组织工程皮肤中黑色素细胞的培养纯化

目的 探讨如何以简便快速的方法获得大量高纯度的黑色素细胞。方法 以包皮组织作为细胞来源，用消化法获得表皮细胞悬液，用该实验室设计的方法对细胞进行纯化，Dopa染色，S010免疫组化和透射电镜鉴定细胞来源。结果 用该实验室的纯化方法培养的黑色素细胞贴壁快，细胞产量高，未见角朊细胞和成纤维细胞污染。结论 结果表明该实验方法可以快速获得大量高纯的黑色素细胞。     

构建组织工程皮肤种子细胞的人血管内皮细胞的培养

目的探讨如何以简便快捷的方法获得大量生长状态良好的人血管内皮细胞,以作为构建含血管组织工程皮肤的种子细胞。方法以新生儿脐带静脉作为细胞来源,用消化法获得血管内皮细胞,用本实验室设计的方法对细胞进行纯化培养,倒置显微镜观察细胞形态,结合免疫组化检测(第Ⅷ因子)和透射电镜(Weibel-Palade小体)鉴定细胞来源。结果用本实验室的纯化方法获得的血管内皮细胞未见成纤维细胞污染,细胞增殖旺盛,处于良好的生长状态。结论结果表明本实验方法可以获得大量处于良好生长状态的高纯度血管内皮细胞,可以用于含血管组织工程皮肤的构建。     

构建组织工程皮肤种子细胞的人血管内皮细胞的培养

目的 探讨如何以简便快捷的方法获得大量生长状态良好的人血管内皮细胞，以作为构建含血管组织工程皮肤的种子细胞。方法 以新生儿脐带静脉作为细胞来源，用消化法获得血管内皮细胞，用本实验室设计的方法对细胞进行纯化培养，倒置显微镜观察细胞形态，结合免疫组化检测(第Ⅷ因子)和透射电留(Weibel-Palade小体)签定细胞来源。结果 用本实验室的纯化方法获得的血管内皮细胞未见成纤维细胞污染，细胞增殖旺盛，处于良好的生长状态。结论 结果表明本实验方法可以获得大量处于良好生长状态的高纯度血管内皮细胞，可以用于含血管组织工程皮肤的构建。     

许旺细胞体外培养纯化的观察

目的:许旺细胞能分泌生长因子,在周围神经再生中起重要作用,获得高纯度许旺细胞是对其研究的重要前提,为此探讨获得高纯度的SD乳鼠的许旺细胞的有效方法。方法:组织块反复种植纯化法、低含量胰酶快速消化和差速贴壁法。结果:经组织块反复种植纯化法、低含量胰酶快速消化和差速贴壁法综合运用,所得许旺细胞的纯度很高,可达98%以上,而且细胞所受到的外界因素影响也较少。结论:通过体外培养方法得到高纯度且受到的外界因素影响小的许旺细胞,可采用组织块反复种植纯化法、低含量胰酶快速消化和差速贴壁法综合运用的方法。     

许旺细胞体外培养纯化的观察

目的：许旺细胞能分泌生长因子，在周围神经再生中起重要作用，获得高纯度许旺细胞是对其研究的重要前提，为此探讨获得高纯度的SD乳鼠的许旺细胞的有效方法。方法：组织块反复种植纯化法、低含量胰酶快速消化和差速贴壁法。结果：经组织块反复种植纯化法、低含量胰酶快速消化和差速贴壁法综合运用，所得许旺细胞的纯度很高，可达98％以上，而且细胞所受到的外界因素影响也较少。结论：通过体外培养方法得到高纯度且受到的外界因素影响小的许旺细胞，可采用组织块反复种植纯化法、低含量胰酶快速消化和差速贴壁法综合运用的方法。     

许旺细胞的体外培养提纯

许旺细胞是组织工程修复神经损伤的种子细胞,能否获得大量且高纯度的许旺细胞对于组织工程学至关重要.许旺细胞的培养纯化技术日趋完善,其经典的培养方法有植块法和酶消化法,在此基础上改进的去除成纤维细胞的方法有差速贴壁法、免疫选择法、阿糖胞苷补充法、刺激因子增殖法等.最近比较新的提纯方法有磁性活细胞分离法、层粘连蛋白纯化法、三维支架联合培养法.另外还有一些因子,如胎牛血清浓度等影响许旺细胞的扩增.这些技术的主要目的就是在体外获得大量的高纯度许旺细胞,以满足组织工程修复神经的需要.许旺细胞的体外培养和提纯方法众多,且已取得初步进展,但在此技术完全成熟之前尚还有许多问题有待解决,如周期长、细胞活性低、在传代培养中细胞生物学特性不稳定等.     

用于临床移植的不同时期人胚胎嗅鞘细胞的形态学变化

目的 建立体外培养纯化人胚嗅鞘细胞的模型,观察不同时期人胚嗅鞘细胞形态学变化. 方法 采用无血清培养技术,差速贴壁+免疫吸附的纯化方法 ,培养并纯化取自 3～ 7个月人胚嗅球的嗅鞘细胞.根据 GFAP免疫组化染色结果统计培养所得嗅鞘细胞纯度. 结果 此培养方法可获得大量、高纯度的嗅鞘细胞. 结论 成功培养了人胚嗅鞘细胞,为进一步临床移植应用研究提供纯度较高的人胚嗅鞘细胞.     

用于临床移植的不同时期人胚胎嗅鞘细胞的形态学变化

目的：建立体外培养纯化人胚嗅鞘细胞的模型，观察不同时期人胚嗅鞘细胞形态学变化。方法：采用无血清培养技术，差速贴壁 免疫吸附的纯化方法，培养并纯化取自3～7个月人胚嗅球的嗅鞘细胞。根据GFAP免疫组化染色结果统计培养所得嗅鞘细胞纯度。结果：此培养方法可获得大量、高纯度的嗅鞘细胞。结论：成功培养了人胚嗅鞘细胞，为进一步临床移植应用研究提供纯度较高的人胚嗅鞘细胞。     

体外纯化乳鼠雪旺细胞的三种方法比较

目的:比较3种体外纯化乳鼠雪旺细胞方法的差异.方法:分别用低浓度胰酶(0.125%)和双十差速贴壁消化法、冷喷注法、磁珠分选法3种不同的方法纯化雪旺细胞,并比较3种方法获得细胞的活力和纯度.结果:差速贴壁消化法所得到的细胞纯度稍低,活力尚可;冷喷注法获得的细胞纯度和活力均较高;磁珠分选法获得的细胞纯度和冷喷注法相当,但是活力稍差.结论:冷喷注法可以获得高纯度和高活力的雪旺细胞.     

新生大鼠高纯度少突胶质前体细胞系细胞的培养与鉴定：改良振荡分离纯化法的特点

目的：观察体外培养少突胶质前体细胞的增殖和分化规律，探索其获得纯度较高细胞的实验方法。方法：实验于2004-03-01／08-01在军事医学科学院基础医学所实验室完成。取新生SD大鼠腑皮质体外培养，利用改良的振荡分离纯化法，振荡可将长于星形胶质细胞层表面的少突胶质前体细胞分离出来，差速贴壁以去除其他细胞，传代细胞分别用无血清和有血清的培养液培养，免疫组织化学法测表面抗原进行细胞鉴定。结果：可获得纯度大于95％不同发育阶段的少突胶质细胞系细胞或2型星形胶质细胞少突胶质前体细胞祖细胞A285，04阳性，不成熟少突胶质细胞04．01阳性，成熟少突胶质细胞髓鞘碱性蛋白阳性，2型星形胶质细胞胶质纤维酸性蛋白阳性。结论：改良的振荡分离纯化法是获取高纯度少突胶质细胞系细胞的有效方法。     

Efficient culturing of human melanocytes from suction blisters

The culturing of normal human melanocytes from the roof of suction blisters of adult oriental volunteers was carried out. Since the epidermal roofs of blisters did not contain any fibroblasts, and since keratinocytes did not attach to the culture dishes in the presence of PMA (phorbol 12-myristate 13-acetate), many melanocytes were obtained which grew well in the presence of PMA. This method is a very simple and easy way to establish pure melanocyte cultures.     

不同培养条件及时相对黑素细胞生物学特性的影响

目的观察不同培养条件及时相对体外培养的正常人黑素细胞生物学特性的影响。方法分离正常成人包皮黑素细胞,分别在添加有碱性成纤维细胞生长因子(bFGF)和霍乱毒素(CT)的RPMI1640培养基及角质形成细胞条件培养基中培养。培养的细胞用L-Dopa染色及S-100蛋白染色鉴定。观察其形态、增殖特性。结果培养细胞经生物学鉴定为黑素细胞。原代培养及条件培养基传代培养中黑素细胞有多个突起,而bFGF/CT培养基中传代培养时黑素细胞多呈梭形。两种培养条件下黑素细胞增殖特性无明显区别。结论两种方法均可稳定获得黑素细胞的体外选择性纯培养,角质形成细胞可通过细胞间接触及分泌细胞外因子影响黑素细胞的树突形成。     

自体黑素细胞体外培养与移植治疗白癜风的研究

[目的]研究和探索黑素细胞培养移植治疗白癜风的方法与疗效.[方法]采用从发疱壁上获取黑素细胞、纯黑素细胞培养与增殖、移植区刮除种植法,进行自体黑素细胞培养移植治疗白癜风.[结果]18例白癜风患者中21块皮损进行了自体黑素细胞培养移植,总有效率为90.48%.[结论]此操作方法较简单、治疗面积大,色素分部均匀,值得临床推广和应用.     

Impact of ultraviolet radiation on dermal and epidermal DNA damage in a human pigmented bilayered skin substitute

Our laboratory has developed a scaffold‐free cell‐based method of tissue engineering to produce bilayered tissue‐engineered skin substitutes (TESs) from epidermal and dermal cells. However, TES pigmentation is absent or heterogeneous after grafting, due to a suboptimal number of melanocytes in culture. Our objectives were to produce TESs with a sufficient quantity of melanocytes from different pigmentation phototypes (light and dark) to achieve a homogeneous color and to evaluate whether the resulting pigmentation was photoprotective against ultraviolet radiation (UVR)‐induced DNA damage in the dermis and the epidermis. TESs were cultured using different concentrations of melanocytes (100, 200, and 1,500 melanocytes/mm²), and pigmentation was evaluated in vitro and after grafting onto an athymic mouse excisional model. Dermal and epidermal DNA damage was next studied, exposing pigmented TESs to 13 and 32.5 J/cm² UVR in vitro. We observed that melanocyte cell density increased with culture time until reaching a plateau corresponding to the cell distribution of native skin. Pigmentation of melanocyte‐containing TESs was similar to donor skin, with visible melanin transfer from melanocytes to keratinocytes. The amount of melanin in TESs was inversely correlated to the UVR‐induced formation of cyclobutane pyrimidine dimer in dermal fibroblasts and keratinocytes. Our results indicate that the pigmentation conferred by the addition of melanocytes in TESs protects against UVR‐induced DNA damage. Therefore, autologous pigmented TESs could ensure photoprotection after grafting.     

皮肤发疱仪的研制与黑素细胞培养移植治疗白癜风

目的：进一步研制皮肤发疱仪．研究和观察局部皮肤发疱与黑素细胞培养后移植治疗白癜风的方法与效果。方法：采用从发疱壁上获取黑素细胞、纯黑素细胞培养与增殖、移植区刮除种植法、黑素细胞培养移植治疗白癜风。结果：25例白癜风患者中31块皮损进行了自体黑素细胞培养移植．总有效率为90％。结论：皮肤发疱仪临床应用效果好，操作方法较简单，黑素细胞培养后移植治疗白癜风面积大，色素分部均匀，值得临床推广和应用。     

Innervation of melanocytes in human skin

Communication between the nervous system and epidermal melanocytes has been suspected on the basis of their common embryologic origin and apparent parallel involvement in several disease processes, but never proven. In this study, confocal microscopic analysis of human skin sections stained with antibodies specific for melanocytes and nerve fibers showed intraepidermal nerve endings in contact with melanocytes. This intimate contact was confirmed by electron microscopy, which further demonstrated thickening of apposing plasma membranes between melanocytes and nerve fibers, similar to synaptic contacts seen in nervous tissue. Since many intraepidermal nerve fibers are afferent nerves that act in a "neurosecretory" fashion through their terminals, cultured human melanocytes were stimulated with calcitonin gene-related peptide (CGRP), substance P, or vasoactive intestinal peptide, neuropeptides known to be present in cutaneous nerves, to examine their possible functions in the epidermal melanin unit. CGRP increased DNA synthesis rate of melanocytes in a concentration- and time-dependent manner. Cell yields after 5 d were increased 25% compared with controls maintained in an otherwise optimized medium. Furthermore, stimulation by CGRP induced rapid and dose-dependent accumulation of intracellular cAMP, suggesting that the mitogenic effect is mediated by the cAMP pathway. These studies confirm and expand a single earlier report in an animal model of physical contact between melanocytes and cutaneous nerves and for the first time strongly suggest that the nervous system may exert a tonic effect on melanocytes in normal or diseased human skin.     

Recombinant human stem cell factor (kit ligand) promotes human mast cell and melanocyte hyperplasia and functional activation in vivo

Stem cell factor (SCF), also known as mast cell growth factor, kit ligand, and steel factor, is the ligand for the tyrosine kinase receptor (SCFR) that is encoded by the c-kit proto-oncogene. We analyzed the effects of recombinant human SCF (r-hSCF, 5-50 micrograms/kg/day, injected subcutaneously) on mast cells and melanocytes in a phase I study of 10 patients with advanced breast carcinoma. A wheal and flare reaction developed at each r-hSCF injection site; by electron microscopy, most dermal mast cells at these sites exhibited extensive, anaphylactic-type degranulation. A 14-d course of r-hSCF significantly increased dermal mast cell density at sites distant to those injected with the cytokine and also increased both urinary levels of the major histamine metabolite, methyl- histamine, and serum levels of mast cell alpha-tryptase. Five subjects developed areas of persistent hyperpigmentation at r-hSCF injection sites; by light microscopy, these sites exhibited markedly increased epidermal melanization and increased numbers of melanocytes. The demonstration that r-hSCF can promote both the hyperplasia and the functional activation of human mast cells and melanocytes in vivo has implications for our understanding of the role of endogenous SCF in health and disease. These findings also indicate that the interaction between SCF and its receptor represents a potential therapeutic target for regulating the numbers and functional activity of both mast cells and cutaneous melanocytes.     

Melanocyte-conditioned medium stimulates while melanocyte/keratinocyte contact inhibits keratinocyte proliferation

The interaction between melanocytes and keratinocytes in epidermal tissue suggest a bidirectional interchange between these two cell types. Although keratinocytes appear to affect melanocyte function, there are no reported effects of melanocytes on keratinocytes. Using cell strains, we examined the effect of melanocytes on keratinocyte proliferation. Two conditioned medium techniques were used: one was a co-culture system, where both cell types, grown on separate surfaces shared a common volume of medium. The second was simply feeding keratinocytes melanocyte-conditioned medium. Mixed cultures (both cell types together in a monolayer) where also studied. Our results showed that melanocyte-conditioned medium and melanocytes in co-culture significantly stimulated keratinocyte proliferation as measured by bromodeoxyuridine incorporation assay. However, growth of both cell types together in culture did not affect the growth rate of either cell type. Our results showed that cultured human melanocytes produce one or more soluble factors that stimulate the growth of cultured keratinocytes.