参见作者原研究论文

本实验方案简略版
Jul 2017

本文章节


 

Ex vivo Culture Assay Using Human Hair Follicles to Study Circadian Characteristics
通过人类毛囊体外培养研究昼夜节律特征   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Ex vivo culture assays of biopsy specimens are advantageous for the experimental evaluation of human circadian characteristics. We developed a simple and non-invasive experimental evaluation method for monitoring the expression of circadian clock genes in an ex vivo culture assay using human hair follicles. This method imposes little burden on subjects. This assay is useful for validating correlations between circadian characteristics in hair follicles and intrinsic characteristics observed in physiological and behavioral studies. While they should be further validated, this ex vivo method constitutes a useful tool for estimating in vivo circadian characteristics.

Keywords: Circadian rhythm (昼夜节律), Clock gene (生物钟基因), Human (人类), Hair follicle (毛囊), Ex vivo (体外), Non-invasive (非侵入式)

Background

Living organisms exhibit circadian rhythms in physiology and behavior that are driven by the circadian clock (Young and Kay, 2001). The circadian clockwork consists of cell-autonomous and clock gene-driven negative feedback loops of transcription (Dunlap, 1999). In mammals, the transcription factors BMAL1 and CLOCK activate the transcription of clock and clock-related genes such as Period (Per) and Cryptochrome (Cry) via E-box elements. PER together with CRY, a potent transcriptional inhibitor, subsequently functions to negatively regulate this complex (Reppert and Weaver, 2002). The in vivo evaluation of individual intrinsic circadian characteristics in humans, either with a constant routine or forced desynchrony protocol, is expensive and labor-intensive. Therefore, evaluation using ex vivo culture assays to estimate in vivo circadian characteristics could present important advantages. For example, several studies have concluded that circadian characteristics in peripheral cells reflect individual circadian preferences, known as chronotype (Brown et al., 2005; Hida et al., 2013). For a simple and non-invasive evaluation of cell-autonomous circadian performance in humans, including intrinsic period length, we developed a method to monitor clock gene expression in real time using an ex vivo culture of hair follicles (Yamaguchi et al., 2017).

Materials and Reagents

  1. (Optional) Keep-warm bag
  2. Sterile sampling tubes (the volume size should be within 0.2 from 1.5 ml) (e.g., 0.2 ml tube, Bio-Bik, catalog number: 133003 ; 1.5 ml tube, VIOLAMO, catalog number: 1-1600-1 )
  3. 35 mm dishes (e.g., IWAKI, catalog number: 1000-035 )
  4. DMEM without Phenol Red (Sigma-Aldrich, catalog number: D2902 )
  5. DMEM (Nacalai, catalog number: 08456-94 )
    Although DMEM from other suppliers is usable as a substitute for these DMEMs, the possibility cannot be excluded that the difference in concentrations of minor ingredients affects success rates and results slightly.
  6. Sodium bicarbonate (Sigma-Aldrich, catalog number: S8761 )
  7. HEPES (Nacalai, catalog number: 17557-94 )
  8. D-Glucose (Sigma-Aldrich, catalog number: G8769 )
  9. Penicillin/streptomycin (Thermo Fisher Scientific, catalog number: 15070-063 )
  10. L-Glutamin (Nacalai, catalog number: 16948-04 )
  11. Sodium pyruvate (Sigma-Aldrich, catalog number: S8636 )
  12. Luciferin (WAKO, catalog number: 126-05116 )
  13. Silicone (Shin-Etsu, catalog number: KS-64 )
    Note: Reagents #6-13: Similar items from other suppliers serve as a substitute.
  14. Adenovirus vectors carrying the luciferase (luc) gene driven by circadian promoter/enhancer elements (e.g., Bmal1-luc, Per2-luc and Per3-luc). Some entry vectors we constructed and deposited are available from the RIKEN bank as follows:
    RDB15083 mouse Period2 (-2.8 ~ +0.1 kb) - Luciferase/pENTR-1A
    RDB15084 human Period3 (-3.1 ~ +0.2 kb) - Luciferase/pENTR-1A
    RDB15085 human Bmal1 (-1.7 ~ +0.1 kb) - Luciferase/pENTR-1A
    The ViraPower Adenoviral Gateway Expression Kit (K4940-00, Thermo Fisher Scientific) is required to generate adenoviruses using these entry vectors. Procedures using adenoviruses must be performed inside P2 areas. Autoclave and discard any infectious wastes
  15. Infection medium (see Recipes)
  16. Pre-incubation medium (see Recipes)
  17. Rinse medium (see Recipes)
  18. Luciferin-containing medium (see Recipes)

Equipment

  1. Non-slip, cosmetic-use tweezers (e.g., Shiseido, eyebrow nippers 211)
  2. Tweezers (e.g., FST, catalog number: 18132-12 )
  3. Block incubator set to 36.5 °C (e.g., ASTEC, model: BI-516C )
  4. Laminar flow cabinet (e.g., SANYO, model: MCV-B131F )
    Note: Equipment #1-4: Similar items from other suppliers serve as a substitute.
  5. Incubator at 35-36.5 °C with 5% CO2 (e.g., ASTEC, model: SCA-165DRS )
    Use a CO2 incubator that utilizes an infrared sensor that is not affected by humidity inside the chamber (see details in Procedure Step 18).
  6. Photomultiplier tube (Hamamatsu, model: LM2400 )
  7. Luminescence microscope (Olympus, model: LV200 )

Software

  1. Cosinor software (freely downloadable from a website: https://www.circadian.org/softwar.html)

Procedure

Ex vivo culture of human hair follicles to investigate circadian gene expression:

  1. Human studies must be conducted in accordance with the Declaration of Helsinki and approved by institutional review boards.
  2. Recruit subjects and obtain informed consent from each subject or the subject's family.
  3. (Optional) Although dependent on research aim, volunteers may be asked to follow a set schedule for one or several weeks. For example, wake-up time and meal times may be set based on the lifestyle habits of each volunteer. They may also be asked to refrain from consuming excess alcohol and snacks and taking long naps. Individual chronotypes can be assessed using the morningness-eveningness questionnaire (MEQ) or the Munich chronotype questionnaire (MCTQ) (Zavada et al., 2005).
  4. Prepare infection medium (see Recipes) by adding an adenovirus carrying the luciferase gene driven by circadian promoter/enhancer elements to fresh DMEM at a 1:20 dilution. The titer of our adenovirus stocks is within a range of 1.24 x 109-1.47 x 109 infectious units/ml. The use of adenovirus vectors carrying Per3-luc may be recommended (see the following note for this reason). For vector construction, we used the ViraPower Adenoviral Gateway Expression Kit (Yamaguchi et al., 2017). Briefly, together with the luciferase gene, the transcription-regulatory region of a clock gene was subcloned into the pENTR-1A entry vector and subsequently introduced into the pAd/PL-DEST vector using LR recombination.
    Note: We previously compared period length, robustness and standard deviation (SD) among three adenovirus vectors carrying Bmal1-luc, Per2-luc and Per3-luc (Yamaguchi et al., 2017). Although the average period length was almost identical among the three vectors, the SD of the period length of Per3 was consistently smaller than that of the others. Additionally, the period length of Per3 was the most robust at the smallest SD values. We therefore preferably used the adenovirus vector carrying Per3-luc in our experimental conditions.
  5. Before sampling hair follicles, prepare sterile sampling tubes containing 80 μl of pre-incubation medium, rinse medium or infection medium (see Recipes for the composition of each medium). One tube containing the respective medium is required per hair follicle. Pre-warm and maintain medium at 36.5 °C using a block incubator.
  6. (Optional) On cold winter days, warming the carotid arteries by attaching pocket warmers to both sides of the neck may improve the success rate of hair follicle sampling.
  7. As shown in Figure 1, collect several strands of scalp (or facial) hair by firmly holding and pulling individual strands of hair by the root using non-slip tweezers (for cosmetic use). To avoid hair follicle cell death by drying, quickly immerse the hair follicle cells into pre-warmed pre-incubation medium without removing the hair shaft. Keep samples at 36.5 °C until the next step (infection: Step 11). Although hair collection does not necessarily require a sterile environment, procedures after Step 11 are recommended to be performed under sterile conditions.


    Figure 1. Collection of strands of scalp hair. A. Firmly hold and pull individual strands of hair by the root using non-slip tweezers. B. Use plucked hairs whose root surface is mostly or fully covered with hair follicle cells. C. Quickly immerse hair follicle cells in pre-warmed pre-incubation medium without removing the hair shaft.

  8. Cut the hair shaft, leaving about 10 to 20 mm.
  9. To obtain a strong bioluminescence signal, use plucked scalp (or facial) hairs whose root surface is mostly or fully covered with hair follicle cells. A representative image showing successful infection of an adenovirus carrying Per2-luc to a hair root mostly covered with hair follicle cells is shown in our original paper (Figure 1B, Yamaguchi et al., 2017). We previously investigated the effect of differences in hair stage on circadian period length and found no significant differences in period length among stages (Yamaguchi et al., 2017).
  10. (Optional) In the case of out-of-lab sampling, maintain samples in a keep-warm bag without CO2 during transport to the laboratory. We confirmed that shipping duration up to approximately 10 h had no obvious effect on the circadian period length of clock gene expression.
  11. Inside a laminar flow cabinet, use sterilized tweezers to pick up the hair shaft, immerse the hair follicles into pre-warmed rinse medium, and transfer and completely immerse the follicles into pre-warmed infection medium on a block incubator (36.5 °C). Procedures using adenoviruses must be performed inside P2 areas. Autoclave and discard any infectious wastes.
  12. Close the lid of tubes and incubate them for 24 h at 36.5 °C with 5% CO2.
  13. The next day, prepare sterile sampling tubes containing pre-warmed rinse medium (at 36.5 °C) for the removal of viruses. Two tubes are required for washing twice.
  14. Syringe and push out silicone into a mound on the bottom of 35 mm culture dishes (Figure 2 and Video 1). Place the silicone somewhat off-center to facilitate locating the hair follicle at the center of the culture dish because reducing the distance between a PMT surface and infected cells enhances bioluminescence counts. Use one dish per hair follicle. Sterilization of silicone is not essential, but we recommend using silicone from an unopened packet designated for culture use. In addition, prepare and pre-warm luciferin-containing medium (see Recipes for the composition of each medium) at a concentration of 0.1 mM.

    Video 1. Placing silicone in a mound on the bottom of the culture dishes

  15. Use sterile tweezers to pick up the hair by the shaft and transfer the infected hair follicles into 80 μl of pre-warmed rinse medium. After one-minute incubation, again transfer infected hair follicles into the same volume of fresh rinse medium and incubate them for the same duration to remove adenoviruses as completely as possible.
  16. Transfer the hair follicle to the silicone mound in a 35 mm culture dish for bioluminescence monitoring (Video 2). To avoid floating during measurement, push the hair shaft into the silicone so that the shaft is stuck to the silicone and is fixed on the bottom of the dish (Figure 2). Position the hair follicle near to the center of the dish for efficient bioluminescence detection.

    Video 2. Transferring a hair follicle to a silicone mound

  17. Completely cover the immobilized hair follicle with 2-3 ml pre-warmed luciferin-containing medium.


    Figure 2. Fixing a hair follicle to the bottom of a culture dish. A. Transfer a hair follicle to a silicone mound in a 35 mm culture dish. Push the hair shaft into the silicone so that the shaft is stuck to the silicone and is fixed to the bottom of the dish. B. Position the hair follicle near to the center of the dish.

  18. As shown in Figure 3, measure bioluminescence in real-time using a photomultiplier tube inside a dark box specifically designed to reduce background noise to detect ultra-weak photon emissions ( LM2400 , Hamamatsu, Japan) or a luminescence microscope optimized for single-cell imaging (Olympus, Japan) at 35 °C with 5% CO2. To avoid rust formation, the LM2400 is located inside a CO2 culture incubator under low humidity conditions. To prevent the culture medium from drying out, instead of using the water tray from the CO2 incubator, use the one inside the LM2400 . Use a CO2 incubator that utilizes an infrared sensor that is not affected by humidity inside the chamber. For measurement, open the top of the LM2400 and simply place culture dishes on the metal tray. Culture dishes without hair follicles should provide reads of 5,000-10,000 counts per minute (background control). In successful cases, bioluminescence from the hair follicles should be greater than the background counts and show circadian oscillation over more than four to five days.


    Figure 3. Monitoring bioluminescence in real time. Bioluminescence can be measured in real time using a photomultiplier tube (top three panels, LM2400 ) or a luminescence microscope optimized for single-cell imaging (bottom three panels, LV200 ) at 35 to 36.5 °C with 5% CO2. Left panels, wide views of the instruments; middle panels, views of sample holders; right panels, views of culture dishes on the sample holders.

Data analysis

Data collection is performed using the associated software “Software for Photon Detection Unit C10749 ( LM2400 v21)-JP”. To analyze circadian parameters, baseline changes need to be removed. As shown in Figure 4, data sets are therefore detrended using Microsoft Excel by subtracting the 24 h running average from the raw data (A, raw data; B, detrended data). Circadian robustness, circadian phase (angle) and circadian period length are calculated using Cosinor software, provided as a gift by Dr. Refinetti. Oscillation data are considered reliable and useful not only when clear circadian oscillation persists for ≥ 4 days but also when circadian robustness is 70% or greater.


Figure 4. Example data. Data sets are detrended by subtracting the 24 h running average from the raw data. A. Raw data (software, Photon Detection Unit C10749 ( LM2400 v21)-JP); B. detrended data (software, Microsoft Excel).

Notes

  1. Although the absolute value of circadian period length in cultured hair follicles differs from those obtained from physiological and behavioral studies, we confirmed that the relative difference in period length among mouse genotypes is similar between clock gene expression in hair follicles and locomotor activity (Yamaguchi et al., 2017). Our ex vivo method may therefore be a useful tool for estimating in vivo circadian characteristics.
  2. Culture medium composition reportedly affects period length (Lee et al., 2011; Noguchi et al., 2012). For example, we have found that the absence of phenol red results in damping and relatively longer periods (Yamaguchi et al., 2017). It is therefore important to use identical medium composition and product lot numbers throughout all sets of experiments for comparison of relative differences between subjects.
  3. Dexamethasone (DEX) is a well-known synchronizer for peripheral clocks. Peripheral clock gene expression is often monitored in vitro and ex vivo after DEX treatment. However, adenoviral infection can also synchronize clock gene expression. To simplify the experimental protocol and reduce cellular stress, DEX treatment is therefore excluded when using adenovirus-infected hair follicles.

Recipes

  1. Infection medium (80 μl or more per hair follicle)
    DMEM (Nacalai, Japan)
    1% penicillin/streptomycin
    5% Adenovirus
  2. Pre-incubation medium (80 μl or more per hair follicle)
    DMEM without Phenol Red (Sigma-Aldrich)
    0.035% sodium bicarbonate
    10 mM HEPES
    4.5 g/L D-Glucose
    1% penicillin/streptomycin
    1 mM L-Glutamin
    1 mM sodium pyruvate
  3. Rinse medium (80 μl or more for each washing)
    DMEM (Nacalai, Japan)
    1% penicillin/streptomycin
  4. Luciferin-containing medium (2-3 ml per hair follicle)
    DMEM (Nacalai, Japan)
    1% penicillin/streptomycin
    0.1 mM luciferin

Acknowledgments

We thank Ai Yamaguchi, Akihiko Okamoto, Ritsuko Matsumura, Miho Sato, Rie Okamitsu and Junko Sumino for their expert technical assistance. We also acknowledge the support of fellowships from the Yamaguchi Gerontology Research Institute, the Akaeda Medical Research Foundation, the SENSHIN Medical Research Foundation, and the Japan Society for the Promotion of Science. The authors declare no competing financial interests. This protocol was originally developed in Yamaguchi et al., 2017.

Competing interests

The authors declare no conflicts of interest or competing interests.

Ethics

This study was conducted in accordance with the Declaration of Helsinki and was approved by the institutional review boards of Yamaguchi University (approval number: H25-81-4). Informed consent was obtained from all subjects.

References

  1. Brown, S. A., Fleury-Olela, F., Nagoshi, E., Hauser, C., Juge, C., Meier, C. A., Chicheportiche, R., Dayer, J. M., Albrecht, U. and Schibler, U. (2005). The period length of fibroblast circadian gene expression varies widely among human individuals. PLoS Biol 3(10): e338.
  2. Dunlap, J. C. (1999). Molecular bases for circadian clocks. Cell 96(2): 271-290.
  3. Hida, A., Kitamura, S., Ohsawa, Y., Enomoto, M., Katayose, Y., Motomura, Y., Moriguchi, Y., Nozaki, K., Watanabe, M., Aritake, S., Higuchi, S., Kato, M., Kamei, Y., Yamazaki, S., Goto, Y., Ikeda, M. and Mishima, K. (2013). In vitro circadian period is associated with circadian/sleep preference. Sci Rep 3: 2074.
  4. Lee, S. K., Achieng, E., Maddox, C., Chen, S. C., Iuvone, P. M. and Fukuhara, C. (2011). Extracellular low pH affects circadian rhythm expression in human primary fibroblasts. Biochem Biophys Res Commun 416(3-4): 337-342.
  5. Noguchi, T., Wang, C. W., Pan, H. and Welsh, D. K. (2012). Fibroblast circadian rhythms of PER2 expression depend on membrane potential and intracellular calcium. Chronobiol Int 29(6): 653-664.
  6. Reppert, S. M. and Weaver, D. R. (2002). Coordination of circadian timing in mammals. Nature 418(6901): 935-941.
  7. Yamaguchi, A., Matsumura, R., Matsuzaki, T., Nakamura, W., Node, K. and Akashi, M. (2017). A simple method using ex vivo culture of hair follicle tissue to investigate intrinsic circadian characteristics in humans. Sci Rep 7(1): 6824.
  8. Young, M. W. and Kay, S. A. (2001). Time zones: a comparative genetics of circadian clocks. Nat Rev Genet 2(9): 702-715.
  9. Zavada, A., Gordijn, M. C., Beersma, D. G., Daan, S. and Roenneberg, T. (2005). Comparison of the munich chronotype questionnaire with the horne-ostberg's morningness-eveningness score. Chronobiol Int 22(2): 267-278.

简介

[摘要] 活检标本的体外培养测定法有利于人体昼夜节律特征的实验评估。我们开发了一种简单而无创的实验评估方法,用于监测人发体外培养法中昼夜节律基因的表达follicles.This方法强加subjects.This测定法是用于验证昼夜CH之间的相关性有用的小负担在毛囊和固有特性在生理和行为studies.While观察aracteristics它们应该被进一步验证,该离体方法用于小的有用工具体内估计 昼夜节律特征。

[背景] 活的生物体表现出在生理和行为的昼夜节律由生物钟驱动(杨和Kay,2001)。该昼夜发条由转录的细胞自主性和时钟基因驱动的负反馈环路(邓拉普,1999 )。在哺乳动物中,转录因子BMAL1和CLOCK 通过E-box元件激活时钟和与时钟相关的基因(例如Period (Per )和Cryptochrome (Cry ))的转录.PER与有效的转录抑制剂CRY一起起作用负性调节这一复杂(里珀特织女,2002年)。在体内评估个体的内在节律特点在人类,要么以恒定的例行或强制去同步化协议,价格昂贵,耗力。因此,评估利用离体培养试验中要估计体内的昼夜节律特征可能具有重要的优势。例如,一些研究已经结束,n个外周细胞反映了个体的昼夜节律偏好,称为计时型(Brown 等人,2005; Hida 等人,2013 )。为了简单,无创地评估人类细胞自主性昼夜节律的表现,包括内在周期长度,我们他开发了一种利用离体毛囊培养物实时监测时钟基因表达的方法(Yamaguchi 等,2017 )。

关键字:昼夜节律, 生物钟基因, 人类, 毛囊, 体外, 非侵入式

材料和试剂


 


(可选)保暖袋
取样管无菌(吨他体积大小不应该从1.5米BE在0.2 升)(例如,0.2米升管,生物碧,目录号的:133003 1.5 米升管,VIOLAMO ,目录号的:1-1600-1)
35mm培养皿(例如,IWAKI,目录号:1000-035)
不含酚红的DMEM(Sigma-Aldrich,目录号:D2902)
DMEM(Nacalai ,目录号:08456-94)
尽管可以使用其他供应商提供的DMEM替代这些DMEM,但不能排除次要成分的浓度差异会影响成功率和结果的可能性。


碳酸氢钠(Sigma-Aldrich,目录号:S8761)
HEPES(Nacalai ,目录号:17557-94)
D-葡萄糖(Sigma-Aldrich,目录号:G8769)
青霉素/链霉素(Thermo Fisher Scientific,目录号:15070-063)
L- 谷氨酰胺(Nacalai ,目录号:16948-04)
丙酮酸钠(Sigma-Aldrich,目录号:S8636)
萤光素(WAKO,货号:126-05116)
矽胶(信越(Shin-Etsu),目录号:KS-64)
注意:试剂#6 -1 3 :其他供应商的类似物品代替。


携带萤光素酶(腺病毒载体LUC )基因通过昼夜启动子驱动/增强子元件(例如,BMAL1-LUC ,Per2基因-luc的和的Per3-luc的),我们构建和沉积。有的进入载体可从RIKEN银行如下:
小鼠RDB15083 Period2 (-2.8〜Tasu0.1 Kb)- 荧光素酶/ PENTR-1A


人RDB15084 Period3 (-3.1〜Tasu0.2 Kb)- 荧光素酶/ PENTR-1A


人RDB15085 Bmal1 (-1.7〜Tasu0.1 Kb)- 荧光素酶/ PENTR-1A


需要使用ViraPower 腺病毒网关表达试剂盒(K4940-00,Thermo Fisher Scientific)才能使用这些进入载体生成腺病毒,必须在P2区域内进行使用腺病毒的程序高压灭菌并丢弃任何传染性废物。


感染介质(请参见食谱)
预培养培养基(请参见食谱)
冲洗介质(请参阅食谱)
含萤光素的培养基(请参见食谱)
 


设备


 


防滑,COS metic使用镊子(例如,资生堂,眉钳211)
镊子(例如,FST,目录号:18132-12)
块培养箱设置为36.5°C(例如,ASTEC,型号:BI-516C)
层流室中(例如,三洋,型号:MCV-B131F)
注意:设备1-4:其他供应商的类似物品可以替代。


在35-36.5°C和5%CO 2的培养箱中(例如,ASTEC,型号:SCA-165DRS)
使用CO 2 培养箱,该培养箱应安装一个不受箱内湿度影响的红外传感器(请参阅步骤18中的详细信息)。


光电倍增管(滨松,型号:LM2400)
发光显微镜(奥林巴斯,型号:LV200)
小号oftware


 


Cosinor 软件(可从网站免费下载:https : //www.circadian.org/softwar.html)。
 


程序


 


体外人类毛囊的文化研究节律基因expressio ñ :


人体研究必须按照《赫尔辛基宣言》进行并得到机构审查委员会的批准。
招募受试者并获得每个受试者或受试者家庭的知情同意。
(可选)尽管取决于研究目标,但可能会要求志愿者遵循既定的计划安排一到几周的时间,例如,可以根据每个志愿者的生活习惯来设置起床时间和进餐时间,或者问从消耗过量的醇和小吃和采取长小睡不要。个别chronotypes可以使用评估克清晨型-eveningness 问卷(MEQ)或慕尼黑chronotype问卷(MCTQ )(Zavada 等人。,2005)。
通过将含有由昼夜节律启动子/ 增强子元件驱动的荧光素酶基因的腺病毒以1:20的稀释度添加到新鲜的DMEM中来制备感染培养基(请参见食谱),我们的腺病毒原液的滴度在1.24 x 10 9 -1.47 范围内×10 9 感染单位/ M 大号。使用腺病毒载体携带的Per3吕克可能被推荐(小号的Ee 下面的N 大手这个原因)。载体构建,我们使用的ViraPower 腺病毒网关表达试剂盒(山口等。,2017)。简而言之,将时钟基因的转录调节区域与萤光素酶基因一起亚克隆到pENTR-1A进入载体中,然后通过LR重组引入pAd / PL-DEST载体中。
注意:我们之前比较了三种携带Bmal1-luc,Per2-luc和Per3-luc的腺病毒载体的周期长度,稳健性和标准差(SD)(Yamaguchi et al。,2017),尽管平均周期长度几乎相同这三个载体的Per3的周期长度的SD始终小于其他载体,另外,Per3的周期长度在最小的SD值时最稳健,因此我们优选使用携带Per3-luc的腺病毒载体我们的实验条件。


在对毛囊进行采样之前,请准备一个无菌采样管,其中应包含80 Myu L 的预培养培养基,冲洗培养基或感染培养基(每种培养基的成分请参见配方),每个毛囊需要一根包含相应培养基的试管。并使用分块培养箱将培养基保持在36.5°C。
(可选)在寒冷的冬季,通过在颈部的两侧安装口袋加热器来加热颈动脉可以提高毛囊采样的成功率。
如图1所示,用防滑镊子(美容用)牢固地握住并拉动根部的单根头发,收集几根头皮(或面部)头发。为避免干燥导致的毛囊细胞死亡,请快速浸泡将毛囊细胞放入未预热的预温育培养基中,不移开发干,将样品保持在36.5°C直至下一步(感染:步骤11)。尽管收集头发不一定需要无菌环境,但步骤11 之后的程序建议在无菌条件下进行。
 






1图。收集绞股Ø ˚F头皮。A.紧握并使用防滑镊子取出每个股的头发逐根。B. 用弹拨毛发的根表面大部分或完全覆盖着毛囊细胞。C.将毛囊细胞快速浸入预热的预温育培养基中,而无需去除毛干。


 


剪掉发干,留下约10至20毫米。
为了获得强的生物发光信号,请使用拔毛的头皮(或面部)毛,其根表面大部分或完全被毛囊细胞覆盖。代表性图像显示成功携带Per2-luc 的腺病毒感染到大部分被毛囊覆盖的发根上细胞在我们的原始论文中显示(图1B,Yamaguchi 等人,2017)。我们之前研究了毛发阶段的差异对昼夜节律周期长度的影响,发现阶段之间的周期长度没有显着差异(Yamaguchi 等人,2017)。)。
(可选)在实验室外采样的情况下,将样品运输到实验室时应将样品放在不带CO 2 的保暖袋中。我们确认,运输时间长达10小时对昼夜周期长度没有明显影响时钟基因表达。  
在层流柜内,使用消毒镊子拾起发干,将毛囊浸入预热的漂洗介质中,然后将毛囊转移并完全浸入块温箱(36.5°C)的预热感染介质中。使用腺病毒的程序必须在P2区域内进行。高压灭菌并丢弃任何传染性废物。
关闭管盖,在36.5°C和5%CO 2 下孵育24小时。
第二天,为制备含预温冲洗介质的无菌取样管(在36.5℃)的祛瘀的人viruses.Two 管所必需的洗涤两次。
注射器并将硅酮推入35毫米培养皿底部的土墩中(图2和视频1)。将硅酮稍微偏心放置,以利于将毛囊定位在培养皿的中央,因为这可以缩短毛囊之间的距离PMT表面和受感染的细胞会增加生物发光计数。每个毛囊只能使用一碟。有机硅的灭菌不是必不可少的,但是我们建议使用未开封包装中指定用于培养的有机硅。此外,准备并预热含萤光素的培养基(参见浓度为0.1 mM 的每种培养基的配方。
 






视频1.在培养皿底部的土堆中放置有机硅


 


使用无菌镊子通过杆柄拾起头发并转移毛囊感染了 80 兆升的预热冲洗液,孵育一分钟后再次转移 将感染的毛囊放入相同体积的新鲜漂洗介质中,并孵育相同的时间,以尽可能完全地去除腺病毒。
将毛囊转移到35毫米培养皿中的硅土堆中以进行生物发光监测(视频2)。为避免在测量过程中漂浮,请将发干推入硅树脂中,以便将发干棒固定在硅树脂上并固定在底部将毛囊放置在靠近培养皿中心的位置,以进行有效的生物发光检测。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-6-5 \ 3638--0101776 MakotoAkashi 552046 \ video 2.jpg


视频2 Transfe [R 环毛囊硅丘


 


COMPLE Tely盖固定化毛囊随着2-3米大号预热荧光素物的培养基。




图2 。Fixin G A毛囊底部培养皿。A.转移的毛囊硅土墩在35毫米的培养皿中。按发干到硅,从而轴粘在有机硅和被B.将毛囊放置在靠近盘子中央的位置。


 


如图3所示,measu 重新生物发光实时- 时间使用特别设计,以减少背景噪声,以检测超弱光子发射(LM2400,滨松,日本)暗箱内的光电倍增管或用于单细胞优化的发光显微镜在5%CO 2 下于35°C进行成像(日本奥林巴斯)。为避免生锈,LM2400位于低湿度条件下的CO 2 培养箱内。为了防止培养基变干,请不要用水从CO托盘2 培养箱中,使用LM2400.Use内的一个在CO 2 孵化器中提取未受到chamber.For测量内部湿度的红外线传感器,打开LM2400的顶部和简单地地方培养皿金属托盘:无毛囊的培养皿每分钟读数应为5,000-10,000 (背景对照)。成功的情况下,毛囊的生物发光应大于背景计数和 超过四到五天的昼夜节律振荡。








图3 。监测生物发光的实时性。生物发光,可以实时测量使用光电倍增管(顶三面围板,LM2400)或荧光显微镜优化了对单细胞成像(底三面围板,LV200)35至36.5℃ 5%的CO 2。左面板,仪器的视角;中间面板,样品架的视角;右面板,样品架上的培养皿的视角。


 


数据分析


 


使用关联的软件“用于光子检测单元的软件C10749(LM2400v21)-JP”执行数据收集,要分析昼夜节律参数,需要删除基线变化,如图4所示,因此使用Microsoft Excel对数据集进行去趋势处理,方法是减去在24 h的速度行驶从原始数据平均值(A,原始数据; B,去趋势数据)。昼夜鲁棒性,昼夜节律阶段(角度)和昼夜周期长度是使用计算余弦软件,如由博士馈赠提供Refinetti .Oscillation 数据不仅在昼夜节律振荡持续≥4天时,而且在昼夜节律鲁棒性为70%或更高时,都被认为是可靠且有用的。


 






4图。实施例数据。数据集去趋势通过减去24 H运行平均从原始的DAT A A。 。原始数据(软件,光子检测单元C10749(LM2400v21 -jP)); B 。除趋势数据(软件,Microsoft Excel中)。


 


笔记


 


尽管培养的毛囊中昼夜节律周期长度的绝对值与生理和行为研究获得的值不同,但我们证实了小鼠基因型的周期长度的相对差异在毛囊中的时钟基因表达与运动能力之间是相似的(Yamaguchi et al。等人,2017)。因此,我们的离体方法可能是估计体内昼夜节律特征的有用工具。
据报道,培养基的组成会影响周期的长短(Lee 等,2011; Noguchi 等,2012),例如,我们发现酚红的缺失会导致阻尼和较长的周期(Yamaguchi 等,2017)。因此,在所有实验组中使用相同的培养基成分和产品批号对于比较受试者之间的相对差异非常重要。
地塞米松(DEX)是外围时钟的著名同步器,在DEX处理后,通常在体外和离体监测外围时钟基因的表达,但是,腺病毒感染也可以同步时钟基因的表达,从而简化了实验方案并减轻了细胞压力因此,当使用腺病毒感染的毛囊时,排除DEX治疗。
 


菜谱


 


中度感染(每个毛囊80 Myu L 或更多)
DMEM(日本Nacalai )


1%青霉素/链霉素


5%腺病毒


中温孵化(每个毛囊80 Myu L 或更多)
不含酚红的DMEM(Sigma-Aldrich)


0.035%碳酸氢钠


10毫米HEPES


4.5克/ 升D-葡萄糖


1%青霉素/链霉素


1 毫米L- 谷氨酰胺


1 mM丙酮酸钠


中度漂洗(每次洗涤80 Myu L 或更多)
DMEM(日本Nacalai )


1%青霉素/链霉素


含萤光素的培养基(2-3 毫升升的毛囊)
DMEM(日本Nacalai )


1%青霉素/链霉素


0.1 mM萤光素


 


致谢


 


我们感谢山口爱依(Ai Yamaguchi),冈本明彦(Akihiko Okamot O),松村梨子(Ritsuko Matsumura),佐藤美穗(Miho Sato),冈上理惠(Rie Okamitsu)和纯野纯子(Junko Sumino)的专业技术援助,并感谢山口老年医学研究所,赤田医学研究基金会,SENSHIN Me Dical的研究金支持。研究基金会和日本科学促进协会,作者宣称没有竞争的经济利益,该协议最初是由Yamaguchi 等人于2017年开发的。


 


利益争夺


 


作者声明没有利益冲突或利益冲突。


 


伦理


 


这项研究是根据赫尔辛基宣言进行,并批准了山口大学的机构审查委员会(批准numbe [R :H25-81-4).Informed 所有受试者均获得同意。






参考文献


布朗,SA,佛罗里达州,奥莱拉,F.,Nagoshi ,E.,Hauser,C.,Juge ,C。,Meier,CA,Chicheportiche ,R。,Dayer ,JM,Albrecht ,U. 和Schibler ,U。 2005)。成纤维细胞昼夜节律基因表达的周期长度在人类之间差异很大(《公共科学图书馆· 生物学》3(10):e338。
Dunlap,JC(1999)。生物钟的分子基础,细胞96(2):271-290。
飞弹,A。,北村,S.,大泽,Y。,榎本,M.,片寄,Y。,本村,Y。,森口,Y。,野崎,K.,渡边,M.,Aritake ,S。, Higuchi,S.,Kato,M.,Kamei,Y.,Yamazaki,S.,Goto ,Y.,Ikeda,M. and Mishima,K.(2013)。体外昼夜节律与昼夜节律/睡眠偏好有关。科学代表3:2074。
李,SK,阿钦,E.,马多克斯,C.,陈,SC,Iuvone ,PM和福原,C。(2011)。细胞外低pH会影响人类初级成纤维细胞的昼夜节律表达。生物化学生物物理学RES COMMUN 416(3- 4):337-342。
Noguchi,T.,Wang,CW,Pan,H.和Welsh,DK(2012)。PER2表达的成纤维细胞昼夜节律取决于膜电位和细胞内钙。Chronobiol Int 29(6):653-664。
Reppert ,SM和Weaver,DR(2002)。哺乳动物昼夜节律的协调。自然418(6901):935-941。
Yamaguchi,A.,Matsumura,R.,Matsuzaki ,T.,Nakamura,W.,Node,K. and Akashi,M.(2017)。一种简单的方法,利用离体培养的毛囊组织研究内在的昼夜节律特征人类。科学代表7(1):6824。
Young,MW和Kay,SA(2001)。时区:生物钟的比较遗传学。Nat Rev Genet 2(9):702-715。
Zavada 。,A,Gordijn ,MC,Beersma ,DG,大安,S和。Roenneberg ,T(2005)。在比较慕尼黑与霍恩-ostberg的清晨型-eveningness得分chronotype问卷。 Chronobiol 诠释22(2):267 -278。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nishida, A., Miyawaki, Y., Node, K. and Akashi, M. (2020). Ex vivo Culture Assay Using Human Hair Follicles to Study Circadian Characteristics. Bio-protocol 10(11): e3638. DOI: 10.21769/BioProtoc.3638.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。