人表皮干细胞体外分离培养及鉴定.doc

人表皮干细胞的体外分离、培养及鉴定 何黎 顾华 昆明医学院第一附属医院皮肤性病科 云南摘要 目的：探索实验条件下人表皮干细胞的体外分离、培养及鉴定。 方法：利用细胞工程方法进行组织分离及细胞培养：通过免疫组化方法，利用角蛋白单克隆抗体对培养的角质形成细胞进行鉴定，并利用表皮干细胞的相对特异标识分子CK19对其进行检测分析。 结果：表皮自真皮较完整分离，电镜及免疫组化证实培养细胞为角质形成细胞，免疫组化结果显示：CK反应阳性，部分细胞CK19阳性，表明有表皮干细胞存在。 结论：体外分离、培养角质形成细胞成功，且分离得到的角质形成细胞中有表皮干细胞存在。 关键词：角质形成细胞 表皮干细胞 细胞培养 角蛋白19对于烧伤、急性创伤、某些疾病导致的皮肤缺损，尤其是大面积烧伤一直以自体皮移植作为首选方案，而自体皮肤不足是临床遇到的主要问题。随着细胞培养技术和组织工程的出现，使许多脏器或组织体外重建成为可能。人工皮肤的研制就是一个典型例子。在这一技术中，种子细胞角质形成细胞的体外培养，以及体外分离到的角质形成细胞中是否有在体外能大量增殖的表皮干细胞决定了能否成功构建人工皮肤。为此本课题采用组织分离方法及细胞培养方法进行角质形成细胞体外分离及培养；通过免疫组化方法，利用人广谱单克隆抗体对培养细胞进行鉴定；并利用CK19对体外培养细胞中是否有表皮干细胞进行检测。材料和方法一、 材料1、 取材选择6-26岁健康男性，行包皮环切术切除的包皮。2、 主要培养基及试剂(1)磷酸盐缓冲溶液（PBS和D-Hanks液）：（2）培养基：K-SFM（Gibco公司），编号：37010，内含2.5ug表皮细胞生长因子（EGF）和25mg小牛垂体(BPE)：（3）分离酶：dispase（4）胰蛋白酶；（5）抗人广谱角蛋白单克隆抗体；（6）CK19单克隆抗体；（7）SP超敏免疫组化试剂盒。二、 方法1、 取材将包皮环切术切除的健康包皮组织放入50ml离心管中（内装10mlDMEM培养基，含青、链酶素及硫酸庆大酶素）。2、 角质形成细胞的分离 将手术切除的包皮组织剪截成0.5大小皮片,75%酒精漂洗3次后，用含硫酸庆大酶素的PBS重复漂洗3次，再用PBS冲洗3-5次,放入装有10mldispase酶的离心管中放入4冰箱过夜。次日晨，用无菌弯头眼科镊轻轻揭下表皮，放入含有5ml0.05% 胰酶及0.01%EDTADE的50ml离心管，置于37消化吸收10min ，加入含有15%小牛血清的DMEM终止消化，滤网过滤残渣，将滤下的液体离心，弃上清，在加入不敷出10MLK-SFM培养基充分混匀后再离心一次，弃上清，加入5ml完全K-SFM培养基，用液管充分吹散细胞团。3、 细胞计数 将10ul细胞悬液与10ul台盼蓝充分混匀后，于计数板上计数，每次收集到的细胞总在11之间，细胞活率90%以上。 4、角质形成细胞原代培养 将制备的角质形成细胞悬液以 1 的密度接种到25 塑料培养瓶中，置于37 的培养箱中培养，48小时后换液，以后每3天换液一次。 5、角质形成细胞的传代培养 当原代培养角质形成细胞铺满瓶底约80%左右时，弃培养基，加入0.05%胰蛋白酶和0.01%EDTA混合液，约3-5min后加入含15%牛血清的DMEM终止消化 ，然后离心弃上清，加入10 完全K-SFM培养基,记数后按 的密度接种到25 塑料培养瓶中,置于37 的培养箱中培养,每2-3天换液一次.代长满培养瓶约80%后,重复上述步骤继续传代培养. 6 角质形成细胞的鉴定(1) 倒置显微镜观察将原带代培养的角质形成细胞接种之后, 倒置显微镜观察细胞形态及动态变化,并于培养后1天3天5天7天9天11天照相.(2) 透射电子显微镜观察将传代培养的角质形成细胞按常规方法处理后,置于100C-X透射电子显微镜下观察并照相.(3) 免疫组化染色 将原代培养的角质形成细胞调整细胞悬液进行免疫组化染色.以光谱的鼠抗人角蛋白单克隆抗体及CK19分别作为一抗,以PBS作为阴性对照.结 果1. 角质形成细胞的分离及培养取出经dipase酶消化后的包皮组织,行HE染色,组织形态学观察显示:表皮自真皮分离较完整,表皮基底层中细胞基本完整.2、倒置显微镜观察结果于培养的第1、3、5、7、9、11天倒置显微镜观察细胞形态并照相3、透过电镜下观察结果电镜下细胞结构清晰，细胞内可见大量的束状张力细丝，证实为角质形成细胞：且其间可见少量的较为幼稚的角质形成细胞（细胞体积较小。细胞核大，核仁明显，常染色质丰富，异染色质教少，胞浆可见少量的张力细丝及较丰富的线粒体）4 、免疫组化染色结果利用抗角蛋白单克隆抗体对培养的细胞进行免疫组化鉴定，细胞呈阳性反应，证实为人角质形成细胞：CK19免疫组化染色显示，有少量CK19阳性细胞存在，并以PBS作为一抗设置阴性对照。讨 论 人正常皮肤的表皮细胞包括角质形成细胞、黑素细胞、Merkel细胞，而角质细胞占表皮细胞的绝大部分。表皮基底层是表皮细胞的生发层，紧靠基底膜，随着表皮基底层细胞复杂而有续的增殖过程，分裂、增殖的细胞由内向外逐层推移，依次形成棘细胞层、颗粒层、透明层、角质层，以维持体表结构、屏障的完整性及内环境的稳定。在表皮中除了基底层棘层的角质形成细胞具有分裂、增殖能力外，其他层细胞均属于终末分化或退化了的细胞，不具备增殖能力，因此，人表皮角质形成细胞的培养，实质上就是收集表皮基底层、棘层细胞并保持其增殖活力。角质形成细胞体外培养成功的关键因素取决于：1 、采用 dipase /胰蛋白酶联合消化分离收集细胞本课题采用dipase和胰蛋白酶/EDTA两步消化法，disase酶是一种中性蛋白酶，研究表明中性蛋白酶主要分解皮肤的IV型胶原蛋白和纤维粘连蛋白，只破坏半桥粒结构，并不表皮细胞间的桥粒结构。在我们的实验中，联合应用两步酶消化法，首先采用2U/ml dispase 酶分离真皮、表皮，再运用胰蛋白酶 / EDTA 离散表皮细胞，制备成单细胞悬液；这样，既缩短了酶消化作用的时间，获得较多具增殖能力的表皮细胞，而且减少了成纤维细胞的污染。 2、应用无血清培养基培养 目前，角质形成细胞体外培养方法主要有三种：1、组织块培养法：2、以3T3 细胞作为滋养层的有血清培养法：3、无血清培养法。角质形成细胞无血清培养方法的建立，去处了3T3细胞的影响，而其中起重要作用的是牛垂体提取物，为表皮细胞的生长提供了必须的一些成分，无血清培养基尚可抑制成纤维细胞的增殖，促进上皮细胞的增长。在我们的实验中，采用Gibco公司生产的角质形成细胞无血清培养基（K-SFM），培养基中没有血清成分，且成分明确，避免了过去有血清培养基中不明成分对细胞的影响；我们对细胞培养增殖进行动态观察，也发现培养至第九天时细胞即可融合连成片铺面瓶底约80%，说明此无血清培养基适宜于角质形成细胞的体外培养及增殖。3、 养角质形成细胞及表皮干细胞鉴定角蛋白是角质形成细胞的一种特异结构蛋白，它们构成直径为10nm 的微丝，在细胞内形成广泛的网状结构。角蛋白由多肽链组成，叫蛋白多肽分子量约为40000-70000。在人表皮中不同分化时期的角质形成细胞表达不同的叫蛋白，CK19 是分子两为40kDa的一种叫蛋白，是叫蛋白家庭中最小的成员，有研究CK19 主要在表皮基底层细胞中表达，且在体外培养是有一定的慢周期性和强增殖能力，具有一定表皮干细胞特征。所以，在我们的实验中，选择具有相对特异性的CK19 作为体外培养角质形成细胞中是否有表皮干细胞存在的检测标记，结果证明，我们联合应用dispase 酶和胰蛋白酶 / EDTA 消化、分离获取的表皮细胞透射电子显微镜下证实在培养的细胞中有一部分较幼稚的角质形成细胞；免疫组化也证实了所培养的细胞有CK19阳性细胞表达，说明了我们分离收集到的表皮角质形成细胞中有表皮干细胞存在。 Isolation, culture and identification of human epidermal stem cell in vitro He Li, Go Haul (Department of dermatology and dendrology, the first subsidiary hospital, Kunming College of Medicine, Yunnan)Abstract: Objective To explore isolation, culture and identification of human epidermal stem cell in vitro under an experimental condition.Methods: Cell engineering used to isolate tissue and cultural cells; with immunhistochemical method, the keratin monoclonal antibody was used to identify the cultural keratinocyte, and the relative specific labeled molecule CK19 was used to analyze and examine.Results: Epidermis was isolated from cutis Vera completely, The electron microscope and immunohistochemical method proved that the cultural cells were the very keratinocyte, The results of immunohistochemist showed: CK19 turned out to be positive, parts of the cell CK19 as positive, and that showed epidermal stem cells had existed.Conclusion: Isolation in vitro and keratinocyte culture were successful, and there were epidermal stem cells in the kerutinocytes, which have been isolated.Key words: keratinocyte, epidermal stem cell, culture CK19 This is always the first plan to cure burns, acute wounds, especially the wide area of burns. But skin breakage is the major problem the clinical doctors met. With the rising of cell culture technique and tissue engineering, it made possible to rebuild many organs and tissue in vitro. Artificial skin is a typical illustration. During the technique, artificial skin is determined by seed cells-Culture kertinocyte in vitro, and the external isolation of keratinoicyte which whether can multiply epidermal stem cells in vitro. So this subject adopted tissue isolation and cell culture to separate and cultivate kerationcyte, and it also identify cultural cells with immunohistochemical method and human broad spectrum monoclonal antibody; CK19 was determined to detect whether there were epidermal stem cell in cultural cells.Material and Method Materials 1.1 Picking up materials several healthy males were selected (age between 6 and 26). 1.2 The main cultural medium and reagent (1) phosphate buff solution (PBS) and D-Hank s solution; (2) cultural medium: K-SEM (Gibco corporation, NO.37010, contained 2.5ug epidermal cell somatomedin (EGF) and hypothesis (BPF);(3) dispase enzyme: dispase (4) trypsin; (5) human broad spectrum monoclonal antibody ;(6) CK19 monoclonal ;(7) SP immunohistochemical method. 2 Method 2.1 materials selection Perdium tissue that was cut by annular excision was putted into centrifuge tube (with DMEM cultural medium which included penicillin, streptomycin, and garasol.)2.2.isolation of kerutinocyteThe peridium dealed with operation was cut into slices (0.5 x 0.5cm2), After it was rinsed by 75% alcohol for three times, and phosphate buffer solution with garasol for there times again. They were washed by PBS for there to five times, and it was placed into icebox of 4oC over a night .The next morning, the epidermis were disclosed slightly by germ free sterile elbowed tweezers, then they were poured into a 50ml 0.05% centrifuge tube with trypsin and 0.01% EDTA. They then were placed 37”C in the water-bath box to digest for 10 min, DMEM was added with 15% BPE to stop digestion, and they were filtered by sieve (and left filter residue out) and centrifuged the filtered fluid, and the upper fluid was poured out. Then 10ml K-SFM cultural medium was added to blend fully and centrifuged once again; then the upper layer fluid was poured out,. Finally 5ml pure K- SFM was added and separated cell masses was dispersed fully with pipet.2.3 Cell counting 10ml suspension and 10ml trypan-blue were mixed .It was counted on the counter, restrained the whole amounts of the cells (1 x 1061 x 107) and survival rate 90%.2.4 The original kerutinocyte culture Prepared kertinoctye suspension with density of 1*100000cm2 was inoculated the in a plastic culture bottle of 25cm2; and then placed under a 37”C incubator with 5% carbon dioxide, Forty-eight hours later, it was changed every three days from then on.2.5 Culture the advanced generation of kerutinocyte When the original generation of kerutinocyte covered 80% of bottom of bottle, the cultural medium was set aside 0.05% try sin and 0.01% EDFA mixed together was added after about 3-5min, added DMEM with 15% BPE to stop digestion, then the upper fluid was poured out after centrifugation, then 10 ml pure K-SFM cultural medium was added, it was inoculated with a density (1 x 105cm2) on 25cm2 plastic culture bottle after counting, and placed under 37”C cultural medium with 5% cordon dioxide to cultivate, and change every two or three days, until it covered four fifth of the bottom, repeated the upper steps to cultivate the advanced generations. 2.6 Identification on kerutinocyte (!) Inversing the electron microscope for an observation after the original of kerutinocyte has been inoculated under the electron microscope to observe the form and dynamic state of the cells then was taken photos on 1, 3,5,7,9day respectively.(2) Observation by electron microscope s transmitting After the advanced generation of keratinocyte was treated in a normal way, it was placed under 100C-X electron microscope to observe and take photos.(3) Immunohistochemist and staining The suspension of the original generation keratinocyte was adjusted to immunohistchemize, classified the human broad-spectrum monoclonal antibody and CK19 as the first antibody, and PBS as negative control.3 Results 3.1 Isolation and culture keratinocyte The peridium tissue, which has been digested, was selected out by dispase enzyme, and stained by HE. The observation of tissue forming showed: the epidermis was almost isolated from cutis Vera; the basal layers cells existed entirely.3.2 Observation by inversing electron microscope The cell structures were observed and taken photos during their culture on the 1,3,5,7,9,11day respectively.3.3 Observation by transmitting electron microscope From the electron microscope, the structure of cells was showed by the electron microscope clearly: there were large quantities of fascicular tonofilaments, which was confirmed to be keratinocyte; and there were a little of young keratinocyte among them (with a small-sized capacity, a large nucleus, a clear nucleolus, a great many of euchromatin, and a little of hetecochromatin, a bit of tonofilaments, and plenty of mitochondria could be seen in the cytolymph.)3.4 The results of immunohistochemistThe culture cells were identified by immunohistochemist keratin monoclonal antibody, the cells turned out to be positive, and was proved to be human keratinocyte; the CK19 immunohistochemist which has been stained showed that there were a bit of CK19 positive cells existed, and PBS as negative control was set up to be the first antibody.4 Discussions Human normal epidermal cell of skin consists of keratinocyte, melancyte langerhans cells, merker cells, but most of them are keratinocyte.The basal layer is the stratun germinativa of epidermis, sticking to the basement membrane with the complicated but orderly multiplication of the basal layer cells, the isolating and multiplying cells removed from inner to outside through every layer, and formed in order: pricle cell layer granular layer. Clear layer, horny layer, the keratinocyte which has finished the terminal inoculation continued to scale down from the skin surface, so as to keep the structure of body surface and barriers entirely, and steady internal environment, apart from the basal layer and pricle layer of skin surface have the abilities to inoculate and to multiply, other layer cells all belong to the cells that split up or degenerate at last, but have no ability to multiply. As a result, the culture of human body surface is truly regarded to be collecting the basal layer, pricle layer, and keep its multiplying with energy. The key factors of culture keratinocyte in vitro 4.1 Digestion, isolation, and collection cells with dispase enzyme and trypsin mixedThis subject was proved by two steps of isolation, one is dispase enzyme, which was a neutral protease, the research showed that the neutral keratin has mainly isolated IV collagen and fiber mucous protein of skin, and it only destroyed semi-desmosome, but it didnt destroy the desmosome between epidermal cell. We blended two steps of enzyme digestion in our experiment, firstly, the cutis Vera and epidermis were separated by 2u/ml dispase enzyme; secondly, the epidermal cells were dispersed by tripsin or EDTA, then they were made into unicells; so that this experiment not only made the time of enzyme digesting shorter, got more epidermal cell which would proliferate by themselves. But also made less fibrocystic contaminations.4.2 Culture by cultural medium without sera At present, there are three types of methods for culture keratinocyte in vitro, (1) tissue masses culture;(2) culture on the trophoderm of 3T3 cells with sera;(3) culture without sera. The establishment of culture keratinocyte without sera, removed the impact of 3T3 cells, but BPE, has played an important role in supplying some necessary components for the growth of epidermal cell; cultural medium without sera was likely to restrain the proliferation of fibrocystic, and accelerated epidermal cells to increase. We used keratinocyte KTSFM made by Gibco Corporation without sera but the components were showed definitely, this material has prevented some unknown components influence on cultural medium with sera in the past; because proliferation and culture cells were observed with the method of morphologic change, we also discovered that the cells cover