从大鼠睾丸支持细胞的分离和管周细胞
Summary
需要进行其免疫特性的炎症或感染，和利用在学习睾丸免疫特权，信号转导原塞尔托利细胞。在这里，我们描述了高度纯化的主支持细胞和从大鼠睾丸管周细胞的分离的基于酶的协议。
Abstract
睾丸，特别是雄配子，挑战以独特的方式免疫系统，因为精子的分化先出现在青春期的时候 – 建立全身免疫耐受十余年后。精细胞表达了许多可能被视为由免疫系统非自身蛋白质。然后在睾丸必须能够建立耐受这些新抗原一方面，但仍然能够保护自身免受感染和肿瘤的发展，另一方面。因此睾丸是体内该容忍外来抗原没有唤起一个有害的炎性免疫应答的少数免疫特权部位之一。支持细胞中发挥关键作用的维持睾丸的这种免疫特权环境，并且还延长cotransplanted细胞存活在外国的环境。因此主要支持细胞是研究睾丸无法通过电子邮件的免疫特权的重要工具通过建立的细胞系或其它细胞模型asily取代。在这里，我们提出了一个详细而全面的协议，用于支持细胞的分离 – 如果需要的管周细胞 – 从大鼠睾丸一天之内。
Introduction
睾丸产生雄配子和性激素， 即雄激素。器官是由两个车厢。在间质车厢，代表总睾丸容积1的约10-12％，类固醇取睾丸间质细胞内进行。管状隔室表示睾丸体积1的约60-80％，并含有生殖细胞和两种类型的体细胞-管周细胞和支持细胞。睾丸是由结缔组织隔膜分成小叶250-300，每个都包含1-3高度错综复杂的曲细精管。这些细管是由基底膜，胶原片，和管周细胞的周层（图1A）包围。
生殖上皮位于基底膜的腔侧。支持细胞是跨越从基底膜管腔整个生殖上皮大细长细胞。他们强烈ATT痛的基底膜，形成通过基底组织紧密连接，从间质闭塞生发上皮和表示血睾屏障的连续的蜂窝片。支持细胞有显着的细胞质预测和后果，使他们能够进入与种属特异性，但固定数量的生殖细胞紧密形态和功能联系。二倍体胚干细胞增殖和分化成精原细胞。
在减数分裂我短暂的四倍体精母细胞产生减数分裂II期间进一步分为四个单倍体精子细胞发展。所有生殖细胞通过细胞质桥，使得它们形成一个蜂窝网互连。精子细胞的成熟过程中的主要事件是细胞质的大部分挤出，形成残体，在一个叫做精子的过程。残体被支持细胞吞噬。然后晚期精子细胞被释放到管状内腔和输送到附睾进一步成熟。支持细胞和生殖细胞似乎相互配合地形精子发生和功能。
个体睾丸细胞类型的启动准备近一个世纪前，当小睾丸片进行培养和细胞类型鉴定显微镜2。通过打开用细尖镊子白膜后肾小管小心剥离它以后可能分离肾小管及间质车厢3。在1975年威尔士和Wiebe的，以便从附着间质组织和胰酶处理以除去外管周细胞层 4的释放管引入一个胶原酶处理。从早期年轻幼鼠（约20天）就在其中所使用的支持细胞构成，因为精子还没有开始的肾小管上皮细胞人口的很大一部分。在这个年龄段大鼠塞尔特OLI细胞停止分裂，和相邻小区之间的紧密连接形成，使血睾屏障建立5。
独立的威尔士和韦博Dorrington 等人介绍的胰蛋白酶结合，并脱氧核糖核酸其次soybeen胰蛋白酶抑制剂和胶原酶的治疗，发表于同年6。两组以便产生用于电镀的均匀的细胞悬浮液，它包含大约70％的支持细胞也使用机械力（通过注射器针头或分别巴斯德吸管吸取消化管状片段反复）。后在培养3天后，使用介质是无血清的，支持细胞的百分比增加至大约90％。这可以在很大程度上归因于污染的生殖细胞的死亡。残余肾小管周围细胞（PTC）的，但是，坚定不移地通过自己的细胞外基质连接。的PTC，但不支持细胞，已知会产生纤连蛋白可以作为标志物蛋白用于与PTC的推定污染。因此，董建华等。理由是，一个附加的透明质酸酶治疗可以改善塞尔托利细胞级分7的纯度。事实上，他们可以表明，附加的处理是能够由PTC的，以减少污染约20倍，当在比较的含有血清的培养基中用于培养纯化塞尔托利细胞，其变得特别明显。从那时起Tung等的改进的过程。成为普遍的协议和外地8（图1B）被广泛地使用由其它主要基团。
在胶原酶处理多数的PTC的被释放并且可以并行地隔离，以支持细胞。而PTC的剧烈增殖，并且不向卵泡刺激素（FSH）反应，塞尔托利细胞不再进行有丝分裂和通过特性的形态变化到FSH响应和增加环磷酸腺苷（cAMP）的浓度9。非常相似的酶消化的协议可用于初级塞尔托利细胞从其他动物，如人10，鼠标11,12，西伯利亚仓鼠13或牦牛14的隔离。用于除去大量污染生殖细胞低渗休克可以在分离步骤15的端部可以采用。这也允许支持细胞的高效分离从成年大鼠睾丸16。一个富集塞尔托利细胞悬浮液，也可以从生殖细胞通过电镀涂有曼陀罗凝集素17凝集素菜悬浮液中分离。仅支持细胞和一些残留的PTC粘附到凝集素板。
原发性睾丸支持细胞培养，主要是从大鼠，已初步用于研究响应激素或建立细胞系，如鼠标支持细胞里NE TM4 18。该细胞系已在一百多个研究被调查直至今日。在平移的方法支持细胞已被用于共培养细胞和类似的在长期移植物存活异种或同种异体胰岛的共移植组织的免疫保护无全身免疫抑制19。分离的塞尔托利细胞已在共培养实验中被还用于研究上皮-间质（Sertoli细胞- PTCC）和体生殖细胞（支持细胞-生殖细胞）的相互作用20,21。最近主要支持细胞已被用于研究Toll样受体的表达和炎性细胞因子的分泌，以及信号转导级联，导致细胞因子感染后非致病性和肾盂肾炎大肠杆菌表达大肠杆菌 22。最近其他调查采用支持细胞为研究睾丸的免疫privilege 23和表明睾酮预处理抑制LPS诱导的炎症反应24。
Protocol
Representative Results
Discussion
The seminiferous tubules are bounded by a circular layer of peritubular cells and a basal lamina on the luminal side. Sertoli cells are resting on the basal lamina, establish the blood testis barrier through formation of occluding junctions between adjacent cells and provide the structural framework for the organisation of the seminiferous epithelium. Sertoli cells and adjacent spermatogenic cells maintain intimate contacts throughout germ cell development providing physical contact and communication alike. Therefore, wh…
Disclosures
The authors have nothing to disclose.
Acknowledgements
笔者想感谢圭多范霍文和鲁Deboel，鲁汶，谁是在吉森的迈进实验室建立初级睾丸支持细胞的分离极有帮助。莫妮卡Fijak，吉森，承认她与图1A和咨询帮助。研究由德意志研究联合会授予通过BH 93 / 1-1（SB）和资助国际研究研究生院吉林大学吉森（德国）/莫纳什大学（澳大利亚墨尔本）1871年GRK（SB）的支持。从黑森州州的赠款被称为“MännlicheInfertilität贝Infektion＆Entzündung”（MIBIE）节目“兰德斯进攻性楚发展协会Wissenschaftlich-ökonomischerExzellenz”（LOEWE）中也获得支持。
Materials
| Actin (smooth muscle) antibody clone 1A4 | Dako | M0851 | Monoclonal mouse anti human antibody. Use 1:100 dilution for immunofluorescence. |
| Albumin bovine fraction V Standard grade, lyophilized |
Serva | 11930.04 | Filter (0.45 µm) BSA solutions used for immunofluorescence. |
| Corning™ Cell Strainers 70 µm | Corning | 431751 | White color |
| Collagenase A from Clostridium histolyticum | Roche | 10103586001 | |
| DAPI mountant ProLong® Gold Antifade | Life technologies | P-36931 | |
| D-glucose | Sigma | G8644 | 100 g/l |
| Dulbecco´s PBS without Ca2+/Mg2+ | Gibco | 14190-094 | |
| DNase I | Roche | 10104159001 | |
| Electron microscope | Zeiss | EM 109S | |
| Fluorescence microscope | Zeiss | Axioplan 2 | |
| Hyaluronidase from bovine testis | Sigma | H3506 | |
| Inverted light microscope | Olympus | CKX41 | Routine cell culture microscope |
| Mouse IgG / IgM (H+L) polyclonal secondary Antibody |
Life technologies | A-10684 | Alexa Fluor® 488 conjugate |
| Oil Red O | Sigma | O0625 | Has replaced Sudan III and Sudan IV because of brighter color. |
| Paraformaldehyde | Sigma | P6148 | |
| Penicillin (5,000 U/ml)/ Streptomycin (5,000 µg/ml) |
Gibco | 15070-063 | 100x solution |
| RPMI-1640 | Gibco | 21875-034 | Contains 300 mg/L L-glutamine |
| Trypsin from porcine pancreas | Sigma | T5266 | |
| Trypan Blue Stain (0.4%) | Gibco | 15250-061 | |
| Trypsin inhibitor from soybean | Sigma | T6522 | The Sigma product is considerably cheaper than the previously used BPTI (Aprotinin) from Roche. |
| Vimentin antibody (V9) | Sigma | V6630 | Monoclonal mouse anti pig vimentin antibody. Use 1:100 dilution for immunofluorescence. |
| Wistar WU rats | Charles River | N/A | Should be 19 days old on day of experiment. |
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