鼠标涎腺干细胞的分离

Biology
 

Summary

分离干细胞从小鼠唾液腺的优化协议描述。该方法采用酶法和机械消化,并允许包含干细胞的特征细胞salispheres的隔离。

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Pringle, S., Nanduri, L. S., Marianne, v. d., Ronald, v. O., Coppes, R. P. Isolation of Mouse Salivary Gland Stem Cells. J. Vis. Exp. (48), e2484, doi:10.3791/2484 (2011).

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Abstract

成熟的人类和小鼠出身的唾液腺包括至少5个细胞类型,其中每一个有利于口腔唾液的生产和排泄。浆液性和粘液性腺泡细胞蛋白和腺体的粘液生产工厂,分别代表唾液分泌的起源。一旦合成,通过一系列的乳腺导管上皮型形态细胞分泌的各种酶和其他唾液中的蛋白质成分,直到最终被驱逐的唾液通过进入腔口的主要管。唾液的组成是由导管上皮细胞在此过程中也修改了。

在疾病,如干燥综合征的表现,并在一些临床情况,如放射治疗头部和颈部癌症,唾液分泌的腺体是大大降低了 1,2。由此产生的口干,口干的主观感觉,不仅影响病人的吞咽和说话的能力,但还鼓励龋齿的发展,并能得到社会的衰弱的患者。

唾液生产的恢复,在上述的临床条件,因此未满足临床需要, 一些研究表明3-5唾液腺的再生能力。继鼠标和人体6-8内的各种组织细胞的干细胞样的人群隔离,我们已经证明使用的描述方法,从老鼠的唾液腺中分离出干细胞,可用于抢救照射唾液唾液生产腺体9,10。这一发现的干细胞疗法治疗人类xerostomic条件的发展铺平了道路的方式,也唾液腺含有与多能自我更新能力的细胞微环境的探索。

Protocol

1。摄政的制备

  1. 缓冲液:1%(W / V)牛血清白蛋白在汉克的平衡盐溶液
  2. 重组酶。透明质酸酶:40毫克/毫升,在缓冲液溶解。胶原酶II:23mg /毫升,在缓冲液溶解。使用每个隔离新鲜新鲜准备的酶的解决方案。当溶解,储存于4℃直至使用的消化。
  3. 50毫米氯化钙蒸馏水。通过0.2微米孔径过滤器过滤消毒。
  4. 小鼠唾液腺(MSG)的培养基:DMEM:F12(100国际单位/毫升)与青霉素,链霉素(100微克/毫升),glutamax(2毫米),表皮生长因子- 2(20毫微克/毫升),成纤维细胞生长因子-2(20毫微克/毫升),N2补充(1%),胰岛素(10微克/毫升)和地塞米松(1μM)。

2。机械及酶组织消化

  1. 称量唾液腺解剖。
  2. 切成一个均匀的纸浆使用无菌弯夹层剪刀在小培养皿中的腺体。
  3. 收集在14毫升管的肉末组织,使用每80毫克颌下腺组织的缓冲液1ml。冲洗干净的培养皿中使用的缓冲区的一些组织。
  4. 添加1毫升,每80毫克组织的缓冲区的另一个25μL,胶原酶II酶液,25μL透明质酸酶液和250μL钙氯化物溶液每80毫克组织。如果大量的组织工作,步骤可以在2.4 - 2.9的T25为方便起见,组织培养瓶中进行。
  5. 在37 ° C时为20分钟摇晃水浴中孵育。删除管和吸管磨碎通过组织混合酶彻底再次。
  6. 再过20分钟替换水浴。
  7. 离心收集,组织400 XG,8分钟。弃上清。
  8. 重悬于2 mL缓冲液中,每80毫克组织,并重复上述酶和氯化钙此外。在摇晃水浴孵育20分钟。删除管和吸管磨碎混合酶彻底。
  9. 最后20分钟摇动水浴孵育。离心收集细胞,如上,弃上清。

3。洗涤步骤

  1. 重悬于2 mL缓冲液和吸液管组织的酶洗中的每一个组织的80毫克。
  2. 以前离心分离收集。弃上清。
  3. 重复清洗使用1 mL缓冲液,每80毫克组织。离心收集,弃上清。

4。过滤

  1. 悬浮在1 mL缓冲液每80毫克组织组织的解决方案。
  2. 添加的解决方案,以100微米孔径的过滤器放置超过50毫升猎鹰管。不要申请超过3 MLS剁碎组织解决方案,每列,如过滤器可能会成为阻止。允许渗入。取下挂吸管过滤器底部的过滤材料,并添加到滤液。
  3. 使用26号针头的注射器,取滤液50毫升管,适用于50微米孔径过滤器5 mL管。允许通过过滤器,松开盖子,如果有必要协助。
  4. 离心管,以前收集。弃上清。

5。电镀和中

  1. 组合成一个卷的所有颗粒。计数使用自动细胞计数或血球。
  2. 细胞密度板0.4 × 10 6细胞,每孔12孔板,或2.67 × 10 6%的T25组织培养瓶中细胞。添加1毫升,味精中每口井或6毫升每个T25烧瓶。
  3. 在37 ° C。球应清晰可见第2天。

6。代表性的成果:

经过2至3天的文化,小聚集的细胞(salispheres)将会明显的文化。 Salispheres将继续在规模增长超过10天的文化期间。代表的salispheres相衬显微镜图像如图1所示。增殖细胞表达干细胞相关的标志蛋白,可从这些领域中分离出,优化之间的天3-5后隔离,并进入功能,唾液生产腺泡细胞的分化能力。

图1
图1。Salisphere 在体外形成。使用本议定书的机械和酶消化后,规模日益扩大的领域中可以找到浮动文化。面板代表的领域相衬显微镜图像从天0(一),4(B)7(C)和10(四)。比例尺= 50微米。

Discussion

这里描述的组织培养方法代表一个隔离的干细胞从老鼠的唾液腺含有salispheres重现协议。使用这种方式分离出的细胞的研究都强调 9涎腺干细胞的再生能力。一个100 c - Kit的+从salispheres源性细胞移植诱导照射小鼠唾液腺的功能恢复。这些数据是令人振奋的,并提供了一​​个干细胞为基础的治疗口干症调查的起点。许多途径仍有待探讨,包括完整的干细胞标记蛋白表达谱,颌下腺救援照射腮腺唾液腺,反之亦然功能的能力, 并在体内的干细胞小生境的假定表征细胞。最终,人体组织样本,并随后口干在治疗人类患者使用分离的细胞的潜力,这一协议的翻译是最令人兴奋的应用程序所描述的方法。

Disclosures

没有利益冲突的声明。

Materials

Name Company Catalog Number Comments
Hyaluronidase Sigma-Aldrich H3506 Store at – 20 °C
Collagenase II GIBCO, by Life Technologies 17101-015 Store at 4 °C
Epidermal Growth Factor-2 Sigma-Aldrich E9644 Make a stock of 10 μg / mL in phosphate buffered saline (PBS). Store at – 20 °C in single use aliquots.
Fibroblast Growth Factor-2 Sigma-Aldrich F0291 Make stock of 25 μg / mL in PBS. Store at – 20 °C in single use aliquots.
N2 supplement GIBCO, by Life Technologies 17502-048 As manufacturer instructions.
Insulin Sigma-Aldrich I6634 Make stock of 2 mg / mL in tap water. Adjust water to pH 2-3 using glacial acetic acid prior to dissolving.
Dexamethasone Sigma-Aldrich D4902
100 μM pore-size sterile cell strainers BD Biosciences 352360
Polystyrene round-bottomed tubes with cell strainer caps (50 μM pore size) BD Biosciences 352235

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References

  1. Vissink, A. Oral Sequelae of Head and Neck Radiotherapy. Crit Rev Oral Biol Med. 14, 199-212 (2003).
  2. Napeñ, as, J, J., Brennan, M. T., Fox, P. C. Diagnosis and treatment of xerostomia (dry mouth). Odontology. 97, 76-83 (2009).
  3. Denny, P. C. Parenchymal cell proliferation and mechanisms for maintenance of granular duct and acinar cell populations in adult male mouse submandibular gland. 235, 475-485 (2005).
  4. Man, Y. G. Persistence of a perinatal cellular phenotype in submandibular glands of adult rat. J. Histochem. Cytochem. 43, 1203-1215 (1995).
  5. Cotroneo, E., Proctor, G. B., Carpenter, G. H. Regeneration of acinar cells following ligation of rat submandibular gland retraces the embryonic-perinatal pathway of cytodifferentiation. Differentiation. 79, 120-130 (2010).
  6. Eirew, P. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability. Nat Med. 14, 1384-1389 (2008).
  7. Gorjup, E. Glandular tissue from human pancreas and salivary gland yields similar stem cell populations. European Journal of Cell Biology. 88, 409-421 (2009).
  8. Alonso, L., Fuchs, E. Stem cells of the skin epithelium. Proc Natl Acad Sci U S A. 100, Suppl 1. 11830-11835 (2003).
  9. Lombaert, I. M. Rescue of salivary gland function after stem cell transplantation in irradiated glands. Plos One. 3, e2063-e2063 (2008).
  10. Coppes, R. P., Goot, A. vander, Lombaert, I. M. A. Stem Cell Therapy to Reduce Radiation-Induced Normal Tissue Damage. Seminars in Radiation Oncology. 19, 112-121 (2009).

Comments

8 Comments

  1. This is an urgent need for patients who had cancer radiation therapy.
    When are clinical trials on humans expected to commence?

    Reply
    Posted by: Anonymous
    April 25, 2011 - 8:43 AM
  2. We are working at the moment on the translation of our stem cell isolation technique to human salivary gland tissue, and are characterising the stem cell-like attributes of these isolated cells. We hope to achieve translation of the technique to the clinical setting for use for therapy of xerostomia in human patients by ²017.

    Reply
    Posted by: Sarah P.
    April 26, 2011 - 7:46 AM
  3. Why not earlier?

    Reply
    Posted by: Anonymous
    May 25, 2011 - 7:38 AM
  4. Mrs Pringle,How do you think ,Is it possible to be created or regenerated salivary glands if they were destroyed.
    I have atrophyc rhinopharyngitis.My salivary glands in the mouth are working ,but these on my throat were destroyed after incorect treatment over them and as a result of this
    I have lost my salivary glands in this area.Due to this I have a lot of health problems, and my hope is the scientists to discover a way of transplantation of salivary glands,using these glands ,that still are working.
    How do you think ,Is it possible?
    Thank you!

    Reply
    Posted by: Anonymous
    October 24, 2011 - 10:43 AM
  5. Dear Jana,

    Sorry to hear about your problems.
    We are focusing our research at the moment on providing at therapy for xerostomia in the major salivary glands of humans. This potential therapy is looking very promising. We work with these glands because they are the ones most commonly affected by radiation treatment. They are also large and easy to locate and work with.

    As far as we are aware, no-one is trying to develop a therapy for the minor salivary glands. This is mostly due to their high number and very small size - there are over 600 of them scattered throughout the oral cavity and throat. This would make a cell transplantation approach very challenging.

    So although I would love to be able to tell you that a stem cell-based therapy for xerostomia resulting from dysfunction of the minor salivary glands is in process, I think this is unlikely, I would not like to leave you with unrealistically high hopes!

    Regards,

    Sarah.

    Reply
    Posted by: Sarah P.
    October 31, 2011 - 4:31 AM
  6. Dear Sarah,
    thank you very much for your replay!

    The news are bad for me and maybe I couldn't see my small children grown up,but I believe that one day
    scientists like you will discover a way of solvetion of this serious problem.
    There are many people that suffer Seogren-sindrom and Empty nose(atrophic rhinitis),there is a web site "Empty nose sindrome association".I think ,if one day will be find a cure,many peopele would be saved!

    One more time,thank you for spared time!
    I wish you all the best and great success at your work!!!!
    regards:Jana

    Reply
    Posted by: Anonymous
    November 2, 2011 - 10:14 AM
  7. Dear Jana,

    My wife was diagnosed with a metastatic undifferentiated nasopharyngeal carcinoma The primary was found deep in her throat at the base of the tongue. Radiation therapy commenced on ²² January ²007 with the last treatment on 5 March ²007. She received a relatively high radiation dose of 6,000 cGy on the sides and 6,500 cGy in the centre. Consequently she had the usual side effects of a permanent dry mouth and throat, sore tongue, sensitive and painful teeth and gums, difficulty swallowing even soft food, etc. The oncologist said that the salivary glands will never recover if they have not recovered even partially over a ² year period.

    During October this year, 4 years and 7 months after her last radiation treatment, her saliva returned in one day; not gradually. The doctors don²17;t know why. One guess is her high protein diet of milk, yogurt, cheese, etc. but its just a guess not based on any science.

    Don²17;t loose hope
    Regards
    Paul http://www.ncbi.nlm.nih.gov/pubmed/²0698985 http://www.biomedcentral.com/content/pdf/1471-²407-10-417.pdf Restoration of radiation therapy-induced salivary gland dysfunction in mice by post therapy IGF-1 administration
    Abstract
    Background: Radiotherapy for head and neck cancer results in severe and chronic salivary gland dysfunction in most individuals. This results in significant side effects including xerostomia, dysphagia, and malnutrition which are linked to significant reductions in patients²17; quality of life. Currently there are few xerostomia treatment approaches that provide long-term results without significant side effects. To address this problem we investigated the potential for post-therapeutic IGF-1 to reverse radiation-induced salivary gland dysfunction.

    Methods: FVB mice were treated with targeted head and neck radiation and significant reductions in salivary function were confirmed 3 days after treatment. On days 4-8 after radiation, one group of mice was injected intravenously with IGF-1 while a second group served as a vehicle control. Stimulated salivary flow rates

    Conclusions: Post-therapeutic IGF-1 treatment restores salivary gland function potentially through normalization of
    cell proliferation and improved expression of amylase. These findings could aid in the rational design of therapy protocols or drugs for the treatment of radiation-induced salivary gland dysfunction in patients who have completed their anti-cancer therapies.

    Insulin-like growth factor 1 (IGF-1) also known as somatomedin C is a protein that in humans is encoded by the IGF1 gene.

    IGF-1 is produced primarily by the liver as an endocrine hormone

    In rat experiments the amount of IGF-1 mRNA in the liver was positively associated with dietary casein and negatively associated with a protein free diet.
    IGF-1 then stimulates systemic body growth, and has growth-promoting effects on almost every cell in the body, especially skeletal muscle, cartilage, bone, liver, kidney, nerves, skin, hematopoietic cell, and lungs. In addition to the insulin-like effects, IGF-1 can also regulate cell growth and development, especially in nerve cells, as well as cellular DNA synthesis.
    ---------
    Casein
    From Wikipedia, the free encyclopedia
    Jump to: navigation, search
    For information about casein usage in artistic painting, see Casein paint.
    Casein (pronunciation: /G²;keɪsiɪn/, from Latin caseus, "cheese") is the name for a family of related phosphoprotein proteins (αS1, αS², β, κ). These proteins are commonly found in mammalian milk, making up 80% of the proteins in cow milk and between 60% and 65% of the proteins in human milk. Casein has a wide variety of uses, from being a major component of cheese, to use as a food additive, to a binder for safety matches. As a food source, casein supplies amino acids; carbohydrates; and two inorganic elements, calcium and phosphorus
    --------------------------------------------

    Reply
    Posted by: Anonymous
    November 2, 2011 - 10:58 AM
  8. Dear Paul,

    thank you for your letter ,your support and advices!
    I fight everyday to make my health condition beter, becouse I have children and they need me!But now I have atrophic rhinitis due to atrophic pharingiitis and day after day it becomes harder and harder to cope with this really
    serious problem!Despite every medical data I don't lose hope,becouse when there aren't any others possibilities ,the faith is the most important thing that can help us and give us strength!

    I hope that your wife is fine now ,after partly recovering of her salivary glands!I wish your family whole the hapiness in the world and the most important thing-"Health",you deserve these thinngs!!!!

    Paul,if you learn something new at this area of medicine,I beg you to write me!Here is my e-mail: tisho²001@mail.bg.

    regards:Jana

    Reply
    Posted by: Anonymous
    November 3, 2011 - 10:18 AM

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