两种方法从子宫切除标本小学建立人子宫内膜基质细胞
Summary
建立基层子宫内膜间质细胞培养系统从子宫切除标本是追求研究的目的繁多前一个有价值的生物技术和关键的一步。在这里，我们描述了两种方法用于建立从人类患者手术切除的子宫内膜组织间质文化。
Abstract
许多努力，一直致力于建立体外细胞培养系统。这些系统被设计来模拟体内过程的广大。从人类子宫内膜样产生的细胞培养系统也不例外。应用范围从正常循环生理过程的子宫内膜病变如妇科癌症，感染性疾病和生殖缺陷。在这里，我们提供了两种方法从手术切除的子宫内膜子宫切除标本建立初级子宫内膜间质细胞。第一种方法被称为“刮法”，并采用机械刮削使用手术或刀片而第二种方法被称为“胰蛋白酶的方法”，这后一种方法使用胰蛋白酶的酶活性，以促进细胞的分离和主细胞生长。我们通过数字图像和显微说明一步一步的方法。我们还providË例子通过实时定量聚合酶链反应（定量PCR）和免疫荧光（IF）验证子宫内膜间质细胞系。
Introduction
人类子宫语料库包括三个层，所述perimetrium（或浆膜），子宫肌层和子宫内膜。区分每一层是建立子宫内膜细胞系的重要一步。该perimetrium是子宫的最外层和薄，浆液性细胞组成。子宫肌层是子宫的厚，中间层和由平滑肌细胞。子宫内膜被确定为子宫的内层和包括上皮和基质细胞群体。
子宫内膜被进一步细分为basalis层的干细胞群是假设重新填充层泛函分析大约每28天1次。人类子宫内膜的一层泛函分析经历响应循环激素显著生化和形态学改变。这些激素是来自垂体和卵巢。
该协调生产和激素的释放结果在一个生殖周期。生殖周期的目的是准备子宫内膜潜在的胚胎着床的事件。在人类中，生殖周期被称为“月经周期”而分为三个阶段 – 增殖，分泌，和月经。增生期涉及子宫内膜泛函分析层的扩散，而分泌期的特点是成熟泛函分析。具体而言，细胞外的改变，分泌物和细胞分化信号的电位注入。如果植入分泌期结束前不会发生，子宫内膜泛函分析层是在月经期脱落。月经和触发泛函分析层脱落事件的重要性仍存在争议。在人类中，已经提出了月经是已知的特定分泌中期阶段分化事件的结果作为“自发蜕膜”2。在这个手稿中，我们提供详细的方法为子宫内膜间质细胞的分离方法，用免疫荧光和数字图像的组合，以证明这些方法的功效。此外，我们采用一种常用的自发蜕膜的体外模型，以确认子宫内膜间质细胞的分离。
Protocol
在这个手稿中使用子宫切除标本的和谐与编号IRB-HSR＃14424一个大学IRB批准的道德协议。 1，从临床来源样品采集开始前获得政府和机构为基础的道德准则和批准文件。 开展在无菌条件下的所有步骤。 在50ml管中保存患者来源组织中的介质（RPMI或DMEM /高糖）中在4℃下，如果样品不能在培养立即处理。样本可以被存储在该状态下，最多24小时。 用1X无?…
Representative Results
所强调的协议部分，一定处理和准备人体组织时进行下政府的所有方法，制度和道德准则。 包括在这个手稿是“刮法”的一般工作流程的示意图（ 图1A）和用于建立初级子宫内膜文化“的胰蛋白酶法”（ 图1B）。这些方法进行了详细的协议部分中描述（参见部分1 – 3）。这两种方法都证明是成功的首要子宫内膜培养物的生长?…
Discussion
其它基团所描述的，并适于方法对子宫内膜间质培养物，其中大部分利用胶原酶4,12,13,15-18的制备。在这个手稿中，我们提供的方法和证据为2的简化主子宫内膜间质培养的方法，这两者都是由本实验室利用出于经济原因和胰蛋白酶和/或刀片的方便可用的。
当我们比较两种方法，既成功地生成可行的原代培养。我们首选的方法是胰蛋白酶方法通常有更高的收益率以较小…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢党邵族博士和她的实验室成员的协同努力利用其成像和显微设备。我们也感谢Biorepository和组织研究基金（BTRF）核心，杰夫·哈珀，居民在弗吉尼亚大学为我们提供子宫组织。我们感谢卡罗尔Szlachta的帮助示意图。
Materials
| 0.25 Trypsin or 0.05% Trypsin | Hyclone | SH3023602 or SH30004202 | |
| 1.7 micro Centrifuge Tube | Genesee Scientific | 22-272A | |
| 1µl,20µl, 200ml and 1000µl Pipette | Genesee Scientific | 24-401,24-402, 24-412, 24-430 | |
| 15ml Conical Tube | Hyclone | 339650 | |
| 50ml Conical Tube | Hyclone | 339652 | |
| 6cm Cell Culture Dish | Thermo scientific | 12-556-002 | |
| 8 well Chambers | Thermo Scientific | AB-4162 | |
| Acetate | Fisher scientific | C4-100 | |
| AMV RT Enzyme/Buffer | Bio Labs | M077L | |
| Bovine Serum Albumin (BSA) | Fisher Scientific | BP-1605-100 | |
| Buffered Zinc Formalin | Thermo | 59201ZF | |
| Charcoal strip FBS | Fisher | NC9019735 | |
| Chloroform | Fisher Scientific | BP1145-1 | |
| Cover slip | Fisher Brand | 12-544D | |
| Cyclic AMP (cAMP) | Sigma | B7880 | |
| DMEM/High Glucose | Hyclone | SH30243FS | |
| dNTP | Bioline | BIO-39025 | |
| Donkey Anti Goat -TRITC | Santa Cruz | SC-3855 | |
| Donkey Serum | Jackson’s lab | 017-000-002 | |
| E Cadherin Antibody | Epitomics | 1702-1 | |
| Ethanol | Fisher Scientific | BP2818-1 | |
| Fetal Bovine Serum (FBS) | Fisher Scientific | 03-600-511 | |
| Fungizone Amphotericin B | Gibco | 15290-018 | |
| GAPDH Probe | Life Technologies | HS99999905 | |
| Glycogen | 5Prime | 2301440 | |
| Goat Anti Mouse -FITC | Jackson’s Lab | 115-096-003 | |
| Isopropanol | Fisher Scientific | BP2618-1 | |
| Kanamycin | Fisher Scientific | BP906-5 | |
| Medroxyprogesterone acetate (MPA) | Sigma | M1629 | |
| MeOH (Methanol) | Fisher Scientific | A4-08-1 | |
| Mounting Media (w/DAPI) | Vector Labratories | H-1500 | |
| N6 DNA Oligos | Invitrogen | ||
| Number 15 Scraper | BD | 371615 | |
| Pan Cytokeratin Mouse mAB | Cell Signaling | 4545 | |
| PBS (phosphate buffered saline) | Fisher Scientific | BP-399-4 | |
| Penicillin-Streptomycin Glutamine Solution 100X | Hyclone | SV30082.01 | |
| PML Anti Goat Anti body | Santa Cruz | SC-9862 | |
| Primer(s) | Eurofins | ||
| RPMI | Hyclone | SH30027FS | |
| RPMI (Phenol free) | Gibco | 11835 | |
| Sybr Green | Thermo Scientific | AB-4162 | |
| Taqman | Thermo | AB-4138 | |
| Trizol | Life Technologies | 15596018 | |
| Vimentin Antibody | Epitomics | 4211-1 |
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Citer Cet Article
Jividen, K., Movassagh, M. J., Jazaeri, A., Li, H. Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens. J. Vis. Exp. (87), e51513, doi:10.3791/51513 (2014).