目的 观察TNF-α在血管内皮细胞向间质细胞转化中的作用,探讨纤维化疾病发生的机制. 方法 取健康胎儿脐带,体外酶消化法分离培养人脐静脉内皮细胞(HUVEC),用免疫荧光法进行鉴定.取第3～5代对数生长期HUVEC分别接种于12孔板和6孔板中,均按随机数字表法分为6组:对照组,培养液中不加任何刺激因素；5、10、25、50、100 ng/mL TNF-α组,分别在培养液中添加相应终浓度TNF-α,每组3个样本.培养72 h后倒置相差显微镜下观察各组细胞形态；免疫荧光法检测12孔板中各组细胞凝血因子Ⅷ和α平滑肌肌动蛋白(α-SMA)的表达,计算2种因子双阳性细胞数比值及吸光度值比值；RT-PCR检测6孔板中各组细胞钙黏蛋白、α-SMA和Ⅰ型胶原mRNA的表达(以灰度值比值表示).对数据行单因素方差分析及LSD检验. 结果(1)原代培养HUVEC为圆形、短梭形或扁平形,传代后细胞呈铺路石样旺盛生长.经第1、2、3、4、5次传代后HUVEC凝血因子Ⅷ阳性表达率分别为(85.5±1.8)％、(88 1±5 0)％、(93.6±3.7)％、(92.9±4 8)％、(89.5±1.1)％,明显高于原代培养HUVEC的(81.4±3.8)％,F值均为7.481,P值均小于0.05.(2)对照组细胞呈圆形、短梭形或扁平形,细胞间连接紧密；5、10、25、50、100 ng/mL TNF-α组随着TNF-α浓度的增加,细胞形态逐渐向长梭形转变,细胞间连接减少、间隙变大.(3)对照组中凝血因子Ⅷ、α-SMA双阳性细胞数比值和吸光度值比值分别为0.055±0 015、0.078±0 017,均显著低于5、10、25、50、100 ng/mL TNF-α组的0 257±0.106、0 280±0.129、0.505±0 059、0.817±0.035、0.929±0.101和0.437±0 040、0 456±0.097、0 496±0.082、0.787±0 131、0.885±0 087,F值分别为45 009、50.099,P值均小于0.01.(4)5、10、25、50、100 ng/mL TNF-α组细胞中钙黏蛋白mRNA水平分别为0.70±0.05、0.63±0 06、0 60±0.10、0.45±0.16、0 26±0.14,后4组显著低于对照组的0 83±0.03,F值均为11.593,P ＜0.05或P＜0.01.5、10、25、50、100 ng/mL TNF-α组细胞α-SMA和Ⅰ型胶原mRNA水平分别为0.45±0.10、0.51±0.16、0.49±0 12、0.60±0.09、0 76±0.03和0.38±0.18、0.45±0.15、0 52±0 12、0 66±0.17、0.76±0.20,后3组2项指标水平显著高于对照组的0.37±0.14、0 31±0 12,F值分别为7.839、2.898,P ＜0.05或P＜0.01. 结论 TNF-α呈明显浓度依赖性促进HUVEC向间质细胞转化,可能是瘢痕形成中纤维化组织内肌成纤维细胞的又一重要来源.
目的 觀察TNF-α在血管內皮細胞嚮間質細胞轉化中的作用,探討纖維化疾病髮生的機製. 方法 取健康胎兒臍帶,體外酶消化法分離培養人臍靜脈內皮細胞(HUVEC),用免疫熒光法進行鑒定.取第3～5代對數生長期HUVEC分彆接種于12孔闆和6孔闆中,均按隨機數字錶法分為6組:對照組,培養液中不加任何刺激因素；5、10、25、50、100 ng/mL TNF-α組,分彆在培養液中添加相應終濃度TNF-α,每組3箇樣本.培養72 h後倒置相差顯微鏡下觀察各組細胞形態；免疫熒光法檢測12孔闆中各組細胞凝血因子Ⅷ和α平滑肌肌動蛋白(α-SMA)的錶達,計算2種因子雙暘性細胞數比值及吸光度值比值；RT-PCR檢測6孔闆中各組細胞鈣黏蛋白、α-SMA和Ⅰ型膠原mRNA的錶達(以灰度值比值錶示).對數據行單因素方差分析及LSD檢驗. 結果(1)原代培養HUVEC為圓形、短梭形或扁平形,傳代後細胞呈鋪路石樣旺盛生長.經第1、2、3、4、5次傳代後HUVEC凝血因子Ⅷ暘性錶達率分彆為(85.5±1.8)％、(88 1±5 0)％、(93.6±3.7)％、(92.9±4 8)％、(89.5±1.1)％,明顯高于原代培養HUVEC的(81.4±3.8)％,F值均為7.481,P值均小于0.05.(2)對照組細胞呈圓形、短梭形或扁平形,細胞間連接緊密；5、10、25、50、100 ng/mL TNF-α組隨著TNF-α濃度的增加,細胞形態逐漸嚮長梭形轉變,細胞間連接減少、間隙變大.(3)對照組中凝血因子Ⅷ、α-SMA雙暘性細胞數比值和吸光度值比值分彆為0.055±0 015、0.078±0 017,均顯著低于5、10、25、50、100 ng/mL TNF-α組的0 257±0.106、0 280±0.129、0.505±0 059、0.817±0.035、0.929±0.101和0.437±0 040、0 456±0.097、0 496±0.082、0.787±0 131、0.885±0 087,F值分彆為45 009、50.099,P值均小于0.01.(4)5、10、25、50、100 ng/mL TNF-α組細胞中鈣黏蛋白mRNA水平分彆為0.70±0.05、0.63±0 06、0 60±0.10、0.45±0.16、0 26±0.14,後4組顯著低于對照組的0 83±0.03,F值均為11.593,P ＜0.05或P＜0.01.5、10、25、50、100 ng/mL TNF-α組細胞α-SMA和Ⅰ型膠原mRNA水平分彆為0.45±0.10、0.51±0.16、0.49±0 12、0.60±0.09、0 76±0.03和0.38±0.18、0.45±0.15、0 52±0 12、0 66±0.17、0.76±0.20,後3組2項指標水平顯著高于對照組的0.37±0.14、0 31±0 12,F值分彆為7.839、2.898,P ＜0.05或P＜0.01. 結論 TNF-α呈明顯濃度依賴性促進HUVEC嚮間質細胞轉化,可能是瘢痕形成中纖維化組織內肌成纖維細胞的又一重要來源.
목적 관찰TNF-α재혈관내피세포향간질세포전화중적작용,탐토섬유화질병발생적궤제. 방법 취건강태인제대,체외매소화법분리배양인제정맥내피세포(HUVEC),용면역형광법진행감정.취제3～5대대수생장기HUVEC분별접충우12공판화6공판중,균안수궤수자표법분위6조:대조조,배양액중불가임하자격인소；5、10、25、50、100 ng/mL TNF-α조,분별재배양액중첨가상응종농도TNF-α,매조3개양본.배양72 h후도치상차현미경하관찰각조세포형태；면역형광법검측12공판중각조세포응혈인자Ⅷ화α평활기기동단백(α-SMA)적표체,계산2충인자쌍양성세포수비치급흡광도치비치；RT-PCR검측6공판중각조세포개점단백、α-SMA화Ⅰ형효원mRNA적표체(이회도치비치표시).대수거행단인소방차분석급LSD검험. 결과(1)원대배양HUVEC위원형、단사형혹편평형,전대후세포정포로석양왕성생장.경제1、2、3、4、5차전대후HUVEC응혈인자Ⅷ양성표체솔분별위(85.5±1.8)％、(88 1±5 0)％、(93.6±3.7)％、(92.9±4 8)％、(89.5±1.1)％,명현고우원대배양HUVEC적(81.4±3.8)％,F치균위7.481,P치균소우0.05.(2)대조조세포정원형、단사형혹편평형,세포간련접긴밀；5、10、25、50、100 ng/mL TNF-α조수착TNF-α농도적증가,세포형태축점향장사형전변,세포간련접감소、간극변대.(3)대조조중응혈인자Ⅷ、α-SMA쌍양성세포수비치화흡광도치비치분별위0.055±0 015、0.078±0 017,균현저저우5、10、25、50、100 ng/mL TNF-α조적0 257±0.106、0 280±0.129、0.505±0 059、0.817±0.035、0.929±0.101화0.437±0 040、0 456±0.097、0 496±0.082、0.787±0 131、0.885±0 087,F치분별위45 009、50.099,P치균소우0.01.(4)5、10、25、50、100 ng/mL TNF-α조세포중개점단백mRNA수평분별위0.70±0.05、0.63±0 06、0 60±0.10、0.45±0.16、0 26±0.14,후4조현저저우대조조적0 83±0.03,F치균위11.593,P ＜0.05혹P＜0.01.5、10、25、50、100 ng/mL TNF-α조세포α-SMA화Ⅰ형효원mRNA수평분별위0.45±0.10、0.51±0.16、0.49±0 12、0.60±0.09、0 76±0.03화0.38±0.18、0.45±0.15、0 52±0 12、0 66±0.17、0.76±0.20,후3조2항지표수평현저고우대조조적0.37±0.14、0 31±0 12,F치분별위7.839、2.898,P ＜0.05혹P＜0.01. 결론 TNF-α정명현농도의뢰성촉진HUVEC향간질세포전화,가능시반흔형성중섬유화조직내기성섬유세포적우일중요래원.
Objective To observe the role of tumor necrosis factor α(TNF-α)in endothelial-mesenchymal transition(EnMT),and to explore the mechanism of fibrosis disease. Methods Human umbilical vein endothelial cells(HUVEC)from umbilical cord of healthy fetus were isolated by enzymatic digestion and identified by immunofluorescence assay.The third to fifth generations of cultured HUVEC in logarithmic phase were harvested and seeded in 12-well plates and 6-well plates,and they were divided into control group(ordinary culture without any stimulation),5,10,25,50,and 100 ng/mL TNF-α groups (5,10,25,50,100 ng/mL of TNF-α was respectively added into the nutrient solution)according to the random number table,with three samples in each group.After being cultured for 72 hours,the cell morphology was observed under inverted phase-contrast microscope; the expression levels of coagulation factor Ⅷ and α smooth muscle actin(α-SMA)were detected by immunofluorescence assay,and the ratios of numbers (absorbance values)of cells with expression of both factors were calculated.The mRNA expression levels of cadherin,α-SMA,and type Ⅰ collagen were detected by RT-PCR(denoted as gray value ratio).Data were processed with one-way analysis of variance and LSD test. Results ( 1 )The shape of primary HUVEC was round,short-spindle,or flat,and ceils grew vigorously in cobblestone appearance after passages.After being subcultured for 1,2,3,4,5 passage(s),the positive rate of coagulation factor Ⅷ of HUVEC was respectively(85.5 ±1.8)％,(88.1 ±5.0)％,(93.6 ±3.7)％,(92.9 ±4.8)％,(89.5 ±1.1)％,and they were significantly higher than that of primary HUVEC [( 81.4 ± 3.8)％,with F values all equal to 7.481,P values all below 0.05 ].(2)As compared with that in control group,the appearance of cells in 5,10,25,50,and 100 ng/mL TNF-α groups was gradually transformed from round,short-spindle,or flat shape to long-spindle shape with reduced intercellular junction and larger intercellular gap along with the increase in the concentration of TNF-α.(3)The ratios of numbers and the absorbance values of coagulation factor Ⅷ and α-SMA double positive cells in control group(0.055 ± 0.015,0.078 ± 0.017)were significantly lower than those in 5,10,25,50,and 100 ng/mL TNF-α groups(0.257 ± 0.106,0.280 ± 0.129,0.505 ±0.059,0.817 ±0.035,0.929 ±0.101 and 0.437 ±0.040,0.456 ±0.097,0.496 ±0.082,0.787 ± 0.131,0.885 ± 0.087,with F value respectively 45.009,50.099,P values all below 0.01 ).(4)The expression levels of cadherin mRNA in 5,10,25,50,and 100 ng/mL TNF-α groups were 0.70 ±0.05,0.63 ± 0.06,0.60 ± 0.10,0.45 ± 0.16,and 0.26 ± 0.14,and it was significantly lower in the latter four groups than in control group(0.83 ±0.03,with F values all equal to 11.593,P ＜0.05 or P ＜0.01 ).The mRNA expression levels of α-SMA and collagen Ⅰ in 5,10,25,50,and 100 ng/mL TNF-α groups were 0.45 ± 0.10,0.51 ± 0.16,0.49 ± 0.12,0.60 ± 0.09,0.76 ± 0.03 and 0.38 ± 0.18,0.45 ± 0.15,0.52 ± 0.12,0.66 ±0.17,0.76 ± 0.20,and they were significantly higher in the latter three groups than in control group(0.37 ±0.14,0.31 ±0.12,with F value respectively 7.839,2.898,P ＜0.05 or P ＜ 0.01 ). Conclusions TNF-α can obviously promote EnMT in a dose-dependent manner.EnMT may be another significant source of myofibroblasts that contributes to fibrotic tissue in scar formation.
