Transplantation of Fecal Microbiota Shaped by Diet
饮食经控制的粪便微生物群的移植   

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Nature Immunology
Apr 2017

 

Abstract

Alterations in diet and gut microbial ecology underlie the pathogenesis of type 1 diabetes (T1D). In the non-obese diabetic (NOD) mouse, we found high concentrations of bacterial metabolites acetate and butyrate in blood and faeces correlated with protection from disease. We reconstituted germ free (GF) NOD mice with fecal bacteria from protected NOD mice fed with high acetate- and butyrate-yielding diets, to test whether the transferred gut microbiota protect against the development of T1D. GF NOD mice that received a microbiota shaped by high acetate- but not butyrate-yielding diet showed a marked protection against diabetes. This fecal transplantation assay demonstrated the potential for a dietary technology to reshape the gut microbiota that enables specific bacteria to transfer protection against T1D.

Keywords: Gut microbiota (肠道微生物群), Bacteria reconstitution (细菌重组), Oral gavage (经口灌胃), Germ-free NOD mice (无菌NOD小鼠), Disease Incidence (疾病发生率), Type 1 diabetes (1型糖尿病)

Background

Changes in the gut microbiota have been observed in a wide variety of illnesses and conditions. A dysbiotic gut microbiota loses homeostatic balance due to changes in the ratios between commensal and pathogenic bacteria. Dysbiosis can be observed when there are large changes in the makeup of the microbiota, with certain species increasing or decreasing in number (Clemente et al., 2012; Rajilic-Stojanovic, 2013). Moreover changes in gut microbiota composition (which may affect metabolites such as SCFAs) associate with many inflammatory diseases (Clemente et al., 2012), including T1D (de Goffau et al., 2013; Endesfelder et al., 2014). For example, patients with, or people who are predisposed to, autoimmune type 1 diabetes typically show a decrease in Firmicutes abundance and an increase in their Bacteroidetes abundance (Giongo et al., 2011). In contrast, various types of inflammatory bowel diseases (IBD), such as ulcerative colitis or Crohn’s disease show the opposite (Frank et al., 2007; Spor et al., 2011). One possibility for treatment of T1D is the use of beneficial bacteria, following their identification and successful trialing. For example, Lactobacillus johnsonii isolated from diabetes resistant rats was able to prevent T1D development in the spontaneous rat model of T1D (Valladares et al., 2010). Likewise, transfer of microbiota from male mice, who are less prone to develop T1D, to female mice reduced their rates of T1D, which correlated with changes in the mouse’s hormone levels (Markle et al., 2013). We used diets that reshape the gut microbiota composition and induced the release of microbial short chain fatty acids (SCFAs). In this study, we have demonstrated that particularly the SCFA acetate and butyrate reduced the onset of T1D in NOD mice (Marino et al., 2017). We wanted to determine if the protective effect was coming directly from the bacteria, or by the produced microbial SCFA acetate or butyrate. We therefore have developed a protocol to harvest the microbiota from diet-fed NOD mice, and transfer it to GF NOD mice, so that we can examine the effects of the altered microbiota and its metabolites on the pathogenesis of T1D.

Materials and Reagents

  1. 1.7 ml microfuge tubes, autoclaved (Corning, Axygen®, catalog number: MCT-175-C )
  2. Sterile Petrie dishes 35 x 10 mm (Corning, Falcon®, catalog number: 351008 )
  3. Sterile 5 ml screw top tubes (Techno Plas, catalog number: P5016SU )
  4. Gloves
  5. Donor SPF female NOD mice (NOD mice were derived from Monash Animal Research Platform, Melbourne Australia)
  6. Recipient germ free female NOD mice, pregnant (GF NOD mice were derived from Germ Free Unit, Walter and Eliza Hall Institute of Medical Research)
  7. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  8. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  9. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: NIST2186II )
  10. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: NIST200B )
  11. Ethanol (Merck, catalog number: 818760 )
  12. 10x phosphate-buffered saline (PBS) (see Recipes)
  13. Sterile deoxygenated 1x PBS (see Recipes)
  14. 70% ethanol (see Recipes)

Equipment

  1. P1000 pipette (Eppendorf, catalog number: 3121000120 )
  2. Surgical tools
    1. Fine Iris Scissors, delicate pattern, 11.5 cm straight (Fine science tools, catalog number: 91460-11 )
    2. Standard pattern forceps, 12 cm straight (Fine Science tools, catalog number: 11000-12 )
    3. Standard pattern forceps, 12 cm curved (Fine Science tools, catalog number: 11001-12 )
    4. Gavaging needle, curved 20 G 38 mm (ABLE Scientific, catalog number: ASGN7910 )
  3. Laminar hood
  4. Tissue homogeniser, IKA T10 basic model (IKA, model: T 10 basic), with S10N-5G Dispersing element (IKA, model: S 10 N - 5 G )
  5. Class II Biological safety cabinet
  6. Autoclave
  7. Tabletop centrifuge for 15 ml conical tubes (Eppendorf, model: 5810 )
  8. Conventional mouse cages

Procedure

  1. Donor mice preparation
    1. Co-house donor SPF female NOD mice in the same cage or randomly swap them between cages 3 times per week prior beginning diet treatment.
    2. Repeat this procedure for 3 weeks prior to starting treatment with the diet.
    3. Feed 10 week-old donor female NOD mice with diets for 5 weeks and collect fecal and cecal contents at 15 weeks of age. The choice of diet is the variable being examined in this protocol, to see if dietary effects work by altering the microbiota.
    4. Collect faeces from all the donor mice within the diet group so as to more widely sample the microbiotas present. For every two mice receiving gavages, collect the contents of one mouse’s caecum. Because multiple microbiota collections and donations will occur during the experiment, pooling the faeces samples from all the donor mice in a treatment group is essential to maintain a consistent microbiota transfer.

  2. Preparation of mixed cecal and fecal samples
    1. Faeces collection
      1. Place individual donor mice that had been fed diets for 5 weeks in a clean empty cage under a laminar hood.
      2. Collect pellets from the bottom of the cage immediately after they are produced by the mouse, and place in a sterile 1.5 ml microtube containing 500 µl of sterile PBS at room temperature (see Recipes).
    2. Cecal content collection
      1. Sacrifice Donor mouse according to ethical guidelines.
      2. Before dissection, submerge mouse in 70% ethanol (see Recipes). Dissect the mouse and collect the cecal content within Laminar hood to avoid external contamination. To do this make a longitudinal incision through the skin and peritoneum of the mouse’s abdomen to expose the peritoneal cavity.
      3. Remove the caecum by cutting it free from the small intestine and colon at the junction of these organs with the caecum (see Figure 1), and place in a sterile Petri dish. 


        Figure 1. Removal of caecum from mice. A. Cutting of the junction between the caecum and the small and large intestines. B. Caecum after being removed.

      4. Cut off the end of the caecum, and expel the contents into a sterile 5 ml tube containing 1 ml of PBS at room temperature. This is best achieved by using one pair of flat tweezers to hold the caecum in the tube, and a second pair of rounded tweezers to push the contents out into the tube (see Video 1).

        Video 1. Removing caecal contents from the caecum

  3. Sample preparation
    1. Add the faeces in 500 µl of sterile PBS to the 5 ml tube containing the caecal contents in 1 ml sterile PBS for a final volume of ~1.5 ml. Adjust with additional sterile PBS if below this volume.
    2. Before homogenising samples thoroughly, clean the homogeniser by rinsing it in 70% ethanol (see Recipes) for 1 min twice and in sterile PBS once.
    3. Resuspend the faeces and caecal contents in the sterile PBS using the homogeniser.
    4. Spin down the homogenate at 805 x g (RCF) for 10 min at room temperature to pellet and remove solid material, and collect the supernatant which will contain the bacteria.

  4. Oral gavage
    1. Diluted homogenates from donors will be administered directly into the stomach of the recipient mice via oral gavage. The diluted cecal content does not generate any risk to the animal’s wellbeing as mice normally eat their own faeces.
    2. Firstly the animal is firmly restrained to immobilize the head. The mouse is kept in an upright (vertical) position and the gavage needle is gently passed through the side of the mouth, following along the roof of the mouth and is advanced through the oesophagus and toward the stomach. It is important to note that the entire length of the needle must be inserted into the mouse, this will confirm that the needle has entered the oesophagus not the trachea.
    3. After the needle is passed to the correct length, the microbiome will be injected. Mice don’t need to fast and volumes administered will not exceed 10 ml/kg in mice. If the animal shows any signs of distress after administration begins, the procedure should stop and the needle should be withdrawn immediately. If it appears that material has been injected into the lungs (if the animal shows signs of choking), then the animal should be euthanized. Recipient mice are left to rest for the next 24 h.
    4. Reconstitute the recipient GF mice with 200 μl of the homogenate supernatant via oral gavage.
    5. Recipient mice should be observed for no less than 15 min after procedure, and each day for the following two days after the procedure for signs of pain or distress, such as gasping, bleeding or frothing at the mouth. If any of these signs are observed, the mouse should be humanely euthanized.
    6. After gavage the pregnant GF Recipient Females will then be left alone with ‘Do Not Disturb’ cards for 2 days and will be housed in clean, autoclaved cages, using sterile food, water and bedding. To handle the GF recipient mice use double gloves and perform all work in a laminar hood. The oral gavage needs to be performed by a person experienced in the technique as it is important to be very delicate to avoid disrupting the mother, leading her to kill her pups.
    7. Inoculate pregnant germ-free NOD female recipient mice with a first oral dose of bacterial mix at the embryonic stage E(13) of pregnancy (measured from observation of plug), and a second oral gavage when pups are 2 weeks-old.
    8. Pups will be ready to be weaned at 21-24 days of age, and placed on normal chow and normal water. Similar to their mothers, 3-4 weeks old pups will receive two gavages with the donor microbiota, one day apart.
    9. Once the pups from the recipient mother’s are past their treatment period, they will be monitored for diabetes onset for up to 30 weeks of age. Diabetes assessment will be done by checking the blood glucose levels, with recordings above 12 mmol/L recorded on two consecutive days considered diabetic as previously described (Marino et al., 2009), and compared between groups. Examples of the results can be found in (Marino et al., 2017)

Notes

  1. Due to the nature of the experiment, focusing on transferring the microbiota as accurately as possible from the donor to the recipient it is necessary to make as effort as possible to avoid external contamination. All collection tubes and media should be autoclaved, samples handled in sterile environments and all mice work conducted under the hood.
  2. Donor mice should be matched to the age and gender of the recipient mice to minimize other variables causing differences in the microbiota beyond diet.
  3. The co-housing of the donor mice prior to the beginning of the experiment is important to standardize the microbiota of all the mice before the diets are added to alter the microbiota in a specific way.
  4. Collection of fresh donor material on the day of the donation is highly recommended as storage of the microbiota may lead to altered bacterial populations being present. Storage in the fridge may lead to some species growing, while others remain static, leading to the relative abundances to no longer represent the environment in the gut. Likewise, storage in a freezer may lead to bacterial death, again altering relative populations.
  5. It is ideal to maintain anaerobic conditions whilst handling the samples, but maintaining this during the experiment is impractical. We took as many practical steps as possible, such as using freshly autoclaved PBS and minimization of the lengths between sample collection and gavage, to try to minimize obligate anaerobes dying off.
  6. We found performing multiple gavages of the desired microbiota helped ensure that the desired microbiota was efficiently transferred as accurately as possible. Because of this, the mixing of faeces samples from the donor groups is important to keep the transferred microbiota consistent.
  7. Assessment of the oral gavage after it has been performed could be done by collecting and culturing faeces from the mice post gavage to see if the formerly GF mice now have bacteria present. However, the best way to determine the success of the gavage is by analysis of the recipient microbiota in comparison to the donor samples through next-gen sequencing.

Recipes

  1. 10x phosphate-buffered saline (PBS)
    80 g NaCl
    2 g KCl
    14.4 g Na2HPO4
    2.4 g KH2PO4
    Prepare 10x PBS by resuspending all ingredients in distilled water to make up to 1 L
    Dilute 10x PBS with distilled water to make 1x PBS
    Autoclave 1x PBS to sterilize and deoxygenate PBS fresh for the experiment
  2. 70% ethanol
    Dilute 99% analytical grade ethanol in distilled water to 70% concentration

Acknowledgments

This work was supported by Juvenile Diabetes Research Foundation (JDRF, 3-2013-94), Diabetes Australia Research Trust (DART, project grant Y14M1-MARE). We would like to thank H.Y. Goh, Y.Yap (Monash University), C. McKenzie (Germ Free Unit, Walter & Eliza Hall Institute of Medical Research) and Monash University Animal Services (MAS) for their assistance. Competing financial interests: Authors declare no competing financial interests.

References

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  2. de Goffau, M. C., Luopajarvi, K., Knip, M., Ilonen, J., Ruohtula, T., Harkonen, T., Orivuori, L., Hakala, S., Welling, G. W., Harmsen, H. J. and Vaarala, O. (2013). Fecal microbiota composition differs between children with β-cell autoimmunity and those without. Diabetes 62(4): 1238-1244.
  3. Endesfelder, D., zu Castell, W., Ardissone, A., Davis-Richardson, A. G., Achenbach, P., Hagen, M., Pflueger, M., Gano, K. A., Fagen, J. R., Drew, J. C., Brown, C. T., Kolaczkowski, B., Atkinson, M., Schatz, D., Bonifacio, E., Triplett, E. W. and Ziegler, A. G. (2014). Compromised gut microbiota networks in children with anti-islet cell autoimmunity. Diabetes 63(6): 2006-2014.
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  5. Frank, D. N., St Amand, A. L., Feldman, R. A., Boedeker, E. C., Harpaz, N. and Pace, N. R. (2007). Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc Natl Acad Sci U S A 104(34): 13780-13785.
  6. Giongo, A., Gano, K. A., Crabb, D. B., Mukherjee, N., Novelo, L. L., Casella, G., Drew, J. C., Ilonen, J., Knip, M., Hyoty, H., Veijola, R., Simell, T., Simell, O., Neu, J., Wasserfall, C. H., Schatz, D., Atkinson, M. A. and Triplett, E. W. (2011). Toward defining the autoimmune microbiome for type 1 diabetes. ISME J 5(1): 82-91.
  7. Macia, L., Tan, J., Vieira, A. T., Leach, K., Stanley, D., Luong, S., Maruya, M., Ian McKenzie, C., Hijikata, A., Wong, C., Binge, L., Thorburn, A. N., Chevalier, N., Ang, C., Marino, E., Robert, R., Offermanns, S., Teixeira, M. M., Moore, R. J., Flavell, R. A., Fagarasan, S. and Mackay, C. R. (2015). Metabolite-sensing receptors GPR43 and GPR109A facilitate dietary fibre-induced gut homeostasis through regulation of the inflammasome. Nat Commun 6: 6734.
  8. Marino, E., Richards, J. L., McLeod, K. H., Stanley, D., Yap, Y. A., Knight, J., McKenzie, C., Kranich, J., Oliveira, A. C., Rossello, F. J., Krishnamurthy, B., Nefzger, C. M., Macia, L., Thorburn, A., Baxter, A. G., Morahan, G., Wong, L. H., Polo, J. M., Moore, R. J., Lockett, T. J., Clarke, J. M., Topping, D. L., Harrison, L. C. and Mackay, C. R. (2017). Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes. Nat Immunol 18(5): 552-562.
  9. Marino, E., Villanueva, J., Walters, S., Liuwantara, D., Mackay, F. and Grey, S. T. (2009). CD4+CD25+ T-cells control autoimmunity in the absence of B-cells. Diabetes 58(7): 1568-1577.
  10. Markle, J. G., Frank, D. N., Mortin-Toth, S., Robertson, C. E., Feazel, L. M., Rolle-Kampczyk, U., von Bergen, M., McCoy, K. D., Macpherson, A. J. and Danska, J. S. (2013). Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity. Science 339(6123): 1084-1088.
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简介

饮食和肠道微生物生态学的改变是1型糖尿病(T1D)发病的基础。 在非肥胖糖尿病(NOD)小鼠中,我们发现血液和粪便中高浓度的乙酸和丁酸的细菌代谢物与防止疾病相关。 我们使用来自受保护的NOD小鼠的粪便细菌重建无菌(GF)NOD小鼠,饲喂高乙酸盐和丁酸盐产生的饮食,以测试转移的肠道微生物群是否防止T1D的发展。 GF NOD小鼠接受由高乙酸盐但不是丁酸盐产生饮食形成的微生物群显示出对糖尿病的显着保护。 这种粪便移植试验表明,饮食技术可以重塑肠道微生物群,使特定的细菌能够转移对T1D的保护。


【背景】已经在各种各样的疾病和病况中观察到肠道微生物群的变化。由于共生菌和致病菌之间的比例变化,肠道益生菌微生物群体失去了体内平衡。当微生物的组成变化很大时,可以观察到生物异常,某些种类的数量增加或减少(Clemente等,2012; Rajilic-Stojanovic,2013)。此外,肠道微生物群组成(其可能影响代谢物如SCFAs)的变化与许多炎性疾病有关(Clemente等人,2012),包括T1D(de Goffau等人 >,2013; Endesfelder 等,2014)。例如,易患自身免疫性1型糖尿病的患者通常表现出厚壁菌属丰度的降低以及其类杆菌丰度的增加(Giongo等人,2011)。相反,各种类型的炎症性肠病(IBD),例如溃疡性结肠炎或克罗恩氏病表现出相反的效果(Frank等人,2007; Spor等人, 2011)。治疗T1D的一种可能性是在鉴定和成功试验之后使用有益菌。例如,从糖尿病抗性大鼠中分离的约氏乳杆菌能够预防T1D自发性大鼠模型中的T1D发展(Valladares等人,2010)。同样,从不易发生T1D的雄性小鼠的微生物群转移到雌性小鼠中降低了与小鼠激素水平变化相关的T1D的发生率(Markle等人,2013) 。我们使用改变肠道微生物组成的饮食并诱导微生物短链脂肪酸(SCFA)的释放。在这项研究中,我们已经证明特别是SCFA醋酸酯和丁酸酯可以减少NOD小鼠T1D的发作(Marino等,2017)。我们想确定保护效果是直接来自细菌,还是产生的微生物SCFA乙酸盐或丁酸盐。因此,我们制定了从饮食喂养的NOD小鼠收获微生物群的方案,并将其转移到GF NOD小鼠,以便我们可以检查改变的微生物群及其代谢物对T1D发病机制的影响。

关键字:肠道微生物群, 细菌重组, 经口灌胃, 无菌NOD小鼠, 疾病发生率, 1型糖尿病

材料和试剂

  1. 1.7 ml微量离心管,高压灭菌(Corning,Axygen ,目录号:MCT-175-C)
  2. 无菌Petrie盘子35 x 10毫米(Corning,Falcon ,产品目录号:351008)
  3. 无菌5毫升螺旋顶管(Techno Plas,目录号:P5016SU)
  4. 手套
  5. 供体SPF雌性NOD小鼠(NOD小鼠来源于澳大利亚墨尔本的Monash动物研究平台)
  6. 受孕的无菌雌性NOD小鼠,怀孕(GF NOD小鼠来自Germ Free Unit,Walter和Eliza Hall医学研究所)
  7. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  8. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  9. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:NIST2186II)
  10. 磷酸二氢钾(KH2PO4)(Sigma-Aldrich,目录号:NIST200B)
  11. 乙醇(Merck,目录号:818760)
  12. 10倍磷酸盐缓冲盐水(PBS)(见食谱)
  13. 无菌无氧脱氧1x PBS(见食谱)
  14. 70%乙醇(见食谱)

设备

  1. P1000移液器(Eppendorf,目录号:3121000120)
  2. 手术工具
    1. 精美的虹膜剪刀,精致的图案,直径11.5厘米(精密科学工具,目录号:91460-11)
    2. 标准模式镊子,12厘米直(精细科学工具,目录号:11000-12)
    3. 标准图案镊子,12厘米弯曲(精细科学工具,目录号:11001-12)
    4. 弯头20 G 38毫米(ABLE Scientific,目录号:ASGN7910)
  3. 层流罩
  4. 使用S10N-5G分散元件(IKA,型号:S 10 N - 5 G)组织匀浆器,IKA T10基本型(IKA,型号:T 10 basic)
  5. 二类生物安全柜
  6. 高压灭菌器
  7. 用于15ml锥形管的台式离心机(Eppendorf,型号:5810)
  8. 传统的小鼠笼

程序

  1. 供体小鼠制备
    1. 在同一个笼子中共饲养供体SPF雌性NOD小鼠,或者在开始饮食治疗之前每周3次在笼子中随机交换它们。
    2. 在开始用饮食治疗之前重复此程序3周。
    3. 饲喂10周龄的供体雌性NOD小鼠与饮食5周并在15周龄时收集粪便和盲肠内容物。饮食的选择是在这个议定书中检查的变量,以查看饮食效果是否通过改变微生物的作用。
    4. 从饮食组内的所有供体小鼠收集粪便以更广泛地采样存在的微生物。接受灌胃的每两只老鼠收集一只老鼠的盲肠内容物。由于在实验过程中会发生多个微生物群的收集和捐赠,所以将来自所有供体小鼠的粪便样本汇集在一个治疗组中对于保持一致的微生物群转移是必不可少的。

  2. 混合盲肠和粪便样品的制备
    1. 粪便收集
      1. 将已经饲喂了5周的个体供体小鼠置于层流罩下的清洁空笼中。

      2. 在小鼠生产后立即收集笼子底部的小球,并置于无菌1.5ml微型管中,室温下含有500μl无菌PBS(见食谱)。
    2. 盲肠内容收集
      1. 牺牲捐助老鼠根据道德准则。
      2. 解剖前,将小鼠浸入70%乙醇中(见食谱)。解剖鼠标并收集层流罩内的盲肠内容物,以避免外部污染。为此,通过腹部的皮肤和腹膜进行纵向切口以暴露腹腔。
      3. 从盲肠切除小肠和结肠,将盲肠切除(见图1),并置于无菌培养皿中。 


        图1.去除小鼠盲肠:一种。切除盲肠与小肠和大肠之间的连接处。 B.贝因以后的盲肠
      4. 切除盲肠末端,并在室温下将内容物排入含有1ml PBS的无菌5ml管中。这最好通过使用一对扁平镊子将盲肠保持在管中,第二对圆形镊子将内容物推入管中(见视频1)。

        视频1

  3. 样品制备
    1. 将500μl无菌PBS中的粪便添加到含1ml无菌PBS中的盲肠内容物的5ml试管中,最终体积为约1.5ml。如果低于这个体积,用额外的无菌PBS调整。
    2. 在彻底均质化样品之前,清洗均质器在70%乙醇(见食谱)中冲洗1分钟两次,在无菌PBS中冲洗一次。

    3. 使用匀浆器将无菌PBS中的粪便和盲肠内容物重新悬浮
    4. 在室温下,将匀浆物在805×g(RCF)下旋转10分钟以沉淀并除去固体物质,并收集含有细菌的上清液。

  4. 口服灌胃
    1. 来自供体的稀释的匀浆将通过口服灌胃直接施用到受体小鼠的胃中。稀释的盲肠内容物不会对动物的健康产生任何风险,因为老鼠通常吃自己的粪便。
    2. 首先,动物被牢固地限制住头部。将鼠标保持在垂直(竖直)位置,并且将灌胃针轻轻地通过嘴的侧面,沿着嘴的顶部,并且通过食道前进到胃。重要的是要注意,针的整个长度必须插入鼠标,这将确认针进入食管而不是气管。
    3. 针头通过正确的长度后,微生物将被注入。小鼠不需要禁食,小鼠体内给药量不会超过10毫升/千克。如果开始给药后动物出现任何痛苦迹象,应停止手术,并立即取下针头。如果材料已经注射到肺部(如果动物出现窒息的迹象),那么动物应该被安乐死。受体小鼠在接下来的24小时内休息。

    4. 用200μl匀浆上清液重建受体GF小鼠
    5. 在手术后不少于15分钟的时间内应该观察受体小鼠,并且在手术之后的每两天,每天观察疼痛或窘迫迹象,例如喘气,出血或口腔发泡。如果观察到这些迹象中的任何一个,则应该对小鼠进行人道安乐死。
    6. 灌胃后,怀孕的GF接受者女性将独自留下2天的“请勿打扰”卡,并使用无菌食品,水和寝具安置在清洁的高压灭菌笼中。为了处理GF接受者,使用双手套并在层流罩中进行所有的工作。口服灌胃需要由技术熟练的人来完成,因为避免打乱母亲,导致她杀死幼崽是非常重要的。
    7. 在怀孕的胚胎阶段E(13)(通过观察塞子测量),用怀孕的无菌NOD雌性受体小鼠接种第一次口服剂量的细菌混合物,并且当幼仔是2周龄时接受第二次口服灌胃。
    8. 小狗将准备在21-24天断奶,并放在正常的食物和正常的水。类似于他们的母亲,3-4周龄的幼崽会在一天之内接受两次供体微生物群的灌胃。
    9. 一旦来自接受者母亲的幼仔超过了他们的治疗期,他们将被监测糖尿病发作长达30周龄。通过检查血糖水平来进行糖尿病评估,如之前描述的(Marino等人,2009),连续两天记录连续两天以上记录的高于12mmol / L的记录,并且在组之间进行比较。结果的例子可以在(Marino ,2017)
      中找到

笔记

  1. 由于实验的性质,将重点放在尽可能准确地将捐赠者的微生物转移到受者身上,因此有必要尽可能避免外部污染。所有收集管和培养基都应该高压灭菌,样品在无菌环境中处理,所有的老鼠都在罩下工作。
  2. 供体小鼠应与受体小鼠的年龄和性别相匹配,以最大限度地减少导致微生物在饮食之外的差异的其他变量。
  3. 在实验开始之前,供体小鼠的共同住房对于在添加饮食以特定方式改变微生物群之前标准化所有小鼠的微生物群是重要的。
  4. 强烈建议在捐赠当天收集新鲜供体材料,因为微生物群的储存可能导致改变的细菌种群存在。在冰箱里储存可能会导致一些物种的生长,而另一些则保持不变,导致相对丰度不再代表肠道中的环境。同样,在冷藏室储存可能导致细菌死亡,再次改变相对人群。
  5. 处理样品时保持厌氧条件是理想的,但在实验过程中保持这一点是不切实际的。我们采取了尽可能多的实际步骤,例如使用新鲜高压灭菌的PBS,并尽量减少样品采集和灌胃之间的长度,尽量减少专性厌氧菌死亡。
  6. 我们发现进行多次灌胃期望的微生物群有助于确保期望的微生物群被有效地转移尽可能准确。因此,来自供体组的粪便样品的混合对于保持转移的微生物群是一致的是重要的。
  7. 进行口服灌胃的评估可以通过从管饲后的小鼠收集和培养粪便来观察以前的GF小鼠现在是否存在细菌。然而,确定灌胃成功的最好方法是通过分析接受者的微生物群与供体样本通过下一代测序相比较。

食谱

  1. 10倍磷酸盐缓冲盐水(PBS)
    80克NaCl
    2克KCl
    14.4克Na 2 HPO 4 4 2.4克KH 2 PO 4 4克/克 通过将所有成分重新悬浮在蒸馏水中来制备10倍PBS以达到1L
    用蒸馏水稀释10倍PBS制成1x PBS
    高压灭菌1x PBS消毒和去氧新鲜PBS的实验
  2. 70%乙醇
    在蒸馏水中稀释99%的分析级乙醇,浓度达到70%

致谢

这项工作得到了青少年糖尿病研究基金会(JDRF,3-2013-94),澳大利亚糖尿病研究信托基金(DART,项目资助Y14M1-MARE)的支持。我们要感谢H.Y.莫纳什大学的Y.Yap,C. McKenzie(Germ Free Unit,Walter& Eliza Hall医学研究所)和Monash University Animal Services(MAS)。竞争的财务利益:作者声明没有竞争的财务利益。

参考

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引用:McLeod, K. H., Mason, L. and Mariño, E. (2018). Transplantation of Fecal Microbiota Shaped by Diet. Bio-protocol 8(1): e2683. DOI: 10.21769/BioProtoc.2683.
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