CAJ | 학술논문

背景:组织细胞三维立体培养是肺纤维化破坏与组织收缩研究的理想模型,特别是在纤维化的进展过程中,几种细胞因子如肿瘤坏死因子、白细胞介素、前列腺素、胰岛素以及渗出的血浆成分可能在纤维化的损伤修复和组织重塑中起重要的作用.目的:观察三维立体培养条件下肺成纤维细胞在血清和血清蛋白、胰岛素和细胞因子前列腺素E2、转移生长因子β、肿瘤坏死因子α的细胞外胶原基质收缩和细胞凋亡,探讨肺纤维化过程中细胞因子、胰岛素、血清及血清蛋白对肺组织重塑和纤维化形成的影响.设计;随机对照实验.单位:解放军兰州军区兰州总医院呼吸内科.材料:实验于2005-08/2006-01在解放军兰州军区兰州总医院呼吸内科实验室完成.人胚肺成纤维细胞(American Type Culture Collection),DMEM培养液和胎牛血清(GIBCO),胰岛素、转移生长因子(R&D),前列腺素E2(Sigma),Ⅰ型胶原提取自大鼠尾部肌腱.方法:为观察初始浓度对成纤维细胞收缩力的影响及细胞数量对胶原收缩的影响,提取大鼠尾部的胶原,与双蒸水、4×DMEM及成纤维细胞混合成胶原含量为0.75～1.5 g/L的立体组织胶原,细胞浓度为0.2×107～4×107 L-1,置入含体积分数0.05的CO2培养箱37 ℃培养.每天测定胶原的面积,最后计算终面积与初始面积的比值.将0.01%～0.5%血清、0.1%的血清白蛋白和0.1%球蛋白分别加入到培养液中,观察胶原的收缩.加入10 mg/L转移生长因子、10 mg/L白细胞介素1、1 mmol/L胰岛素和0.1 μmol/L前列腺素E2,观察细胞因子对成纤维细胞介导胶原收缩的影响,对胶原中DNA含量进行测定及细胞存活率检测.主要观察指标:不同细胞因子或血清成分对肺成纤维细胞介导胶原收缩的影响,胶原内成纤维细胞数量对胶原收缩的影响,以及不同浓度胶原对胶原收缩和对细胞增殖和凋亡的影响.结果:①胶原的收缩有细胞依赖性,细胞浓度越高则胶原收缩越强烈.②血清和白蛋白的加入引起了剧烈的胶原收缩.③转化生长因子和胰岛素增强胶原的收缩,前列腺素E和白细胞介素1抑制胶原的收缩.④胶原的初始浓度越低,胶原收缩的速度越快越强烈,胶原终面积就越小,其中的成纤维细胞凋亡就越多.结论:肺成纤维细胞介导的胶原收缩可被白细胞介素1和前列腺素E抑制,胰岛素和转化生长因子则促进了胶原收缩.血清及血清成分在受损肺组织的渗出可以导致肺组织胶原的稀释与强烈收缩,而导致成纤维细胞增殖的抑制和凋亡的增加,肺纤维化进一步发展,成为无细胞胶原成分. 揹景:組織細胞三維立體培養是肺纖維化破壞與組織收縮研究的理想模型,特彆是在纖維化的進展過程中,幾種細胞因子如腫瘤壞死因子、白細胞介素、前列腺素、胰島素以及滲齣的血漿成分可能在纖維化的損傷脩複和組織重塑中起重要的作用.目的:觀察三維立體培養條件下肺成纖維細胞在血清和血清蛋白、胰島素和細胞因子前列腺素E2、轉移生長因子β、腫瘤壞死因子α的細胞外膠原基質收縮和細胞凋亡,探討肺纖維化過程中細胞因子、胰島素、血清及血清蛋白對肺組織重塑和纖維化形成的影響.設計;隨機對照實驗.單位:解放軍蘭州軍區蘭州總醫院呼吸內科.材料:實驗于2005-08/2006-01在解放軍蘭州軍區蘭州總醫院呼吸內科實驗室完成.人胚肺成纖維細胞(American Type Culture Collection),DMEM培養液和胎牛血清(GIBCO),胰島素、轉移生長因子(R&D),前列腺素E2(Sigma),Ⅰ型膠原提取自大鼠尾部肌腱.方法:為觀察初始濃度對成纖維細胞收縮力的影響及細胞數量對膠原收縮的影響,提取大鼠尾部的膠原,與雙蒸水、4×DMEM及成纖維細胞混閤成膠原含量為0.75～1.5 g/L的立體組織膠原,細胞濃度為0.2×107～4×107 L-1,置入含體積分數0.05的CO2培養箱37 ℃培養.每天測定膠原的麵積,最後計算終麵積與初始麵積的比值.將0.01%～0.5%血清、0.1%的血清白蛋白和0.1%毬蛋白分彆加入到培養液中,觀察膠原的收縮.加入10 mg/L轉移生長因子、10 mg/L白細胞介素1、1 mmol/L胰島素和0.1 μmol/L前列腺素E2,觀察細胞因子對成纖維細胞介導膠原收縮的影響,對膠原中DNA含量進行測定及細胞存活率檢測.主要觀察指標:不同細胞因子或血清成分對肺成纖維細胞介導膠原收縮的影響,膠原內成纖維細胞數量對膠原收縮的影響,以及不同濃度膠原對膠原收縮和對細胞增殖和凋亡的影響.結果:①膠原的收縮有細胞依賴性,細胞濃度越高則膠原收縮越彊烈.②血清和白蛋白的加入引起瞭劇烈的膠原收縮.③轉化生長因子和胰島素增彊膠原的收縮,前列腺素E和白細胞介素1抑製膠原的收縮.④膠原的初始濃度越低,膠原收縮的速度越快越彊烈,膠原終麵積就越小,其中的成纖維細胞凋亡就越多.結論:肺成纖維細胞介導的膠原收縮可被白細胞介素1和前列腺素E抑製,胰島素和轉化生長因子則促進瞭膠原收縮.血清及血清成分在受損肺組織的滲齣可以導緻肺組織膠原的稀釋與彊烈收縮,而導緻成纖維細胞增殖的抑製和凋亡的增加,肺纖維化進一步髮展,成為無細胞膠原成分.
배경:조직세포삼유입체배양시폐섬유화파배여조직수축연구적이상모형,특별시재섬유화적진전과정중,궤충세포인자여종류배사인자、백세포개소、전렬선소、이도소이급삼출적혈장성분가능재섬유화적손상수복화조직중소중기중요적작용.목적:관찰삼유입체배양조건하폐성섬유세포재혈청화혈청단백、이도소화세포인자전렬선소E2、전이생장인자β、종류배사인자α적세포외효원기질수축화세포조망,탐토폐섬유화과정중세포인자、이도소、혈청급혈청단백대폐조직중소화섬유화형성적영향.설계;수궤대조실험.단위:해방군란주군구란주총의원호흡내과.재료:실험우2005-08/2006-01재해방군란주군구란주총의원호흡내과실험실완성.인배폐성섬유세포(American Type Culture Collection),DMEM배양액화태우혈청(GIBCO),이도소、전이생장인자(R&D),전렬선소E2(Sigma),Ⅰ형효원제취자대서미부기건.방법:위관찰초시농도대성섬유세포수축력적영향급세포수량대효원수축적영향,제취대서미부적효원,여쌍증수、4×DMEM급성섬유세포혼합성효원함량위0.75～1.5 g/L적입체조직효원,세포농도위0.2×107～4×107 L-1,치입함체적분수0.05적CO2배양상37 ℃배양.매천측정효원적면적,최후계산종면적여초시면적적비치.장0.01%～0.5%혈청、0.1%적혈청백단백화0.1%구단백분별가입도배양액중,관찰효원적수축.가입10 mg/L전이생장인자、10 mg/L백세포개소1、1 mmol/L이도소화0.1 μmol/L전렬선소E2,관찰세포인자대성섬유세포개도효원수축적영향,대효원중DNA함량진행측정급세포존활솔검측.주요관찰지표:불동세포인자혹혈청성분대폐성섬유세포개도효원수축적영향,효원내성섬유세포수량대효원수축적영향,이급불동농도효원대효원수축화대세포증식화조망적영향.결과:①효원적수축유세포의뢰성,세포농도월고칙효원수축월강렬.②혈청화백단백적가입인기료극렬적효원수축.③전화생장인자화이도소증강효원적수축,전렬선소E화백세포개소1억제효원적수축.④효원적초시농도월저,효원수축적속도월쾌월강렬,효원종면적취월소,기중적성섬유세포조망취월다.결론:폐성섬유세포개도적효원수축가피백세포개소1화전렬선소E억제,이도소화전화생장인자칙촉진료효원수축.혈청급혈청성분재수손폐조직적삼출가이도치폐조직효원적희석여강렬수축,이도치성섬유세포증식적억제화조망적증가,폐섬유화진일보발전,성위무세포효원성분.
BACKGROUND:Contraction of three-dimensional collagen gels has been used as a model for the contraction which characterizes both normal wound healing and the development of fibrosis in the tissue. Several factors and cytokines,such as tumor necrosis factor alpha (TNF-α), interleukin-1, prostaglandin E2 and insulin have been proved to play important roles in collagen remodeling in vitro as well as serum extravasation during the fibrotic progress.OBJECTIVE: To observe extracellular collagen matrix contraction and apoptosis of fetal lung fibroblasts in TNF-α,interleukin-1, insulin, prostaglandin E2, albumin and globum under three-dimensional culture, and investigate the effects of cytokines, insuin, serum and serum protein on the remodeling and fibrotic formation of lung tissue.DESIGN: A randomized controlled experiment.SETTING: Department of Respiratory Medicine, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA.MATERIALS: The experiment was carried out The experiments were carried out in the respiratory laboratory of Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from August 2005 to January 2006. Human fetal lung fibroblasts (American Type Culture Collection), Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum, insulin, transforming growth factor (TGF) (R&D), prostaglandin E2, type Ⅰ collagen was extracted from rat-tail tendons.METHODS: In order to investigate the effect of initial collagen concentration in the gels on the contractility of fibroblasts,the appropriate amount of collagen was mixed with distilled water, four fold-concentrated DMEM, and cells were suspended so that the mass concentration was 0.75-2.0 g/L, with a physiological ionic strength and the desired cell concentration. In order to investigate the effect of cell number in the gels on the contraction, the cellular concentration fibroblasts in the gels were prepared to 0.2×107-4×107 L-1. The areas of floating gels were measured daily and the contraction was calculated by contrasting the initial size (％ of initial area). Different serum concentrations (0.01％-0.5％)in the medium were prepared, the serum albumin (0.1％) or globulin (0.1％) were added to the serum-free culture medium to observe the gel contraction. TGF (10 mg/L), interleukin-1 (10 mg/L), insulin (1 mg/L) and prostaglandin E2 (0.1 μmol/L) were added to observe the effects of cytokines and insulin on fibroblasts-mediated collagen gel contraction.The DNA content and cellular survivability in gels in collagen were determined.MAIN OUTCOME MEASURES: Effect of lung fibroblasts on collagen contraction with or without the presence of cytokines in three-dimensional culture; Effect of collagen with different concentration on the proliferation and apoptosis.RESULTS: ① Collagen gel contraction showed a dependence on the number of fibroblasts. ② Collagen gel contraction was augmented by increasing serum concentration in culture medium, and albumin increased the contraction dramatically. ③TGF and insulin significantly increased the contraction, whereas prostaglandin E2 and interleukin-1 significantly inhibited the gel contraction. ④ The lower the initial collagen concentration was, the more gel contracted and smaller final size were observed, and cell apoptosis increased.CONCLUSION: During the fibrotic process, fibroblast population migrated into the injured lung tissue may play an important role in the development of pulmonary fibrosis and serum infiltrating into injury lung tissue may play an role in stimulating the fibrotic progress. Infiltrating fluids and edema result in the dilution of the collagen concentration in the pulmonary interstitial which may lead to stronger contraction and serious fibrosis. In the dense fibrosis area, cells were hard to survive. In consequence, the final structure of fibrotic lung could not been changed and lung fibrosis progressed.