Depletion of epidermal langerhans cells and Ia immunogenicity from tape-stripped mouse skin

To explore the relationships among Ia antigen expression, epidermal Langerhans cells, and the immunogenicity of skin allografts, cellophane tape-stripping was used in H-2 congenic and recombinant mice of defined immunogenetic disparity. Tape-stripping of murine abdominal wall skin achieved almost complete depletion of epidermal Langerhans cells within a few hours of application, as measured by cell surface ATPase and expression of Ia antigens. Tape-stripping also reduced, to a considerable degree (but not absolutely), the Ia immunogenicity of skin allografts prepared from stripped surfaces. No comparable reduction in immunogenicity of class I major histocompatibility determinants was observed, suggesting that Langerhans cells are relatively unimportant in the presentation of H-2K antigens in skin grafts. Langerhans cells reappear within 24 h of tape-stripping to anatomically intact skin, but are detectable in orthotopically grafted only after the graft has been in residence for 4 d, i.e., shortly after it has acquired a blood supply. Repopulating Langerhans cells at that time and thereafter are exclusively of host origin. These results indicate that the traffic of Langerhans cells to the skin can be extremely dynamic, especially when the epidermal surface has been markedly disturbed, and the data imply that, under normal circumstances, large numbers of Langerhans cells can be mobilized readily from an available pool of precursors.     

人表皮干细胞体外分选和培养对构建组织工程皮肤种子细胞的可能性

目的：探讨人表皮干细胞在体外分选和培养的叮能性，为构建组织工程皮肤寻找理想的种子细胞。方法：实验于2002-01／12在中山大学基础医学院动物中心完成。根据表皮干细胞与Ⅳ型胶原蛋白的高黏附的特性，运用黏附实验将干细胞从培养在鼠Ⅳ型胶原包被的培养瓶的表皮细胞中分选出来，并从形态学、超微结构及免疫组化等方面进行了鉴定。结果：分选出来培养的细胞符合表皮千细胞的特征，说明分选培养的细胞是表皮干细胞。结论：预先在体外对表皮干细胞进行分选和培养是可行的，该细胞群有望成为构建组织工程皮肤的种子细胞，为下一步应用表皮干细胞构建组织工程皮肤奠定了牢固的基础。     

人表皮干细胞体外分选和培养对构建组织工程皮肤种子细胞的可能性

目的:探讨人表皮干细胞在体外分选和培养的可能性,为构建组织工程皮肤寻找理想的种子细胞。方法:实验于2002-01/12在中山大学基础医学院动物中心完成。根据表皮干细胞与Ⅳ型胶原蛋白的高黏附的特性,运用黏附实验将干细胞从培养在鼠Ⅳ型胶原包被的培养瓶的表皮细胞中分选出来,并从形态学、超微结构及免疫组化等方面进行了鉴定。结果:分选出来培养的细胞符合表皮干细胞的特征,说明分选培养的细胞是表皮干细胞。结论:预先在体外对表皮干细胞进行分选和培养是可行的,该细胞群有望成为构建组织工程皮肤的种子细胞,为下一步应用表皮干细胞构建组织工程皮肤奠定了牢固的基础。     

皮肤组织工程中表皮干细胞培养的实验研究

目的:通过表皮干细胞对基底膜的黏附性,利用层粘连蛋白和Ⅳ型胶原作为递质,探索提取和培养表皮干细胞的技术方法。方法:取新生1～3d的Wistar大鼠全层皮肤,通过酶消化法获得幼鼠表皮,并制成单表皮细胞悬液。以层粘连蛋白和Ⅳ型胶原作为基底膜的替代物,利用表皮干细胞对基底膜的吸附性筛选表皮干细胞。结果:与角质细胞对照,表皮干细胞对层粘连蛋白和Ⅳ型胶原的吸附性很好,克隆形成率较高。用SABC法对其进行整合素β1单抗的免疫组化染色;广谱角蛋白单抗的免疫组化染色观察细胞克隆的细胞浆内出现棕黄色的阳性表达。结论:用层粘连蛋白和Ⅳ型胶原快速黏附法可从大鼠皮肤分离和富集表皮干细胞。     

Efficacy of acyclovir against mouse cytomegalovirus in vivo.

Acyclovir reduced mortality and organ virus titers in mice inoculated intraperitoneally with 10 50% lethal doses of mouse cytomegalovirus. This susceptibility to acyclovir of a herpesvirus which lacks thymidine kinase is surprising. Alternative phosphorylating enzymes may account for this susceptibility.     

In vivo inflammatory activity of epidermal cell-derived thymocyte activating factor and recombinant interleukin 1 in the mouse.

Epidermal cell-derived thymocyte activating factor (ETAF), a cytokine produced by keratinocytes, has previously been shown to be biochemically and functionally very similar, if not identical, to interleukin 1 (IL-1). Both ETAF and IL-1 have been demonstrated to be chemotactic for neutrophils and mononuclear cells in vitro. In order to demonstrate that this activity has physiological relevance we have used a simple in vivo model. The present study demonstrates that injection of high-titer ETAF or purified recombinant murine IL-1 into the mouse footpad results in an influx of neutrophils into the site with peak accumulation at 4 h. Footpad swelling also occurs with a time course roughly paralleling that of the neutrophil accumulation. Injection of control proteins failed to reproduce this phenomenon. Margination of neutrophils within blood vessels was seen within 1 h of injection of ETAF or IL-1, followed by entry into the stroma by 4 h. This suggests that chemotactic activity and not merely increased adherence or inhibition of migration is occurring. 5-10 d of daily, subcutaneous injection of ETAF on the mouse flank resulted in an infiltrate of neutrophils, and to a lesser degree, mononuclear cells in association with epidermal hyperplasia, subcutaneous fibrosis, and focal muscle necrosis in the panniculus carnosus. These findings were not seen in control sites injected with media. These findings provide direct in vivo experimental evidence suggesting a physiologic role for ETAF/IL-1 in local inflammation.     

Induction of hair follicle neogenesis with cultured mouse dermal papilla cells in de novo regenerated skin tissues

De novo skin regeneration with human keratinocytes amplified in culture is a life‐saving procedure for patients with extensive skin loss and chronic wounds. It also provides a valuable platform for gene function and therapeutic assessments. Nevertheless, tissues generated in this manner lack hair follicles that are important for skin homeostasis, barrier function, and repair. In this study, we generated skin tissues with human keratinocytes combined with dermal papilla (DP) cells isolated from mouse whisker hair. For this, cultured keratinocytes and mouse DP (mDP) cells were mixed at 10:1 ratio and seeded onto devitalized human dermal matrix derived from surgically discarded human abdominoplasty skin. After 1 week in submerged culture, the cell/matrix composites were grafted onto the skin wound beds of immunocompromised NSG.SCID mice. Histological analysis of 6‐week‐old skin grafts showed that tissues generated with the addition of mDP cells contained Sox2‐positive dermal condensates and well‐differentiated folliculoid structures that express human keratinocyte markers. These results indicate that cultured mDP cells can induce hair follicle neogenesis in the de novo regenerated skin tissues. Our method offers a new experimental system for mechanistic studies of hair follicle morphogenesis and tissue regeneration and provides insights to solving an important clinical challenge in generation of fully functional skin with a limited source of donor cells.     

应用表皮干细胞构建组织工程皮肤及移植实验

目的:探讨应用人表皮干细胞和猪脱细胞真皮构建组织工程皮肤修复全层皮肤缺损的可行性。方法:实验于2004-06/2005-12在南昌大学医学院烧伤研究所完成。①取环状切除后幼儿包皮(家属知情同意)用中性蛋白酶与胰蛋白酶混合消化液消化,收集细胞,接种在已包被Ⅳ型胶原的培养皿中,快速黏附,保留快速黏附细胞继续培养,培养体系为低钙高糖DMEM培养基,待融和状态时,再次消化,离心收集细胞,再次经过Ⅳ型胶原快速黏附,保留黏附细胞,培养20d左右,用作构建表皮干细胞并进行细胞鉴定。②将第2代细胞接种于铺好Ⅳ型胶原的猪无细胞真皮上,每3天换液。10d后将其转移至不锈钢网架上,行气液界面培养5d左右,构建复合皮。③取SD大鼠20只,随机分2组。于动物背部尾侧正中作2.5cm×2.5cm的全层皮肤至肌筋膜的切除。实验组10只创面覆盖表皮干细胞构建的复合皮移植物,对照组10只覆盖无细胞真皮移植物。④移植后7,10,14,21和35d,两组动物均活检取材,进行创面大体观察、组织学观察及免疫组织化学、免疫荧光鉴定等。结果:①表皮干细胞的分离、扩增及鉴定:在扩增的细胞拟接种于无细胞真皮前,进行角蛋白19免疫组织化学鉴定、以流式细胞术进行α6、CD71鉴定。经胶原Ⅳ黏附的细胞培养后形成较大集落,拟接种的种子细胞中α6briCD71dim细胞占总细胞的(39±7)%;鼠抗人角蛋白19阳性细胞率为(53±6)%。②组织工程皮肤的建立结果:表皮干细胞种植于无细胞真皮上1d后,细胞便贴壁并开始生长增殖,8d左右透过间隙见无细胞真皮上表皮干细胞渐增殖融合成片;2周时无细胞真皮表面可见菲薄皮片状生长的细胞膜片物。复合皮组织学检查可见无细胞真皮表面覆盖多层融合的表皮层细胞,无细胞真皮内细胞成分去除完全。③创面愈合情况:术后2周,实验组移植物成活,创面上已上皮化,移植物柔软,而对照组无细胞真皮表面未上皮化,较干燥,色暗、触之较硬,两组创面均未见明显收缩。4周,实验组创面愈合,未见明显排斥反应。对照组无细胞真皮干燥脱落而创面暴露,创面收缩明显,创面边缘因自体表皮细胞爬行而缩小创面。④组织学检查及免疫组织化学染色结果:实验组2周时创面已全部上皮化,但细胞分层少;3,4周表皮层分化良好,多层结构,真皮纤维内见成纤维细胞长入,见少许炎症细胞,大鼠自体正常皮肤见毛囊及毛发结构。对照组2周创面尚未上皮化,无细胞真皮干燥变性,3,4周后创面仍未愈合,可见大量炎症细胞。实验组愈合创面抗人人类白细胞抗原-Ⅰ型抗原直接免疫荧光染色呈阳性,证明新生表皮由移植的人表皮细胞形成。而对照组阴性。实验组愈合创面广谱角蛋白免疫组织化学染色见整个表皮层细胞胞浆染呈棕黄,为阳性反应。结论:利用体外纯化扩增的表皮干细胞及制备的猪脱细胞真皮可体外联合构建具有复层结构的组织工程皮肤,该组织工程皮肤可用于修复动物全层皮肤缺损。     

D-[3H]glucosamine labelling of epidermal and dermal glycosaminoglycans in cultured human skin

An in vitro model is presented for the study of glycosaminoglycans in human skin. The synthesis of six glycosaminoglycan species in both dermis and epidermis was measured by D-[3H]glucosamine labelling. Punched biopsies (epidermis + entire dermis) of 3 mm in diameter were cultured at 37 degrees C in 5% carbon dioxide-95% air. When the label was added 18 h after explantation, the incorporation started immediately, and for all glycosaminoglycans the time-dependent incorporation was linear for 16 h. The experimental variation was minimized by expressing the measurements in epidermis "per explant" and in dermis "per mg of wet explant". A ratio to dermal hydroxyproline did not improve the precision. Most of the variation arose "before" isolation and separation of the glycosaminoglycans. The labelled products were macromolecules and were converted to small molecules by chondroitinase ABC + heparinase. The total incorporation in dermis was 2 1/2 times higher than in epidermis. Hyaluronic acid was the predominant synthesized product in dermis, and hyaluronic acid and heparan sulphate were the predominant products in epidermis. The proportions (%) in dermis/epidermis were as follows: hyaluronic acid, 61/44; heparan sulphate, 18/31; dermatan sulphate, 5/8; chondroitin 4/6-sulphate, 10/7 and heparin-like glycosaminoglycan, 1/2. The same species were also demonstrated as native constituents of uncultured human skin. Hyaluronic acid and dermatan sulphate predominated in dermis, whereas no single species predominated in epidermis. Their concentrations in uronic acid equivalents per mg of wet skin (pmol/mg of epidermis + dermis) were as follows in dermis/epidermis: hyaluronic acid, 243/0.48; heparan sulphate, 22/0.44; dermatan sulphate, 170/0.56; chondroitin 4/6-sulphate, 72/0.50; and heparin-like glycosaminoglycan, 5/0.22. Thus, only 0.4% of the in vivo synthesized glycosaminoglycan was present in epidermis.     

Inhibition of doxorubicin toxicity in cultured neonatal mouse cardiomyocytes with elevated metallothionein levels

Controversial results have been reported regarding whether metallothionein (MT) functions in doxorubicin (DOX) detoxification in the heart. To determine unequivocally the role of MT in cardiac protection against the toxicity of DOX, ventricular cardiomyocytes isolated from 1- to 3-day neonatal transgenic mice with high levels of cardiac MT and from nontransgenic control animals were applied. On the 6th day of culturing, MT concentrations in the transgenic cardiomyocytes were about 2-fold higher than those in the nontransgenic cells. DOX was added directly into the cultures. Compared with nontransgenic controls, transgenic cardiomyocytes displayed a significant (p <.05) resistance to DOX cytotoxicity, as measured by morphological alterations, cell viability, and lactate dehydrogenase leakage from the cells. This cytoprotective effect of MT correlated with its inhibition of DOX-induced lipid peroxidation. These observations demonstrate unequivocally that elevation of MT concentrations in the cardiomyocytes of 2-fold higher than normal provides efficient protection against DOX toxicity.     

Interleukin 1 and lipopolysaccharide induce an inhibitor of tissue-type plasminogen activator in vivo and in cultured endothelial cells

Human IL-1, recombinant murine IL-1 and E. coli LPS were found to be potent inducers of plasminogen activator (PA)-inhibitor activity, both in vivo, in rats, as well as in cultured human endothelial cells. In vivo, LPS rapidly and dose-dependently (0.01-1,000 micrograms/kg) increased plasma PA-inhibitor activity. Infusion of IL-1 into rats resulted in a small but significant increase in PA-inhibitor activity in rat plasma. Likewise, in cultured human umbilical vein endothelial cells, LPS and IL-1 induced increased synthesis of PA-inhibitor. We suggest that the induced rat plasma inhibitor might be of endothelial origin.     

小鼠精原干细胞体外培养诱导成骨的能力

背景：精原干细胞具有自我更新及多向分化潜能,并可将其体外定向诱导为特异性细胞,提示精原干细胞亦可能具有向成骨细胞转化的可能性,但有关这方面的研究尚未见报道。目的：观察小鼠精原干细胞在体外成骨细胞培养条件下的生物学特征变化。方法：取生后15-20 d小鼠胫骨骨髓,分离、培养骨髓基质细胞,5 d后丝裂霉素C处理制备成饲养层;取生后七至八天小鼠睾丸,经酶消化后制成单细胞悬液并接种于饲养层上。3 d后实验组和对照组分别用条件培养液和基本培养液进行诱导培养,通过倒置相差显微镜观察培养细胞生长状况及形态变化,并结合碱性磷酸酶染色、Ⅰ型胶原免疫荧光染色等方法进行结果检测。结果与结论：实验组精原干细胞在条件培养液中较对照组细胞贴壁生长快,条件培养3-6 d后见细胞生长迅速,呈集落样增长,细胞立体感增强,细胞团、簇的大小继续增加,细胞外基质增多,但未见明显细胞间桥。培养15 d后,对两组细胞进行碱性磷酸酶染色可见胞质、胞膜染为黑色,呈阳性。在荧光显微镜下可见实验组细胞浆内出现绿色荧光,呈阳性荧光反应;对照组细胞呈阴性反应,说明实验组细胞Ⅰ型胶原染色阳性,这与成骨细胞的生物学特性相似。提示精原干细胞在一定的诱导条件下具有成骨能力,有望为骨组织工程学研究提供种子细胞。     

Role of glutathione in the radiation response of mammalian cells in vitro and in vivo

Radiation interacts with biological systems to produce many types of molecular lesions. Much of the molecular damage is of little consequence with regard to cell killing. The lesions that are most likely to contribute to cell killing are DNA lesions produced by clusters of radicals. The formation of clusters of radicals is characteristic of ionizing radiation and accounts for its high efficiency as a cytotoxic agent. The mechanism by which these lesions kill cells is probably the formation of DNA double-strand breaks, ultimately resulting in chromosomal breaks. There is a possibility that some of the other types of molecular lesions produced by radiation may participate in more subtle mechanisms of cell damage. For instance, radiation induces a self-destructive process (apoptosis) in certain cell types, and the molecular lesions that initiate this process have not been identified. Glutathione (GSH) is a versatile protector. Several distinct mechanisms of radioprotection by GSH can be identified. These include radical scavenging, restoration of damaged molecules by hydrogen donation, reduction of peroxides and maintenance of protein thiols in the reduced state. Of these mechanisms, hydrogen donation to DNA radicals is probably the most important. Since competing reactions are very rapid, this mechanism requires a high concentration of GSH. Radioprotection by hydrogen donation to DNA radicals is not effective in oxygenated cells because the normal intracellular GSH concentration is not sufficient for effective competition with oxygen. Consequently, moderate depletion of GSH has no effect on the radiosensitivity of oxygenated cells. Under hypoxic conditions GSH becomes more competitive, and GSH depletion can markedly affect radiosensitivity. The radiosensitivity of hypoxic cells is most affected by GSH depletion in the presence of low concentrations of radiosensitizers. Since hypoxic cells are a characteristic feature of tumors, moderate depletion of GSH in combination with treatment with hypoxic cell radiosensitizers appears to be a promising strategy for selective tumor sensitization in radiation therapy. Oxidation of GSH can result in radiosensitization of both hypoxic and oxygenated cells. The mechanism of this effect appears to involve oxidation of protein thiols which are important for DNA repair. In principle, modification of DNA repair could have a greater impact on radiation therapy than modification of the number of lesions produced by radiation. However, a strategy for modification of GSH or protein thiol redox state in vivo has not yet been devised.     

Metabolism and toxicity of coumarin on cultured human, rat, mouse and rabbit hepatocytes

Summary— We compared the cytotoxic effect of coumarin and its derivatives, 7-hydroxycoumarin (7-OHC), 4-hydroxycoumarin (4-OHC), o -hydroxyphenyl acetic acid (OHPAA) and o -coumaric acid (CA), on cultured hepatocytes from human, rat, mouse and rabbit liver. At 10⁻⁵ and 5 × 10⁻⁵ M, coumarin and its derivatives did not give rise to any signs of toxicity on cultured hepatocytes of the four species. At 10⁻⁴ M, coumarin, but not its derivatives, induced release of lactate dehydrogenase (LDH) into the medium, especially in rat hepatocyte cultures. Intracellular LDH activities were correspondingly reduced. The cytotoxic effect of coumarin in cultured rat hepatocytes was evidenced on morphological examination and from the results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction test. At higher concentrations (5 × 10⁻⁴ M), 7-OHC and CA were also found to be cytotoxic in cultured rat hepatocytes. The cytotoxic effect of coumarin (5 × 10⁻⁴ M) was decreased in the presence of SKF 525-A, a cytochrome P450 inhibitor. Interspecies comparisons showed that rat hepatocytes were the most sensitive to the toxicity of coumarin and its derivatives, whereas human hepatocytes were the most resistant. Our results suggest that the cytotoxicity of coumarin is metabolism and species-dependent. Thus, the rat may not be a suitable model for evaluating the pharmacological hazards of coumarin in humans.     

Turnover of epidermal growth factor binding sites in mouse mammary epithelial cells

An in vitro method of studying epidermal growth factor (EGF) receptors in mouse mammary epithelial cells in serum-free collagen gel culture has been developed. Binding of EGF averaged 108 +/- 19 fmol/mg DNA in cells isolated from freshly dissociated virgin mammary tissue. Initial binding values were maintained in cells cultured in the presence of 0.1 ng/ml EGF, but decreased in either 0 ng/ml or 10 ng/ml EGF. Addition of either chloroquine (100 microM) or ammonium chloride (10 mM) to the culture medium increased receptor levels 10-fold. Cycloheximide (0.1 microgram/ml), ouabain (3 mM), and actinomycin D (5 x 10(-2) micrograms/ml) each decreased receptor levels, in some cases by as much as 80%. Both methylamine (10 mM) and dinitrophenol (0.1 mM) had no significant effect. These studies suggest that the net level of EGF receptors in these target cells is the result of an equilibrium between synthesis and degradation. The difference between the effects of the compounds tested on either receptor degradation or synthesis in comparison to cell growth, may be indicative that receptor degradation is not linked to cell proliferation.     

人Stro-1~+间充质干细胞诱导小鼠皮肤移植免疫耐受的初步研究

本研究探讨人间充质干细胞(MSC)及其Stro-1+MSC亚群对小鼠皮肤移植模型的诱导移植免疫耐受作用。从健康成人骨髓单个核细胞中培养MSC,并筛选Stro-1+MSC。建立小鼠同种异体皮肤移植模型,供体为雌性C57BL/6小鼠,获取皮片,移植给受体雌性BALB/c小鼠。动物分为4组:①Stro-1+MSC组:照射受鼠移植前输注2×106Stro-1+MSC;②MSC组:照射受鼠移植前输注2×106MSC;③照射对照组:受鼠照射后直接进行皮肤移植;④同系对照组:BALB/c小鼠照射后接受同系小鼠皮肤移植。检测项目包括:移植皮片存活时间,HE染色观察移植皮片病理改变,ELISA检测移植前后受鼠血浆转化生长因子β1(TGF-β1)浓度的变化。结果显示:MSC组皮肤移植物存活时间为(12.13±3.34)d,较照射对照组(11.38±1.01)d未见明显延长(P>0.05);Stro-1+MSC组皮肤移植物存活时间为(30.68±5.89)d,较照射对照组及MSC组明显延长(P<0.05),移植皮肤病理检查显示皮片结构清晰。在同系对照组和照射对照组,移植前后受鼠血浆中TGF-β1浓度无显著变化,而在MSC组和Stro-1+MSC组移植后TGF-β1浓度均有显著增高(P<0.05)。结论:Stro-1+MSC较未分选的MSC具有更强的诱导移植免疫耐受的功能,在体内可显著延长小鼠皮片的存活时间,而这种作用似与TGF-β1的表达无关。