细菌内毒素对人皮肤成纤维细胞生物学特性的影响
发布时间：2018-08-14 08:59
【摘要】： 目的：1．通过内毒素(lipopolysaccharide，LPS)刺激实验，，观察LPS对正常皮肤成纤维细胞增殖和胶原合成等生物学特性的影响，探讨LPS在早期创面愈合中的可能作用。2．使用特定浓度LPS刺激正常皮肤成纤维细胞后连续传代，观察其形态学、生物学特性及基因表达谱改变，并与自身增生性瘢痕组织成纤维细胞进行比较，探讨LPS对后期瘢痕形成的影响及可能机制。 方法：随机选取增生性瘢痕患者20例(处于增生期)，取其瘢痕组织及正常皮肤，采用组织块法行成纤维细胞原代培养。对纯化至第3代的正常皮肤成纤维细胞用不同浓度的LPS(E. coli055：B5)进行刺激，实验分组如下：(1)0.000ug/ml LPS刺激组(阴性对照组)；(2)0.005ug/ml LPS刺激组；(3)0.010ug／ml LPS刺激组；(4)0.050ug／ml LPS刺激组；(5)0.100ug/ml LPS刺激组；(6)0.500ug／ml LPS刺激组；(7)1.000ug/ml LPS刺激组。采用下列方法检测成纤维细胞生物学特性(细胞增殖和胶原合成)：细胞计数法、MTT比色法及细胞增殖周期检测成纤维细胞增殖活性，~3H-脯氨酸掺入、胃蛋白酶消化法检测成纤维细胞内胶原合成量，将LPS刺激作用最为明显组成纤维细胞进行连续传代培养，采用透射电镜观察(第4、6、8、10代)成纤维细胞超微结构的变化，其中自第8代开始成纤维细胞超微结构与自身增生性瘢痕组织成纤维细胞超微结构接近，同时采用HE染色、免疫组织化学染色检测形态学及表型变化，结果也具有一致性。因此，选择(第8代)成纤维细胞进行实验，实验分组如下：(1)瘢痕组织成纤维细胞组(阳性对照组)；(2)阴性对照组(正常皮肤成纤维细胞不予LPS刺激)；(5)LPS刺激组(正常皮肤成纤维细胞给予LPS刺激)。采用下列方法检测成纤维细胞增殖和分泌特性：MTT比色法检测成纤维细胞增殖活性；~3H-脯氨酸掺入、胃蛋白酶消化法检测成纤维细胞内胶原合成量，同时采用酶联免疫吸附试验(ELISA)检测成纤维细胞培养上清液中Ⅰ、Ⅲ型胶原含量。采用下列方法检测成纤维细胞基因学指标：逆转录-聚合酶链反应技术(RT-PCR)检测成纤维细胞Ⅰ、Ⅲ型前胶原及胶原酶mRNA表达，基因芯片技术检测成纤维细胞基因表达谱的变化，通过与自身增生性瘢痕组织成纤维细胞相关基因进行对比，挑选差异基因，采用RT-PCR法进行验证。除了上述指标以外，我们还采用ELISA法检测成纤维细胞培养上清液中TGF-β_1、IFN-γ的含量，探讨LPS对成纤维细胞的可能作用机制。 结果：1．LPS对成纤维细胞增殖活性和胶原合成的影响LPS刺激浓度在0.005μg／ml～0.5μg/ml时，促进成纤维细胞增殖和胶原合成(P＜0.05)，以0.1μg／mlLPS刺激作用最为明显，LPS刺激浓度为1.0μg／ml时，则抑制成纤维细胞增殖和胶原合成(P＜0.05)。2．0.1μg/ml LPS刺激并传代后不同代次成纤维细胞的病理学变化自第8代开始成纤维细胞超微结构与自身增生性瘢痕组织成纤维细胞超微结构近似，同时采用HE染色、免疫组织化学染色检测形态学及表型变化，结果也具有一致性。3．0.1μg/ml LPS刺激并传代后成纤维细胞增殖活性的变化0.1μg／mlLPS刺激并传代后成纤维细胞增殖率增加，且其增殖率与自身增生性瘢痕组织成纤维细胞增殖率近似，统计学分析无显著差异(P＞0.05)。4．0.1μg/ml LPS刺激并传代后成纤维细胞胶原合成及Ⅰ、Ⅲ型胶原分泌的变化0.1μg／ml LPS刺激并传代后成纤维细胞胶原合成及各时间点Ⅰ、Ⅲ型胶原的分泌增加、Ⅰ／Ⅲ型胶原比例提高，且与自身增生性瘢痕组织成纤维细胞胶原合成及Ⅰ、Ⅲ型胶原的分泌量、Ⅰ／Ⅲ型胶原比例近似，统计学分析无显著差异(P＞0.05)。5．0.1μg／ml LPS刺激并传代后成纤维细胞Ⅰ、Ⅲ型前胶原及胶原酶mRNA表达的变化0.1μg／ml LPS刺激并传代后成纤维细胞Ⅰ、Ⅲ型前胶原mRNA的表达升高、胶原酶mRNA表达下降，且Ⅰ、Ⅲ型前胶原及胶原酶mRNA表达量与自身增生性瘢痕组织成纤维细胞表达量近似(P＞0.05)。6．0.1μg／ml LPS刺激并传代后成纤维细胞基因表达谱的改变0.1μg/ml LPS刺激并传代后成纤维细胞基因表达谱发生改变，其中与胶原代谢相关的一些基因(Ⅰ型胶原、c-myc、TGF-β1)表达均上调，经RT-PCR验证，这些基因表达与自身增生性瘢痕组织成纤维细胞表达量近似(P＞0.05)。7．0.1μg/ml LPS刺激并传代后成纤维细胞培养上清液中TGF-β_1、IFN-γ含量的变化0.1μg/mlLPS刺激并传代后各时间点成纤维细胞TGF-β_1的分泌增加、IFN-γ的分泌减少，与自身瘢痕组织成纤维细胞分泌量近似，相比无统计学差异(P＞0.05)。 结论：1、一定浓度的LPS刺激后可使成纤维细胞的生物学特性发生改变：细胞增殖和胶原合成增加，过高浓度则呈抑制作用，提示适度的LPS存在可能不影响甚至有利于创面愈合，LPS过多则可能延缓创面愈合时间。2、特定浓度的LPS刺激并传代后，成纤维细胞具备自身增生性瘢痕组织成纤维细胞的形态学特征及某些生物学特性：超微结构、形态学、表型一致，细胞增殖加快和胶原合成增加，Ⅰ、Ⅲ型胶原分泌增加，Ⅰ、Ⅲ型前胶原mRNA表达上调，胶原酶mRNA表达下调，同时基因表达谱也进一步证实了基因学变化。提示LPS可能诱导正常皮肤成纤维细胞转化为增生性瘢痕组织成纤维细胞，参与增生性瘢痕形成。LPS的作用机制可能与其促进TGF-β_1的分泌，而抑制IFN-γ的分泌有关。
[Abstract]:OBJECTIVE: 1. To observe the effects of LPS on proliferation and collagen synthesis of normal skin fibroblasts by lipopolysaccharide (LPS) stimulation test, and to explore the possible role of LPS in early wound healing. 2. To observe the morphology and proliferation of normal skin fibroblasts after stimulating the passage of fibroblasts with specific concentration of LPS. The changes of biological characteristics and gene expression profiles were compared with those of fibroblasts from hypertrophic scar tissues to explore the effect of LPS on the formation of late scar and its possible mechanism.
Methods: Twenty patients with hypertrophic scar were randomly selected and their scar tissues and normal skin were taken out for primary culture by tissue block method. Sex control group; (2) 0.005ug/ml LPS stimulation group; (3) 0.010ug/ml LPS stimulation group; (4) 0.050ug/ml LPS stimulation group; (5) 0.100ug/ml LPS stimulation group; (6) 0.500ug/ml LPS stimulation group; (7) 1.000ug/ml LPS stimulation group. Proliferative activity of fibroblasts was detected by colorimetry and cell proliferation cycle. Collagen synthesis in fibroblasts was detected by ~3H-proline incorporation and pepsin digestion. Fibroblasts with the most obvious LPS stimulation were subcultured continuously. Ultrastructural changes of fibroblasts were observed by transmission electron microscopy (4th, 6th, 8th and 10th passages). Since the 8th generation, the ultrastructure of fibroblasts is similar to that of fibroblasts in hypertrophic scar tissues. At the same time, the morphological and phenotypic changes of fibroblasts were detected by HE staining and immunohistochemical staining. Fibroblast group (positive control group); (2) negative control group (normal skin fibroblasts were not stimulated by LPS); (5) LPS stimulation group (normal skin fibroblasts were stimulated by LPS). The proliferation and secretion characteristics of fibroblasts were detected by MTT colorimetry; ~ 3H-proline incorporation, stomach. Collagen synthesis in fibroblasts was measured by protease digestion and collagen type I and type III content in fibroblast culture supernatant was detected by enzyme linked immunosorbent assay (ELISA). Genomic parameters of fibroblasts were detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of procollagenase mRNA and collagenase mRNA were detected by microarray. The differentially expressed genes were selected by comparing with fibroblast-related genes in hypertrophic scar tissues and verified by RT-PCR. In addition to the above indexes, the supernatant of fibroblast culture was detected by ELISA. The contents of TGF- beta _1 and IFN- gamma in the medium were investigated to explore the possible mechanism of LPS on fibroblasts.
Results: 1. LPS stimulated fibroblast proliferation and collagen synthesis at the concentration of 0.005 ug/ml to 0.5 ug/ml (P < 0.05). LPS stimulated fibroblast proliferation and collagen synthesis at the concentration of 0.1 ug/ml (P < 0.05). LPS stimulated fibroblast proliferation and collagen synthesis at the concentration of 1.0 ug/ml (P < 0.05). 2.0.1 ug/ml LPS stimulation and passage of different passages of fibroblasts after pathological changes since the eighth generation of fibroblasts ultrastructure and fibroblasts of hyperplastic scar tissue ultrastructure similar, at the same time using HE staining, immunohistochemical staining to detect morphological and phenotypic changes, the results were also consistent.3.0.1 ug. After stimulation and passage with g/ml LPS, the proliferation rate of fibroblasts increased after stimulation and passage with 0.1 ug/ml LPS. The proliferation rate of fibroblasts was similar to that of fibroblasts from hypertrophic scar tissue. There was no significant difference between the two groups (P > 0.05). 4.0.1 ug/ml LPS stimulation and passage with fibroblasts collagen. Changes of collagen synthesis and type I and type III secretion in fibroblasts stimulated and subcultured by 0.1 ug/ml LPS increased collagen synthesis and the secretion of type I and type III collagen at various time points, and the ratio of type I/III collagen increased. The ratio of collagen synthesis and type I and type III collagen secretion in fibroblasts from hypertrophic scar tissue was similar to that in fibroblasts from hypertrophic scar tissue. Similarly, there was no significant difference (P > 0.05). 5.0.1 ug/ml LPS stimulated and subcultured fibroblasts type I, III procollagen and collagenase mRNA expression changes 0.1 ug/ml LPS stimulated and subcultured fibroblasts type I, III procollagen mRNA expression increased, collagenase mRNA expression decreased, and type I, III procollagen and collagenase mRNA expression increased. The expression level was similar to that of fibroblasts from hypertrophic scar tissues (P > 0.05). 6.0.1 ug/ml LPS stimulated and subcultured fibroblasts changed gene expression profiles by 0.1 ug/ml LPS stimulation and passage. Some genes related to collagen metabolism (collagen type I, c-myc, TGF-beta 1) The expression of these genes was up-regulated. RT-PCR showed that the expression of these genes was similar to that of fibroblasts from hypertrophic scar tissue (P > 0.05). 7.0.1 ug/ml LPS stimulated and passaged fibroblasts culture supernatant TGF-beta_1, IFN-gamma content changes 0.1 ug/ml LPS stimulated and passaged fibroblasts at various time points TGF-beta_1 secretion increased. In addition, the secretion of IFN-gamma was decreased, similar to the secretion of fibroblasts from scar tissue, but there was no significant difference (P > 0.05).
Conclusion: 1. A certain concentration of LPS can change the biological characteristics of fibroblasts. Cell proliferation and collagen synthesis increase, but excessive concentration of LPS can inhibit the growth of fibroblasts. After passage, fibroblasts possess morphological and biological characteristics of fibroblasts from hypertrophic scar tissues: ultrastructure, morphology, phenotype, cell proliferation and collagen synthesis increased, type I and III collagen secretion increased, type I and III procollagen mRNA expression up-regulated, collagenase mRNA expression down-regulated, and gene surface down-regulated. It is suggested that LPS may induce normal skin fibroblasts to transform into hypertrophic scar tissue fibroblasts and participate in hypertrophic scar formation. The mechanism of LPS may be related to its promotion of TGF-beta_1 secretion and inhibition of IFN-gamma secretion.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329;R644
本文编号：2182343
[Abstract]:OBJECTIVE: 1. To observe the effects of LPS on proliferation and collagen synthesis of normal skin fibroblasts by lipopolysaccharide (LPS) stimulation test, and to explore the possible role of LPS in early wound healing. 2. To observe the morphology and proliferation of normal skin fibroblasts after stimulating the passage of fibroblasts with specific concentration of LPS. The changes of biological characteristics and gene expression profiles were compared with those of fibroblasts from hypertrophic scar tissues to explore the effect of LPS on the formation of late scar and its possible mechanism.
Methods: Twenty patients with hypertrophic scar were randomly selected and their scar tissues and normal skin were taken out for primary culture by tissue block method. Sex control group; (2) 0.005ug/ml LPS stimulation group; (3) 0.010ug/ml LPS stimulation group; (4) 0.050ug/ml LPS stimulation group; (5) 0.100ug/ml LPS stimulation group; (6) 0.500ug/ml LPS stimulation group; (7) 1.000ug/ml LPS stimulation group. Proliferative activity of fibroblasts was detected by colorimetry and cell proliferation cycle. Collagen synthesis in fibroblasts was detected by ~3H-proline incorporation and pepsin digestion. Fibroblasts with the most obvious LPS stimulation were subcultured continuously. Ultrastructural changes of fibroblasts were observed by transmission electron microscopy (4th, 6th, 8th and 10th passages). Since the 8th generation, the ultrastructure of fibroblasts is similar to that of fibroblasts in hypertrophic scar tissues. At the same time, the morphological and phenotypic changes of fibroblasts were detected by HE staining and immunohistochemical staining. Fibroblast group (positive control group); (2) negative control group (normal skin fibroblasts were not stimulated by LPS); (5) LPS stimulation group (normal skin fibroblasts were stimulated by LPS). The proliferation and secretion characteristics of fibroblasts were detected by MTT colorimetry; ~ 3H-proline incorporation, stomach. Collagen synthesis in fibroblasts was measured by protease digestion and collagen type I and type III content in fibroblast culture supernatant was detected by enzyme linked immunosorbent assay (ELISA). Genomic parameters of fibroblasts were detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of procollagenase mRNA and collagenase mRNA were detected by microarray. The differentially expressed genes were selected by comparing with fibroblast-related genes in hypertrophic scar tissues and verified by RT-PCR. In addition to the above indexes, the supernatant of fibroblast culture was detected by ELISA. The contents of TGF- beta _1 and IFN- gamma in the medium were investigated to explore the possible mechanism of LPS on fibroblasts.
Results: 1. LPS stimulated fibroblast proliferation and collagen synthesis at the concentration of 0.005 ug/ml to 0.5 ug/ml (P < 0.05). LPS stimulated fibroblast proliferation and collagen synthesis at the concentration of 0.1 ug/ml (P < 0.05). LPS stimulated fibroblast proliferation and collagen synthesis at the concentration of 1.0 ug/ml (P < 0.05). 2.0.1 ug/ml LPS stimulation and passage of different passages of fibroblasts after pathological changes since the eighth generation of fibroblasts ultrastructure and fibroblasts of hyperplastic scar tissue ultrastructure similar, at the same time using HE staining, immunohistochemical staining to detect morphological and phenotypic changes, the results were also consistent.3.0.1 ug. After stimulation and passage with g/ml LPS, the proliferation rate of fibroblasts increased after stimulation and passage with 0.1 ug/ml LPS. The proliferation rate of fibroblasts was similar to that of fibroblasts from hypertrophic scar tissue. There was no significant difference between the two groups (P > 0.05). 4.0.1 ug/ml LPS stimulation and passage with fibroblasts collagen. Changes of collagen synthesis and type I and type III secretion in fibroblasts stimulated and subcultured by 0.1 ug/ml LPS increased collagen synthesis and the secretion of type I and type III collagen at various time points, and the ratio of type I/III collagen increased. The ratio of collagen synthesis and type I and type III collagen secretion in fibroblasts from hypertrophic scar tissue was similar to that in fibroblasts from hypertrophic scar tissue. Similarly, there was no significant difference (P > 0.05). 5.0.1 ug/ml LPS stimulated and subcultured fibroblasts type I, III procollagen and collagenase mRNA expression changes 0.1 ug/ml LPS stimulated and subcultured fibroblasts type I, III procollagen mRNA expression increased, collagenase mRNA expression decreased, and type I, III procollagen and collagenase mRNA expression increased. The expression level was similar to that of fibroblasts from hypertrophic scar tissues (P > 0.05). 6.0.1 ug/ml LPS stimulated and subcultured fibroblasts changed gene expression profiles by 0.1 ug/ml LPS stimulation and passage. Some genes related to collagen metabolism (collagen type I, c-myc, TGF-beta 1) The expression of these genes was up-regulated. RT-PCR showed that the expression of these genes was similar to that of fibroblasts from hypertrophic scar tissue (P > 0.05). 7.0.1 ug/ml LPS stimulated and passaged fibroblasts culture supernatant TGF-beta_1, IFN-gamma content changes 0.1 ug/ml LPS stimulated and passaged fibroblasts at various time points TGF-beta_1 secretion increased. In addition, the secretion of IFN-gamma was decreased, similar to the secretion of fibroblasts from scar tissue, but there was no significant difference (P > 0.05).
Conclusion: 1. A certain concentration of LPS can change the biological characteristics of fibroblasts. Cell proliferation and collagen synthesis increase, but excessive concentration of LPS can inhibit the growth of fibroblasts. After passage, fibroblasts possess morphological and biological characteristics of fibroblasts from hypertrophic scar tissues: ultrastructure, morphology, phenotype, cell proliferation and collagen synthesis increased, type I and III collagen secretion increased, type I and III procollagen mRNA expression up-regulated, collagenase mRNA expression down-regulated, and gene surface down-regulated. It is suggested that LPS may induce normal skin fibroblasts to transform into hypertrophic scar tissue fibroblasts and participate in hypertrophic scar formation. The mechanism of LPS may be related to its promotion of TGF-beta_1 secretion and inhibition of IFN-gamma secretion.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329;R644
【引证文献】
相关硕士学位论文 前1条
1 战孝光;丹参酮ⅡA对兔良性狭窄胆管成纤维细胞的影响[D];天津医科大学;2009年
本文编号：2182343
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