炎症浸润细胞及基质金属蛋白酶参与光老化发生机制研究
摘要
光老化指皮肤由于反复光暴露引起的提前老化,其特征性组织学变化是弹性组织变性物质的沉积和胶原的嗜碱性变性,伴有Ⅰ型和Ⅲ型前胶原的降解。虽然其发生机制尚未完全明了,但是基质金属蛋白酶(matrix metalloproteinases,MMPs)增高或活性增强是胶原降解的重要机制,另外,某些细胞、细胞因子、代谢物质等也参与了光老化的发生和发展。已有研究证实,紫外线辐射皮肤角质形成细胞和成纤维细胞后激活多种细胞表面受体,经过一系列信号传导和多种成分的级联反应,诱导MMPs合成增加从而促进细胞外基质的降解,其中以MMP-1、MMP-3、MMP-9的作用最为重要。有形态学研究发现,曝光部位皮肤中炎症浸润细胞数目增加,因此,这些炎症浸润细胞在光老化发生、发展中的作用也逐渐受到关注。为了进一步探讨炎症浸润细胞是否及如何参与光老化的发生,本研究比较了曝光与非曝光部位炎症浸润细胞数目、MMPs表达程度,并将外周血单一核细胞(peripheral bloodmononuclear cell,PBMC)活化,观察其表达MMPs情况及对成纤维细胞表达MMPs的影响,为研究光老化的发生及防治提供理论基础。全文分为三章:
第一章炎症浸润细胞在曝光和非曝光皮肤中的组织学表现
目的观察、比较曝光及非曝光部位皮肤中炎症浸润细胞的类型、数目,探讨这些细胞在光老化发生中的作用。方法应用免疫组化方法分别对23例女性健康志愿者前臂伸侧(曝光)和上臂内侧(非曝光部位)皮肤石蜡标本中的细胞表面抗原CD3、CD45RO、CD68进行检测,计数阳性细胞数目,采用配对t检验、Pearson相关分析进行统计学处理。结果曝光部位皮肤组织CD3、CD45RO、CD68阳性细胞数分别为48.91±13.173、46.83±12.915、85.43±22.346/mm~2,非曝光部位分别为40.61±11.571、38.00±10.109、73.48±16.208/mm~2,曝光部位皮肤组织CD3、CD45RO、CD68阳性细胞数显著高于非曝光部位(前二者P＜0.01,后者P＜0.05)。曝光部位皮肤组织CD3、CD45RO阳性细胞数与年龄呈正相关(r=0.557、0.555,P＜0.01),曝光部位CD68及非曝光部位皮肤组织CD3、CD45RO、CD68阳性细胞数与年龄无相关性(r=0.323、0.308、0.217、0.215,P＞0.05)。结论曝光部位CD3、CD45RO、CD68阳性细胞数显著高于非曝光部位,且曝光部位皮肤组织CD3、CD45RO阳性细胞数与年龄呈正相关,T淋巴细胞、巨噬细胞可能在光老化过程中发挥作用。
第二章基质金属蛋白酶在曝光及非曝光部位皮肤中的表达
目的检测基质金属蛋白酶-1、-3、-9(MMP-1、-3、-9)在曝光部位和非曝光部位皮肤中的表达,探讨它们在光老化机制中的作用。方法应用免疫组化方法分别对23例女性健康志愿者前臂伸侧(曝光)和上臂内侧(非曝光部位)皮肤石蜡标本中的MMP-1、MMP-3、MMP-9进行检测,表达强度以细胞染色强度和阳性细胞比例得分相乘即免疫反应强度分布指数(IRIDI)表示,采用Wilcoxon符号秩和检验、Mann-Whitney秩和检验及Spearman秩相关分析进行统计学处理。结果MMP-1、MMP-3、MMP-9在曝光和非曝光部位均表达,曝光部位皮肤组织中MMP-1、MMP-3、MMP-9的IRIDI均值(范围)分别为7.70(3～12)、9.22(6～12)、8.30(6～12),非曝光部位分别为4.26(2～6)、5.39(2～9)、4.04(1～6),曝光部位MMP-1、MMP-3、MMP-9的IRIDI均显著高于非曝光部位(均P＜0.01)。50岁以上组曝光部位皮肤组织MMP-1、MMP-3、MMP-9的IRIDI分别为9.17(6～12)、10.58(8～12)、8.92(8～12),非曝光部位分别为4.75(2～6)、6.42(4～9)、4.33(3～6),曝光部位均显著高于非曝光部位(P＜0.05);50岁以下组曝光部位皮肤组织MMP-1、MMP-3、MMP-9的IRIDI分别为6.09(3～8)、7.73(6～9)、7.64(6～12),非曝光部位分别为3.73(2～6)、4.27(2～8)、3.73(1～6),曝光部位均显著高于非曝光部位(P＜0.05);50岁以上组曝光部位IRIDI值均显著高于50岁以下组(P＜0.05),而两组非曝光部位皮肤组织MMP-1、MMP-9的IRIDI值差异无统计学意义(P＞0.05)。曝光部位皮肤组织MMP-1、MMP-3、MMP-9的IRIDI值与年龄呈正相关(r=0.656、0.691、0.742,P＜0.01),非曝光部位皮肤组织MMP-1、MMP-9的IRIDI值与年龄无相关性。结论MMP-1、MMP-3、MMP-9在曝光部位表达显著高于非曝光部位,三者在光老化的发生发展中均起作用,但作用机制可能不同。
第三章外周血单一核细胞活化及对成纤维细胞的影响
目的研究不同活化剂对PBMC产生MMPs的影响以及PBMC活化后上清液(conditioned media,CM)对培养的皮肤成纤维细胞增殖活性、分泌MMPs的影响。方法抽取、分离外周血单一核细胞,用活化剂PHA及CD3、CD28抗体活化细胞,同时以含10%胎量问血清的RPMI1640培养基作为阴性活化剂,获得CM后将其加入培养的成纤维细胞中,MTT法检测细胞增殖能力,ELISA法检测上清液中IL-6、MMPs水平,RT-PCR法检测细胞MMP-1、MMP-3 mRNA表达水平,采用单因素方差分析等方法进行统计学处理。结果不同处理组PBMC的增殖能力、活化后上清液中IL-6和MMP-3浓度比较有显著性差异(P＜0.05),MMP-1 mRNA表达有显著性差异(P＜0.01),以PHA刺激组最强,不加活化剂组最弱,MMP-3 mRNA无表达,活化后上清液中未检测到MMP-1、MMP-9蛋白。采用不同浓度的CM刺激成纤维细胞后其上清液中MMP-3浓度随着CM刺激浓度的增加而增加,但均低于基础CM,上清液中未检测到MMP-1、MMP-9蛋白,不同处理组成纤维细胞的增殖能力及MMP-1、MMP-3 mRNA表达无显著性差异(P＞0.05)。结论PBMC活化后可分泌MMP-3及表达MMP-1 mRNA,PBMC活化后上清液不能刺激成纤维细胞分泌MMP-1、MMP-3、MMP-9,提示炎症细胞可能通过自身分泌MMPs参与光老化。
Photoaging is described as a premature skin aging due to repeated exposure to ultraviolet,histologically characterized by excessive deposition of degenerative elastotic material and basophilic degeneration of collagen,and accompanied by degradation of typesⅠandⅢprocollagen. This impairment is thought to result from breakdown of collagen by increased matrix metalloproteinases(MMPs) expression,which may involve multiple materials such as cells,cytokines and metabolites,but the mechanisms and pathogenesis of photoaging are not well understood.Previous evidence has indicated that the ultraviolet irradiation promotes MMPs production by activation of multiple cell surface receptors on keratinocytes and fibroblasts and altered downstream signal transduction pathways,among which MMP-1,-3 and -9 are the most significant.In recent years the effect of infiltrating cells in the photoaging process has been noticed,showing histological increase in inflammatory infiltrate in sun-exposed skin.In order to further explore whether and how the inflammatory cells involved in the process of photoaging,the number of infiltrating cells and expression of MMPs·in sun-exposed and sun-unexposed skin would be compared,and the induction of MMPs and the effect on the human fibroblasts after activation of peripheral blood mononuclear cell(PBMC) would be studied,which providing a fundamental basis for occurrence,prevention and treatment of photoaging.The paper consists of three chapters.
ChapterⅠHistological manifestation of infiltrating cells in sun-exposed and sun-unexposed skin
Objective To investigate the effect of infiltrating cells on the photoaging by comparing types and number of infiltrating cells in sun-exposed and sun-unexposed skin.Methods The expression of CD3,CD45RO and CD68 was detected by immunohistochemical staining in 46 paraffine samples from extensor forearm(sun-exposed) and upper-inner arm(sun-unexposed) skin of 23 healthy female volunteers,and the number of positive cells was counted. The above data were analyzed by paired-samples t test and Pearson correlation analysis.Results The mean±SD number of positive cells for CD3,CD45RO and CD68 in the sun-exposed skin sections(48.91±13.173,46.83±12.915 and 85.43±22.346 cells/mm~2,respectively) was significantly higher than that in the sun-unexposed skin sections(40.61±11.571,38.00±10.109 and 73.48±16.208/mm~2,respectively)(the two former P＜0.01,the latter P＜0.05, respectively).The number of positive cells for CD3 and CD45RO in the sun-exposed skin sections increased significantly with age(r=0.557,0.555, all P＜0.01),but the number was uncorrelated with age in the sun-unexposed skin,and the number of positive cells for CD68 was uncorrelated with age in the sun-exposed and sun-unexposed skin.Conclusions The number of positive cells for CD3,CD45RO and CD68 in the sun-exposed skin sections were significantly higher than that in the sun-unexposed skin sections,and the number of positive cells for CD3 and CD45RO in the sun-exposed skin sections was positively correlated with age,implying that T lymphocytes and macrophages may play a role in the process of photoaging.
ChapterⅡExpression of MMPs in sun-exposed and sun-unexposed skin Objective To study the expression of MMP-1,-3 and -9 in sun-exposed skin and -unexposed skin as well as its significance in the mechanism of photoaging.Methods Skin samples were resected from the extensor side of forearm(sun-exposed area) and upper-inner arm(sun-unexposed area) of 23 healthy female volunteers.The expression of MMP-1,-3 and -9 was detected by immunohistochemical staining in 46 skin samples.Immunoreactivity intensity distribution index(IRIDI) based on the intensity of immunoreactivity and proportion of immunoreactive cells was calculated to assess the expression of MMP-1,-3 and -9.Wilcoxon signed ranks test, Mann-Whitney U-test and Spearman rank correlation analysis were performed. Results MMP-1,-3 and -9 were expressed in both sun-exposed and -unexposed skin.The average IRIDI value for MMP-1,-3 and -9 was 7.70(range,3 to 12),9.22(range,6 to 12),8.30(range,6 to 12) in sun-exposed skin,and 4.26(range,2 to 6),5.39(range,2 to 9),4.04(range,1 to 6) in sun-unexposed skin,respectively;significant difference existed between sun-exposed and -unexposed skin in the three parameters(all P＜0.01).A significant increase was observed in the average IRIDI value for MMP-1,-3 and -9 in sun-exposed skin vs.sun-unexposed skin in women aged above 50 years(9.17 vs 4.75,10.58 vs 6.42,8.92 vs 4.33,respectively,all P＜0.05). In women younger than 50 years,the average IRIDI value for MMP-1,-3 and -9 was 6.09(range,3 to 8),7.73(range,6 to 9),7.64(range,6 to 12) in sun-exposed skin,significantly higher than that in sun-unexposed skin[3.73 (range,2 to 6),4.27(range,2 to 8),3.73(range,1 to 6),all P＜0.05]. Increased IRIDI scores of MMP-1,-3 and -9 were noticed in sun-exposed skin in women aged above 50 years vs.those younger than 50 years,but there was no statistical difference in MMP-1 or MMP-9 between the two aged groups in sun-unexposed skin(all P＞0.05).The IRIDI scores of MMP-1,-3 and -9 were positively correlated with age(r=0.656,0.691,0.742,P＜0.01) in sun-exposed skin,but the IRIDI scores of MMP-1 and MMP-9 uncorrelated with age in sun-unexposed skin.Conclusions There is an elevated expression of MMP-1,-3 and -9 in sun-exposed skin vs.sun-unexposed skin,hinting that these three MMPs play a role in the occurrence and development of photoaging, but their biological mechanism may be different.
ChapterⅢActivation of PBMC and the effect of conditioned media(CM) on the cultured human fibroblasts
Objective To study the effect of different stimulator on the induction of MMPs from PBMC,and the effect of conditioned media on human fibroblasts' proliferation and the induction of MMPs.Methods PBMC were isolated from human venous blood and stimulated by PHA or antibodies against CD3 and CD28,simultaneously RPMI1640 medium with 10%fetal calf serum serving as negative stimulator,after which CM were obtained,then CM were added to human skin fibroblasts.Cells' proliferation was measured by MTT assay,the level of IL-6 and MMPs in the supernatant media was detected by ELISA,and the expression of MMP-1 and MMP-3 mRNA was measured by RT-PCR. Analysis of variance(One-Way ANOVA) was mainly used.Results There were significant differences in different stimulated cells about the proliferation of PBMC,the level of IL-6,MMP-3 in the supernatant media (all P＜0.05),and the expression of MMP-1 mRNA(P＜0.05),showing the highest level in cells stimulated by PHA and lowest level in untreated cells.But the expression of MMP-3 mRNA was negative,and there were no MMP-1 and MMP-9 proteins in the supernatant after activation.Using different concentrations of CM to stimulate cultured fibroblast,the level of MMP-3 in different fibroblasts' supernatant media increased with stimulus concentration of CM,but was lower than that in baseline CM.There were no significant differences in different treated fibroblasts about the proliferation,MMP-1 and MMP-3 mRNA expression(all P＞0.05),and no MMP-1 and MMP-9 proteins in the supernatant after stimulation.Conclusions MMP-1 mRNA could be expressed and MMP-3 secreted from PBMC after activation.The supernatant media obtained from PBMC activation have no capacity to improve the induction of MMP-1,-3 and -9 from fibroblasts,suggesting that the inflammatory cells may be involved in the process of photoaging by production of MMPs themselves.
第一章炎症浸润细胞在曝光和非曝光皮肤中的组织学表现
目的观察、比较曝光及非曝光部位皮肤中炎症浸润细胞的类型、数目,探讨这些细胞在光老化发生中的作用。方法应用免疫组化方法分别对23例女性健康志愿者前臂伸侧(曝光)和上臂内侧(非曝光部位)皮肤石蜡标本中的细胞表面抗原CD3、CD45RO、CD68进行检测,计数阳性细胞数目,采用配对t检验、Pearson相关分析进行统计学处理。结果曝光部位皮肤组织CD3、CD45RO、CD68阳性细胞数分别为48.91±13.173、46.83±12.915、85.43±22.346/mm~2,非曝光部位分别为40.61±11.571、38.00±10.109、73.48±16.208/mm~2,曝光部位皮肤组织CD3、CD45RO、CD68阳性细胞数显著高于非曝光部位(前二者P＜0.01,后者P＜0.05)。曝光部位皮肤组织CD3、CD45RO阳性细胞数与年龄呈正相关(r=0.557、0.555,P＜0.01),曝光部位CD68及非曝光部位皮肤组织CD3、CD45RO、CD68阳性细胞数与年龄无相关性(r=0.323、0.308、0.217、0.215,P＞0.05)。结论曝光部位CD3、CD45RO、CD68阳性细胞数显著高于非曝光部位,且曝光部位皮肤组织CD3、CD45RO阳性细胞数与年龄呈正相关,T淋巴细胞、巨噬细胞可能在光老化过程中发挥作用。
第二章基质金属蛋白酶在曝光及非曝光部位皮肤中的表达
目的检测基质金属蛋白酶-1、-3、-9(MMP-1、-3、-9)在曝光部位和非曝光部位皮肤中的表达,探讨它们在光老化机制中的作用。方法应用免疫组化方法分别对23例女性健康志愿者前臂伸侧(曝光)和上臂内侧(非曝光部位)皮肤石蜡标本中的MMP-1、MMP-3、MMP-9进行检测,表达强度以细胞染色强度和阳性细胞比例得分相乘即免疫反应强度分布指数(IRIDI)表示,采用Wilcoxon符号秩和检验、Mann-Whitney秩和检验及Spearman秩相关分析进行统计学处理。结果MMP-1、MMP-3、MMP-9在曝光和非曝光部位均表达,曝光部位皮肤组织中MMP-1、MMP-3、MMP-9的IRIDI均值(范围)分别为7.70(3～12)、9.22(6～12)、8.30(6～12),非曝光部位分别为4.26(2～6)、5.39(2～9)、4.04(1～6),曝光部位MMP-1、MMP-3、MMP-9的IRIDI均显著高于非曝光部位(均P＜0.01)。50岁以上组曝光部位皮肤组织MMP-1、MMP-3、MMP-9的IRIDI分别为9.17(6～12)、10.58(8～12)、8.92(8～12),非曝光部位分别为4.75(2～6)、6.42(4～9)、4.33(3～6),曝光部位均显著高于非曝光部位(P＜0.05);50岁以下组曝光部位皮肤组织MMP-1、MMP-3、MMP-9的IRIDI分别为6.09(3～8)、7.73(6～9)、7.64(6～12),非曝光部位分别为3.73(2～6)、4.27(2～8)、3.73(1～6),曝光部位均显著高于非曝光部位(P＜0.05);50岁以上组曝光部位IRIDI值均显著高于50岁以下组(P＜0.05),而两组非曝光部位皮肤组织MMP-1、MMP-9的IRIDI值差异无统计学意义(P＞0.05)。曝光部位皮肤组织MMP-1、MMP-3、MMP-9的IRIDI值与年龄呈正相关(r=0.656、0.691、0.742,P＜0.01),非曝光部位皮肤组织MMP-1、MMP-9的IRIDI值与年龄无相关性。结论MMP-1、MMP-3、MMP-9在曝光部位表达显著高于非曝光部位,三者在光老化的发生发展中均起作用,但作用机制可能不同。
第三章外周血单一核细胞活化及对成纤维细胞的影响
目的研究不同活化剂对PBMC产生MMPs的影响以及PBMC活化后上清液(conditioned media,CM)对培养的皮肤成纤维细胞增殖活性、分泌MMPs的影响。方法抽取、分离外周血单一核细胞,用活化剂PHA及CD3、CD28抗体活化细胞,同时以含10%胎量问血清的RPMI1640培养基作为阴性活化剂,获得CM后将其加入培养的成纤维细胞中,MTT法检测细胞增殖能力,ELISA法检测上清液中IL-6、MMPs水平,RT-PCR法检测细胞MMP-1、MMP-3 mRNA表达水平,采用单因素方差分析等方法进行统计学处理。结果不同处理组PBMC的增殖能力、活化后上清液中IL-6和MMP-3浓度比较有显著性差异(P＜0.05),MMP-1 mRNA表达有显著性差异(P＜0.01),以PHA刺激组最强,不加活化剂组最弱,MMP-3 mRNA无表达,活化后上清液中未检测到MMP-1、MMP-9蛋白。采用不同浓度的CM刺激成纤维细胞后其上清液中MMP-3浓度随着CM刺激浓度的增加而增加,但均低于基础CM,上清液中未检测到MMP-1、MMP-9蛋白,不同处理组成纤维细胞的增殖能力及MMP-1、MMP-3 mRNA表达无显著性差异(P＞0.05)。结论PBMC活化后可分泌MMP-3及表达MMP-1 mRNA,PBMC活化后上清液不能刺激成纤维细胞分泌MMP-1、MMP-3、MMP-9,提示炎症细胞可能通过自身分泌MMPs参与光老化。
Photoaging is described as a premature skin aging due to repeated exposure to ultraviolet,histologically characterized by excessive deposition of degenerative elastotic material and basophilic degeneration of collagen,and accompanied by degradation of typesⅠandⅢprocollagen. This impairment is thought to result from breakdown of collagen by increased matrix metalloproteinases(MMPs) expression,which may involve multiple materials such as cells,cytokines and metabolites,but the mechanisms and pathogenesis of photoaging are not well understood.Previous evidence has indicated that the ultraviolet irradiation promotes MMPs production by activation of multiple cell surface receptors on keratinocytes and fibroblasts and altered downstream signal transduction pathways,among which MMP-1,-3 and -9 are the most significant.In recent years the effect of infiltrating cells in the photoaging process has been noticed,showing histological increase in inflammatory infiltrate in sun-exposed skin.In order to further explore whether and how the inflammatory cells involved in the process of photoaging,the number of infiltrating cells and expression of MMPs·in sun-exposed and sun-unexposed skin would be compared,and the induction of MMPs and the effect on the human fibroblasts after activation of peripheral blood mononuclear cell(PBMC) would be studied,which providing a fundamental basis for occurrence,prevention and treatment of photoaging.The paper consists of three chapters.
ChapterⅠHistological manifestation of infiltrating cells in sun-exposed and sun-unexposed skin
Objective To investigate the effect of infiltrating cells on the photoaging by comparing types and number of infiltrating cells in sun-exposed and sun-unexposed skin.Methods The expression of CD3,CD45RO and CD68 was detected by immunohistochemical staining in 46 paraffine samples from extensor forearm(sun-exposed) and upper-inner arm(sun-unexposed) skin of 23 healthy female volunteers,and the number of positive cells was counted. The above data were analyzed by paired-samples t test and Pearson correlation analysis.Results The mean±SD number of positive cells for CD3,CD45RO and CD68 in the sun-exposed skin sections(48.91±13.173,46.83±12.915 and 85.43±22.346 cells/mm~2,respectively) was significantly higher than that in the sun-unexposed skin sections(40.61±11.571,38.00±10.109 and 73.48±16.208/mm~2,respectively)(the two former P＜0.01,the latter P＜0.05, respectively).The number of positive cells for CD3 and CD45RO in the sun-exposed skin sections increased significantly with age(r=0.557,0.555, all P＜0.01),but the number was uncorrelated with age in the sun-unexposed skin,and the number of positive cells for CD68 was uncorrelated with age in the sun-exposed and sun-unexposed skin.Conclusions The number of positive cells for CD3,CD45RO and CD68 in the sun-exposed skin sections were significantly higher than that in the sun-unexposed skin sections,and the number of positive cells for CD3 and CD45RO in the sun-exposed skin sections was positively correlated with age,implying that T lymphocytes and macrophages may play a role in the process of photoaging.
ChapterⅡExpression of MMPs in sun-exposed and sun-unexposed skin Objective To study the expression of MMP-1,-3 and -9 in sun-exposed skin and -unexposed skin as well as its significance in the mechanism of photoaging.Methods Skin samples were resected from the extensor side of forearm(sun-exposed area) and upper-inner arm(sun-unexposed area) of 23 healthy female volunteers.The expression of MMP-1,-3 and -9 was detected by immunohistochemical staining in 46 skin samples.Immunoreactivity intensity distribution index(IRIDI) based on the intensity of immunoreactivity and proportion of immunoreactive cells was calculated to assess the expression of MMP-1,-3 and -9.Wilcoxon signed ranks test, Mann-Whitney U-test and Spearman rank correlation analysis were performed. Results MMP-1,-3 and -9 were expressed in both sun-exposed and -unexposed skin.The average IRIDI value for MMP-1,-3 and -9 was 7.70(range,3 to 12),9.22(range,6 to 12),8.30(range,6 to 12) in sun-exposed skin,and 4.26(range,2 to 6),5.39(range,2 to 9),4.04(range,1 to 6) in sun-unexposed skin,respectively;significant difference existed between sun-exposed and -unexposed skin in the three parameters(all P＜0.01).A significant increase was observed in the average IRIDI value for MMP-1,-3 and -9 in sun-exposed skin vs.sun-unexposed skin in women aged above 50 years(9.17 vs 4.75,10.58 vs 6.42,8.92 vs 4.33,respectively,all P＜0.05). In women younger than 50 years,the average IRIDI value for MMP-1,-3 and -9 was 6.09(range,3 to 8),7.73(range,6 to 9),7.64(range,6 to 12) in sun-exposed skin,significantly higher than that in sun-unexposed skin[3.73 (range,2 to 6),4.27(range,2 to 8),3.73(range,1 to 6),all P＜0.05]. Increased IRIDI scores of MMP-1,-3 and -9 were noticed in sun-exposed skin in women aged above 50 years vs.those younger than 50 years,but there was no statistical difference in MMP-1 or MMP-9 between the two aged groups in sun-unexposed skin(all P＞0.05).The IRIDI scores of MMP-1,-3 and -9 were positively correlated with age(r=0.656,0.691,0.742,P＜0.01) in sun-exposed skin,but the IRIDI scores of MMP-1 and MMP-9 uncorrelated with age in sun-unexposed skin.Conclusions There is an elevated expression of MMP-1,-3 and -9 in sun-exposed skin vs.sun-unexposed skin,hinting that these three MMPs play a role in the occurrence and development of photoaging, but their biological mechanism may be different.
ChapterⅢActivation of PBMC and the effect of conditioned media(CM) on the cultured human fibroblasts
Objective To study the effect of different stimulator on the induction of MMPs from PBMC,and the effect of conditioned media on human fibroblasts' proliferation and the induction of MMPs.Methods PBMC were isolated from human venous blood and stimulated by PHA or antibodies against CD3 and CD28,simultaneously RPMI1640 medium with 10%fetal calf serum serving as negative stimulator,after which CM were obtained,then CM were added to human skin fibroblasts.Cells' proliferation was measured by MTT assay,the level of IL-6 and MMPs in the supernatant media was detected by ELISA,and the expression of MMP-1 and MMP-3 mRNA was measured by RT-PCR. Analysis of variance(One-Way ANOVA) was mainly used.Results There were significant differences in different stimulated cells about the proliferation of PBMC,the level of IL-6,MMP-3 in the supernatant media (all P＜0.05),and the expression of MMP-1 mRNA(P＜0.05),showing the highest level in cells stimulated by PHA and lowest level in untreated cells.But the expression of MMP-3 mRNA was negative,and there were no MMP-1 and MMP-9 proteins in the supernatant after activation.Using different concentrations of CM to stimulate cultured fibroblast,the level of MMP-3 in different fibroblasts' supernatant media increased with stimulus concentration of CM,but was lower than that in baseline CM.There were no significant differences in different treated fibroblasts about the proliferation,MMP-1 and MMP-3 mRNA expression(all P＞0.05),and no MMP-1 and MMP-9 proteins in the supernatant after stimulation.Conclusions MMP-1 mRNA could be expressed and MMP-3 secreted from PBMC after activation.The supernatant media obtained from PBMC activation have no capacity to improve the induction of MMP-1,-3 and -9 from fibroblasts,suggesting that the inflammatory cells may be involved in the process of photoaging by production of MMPs themselves.
引文
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