紫外线照射后生成的微囊泡对成纤维细胞氧化损伤及凋亡的作用

目的：明确对UVA及UVB照射后皮肤成纤维细胞生成的微囊泡对成纤维细胞氧化损伤及凋亡的作用。方法：紫外线照射人皮肤成纤维细胞,提取细胞上清液中的微囊泡,利用光散射分析技术鉴定分析微囊泡的大小及数量。将紫外线照射后生成的微囊泡与正常成纤维细胞共孵育,荧光酶标仪定量检测活性氧含量,流式细胞仪检测细胞凋亡率。结果：UVA及UVB照射后皮肤成纤维细胞释放的微囊泡数量及大小明显高于正常成纤维细胞释放的微囊泡。正常纤维细胞、UVA和UVB照射后的成纤维细胞与微囊泡共孵育后活性氧荧光值分别为（52.76±1.4347）、（82.60±4.082）和（85.94±6.264）,凋亡率分别为（3.260±1.732）%,（28.94±2.430）%和（34.48±2.718）%,细胞的氧化损伤和凋亡可被抗氧化剂逆转。结论：急性中长波紫外线照射可诱导皮肤成纤维细胞释放微囊泡进一步介导细胞的氧化损伤和凋亡。     

白藜芦醇对UVB照射的人皮肤成纤维细胞MMP-1水平及细胞凋亡的影响

目的初步探寻红葡萄酒中主要活性成分白藜芦醇对紫外线照射皮肤保护作用的可能机制。方法白藜芦醇对UVB照射的人皮肤成纤维细胞基质金属蛋白酶(MMP)-1水平及细胞凋亡的影响。将培养的人皮肤成纤维细胞分为5组,A组:无UVB照射无干预组,B组:UVB照射无干预组,C组:UVB照射1μmol/L白藜芦醇干预组,D组:UVB照射10μmol/L白藜芦醇干预组,E组:UVB照射100μmol/L白藜芦醇干预组。UVB照射剂量均为30mJ/cm2。用免疫组织化学方法分别检测5组细胞中MMP-1水平,用磷脂酰丝氨酸外翻分析(AnnexinV法)检测细胞凋亡率。结果 A～E组5组细胞MMP-1阳性细胞评分及凋亡率差异均有统计学意义(P均<0.05)。A组评分及凋亡率均明显低于B组,两组比较差异有统计学意义(P均<0.05),B组与C,D,E组评分及凋亡率比较差异均有统计学意义(P均<0.05),C～E组3组评分及凋亡率比较差异亦有统计学意义(P<0.05)。结论白藜芦醇可以抑制UVB照射诱导的人皮肤成纤维细胞MMP-1水平的升高,并可减少细胞因UVB照射引起的凋亡,白藜芦醇可能通过此机制对紫外线照射皮肤起到保护作用。     

九味活血化瘀中药对正常人皮肤成纤维细胞胶原合成的影响

成纤维细胞是人体合成胶原的主要场所,探讨药物对成纤维细胞胶原合成的影响,是研究药物治疗胶原合成异常性疾病药理机制的重要步骤。国内治疗系统性硬皮病等胶原异常增生性疾病常选用活血化瘀中药[1]。李明等[2]报道多数活血化瘀中药对正常人皮肤成纤维细胞的增生具有抑制作用,但这些中药能否抑制该细胞的胶原合成尚不清楚。本实验拟观察活血化瘀中药对正常人皮肤成纤维细胞胶原合成的影响,为今后临床中药组方选择药物时提供参考。1材料与方法1.1材料1.1.1正常人皮肤成纤维细胞系的建立:在局麻下分别切取2例女性(25岁、40岁)、1例男性(47岁)…     

成纤维细胞自体填充移植研究进展

成纤维细胞是真皮主要功能细胞,在皮肤抗衰老过程中起重要作用.基础研究表明,自体成纤维细胞移植具有高度的细胞存活率和良好的安全性.移植的微环境以及其他细胞、基因、蛋白、细胞因子等因素可影响成纤维细胞的活性和功能.临床研究表明,自体成纤维细胞填充移植可有效改善皱纹、治疗凹陷性瘢痕、溃疡等损害.     

碱性成纤维细胞生长因子对皮肤萎缩性瘢痕中成纤维细胞的作用

目的探讨人碱性成纤维细胞生长因子(bFGF)对豚鼠皮肤萎缩性瘢痕中成纤维细胞的作用。方法取实验用成年豚鼠20只,于脊柱旁两侧A,B,C三处皮肤分别人工造成萎缩性瘢痕后,实验组在A处真皮层注射50μg/L的bFGF0.1mL,隔日1次,共4次;在C处真皮层注射同剂量生理盐水作阴性对照,B处不作任何处理做空白对照。术后第21天切取瘢痕组织行病理切片,应用鼠抗人ki-67单克隆抗体行免疫组化,显微镜下计算增殖成纤维细胞所占百分率。结果实验组、阴性对照组和空白对照组切口瘢痕增殖成纤维细胞百分率分别为(7.63±1.42)%,(0.98±0.33)%和(1.22±0.34)%。实验组增殖成纤维细胞百分率与阴性对照组及空白对照组比较显著增高,差异有统计学意义(P<0.05)。结论 bFGF可以促进豚鼠皮肤萎缩性瘢痕中的成纤维细胞增殖。     

8-MOP/UVA诱导培养真皮成纤维细胞衰老的作用

目的　探讨以 8 MOP/UVA作用于培养真皮成纤维细胞建立皮肤光老化模型的可行性。方法　采用光镜、电镜、流式细胞仪、酶组织化学、免疫组织化学等方法检测 8 MOP/UVA对培养真皮成纤维细胞多项细胞衰老相关指标的影响。结果　 8 MOP/UVA作用后 ,培养真皮成纤维细胞迅速出现细胞衰老特征性的形态学及生物学特性改变 :细胞由分裂表型转化为无分裂活性表型 ,SA β Gal表达增加、p16蛋白表达增加。结论　 8 MOP/UVA可诱导培养真皮成纤维细胞迅速出现具有细胞衰老特征性的形态学及生物学特性改变 ,以 8 MOP/UVA作用于培养真皮成纤维细胞建立皮肤光老化模型是可行的     

咪喹莫特对人皮肤成纤维细胞活性和凋亡的影响

目的：观察咪喹莫特（imiquimod）溶液对人真皮成纤维细胞（FB）细胞毒性、细胞增殖及细胞凋亡率的影响，探讨其治疗瘢痕疙瘩的可能机制。方法：分离培养人真皮成纤维细胞，用四甲基偶氮唑蓝（MTF）法检测不同浓度咪喹莫特溶液对成纤维细胞毒性及细胞增殖的影响：用流式细胞技术检测咪喹莫特对成纤维细胞凋亡率的影响。结果：不同浓度咪喹莫特加入成纤维细胞培养体系孵育24h后，成纤维细胞形态均有不同程度受损，且细胞增殖活性下降，尤以20mg/L浓度时细胞活性下降明显；各浓度咪喹莫特处理组细胞凋亡率均高于对照组（P〈0．05），但在不同时间点检测的细胞凋亡率差异无统计学意义（P〉0．05）。结论：咪喹莫特可以降低成纤维细胞增殖活性并增加细胞凋亡率，这些可能是其促进皮肤瘢痕组织消退的相关机制。     

Caspase-3在UVB诱导人皮肤成纤维细胞凋亡中的作用

目的：评价caspase-3在UVB诱导皮肤成纤维细胞凋亡中的作用。方法：皮肤成纤维细胞经150mJ／cm2 UVB照射后,用MTF法检测细胞活性,用Hoechst33258染色法检测细胞凋亡,用抑制剂Z-DEVD-FMK抑制caspase-3活性后,检测凋亡细胞数量。结果：UVB明显抑制成纤维细胞的活性,并导致细胞凋亡,并呈时间-效应关系;加入抑制剂Z-DEVD-FMK抑制了UVB导致的细胞凋亡。结论：Caspase-3在UVB照射诱导皮肤成纤维细胞凋亡中发挥重要作用。     

染料木素对UVA诱导成纤维细胞急性光损伤的防护作用

目的：明确染料木素对UVA诱导成纤维细胞急性光损伤的防护作用。方法：将体外培养的成纤维细胞分为空白对照组,UVA照射对照组及UVA+染料木素（0.01,0.1,1,10,100umol/L）组。采用CCK8法测定细胞增殖情况,流式细胞术检测细胞凋亡情况, RT-PCR检测Sirt1mRNA的表达水平。结果：与UVA组比较,UVA+0.01,0.1,1,10 μmol/L染料木素组细胞增值活性及Sirt1mRNA表达明显升高,细胞凋亡率降低（均P<0.05）;而UVA+100μmol/L染料木素,细胞凋亡率无明显差异（P>0.05）;结论：0.01～10μmol/L染料木素对UVA诱导成纤维细胞的急性光损伤有防护作用。     

组织蛋白酶B在光老化皮肤中的表达及意义

目的 探讨组织蛋白酶B在光老化皮肤中的表达意义。方法 6例成人曝光和非曝光皮肤标本,采用免疫组化法定位及对比组织蛋白酶B的表达。体外培养原代人皮肤成纤维细胞,甲氧沙林 ＋ UVA法体外诱导培养细胞光老化。衰老相关-β-半乳糖苷酶染色证明老化诱导成功。Western印迹技术及RT-PCR对比检测光老化成纤维细胞及正常成纤维细胞组织蛋白酶B蛋白及基因表达。结果 6例成人曝光和非曝光活体皮肤均见组织蛋白酶B阳性染色,和非曝光部位相比,曝光部位皮肤阳性染色A值降低。Western印迹结果示,成纤维细胞光老化诱导组蛋白表达较UVA诱导组、甲氧沙林孵育组及空白组明显下调。光老化成纤维细胞诱导后1周,组织蛋白酶B与内参的灰度比由28.099 ± 0.054下降为25.103 ± 0.102,诱导后3周灰度值进一步下降为17.693 ± 0.099。实时定量RT-PCR结果示,光老化细胞组织蛋白酶B mRNA表达下调为正常组的64％（P < 0.05）。结论 组织蛋白酶B在光老化皮肤及老化成纤维细胞中表达降低,且有时间依赖性,与光老化皮肤自我修复能力下降有关。     

Tumor-derived CCL20 affects B16 melanoma growth in mice

BackgroundChemokine ligand-20 (CCL20) expressed in the epidermis is a potent impetus for the recruitment of CC-chemokine receptor 6 (CCR6)-expressing subsets of DCs, B-cells and memory T-cells into the skin. CCL20 and CCR6+ immune cells have been detected in chronic inflammatory skin diseases and several malignancies, including melanoma. Yet, the functional contribution of the CCR6/CCL20 axis for melanoma progression remains controversial.ObjectiveThe functional contribution of CCR6-expressing immune cell subsets and local CCL20 in the tumor microenvironment for the immune control of melanoma was studied.MethodsHomeostatic and inducible CCL20 secretion of murine (B16, Ret) and human (A375, C32) melanoma cells was analyzed by ELISA. To assess the functional relevance of CCR6/CCL20 interactions on local tumor progression, prestimulated or retrovirally transduced B16/F1 melanoma cells overexpressing CCL20 (B16-CCL20) were injected subcutaneously into C57BL/6 Wt mice and congenic CCR6-deficient (CCR6^−/−) mice. Infiltrating leucocytes were examined by flow cytometry in tumors and draining lymph nodes (DLNs).ResultsMelanoma cell lines up-regulate CCL20 secretion upon stimulation with pro-inflammatory cytokines in vitro. While only moderate changes in phenotype and composition of leucocytes were detected in advanced tumors and DLNs, mice injected with CCR6+ B16-CCL20 cells developed smaller tumors compared to B16-Control injected littermates, with CCR6-/- mice displaying the most pronounced reduction in tumor growth and incidence.ConclusionOur results suggest that CCR6/CCL20 interactions and individual independent effects of CCL20 and CCR6 in the microenvironment may be essential for melanoma progression and suggest a decisive role of this chemokine axis for melanoma pathogenesis beyond chemoattraction.     

黑素瘤B16-F1体外诱导淋巴管增生的研究

目的诱导建立小鼠淋巴管内皮细胞构成的良性肿瘤模型,观察小鼠黑素瘤细胞系B16F1体外对淋巴管增生的影响。方法8周龄C57BL/6小鼠腹腔注射弗氏不完全佐剂,对诱导出的小鼠腹腔淋巴管瘤进行病理组织学观察,用免疫组化方法检测淋巴管内皮细胞标志性抗原VEGFC和Flt4。分离、分切肿物后,于纤维蛋白凝胶内用B16F1细胞条件培养液培养,倒置显微镜下观察。结果实验小鼠中膈肌腹腔面、肝脏表面及侧腹壁腹腔面可见边界清楚的散在分布白色肿瘤样组织,常规和超微病理发现为由内皮细胞构成的多管腔囊性结构,并表达淋巴管内皮细胞标志性抗原VEGFC和Flt4。倒置显微镜下可观察到从淋巴管瘤块长入纤维蛋白凝胶内的微淋巴管,B16F1细胞条件培养液可促进淋巴管的生成。结论小鼠腹腔注射弗氏不完全佐剂可稳定诱导小鼠腹腔淋巴管瘤,B16F1细胞对淋巴管的生成有促进作用,黑素瘤的转移与淋巴管生成的关系有必要进一步研究。     

Combined treatment with intratumoral injection of dendritic cells and topical application of imiquimod for murine melanoma

BACKGROUND: The maturation state of dendritic cells is one of the factors that affect their capacity to induce antigen-specific cytotoxic T lymphocytes. Topical cutaneous application of imiquimod can induce the maturation and migration of cutaneous dendritic cells. OBJECTIVES: To evaluate the synergistic effect of topical application of imiquimod plus intratumoral injection of syngeneic bone marrow-derived dendritic cells in the treatment of melanoma. METHODS: For the B16F10 melanoma model, naive C57BL/6 mice were inoculated intradermally with 2x10(3) B16F10 melanoma cells in the right upper flank. Four groups (untreated control, dendritic cells alone, imiquimod alone and imiquimod plus dendritic cells) were included in the animal study, with five mice in each group. Tumour size was measured every 2 weeks, and histochemical and immunohistochemical staining carried out. ELISpot and PKH assays were performed to assess immune activity. RESULTS: Combined treatment of topical application of imiquimod and intratumoral injection of dendritic cells led to significant tumour regression, in contrast to partial eradication of the tumours with imiquimod or dendritic cells alone. CONCLUSION: These findings suggest that combination therapy with topical application of imiquimod and intratumoral administration of dendritic cells is a potent strategy for the treatment of melanoma.     

Dual mode reflectance and fluorescence confocal laser scanning microscopy for in vivo imaging melanoma progression in murine skin

A system was designed and developed for simultaneous fluorescence and reflectance contrast in vivo confocal imaging of murine skin using 488 nm (fluorescence mode) and 830 nm (reflectance mode) laser light sources. B16 melanoma cells and B16-enhanced green fluorescent protein (EGFP) cells were inoculated intradermally into transgenic C57BL/6-TgN (ACTbEGFP) 10sb and non-transgenic C57BL/6 mice, respectively. The inoculation sites were imaged sequentially over a 20 d period. The in vivo confocal images were correlated with ex vivo conventional microscopy. The combined modality system provided single-cell resolution and adequate image registration. In fluorescence mode, B16 melanoma cells appeared as dark objects in the bright background of the GFP expressing murine cells of the C57BL/6 transgenic mouse, and the B16-EGFP melanoma cells had a bright signal within a dark background in C57BL/6 mice. In the C57BL/6 transgenic mouse, a population of fluorescent dendritic cells was observed in the vicinity of the tumor cells. The reflectance images provide a useful reference for those areas in the dermal tissues lacking a fluorescent signal. Combined reflectance/fluorescence in vivo confocal laser scanning microscopy holds significant promise for studies of tumor progression in murine skin.     

Topical CpG enhances the response of murine malignant melanoma to dacarbazine

Malignant melanoma is a potentially fatal skin cancer that is increasing in incidence. Standard chemoimmunotherapy consisting of dacarbazine (DTIC) given with IFN-alpha has had disappointing results. We describe a chemoimmunotherapy protocol for cutaneous melanoma that combines the administration of DTIC with the topical application of CpG oligodinucleotide (ODN). Subcutaneous B16 melanoma tumors in C57BL/6 mice were treated with intraperitoneal injections of DTIC followed by the topical application of CpG-ODN over the tumors. This therapeutic approach abrogated the growth of established tumors and significantly enhanced survival. Topical CpG application was more effective than intratumoral CpG. Cell depletion studies indicated that the antitumor effect was dependent on both CD4(+) and CD8(+) cells but not on natural killer (NK) cells. Tumor-specific cytotoxic T-lymphocyte activity was generated in treated animals and was highest in topically treated animals. Immunohistochemical analysis revealed that DTIC, but not CpG, enhanced tumor cell apoptosis. Further, topical CpG induced an expansion of a B220(+)CD8(+) subset of dendritic cells and a subset of NK1.1(+) CD11c(+) cells within the tumors. By enhancing both tumor cell death and local immune activation, DTIC/topical CpG chemoimmunotherapy induced an effective T-cell-dependent host-immune response against melanoma.     

Ultraviolet A exposure might increase metastasis of mouse melanoma: a pilot study

BACKGROUND: The major sources of long-wave ultraviolet A radiation (UVA; 320-400 nm) exposure are extensive sunbathing and tanning in solaria. While the carcinogenic effects of mid-wave ultraviolet B radiation (UVB; 280-320 nm) are well recognized, the potentially hazardous effects of UVA are less understood. Several studies have shown that a variety of physiological processes in the cell are modified by UVA exposure, some of which might be involved in the regulation of tumor metastasis. In this study we suggest that UVA radiation could lead to the increase of metastatic capability of melanoma cells in mice. METHOD/RESULT: A pilot in vivo study was executed using C57BL/6 mice and syngeneic B16 melanoma cell lines. Mice were intravenously (i.v.) injected with either B16-F1 or B16-F10 melanoma cells into the tail vein and then immediately exposed to UVA. Fourteen days after melanoma injection, lungs were collected and the quantity and quality of metastases were determined under a dissecting microscope. As an outcome of the pilot study we observed that i.v. injected melanoma cells formed more lung metastases in the UVA-exposed mice in comparison with the control mice. CONCLUSION: This result suggests that the UVA exposure of mice, with melanoma cells present in blood circulation, increases the formation of melanoma metastases in lungs. Further studies should determine whether a similar pro-metastatic effect, as observed in mice, could occur in humans and whether other than melanoma tumors might be susceptible.     

Tumor regression of human and murine melanoma after intratumoral injection of IL-12-encoding plasmid DNA in mice

DNA coding for murine interleukin 12 (IL-12) prevents the formation of B16-melanoma metastasis when administered intramuscularly. Here, the antitumor effect of IL-12-encoding DNA on established mouse B16 melanoma and human melanoma tumors was investigated in vivo using two animal models: B16 melanoma in C57B/6 mice and human melanoma in nude mice. In B16 melanoma, intratumoral injections of IL-12-encoding DNA resulted in highly significant growth retardation when compared with mice injected with control vector. In the case of the human melanoma model, treatment with DNA coding for IL-12 induced regression of tumors in all cases, with complete disappearance of the tumor in two out of five animals. DNA treatment did not induce systemic side-effects. In the animals injected with control vector the human melanoma tumors grew expansively. The therapeutic effect of the DNA injection was mediated in part by natural killer (NK) cells as shown by NK-depletion experiments. An antivascular effect of IL-12 treatment was evident in histological examination with endothelial thickening and abrupt changes in vessel diameters. These results suggest that intratumoral plasmid DNA coding for IL-12 holds some promise as a new therapeutic tool for accessible melanoma lesions and should be tested in clinical trial.     

Retention of antimelanoma effect of polyinosinic-polycytidylic acid in neonatally thymectomized, irradiated, leukopenic mice.

Polyinosinic-polycytidylic acid (PIC), a synthetic polyribonucleotide, inhibits the growth of B16 malignant melanoma in C57BL/6 mice. Since PIC has been reported to augment immune responses, we tested the hypothesis that the antitumor effect of PIC against B16 melanoma is via immune stimulation. Mice were neonatally thymectomized or neonatally thymectomized and subsequently irradiated to suppress their immune reactivity. In such animals PIC retained its ability to inhibit the growth of B16 melanoma, in the face of profound leukopenia and lymphopenia, suggesting that its antimelanoma effect is probably not mediated by augmentation of the host's immune antitumor response.     

A human placental extract: in vivo and in vitro assessments of its melanocyte growth and pigment-inducing activities

BACKGROUND: The authenticity of various prototype human placental extracts with biological activity, such as that inducing vitiligo repigmentation, is under serious criticism, mainly due to a lack of demonstration at the cellular level. Considering the present worldwide scenario with regard to the occurrence and treatment of vitiligo, a thorough scientific exploration of such extracts should be undertaken. METHOD: One such prototype placental preparation was prepared, and was evaluated with regard to its melanogenic action in C57BL/6J mice in vivo and its mitogenic and melanogenic activity on B16F10 mouse melanoma cells and normal human melanocytes in vitro. The extract was applied topically to mice with age-induced prolonged telogenic phase of hair growth (grey body coat hair). Standard 3H-thymidine incorporation and spectrophotometric methods were followed to illustrate mitogenic and melanogenic effects at the cellular level. RESULTS: The resurgence of blue skin, followed by shiny black hair, at the regions of application of the extract demonstrated the reversal of the age-induced prolonged telogenic phase of hair growth to the anagenic phase after topical application of the extract on C57BL/6J mice. Further support was obtained from histology where, at the extract-treated sites, the development of new melanogenic centers and hair follicles was observed. During in vitro studies, the vehicle-free extract constituents stimulated both mitogenesis and melanogenesis of B16F10 mouse melanoma cells in a concentration-dependent manner. The cell morphology and extent of melanogenesis also showed significant changes. In addition, two known melanocyte activity-modulating peptides, endothelin-1 (ET-1) and adrenocorticotropic hormone (ACTH), were determined in the extract, chiefly in the total lipid fraction, indicating their effective cutaneous permeation. CONCLUSIONS: The extract was found to be a potent mitogen in the in vitro condition and a potent melanogen in both the in vitro and in vivo situations. This strongly suggests its therapeutic potential for the repigmentation of vitiligo patches.     

Modulation of Growth of Melanoma

Combined heparin-cortisone treatment induces regression of growth in a variety of murine tumors including melanoma. We injected 92 inbred C 57 b1/6 male mice each with 5 X 10(5) melanoma cells (B16, B16 F1, and B16 A6 lines) with different metastatic potential. Heparin (400 U/ml) and cortisone acetate (250 mg/kg SC injections) were given daily. Control experiments were performed both with the administration of no drugs and with administration of cortisone alone. Plasminogen activator activity, which is notoriously related to tumor growth, was evaluated using fibrin plate technique in 10 fragments taken before and 20 days after the combined heparin-cortisone treatment of B16 F1 and B16 A6 melanomas. The combined heparin-cortisone treatment slowed tumor growth, but no tumour regression was observed. Cutaneous fibrinolytic activity appeared increased in all specimens after the treatment.