【摘要】： 目的：1．通過內(nèi)毒素(lipopolysaccharide，LPS)刺激實驗，，觀察LPS對正常皮膚成纖維細胞增殖和膠原合成等生物學特性的影響，探討LPS在早期創(chuàng)面愈合中的可能作用。2．使用特定濃度LPS刺激正常皮膚成纖維細胞后連續(xù)傳代，觀察其形態(tài)學、生物學特性及基因表達譜改變，并與自身增生性瘢痕組織成纖維細胞進行比較，探討LPS對后期瘢痕形成的影響及可能機制。 方法：隨機選取增生性瘢痕患者20例(處于增生期)，取其瘢痕組織及正常皮膚，采用組織塊法行成纖維細胞原代培養(yǎng)。對純化至第3代的正常皮膚成纖維細胞用不同濃度的LPS(E. coli055：B5)進行刺激，實驗分組如下：(1)0.000ug/ml LPS刺激組(陰性對照組)；(2)0.005ug/ml LPS刺激組；(3)0.010ug／ml LPS刺激組；(4)0.050ug／ml LPS刺激組；(5)0.100ug/ml LPS刺激組；(6)0.500ug／ml LPS刺激組；(7)1.000ug/ml LPS刺激組。采用下列方法檢測成纖維細胞生物學特性(細胞增殖和膠原合成)：細胞計數(shù)法、MTT比色法及細胞增殖周期檢測成纖維細胞增殖活性，~3H-脯氨酸摻入、胃蛋白酶消化法檢測成纖維細胞內(nèi)膠原合成量，將LPS刺激作用最為明顯組成纖維細胞進行連續(xù)傳代培養(yǎng)，采用透射電鏡觀察(第4、6、8、10代)成纖維細胞超微結(jié)構(gòu)的變化，其中自第8代開始成纖維細胞超微結(jié)構(gòu)與自身增生性瘢痕組織成纖維細胞超微結(jié)構(gòu)接近，同時采用HE染色、免疫組織化學染色檢測形態(tài)學及表型變化，結(jié)果也具有一致性。因此，選擇(第8代)成纖維細胞進行實驗，實驗分組如下：(1)瘢痕組織成纖維細胞組(陽性對照組)；(2)陰性對照組(正常皮膚成纖維細胞不予LPS刺激)；(5)LPS刺激組(正常皮膚成纖維細胞給予LPS刺激)。采用下列方法檢測成纖維細胞增殖和分泌特性：MTT比色法檢測成纖維細胞增殖活性；~3H-脯氨酸摻入、胃蛋白酶消化法檢測成纖維細胞內(nèi)膠原合成量，同時采用酶聯(lián)免疫吸附試驗(ELISA)檢測成纖維細胞培養(yǎng)上清液中Ⅰ、Ⅲ型膠原含量。采用下列方法檢測成纖維細胞基因?qū)W指標：逆轉(zhuǎn)錄-聚合酶鏈反應技術(RT-PCR)檢測成纖維細胞Ⅰ、Ⅲ型前膠原及膠原酶mRNA表達，基因芯片技術檢測成纖維細胞基因表達譜的變化，通過與自身增生性瘢痕組織成纖維細胞相關基因進行對比，挑選差異基因，采用RT-PCR法進行驗證。除了上述指標以外，我們還采用ELISA法檢測成纖維細胞培養(yǎng)上清液中TGF-β_1、IFN-γ的含量，探討LPS對成纖維細胞的可能作用機制。 結(jié)果：1．LPS對成纖維細胞增殖活性和膠原合成的影響LPS刺激濃度在0.005μg／ml～0.5μg/ml時，促進成纖維細胞增殖和膠原合成(P＜0.05)，以0.1μg／mlLPS刺激作用最為明顯，LPS刺激濃度為1.0μg／ml時，則抑制成纖維細胞增殖和膠原合成(P＜0.05)。2．0.1μg/ml LPS刺激并傳代后不同代次成纖維細胞的病理學變化自第8代開始成纖維細胞超微結(jié)構(gòu)與自身增生性瘢痕組織成纖維細胞超微結(jié)構(gòu)近似，同時采用HE染色、免疫組織化學染色檢測形態(tài)學及表型變化，結(jié)果也具有一致性。3．0.1μg/ml LPS刺激并傳代后成纖維細胞增殖活性的變化0.1μg／mlLPS刺激并傳代后成纖維細胞增殖率增加，且其增殖率與自身增生性瘢痕組織成纖維細胞增殖率近似，統(tǒng)計學分析無顯著差異(P＞0.05)。4．0.1μg/ml LPS刺激并傳代后成纖維細胞膠原合成及Ⅰ、Ⅲ型膠原分泌的變化0.1μg／ml LPS刺激并傳代后成纖維細胞膠原合成及各時間點Ⅰ、Ⅲ型膠原的分泌增加、Ⅰ／Ⅲ型膠原比例提高，且與自身增生性瘢痕組織成纖維細胞膠原合成及Ⅰ、Ⅲ型膠原的分泌量、Ⅰ／Ⅲ型膠原比例近似，統(tǒng)計學分析無顯著差異(P＞0.05)。5．0.1μg／ml LPS刺激并傳代后成纖維細胞Ⅰ、Ⅲ型前膠原及膠原酶mRNA表達的變化0.1μg／ml LPS刺激并傳代后成纖維細胞Ⅰ、Ⅲ型前膠原mRNA的表達升高、膠原酶mRNA表達下降，且Ⅰ、Ⅲ型前膠原及膠原酶mRNA表達量與自身增生性瘢痕組織成纖維細胞表達量近似(P＞0.05)。6．0.1μg／ml LPS刺激并傳代后成纖維細胞基因表達譜的改變0.1μg/ml LPS刺激并傳代后成纖維細胞基因表達譜發(fā)生改變，其中與膠原代謝相關的一些基因(Ⅰ型膠原、c-myc、TGF-β1)表達均上調(diào)，經(jīng)RT-PCR驗證，這些基因表達與自身增生性瘢痕組織成纖維細胞表達量近似(P＞0.05)。7．0.1μg/ml LPS刺激并傳代后成纖維細胞培養(yǎng)上清液中TGF-β_1、IFN-γ含量的變化0.1μg/mlLPS刺激并傳代后各時間點成纖維細胞TGF-β_1的分泌增加、IFN-γ的分泌減少，與自身瘢痕組織成纖維細胞分泌量近似，相比無統(tǒng)計學差異(P＞0.05)。 結(jié)論：1、一定濃度的LPS刺激后可使成纖維細胞的生物學特性發(fā)生改變：細胞增殖和膠原合成增加，過高濃度則呈抑制作用，提示適度的LPS存在可能不影響甚至有利于創(chuàng)面愈合，LPS過多則可能延緩創(chuàng)面愈合時間。2、特定濃度的LPS刺激并傳代后，成纖維細胞具備自身增生性瘢痕組織成纖維細胞的形態(tài)學特征及某些生物學特性：超微結(jié)構(gòu)、形態(tài)學、表型一致，細胞增殖加快和膠原合成增加，Ⅰ、Ⅲ型膠原分泌增加，Ⅰ、Ⅲ型前膠原mRNA表達上調(diào)，膠原酶mRNA表達下調(diào)，同時基因表達譜也進一步證實了基因?qū)W變化。提示LPS可能誘導正常皮膚成纖維細胞轉(zhuǎn)化為增生性瘢痕組織成纖維細胞，參與增生性瘢痕形成。LPS的作用機制可能與其促進TGF-β_1的分泌，而抑制IFN-γ的分泌有關。
[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.
【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329;R644

【引證文獻】

相關碩士學位論文 前1條

1 戰(zhàn)孝光;丹參酮ⅡA對兔良性狹窄膽管成纖維細胞的影響[D];天津醫(yī)科大學;2009年

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