1-2小时，在37°C在50毫升管。试着轻轻地旋转管每20分钟，以帮助细胞解离。 </li><li>在显微镜下检查细胞解离。当单细胞胶原酶介质中可以检测到，孵育样品需额外的30分钟，以提高细胞产量。在一般情况下，从一个鼠标的表皮可以产生高达10 7个细胞。 </li><li>过滤通过细胞过滤器（孔径70微米），自旋细胞组织液通过台式离心1500转，并用PBS洗一次时间。 </li></ol> 6。标签细胞进行流式细胞仪分析<ol><li>重悬细胞在10％FBS的在PBS中30分钟，在2.5×10 6个细胞/ ml，以阻断非特异性结合（在冰上） </li><li>径各1.5毫升的微管中的特定抗体标记细胞的等分试样。每个试管中添加不同的抗体，使最终浓度为2微克/毫升。混合抗体，细胞通过一个温柔的顶点几秒钟，让他们在冰上30分钟，然后用1毫升PBS洗。 BCC标本，我们采用了以下的生物标志物CD11b的PE加Gr1的APC（髓细胞或髓源性抑制细胞）;波形蛋白-FITC（成纤维细胞）（见试剂细节）抗体。 </li><li>细胞内染色，我们使用了一个工具包，并按照厂商的说明书（见试剂细节）。我们用新鲜的细胞，分析细胞表面标志。表面标记物染色后，洗涤细胞，并在PBS重悬。加入DAPI染色的细胞混合物中的终浓度为1微克/毫升。 DAPI染色死细胞，将门从进一步的分析。 </li><li>分析具体数据C软件。我们首先选择根据FSC与SSC情节的单细胞，并用DAPI人口负（活细胞）作进一步的分析。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

我们已经描述了一种用于在基底细胞癌的细胞群体分析。肿瘤组织中的许多细胞类型相一致的，不同的细胞类型不同的行为的发生在给定时间，这是非常困难的，即使在体外条件下的最夺回。使用此协议，我们发现，基底细胞癌的发展是伴随着扩张的表皮干细胞以及招聘髓细胞。比较，与免疫组织化学或免疫荧光分析，细胞种群分析更定量评估到多种细胞类型特异性抗体的细胞群的变化，因为现在可用。 对于这个协议中，需要完成10周诱导基因的表型变化的研究，因为需要时间。因此，它是重要的规划实验的时间提前。细胞的分离和分析，可能需要整整两天。由于用于活细胞分析产品上课，重要的是，以预定的时间提前一个FACSCALIBUR。我们还列出了常见问题的工作时，这个协议（见下文）。 根据我们的经验，下面是我们遇到的常见问题: <ol><li>分离表皮差:这可能是由于不完整的中性蛋白酶治疗（请增加时间diapase治疗）或的中性蛋白酶不足的解决方案（皮肤需要完全沉浸在中性蛋白酶溶液）。我们使用中性蛋白酶溶液5毫升至少一个鼠标。该卷可能会发生变化，根据皮肤的大小。 </li><li>手机号码:这可能由于胶原酶消化不完全的低收益率。请首先检查的胶原酶浓度或到期日。这也可以使用不足量的表皮造成的。此外，应进行酶消化至37℃振荡（请在调整进行消化之前的温度）。 </li><li>细胞团块:这是很常见的皮肤细胞（移液器细胞了几次后，通过过滤器（离心之前），或存在的Mg 2 +和Ca 2 +的溶液中的（请使用镁2 +与Ca 2 +的PBS洗涤细胞）。 </li><li>太多的死细胞退化的组织，这可能是一个结果（请使用新鲜组织），或过度消化（避免扩展潜伏期与中性蛋白酶和胶原酶）。 </li></ol>这个协议可以结合骨髓移植或组织移植研究的细胞来源。例如，移植的GFP表达到致死剂量照射小鼠的骨髓细胞，让我们检查骨髓细胞肿瘤相关成纤维细胞和骨髓细胞的贡献。同样，皮肤肿瘤可以移植到一个新的鼠标主机检查肿瘤 - 宿主相互作用。 除了检测细胞癌变过程中的人口变化，该协议也将允许研究人员，研究药物治疗的效果，肿瘤环境中的细胞。虽然这个协议被设计为使用在皮肤的细胞群体分析癌变，稍加修改就可以对其他类型的癌症。 综上所述，皮肤癌小鼠模型中的细胞群体分析可以给我们的动态细胞癌变过程的变化，从而更好地了解细胞生物学，肿瘤转化，在转化过程中不同的细胞活动。

基底细胞癌（BCC）作为最常见的类型的人类癌症，影响约200万美国人每年1。越来越多的证据表明，刺猬（Hh）信号异常激活相关:BCC的发展（2,3）的动力。 Hh信号在基底细胞癌的改变包括失活突变的PTCH1 4-6，SMO 7-9增益功能的突变，异常表达Hh信号通路的转录因子GLI1 10 GLI2 11或负调节素（富罕见的灭活突变）12。通过所有这些和其他研究，美国联邦药物管理局（FDA）近日批准使用的Hh信号抑制剂Vismodegib治疗转移性和局部晚期基底细胞癌13-15。 尽管所有这些成就16，我们仍然不明白Hh信号驱动致癌物的分子和细胞机制inogenesis。建立动物模型，利用组织特异性激活Hh信号仍然是重要的，这些研究对于我们理解的耐药性。在小鼠中，野生型小鼠不发展基底细胞癌，即使在大量的致癌物质，紫外线或电离辐射的剂量。 PTCH1 + / -小鼠与此相反，容易受到到BCC开发17,18的影响 。 PTCH1 BCC发展渗透率+ / -小鼠是超过50％，18,19虽然PTCH1 + / -小鼠很少发展为完全成熟的基底细胞癌，如果在正常情况下保持。 PTCH1胚胎致死- / - ，组织特异性的基因敲除PTCH1通常用于研究20。此外，有条件的皮肤特异表达的致癌:YFP SmoM2（KRT14 CREER:中R26-SmoM2 YFP或KRT14-CRE:R26-SmoM2 YFP）导致多个微观基底细胞癌的形成在一个非常早期的年龄，提供一个简单的基因检测Hh信号SMO 21 wnstream。 主要有三个问题，我们的知识BCC生物学。首先，基底细胞癌的细胞起源目前尚不完全清楚。虽然一些研究支持毛囊干细胞网站22-24让步，优素福等 25个本地化的小鼠皮肤HH-驱动肿瘤起源细胞在滤泡间表皮区域，而不是在毛囊通过采用细胞特异性表达Cre重组酶的激活表达的一个驱动ROSA26的转基因突变SMO。二，细胞相互作用BCC发展过程中不能很好地理解。它是已知的，角质形成细胞的激活Hh信号。可以导致基底细胞癌的形成，但不理解其他细胞的变化。此外，治疗的SMO拮抗剂在小鼠可以导致耐药26-28。 BCC因此，新的目标，是非常必要的。了解这三个问题，需要在小鼠的细胞变化分析carcinog香根草。角质细胞培养，流式细胞术及皮肤干细胞分离29-31虽然已经出版的几种方法，目前还没有完备的程序在小鼠基底细胞癌的细胞分析。基底细胞癌的小鼠模型，分离表皮，产生的单细胞组织和细胞分析相结合的方法，我们会为读者提供了一套程序来研究细胞信号传导，细胞和分子间的相互作用，在BCC发展。我们相信，这个协议将使读者看到每个过程是如何完成的。此外，我们还提供了一些数据来说明什么样的结果应该看起来像，故障排除，以帮助读者克服困难，在执行这些程序。

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cell population analyses during skin carcinogenesis

癌症的发展是一个多步骤的过程，涉及到许多类型的细胞，包括肿瘤细胞前体细胞，免疫细胞，成纤维细胞和内皮细胞。每种类型的细胞癌变过程中，经过信令和功能上的变化。许多癌症研究人员目前的挑战是剖析这些变化在多步骤的过程中体内不同的细胞类型。在过去的几年中，作者已经开发了一套程序，来隔离不同的细胞群，在皮肤癌的发展过程中，使用YFP的小鼠K14creER/R26-SmoM2只。的程序被划分成6份:1）生成适当的小鼠为研究对象（+与R26-SmoM2 YFP +老鼠在这个协议中）; 2）诱导SmoM2 YFP的表达在小鼠皮肤中; 3 K14creER）制备小鼠皮肤活组织切片检查; 4）隔离从皮肤的表皮; 5）制备单细胞从表皮; 6）标签流式细胞仪分析单细胞群。生成足够数量的合适的基因型的小鼠，在这个协议中的限制步骤，这可能需要长达两个月。其余的步骤需要几个小时到几天。在这个协议中，我们还包括一节进行故障排除。虽然我们侧重于皮肤癌，该协议可以被修改，以适用于其他的人类疾病的动物模型。

它是已知的小鼠没有发展基底细胞癌，除了与Hh信号激活。 图1显示了平滑化的致癌基因的诱导表达后的皮肤的表型，SmoM2 YFP 图2显示了在正常和荷瘤小鼠的骨髓细胞分析。细胞CD11b + Gr1的+细胞是从骨髓细胞，其中一些是髓源性抑制细胞。在正常情况下，细胞CD11b + GR1 +细胞群体是很难察觉，但会增加炎症或肿瘤的发展。细胞CD11b + Gr1的+细胞增加，我们在皮肤结果表明，诱导表达的SmoM2YFP的。目前，这种变化在基底细胞癌的分子机制尚不清楚。 <img alt="图1" src="/files/ftp_upload/50311/50311fig1.jpg" /> 图1。 Morphologica升后诱导小鼠皮肤SmoM2YFP。小鼠基因型的顶部示出了示。他莫昔芬治疗K14creER +和R26 SmoM2的YFP +鼠标中间面板:显示严重异常（右）在皮肤以及皮肤肿瘤的H＆E部分（左）。底部面板的鼠标没有SmoM2YFP感应显示正常的皮肤（右）与H＆E段（左）无异常形态。 <img alt="图2" src="/files/ftp_upload/50311/50311fig2.jpg" /> 图2。流分析的CD11b + Gr1的+细胞的CD11b + Gr1的细胞 ，为了研究本皮肤癌的发展过程中，我们分离单细胞和染色的荧光标记的抗体CD11b和Gr1的。 FlowJo分析后，我们发现这种细胞人口增长从他莫昔芬治疗的小鼠皮肤组织。

Watch this Scientific Journal Video about Cell Population Analyses During Skin Carcinogenesis at JoVE.com

Cell Population Analyses During Skin Carcinogenesis

癌变是一个涉及癌细胞和肿瘤微环境之间的相互作用的方法。要解剖的分子事件，一个需要，隔离癌症的发展过程中的不同阶段的不同的细胞群。利用小鼠模型基底细胞癌癌变过程中，我们描述了一个协议，用于细胞分析。

在皮肤癌变细胞群分析

Cancer development is  a multiple-step process involving many cell types including cancer precursor  cells, immune cells, fibroblasts and endothelial ...

cell-population-analyses-during-skin-carcinogenesis

Research

JoVE Journal

Medicine

12.3K 次观看.  Indiana University. 癌变是一个涉及癌细胞和肿瘤微环境之间的相互作用的方法。要解剖的分子事件，一个需要，隔离癌症的发展过程中的不同阶段的不同的细胞群。利用小鼠模型基底细胞癌癌变过程中，我们描述了一个协议，用于细胞分析。

视频: Cell Population Analyses During Skin Carcinogenesis

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

使用全血刺激试验的促炎性细胞因子IL -1β的精确和简单的测量

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

科研

医学

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

肾小囊和异种移植皮下颗粒植入前列腺癌发生和良性前列腺增生症的评价

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

干生生物标志物的血斑收藏最大限度地参与人口研究

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

在前列腺癌的临床组织中miRNA的表达分析

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

A Method of Trigonometric Modelling of Seasonal Variation Demonstrated with Multiple Sclerosis Relapse Data

This report describes a novel Stata-based application of trigonometric regression modelling to 55 years of multiple sclerosis relapse data from 46 clinical centers across 20 countries located in both hemispheres. Central to the success of this method was the strategic use of plot analysis to guide and corroborate the statistical regression modelling. Initial plot analysis was necessary for establishing realistic hypotheses regarding the presence and structural form of seasonal and latitudinal influences on relapse probability and then testing the performance of the resultant models. Trigonometric regression was then necessary to quantify these relationships, adjust for important confounders and provide a measure of certainty as to how plausible these associations were. Synchronization of graphing techniques with regression modelling permitted a systematic refinement of models until best-fit convergence was achieved, enabling novel inferences to be made regarding the independent influence of both season and latitude in predicting relapse onset timing in MS. These methods have the potential for application across other complex disease and epidemiological phenomena suspected or known to vary systematically with season and/or geographic location.

Combining plot analysis with trigonometric regression is a robust method for exploring complex, cyclical phenomena such as relapse onset timing in multiple sclerosis (MS). This method enabled unbiased characterisation of seasonal trends in relapse onset permitting novel inferences around the influence of seasonal variation, ultraviolet radiation (UVR) and latitude.

季节变化的三角建模的方法证明与多发性硬化症复发数据

This report describes a novel Stata-based application of trigonometric regression modelling to  55 years of multiple sclerosis relapse data from 46 ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

皮肤去神经外科手术:一种用于皮肤病的小鼠模型测试要求的神经方法

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

Assessment of Child Anthropometry in a Large Epidemiologic Study

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to understand health effects of child growth and adiposity. Gold standard methods of measuring adiposity, such as dual X-ray absorptiometry (DXA), may not be feasible in large field studies. Research staff can, however, complete anthropometric measurements, such as body circumferences and skinfold measurements, using inexpensive portable equipment. In this protocol we detail how to obtain manual anthropometric measurements from children, including standing and sitting height, weight, waist circumference, hip circumference, mid-upper arm circumference, triceps skinfold thickness, and subscapular skinfold thickness, and procedures to assess the quality of these measurements. To demonstrate accuracy of these measurements, among 1,110 school-aged children in the pre-birth cohort Project Viva we calculated Spearman correlation coefficients comparing manual anthropometric measurements with a gold standard measure of body fat, DXA fat mass1. To address reliability, we evaluate intra-rater technical error of measurement at a quality control session conducted on adult female volunteers.

In epidemiologic studies of children, well-trained research staff can accurately and precisely assess weight, height, sitting height, skinfold thicknesses, and body circumferences.

大型流行病学研究儿童人体测量评估

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to ...