油酸对人正常和瘢痕成纤维细胞增殖和分泌炎症介质的影响

目的 探讨油酸对人正常Fb和瘢痕Fb增殖和分泌炎症介质的影响. 方法 体外培养人正常Fb和瘢痕Fb,分别按照随机数字表法分为7组(各组样本数为8):空白对照组,除常规成分外培养液中不再添加其他物质;乙醇对照组,培养液中加入终浓度为体积分数2％无水乙醇;不同浓度油酸组,即0.25、0.50、1.00、2.00以及4.00 mmol/L油酸组,培养液中分别加入相应终浓度的油酸(以含体积分数2％无水乙醇培养液配制).采用锥虫蓝染色法观察各组细胞培养1～5d的生长情况.于倒置相差显微镜和透射电镜下观察2种细胞2个对照组、1.00 mmol/L油酸组细胞培养2d的细胞结构,采用流式细胞仪检测2种细胞2个对照组及1.00 mmol/L油酸组细胞培养2d的细胞周期.噻唑蓝法检测各组细胞培养2d的增殖情况.取各组细胞培养2d时的培养上清液,采用改良Griess法测定NO含量,ELISA法检测TNF-α、IL-6、IL-1β、IL-8含量.对数据进行多因素方差分析及重复测量设计的方差分析. 结果 (1)正常Fb和瘢痕Fb的空白对照组与乙醇对照组比较,各指标均无明显差别.(2)培养2～5d,正常Fb和瘢痕Fb 2.00、4.00 mmol/L油酸组细胞数量均低于对应的2个对照组(F值分别为13.773、11.344,P值均小于0.01).(3)培养2d,正常Fb和瘢痕Fb1.00 mmol/L油酸组细胞数量明显减少,部分细胞开始堆积、变圆易脱落;细胞膜不完整,线粒体空泡变性,核固缩,胞内可见脂滴.(4)正常Fb 1.00 mmol/L油酸组的G0/G1期和G2/M期细胞百分比[(93.56±9.98)％、(2.01士0.75)％]显著高于空白对照组[(84.23±10.96)％、(0.37±0.16)％],F值分别为3.026、34.751,P＜0.05或P＜0.01;S期细胞百分比为(4.42 ±0.87)％,明显低于空白对照组的(16.06±1.74)％,F=136.120,P＜0.01.瘢痕Fb 1.00 mmol/L油酸组G0/G1期和G2/M期细胞百分比分别为(93.86±13.90)％、(1.89±0.66)％,显著高于空白对照组[(83.88±10.42)％、(0.41±0.17)％],F值分别为3.529、32.710,P＜0.05或P＜0.01;S期细胞百分比为(3.87±0.63)％,明显低于空白对照组的(15.89±2.02)％,F=116.508,P＜0.01.(5)正常Fb和瘢痕Fb 0.50 ～4.00 mmol/L油酸组细胞增殖率显著低于对应的2个对照组(F值分别为215.945、194.555,P＜0.05或P＜0.01).(6)正常Fb各浓度油酸组细胞分泌NO水平明显高于2个对照组(F=30.240,P＜0.05或P＜0.01);瘢痕Fb 1.00～ 4.00 mmol/L油酸组细胞分泌NO水平明显高于2个对照组(F=12.495,P＜0.01).正常Fb和瘢痕Fb 2.00、4.00 mmol/L油酸组细胞分泌TNF-α、IL-6水平明显高于对应的2个对照组(F TNF-α值分别为6.911、3.818,FIL-6值分别为16.939、11.600,P ＜0.05或P＜0.01).正常Fb和瘢痕Fb各浓度油酸组细胞分泌IL-1β水平显著高于对应的2个对照组(F值分别为25.117、9.137,P值均小于0.01).正常Fb 1.00～4.00 mmol/L油酸组细胞分泌IL-8水平明显高于2个对照组(F=2.717,P＜0.05或P＜0.01),瘢痕Fb 2.00、4.00 mmol/L油酸组细胞分泌的IL-8水平明显高于2个对照组(F=3.338,P＜0.05).正常Fb和瘢痕Fb相同浓度油酸组各炎症因子水平比较无明显差异(F值为0.120 ～3.766,P值均大于0.05). 结论 高浓度油酸虽能抑制瘢痕Fb增殖,但同时也抑制正常Fb增殖,且高浓度油酸能同时促进正常Fb和瘢痕Fb分泌炎症介质,从而导致过度、持续的炎症反应,不利于创面愈合.     

氧化苦参碱对瘢痕疙瘩成纤维细胞增殖及凋亡的影响

目的 研究氧化苦参碱(OMT)对体外培养的人瘢痕疙瘩成纤维细胞(HKFB)的影响及其机制.方法 将不同浓度的OMT作用于HKFB,分别培养24、48、72h后采用四唑盐比色法(MTT)检测HKFB增殖活性,末端标记法(TUNEL)检测细胞凋亡.结果 当氧OMT浓度大于1.0mg/ml时,体外培养HKFB增殖受到抑制、出现凋亡;当OMT浓度小于1.0mg/ml时,其对HKFB的增殖及凋亡无明显影响.结论 一定浓度的OMT可以抑制HKFB增殖并促进其凋亡,提示OMT具有潜在的治疗瘢痕疙瘩作用.     

正常皮肤提取物对成纤维细胞增殖和胶原合成的影响

目的 探讨自体成纤维细胞注射移植后是否出现过度增殖和胶原异常分泌.方法 按照Engel-Catchpole程序制备正常皮肤提取物,在浓度分别为0.0625、0.1250、0.2500、0.5000、1.0000 mg/mlDMEM培养基下,观察对正常成纤维细胞增殖和胶原合成的影响.结果 正常皮肤提取物抑制正常皮肤成纤维细胞的增殖,细胞外胶原的分泌也受一定的影响,受抑制的程度与培养基中皮肤提取物的浓度呈正相关.结论 正常皮肤内存在成纤维细胞增殖和胶原分泌功能的负调节机制,有可能在进行自体成纤维细胞注射移植时发挥作用.     

Effects of histamine on lung permeability in normal and asthmatic subjects.

The permeability of respiratory mucosa, as measured by clearance of diethylenetriamine penta-acetate (DTPA) labelled with technetium 99m, was similar in seven normal and nine asthmatic subjects. Histamine induced bronchoconstriction was associated with a 50% increase in permeability in both groups of subjects. In normal subjects inhaled salbutamol, given as 1 mg acutely or as 200 micrograms four times daily for two weeks, had no effect on pulmonary permeability. Salbutamol, given before histamine challenge, prevented bronchoconstriction, but did not affect the increase in permeability seen in normal subjects. Low doses of histamine, sufficient to cause bronchoconstriction in the asthmatic subjects, produced little bronchoconstriction in normal subjects but caused increases in lung permeability similar to those seen in asthmatic subjects. These studies suggest that these two effects of inhaled histamine, bronchoconstriction and increased permeability, are independent.     

Effect of L-lysine on cytosolic calcium homeostasis in cultured human normal fibroblasts

Summary L-lysine, a cationic essential amino acid, has been reported to affect calcium transport in both intestine and kidney. In order to investigate whether this effect is associated with changes in cytosolic calcium homeostasis, we studied the effect of L-lysine deprivation on intracellular calcium concentration ([Ca²⁺]i), as well as⁴⁵Ca efflux and accumulation in normal human fibroblasts. Steady state [Ca²⁺]i, measured using fura-2 fluorescence in cells cultured for 18 hours in a L-lysine-free medium, was significantly higher than in cells grown in the presence of as little as 4 μM L-lysine. L-lysine deprivation also led to a significant decrease of⁴⁵Ca fractional efflux compared with cells grown in complete medium. This effect was paralleled by a significant decrease in⁴⁵Ca accumulation. Lack of L-arginine from the growth medium for the same time period had no effect on either [Ca²⁺]i,⁴⁵Ca efflux, or⁴⁵Ca accumulation rate. Presumably, the lack of L-lysine for a significant amount of time impairs the active mechanisms of calcium extrusion, which is only partially compensated by a reduction of calcium accumulation rate. This leads to an increased steady state [Ca²⁺]i. It is concluded that L-lysine is an important modulator of cytosolic calcium homeostasis.     

褪黑激素对人增生性瘢痕成纤维细胞增殖和凋亡的影响

目的 探讨褪黑激素对人增生性瘢痕Fb增殖和凋亡的影响与机制. 方法 采用组织块法分离培养人增生性瘢痕Fb.按随机数字表法将细胞分为低、中、高浓度组和对照组.前3组细胞分别采用含1 × 10-5、1×10-3、1 mmol/L褪黑激素的培养液培养,对照组不加褪黑激素常规培养.处理后24 h进行如下检测:对各组细胞进行形态学观察;用四氮唑复合物( XTT)-硫酸酚嗪甲酯(PMS)比色法检测细胞增殖活性;对细胞行膜联蛋白V-异硫氰酸荧光素(FITC)和碘化丙啶(PI)双染色后,用流式细胞仪分析细胞周期和凋亡情况;荧光定量RT-PCR法检测细胞周期蛋白E(cyclin E)、p53和Fas mRNA表达量.对数据行方差分析和LSD检验. 结果 形态学观察显示,对照组Fb为长梭形,呈集落分布;3个浓度组Fb随着褪黑激素浓度升高,细胞逐渐分散,胞体变形缩小,胞膜皱缩,核质比例减小.对照组、低浓度组、中浓度组、高浓度组Fb增殖活性(吸光度值)依次下降,分别为1.79±0.10、1.49±0.15、1.24±0.20、0.92±0.09(F=67.61,P ＜0.05);S期细胞百分比依次下降,分别为(16.9±1.3)％、(10.6±1.1)％、(6.1±1.2)％、(3.2±0.8)％(F=286.10,P ＜0.05);G2/M期细胞百分比依次下降,分别为(16.7±1.6)％、(13.5±1.1)％、(9.8±1.0)％(6.0±0.7)％(F=162.69,P＜0.05);早、晚期凋亡细胞百分比依次升高(F值分别为424.05、236.44,P值均小于0.05).对照组、低浓度组、中浓度组、高浓度组细胞cyclin E的mRNA表达量依次下降,分别为2.90±0.30、1.58±0.21、0.90±0.20、0.24±0.12(F=266.79,P＜0.05);p53和Fas mRNA表达量依次升高(F值分别为10.11、12.03,P值均小于0.05). 结论 褪黑激素可通过影响细胞cyclin E、p53和Fas基因的表达,抑制增生性瘢痕Fb增殖并诱导该细胞凋亡.     

Effect of keratinocyte growth factor—2 on proliferation of human adult keratinocytes

OBJECTIVE: To investigate the proliferative effect of keratinocyte growth factor (KGF-2) on human adult keratinocytes. METHODS: The standard medium was keratinocyte growth medium without bovine pituitary extract (BPE), hydrocortisone or epidermal growth factor (EGF). Keratinocytes from a 48-year-old subject were cultured and seeded on dishes with standard medium of EGF in cell density of 2 x 10(4)/32 mm(2). After 24 hours, the medium was replaced by the standard medium with 0, 4, 16, 125 and 500 ng/ml KGF-2, respectively. The standard medium with EGF was used as the positive control and the standard medium without EGF or KGF-2 was used as the negative controls. The growth of keratinocytes was monitored by 3-(4,5-dimethythiazol-2-yl)-2,5 dipheyl tetrazolium bromide (MTT) assay and by photographs on days 3, 5 and 7, respectively. RESULTS: KGF-2 in concentrations of 4-500 ng/ml showed a significant proliferative effect on days 5 and 7 as compared with that of the negative controls (P < 0.01). On day 3 the cells were proliferated to 1.5-2.5-fold, on day 5 to 3-5-fold and on day 7 to 3-12-fold in KGF-2 medium as that of the negative controls. The optimal response occurred when the concentration of KGF-2 was 125 ng/ml on day 7. Cell proliferation was also consistently higher in all KGF-2 concentrations as compared with that of the positive controls. CONCLUSIONS: KGF-2 has significant effects on the proliferation of adult keratinocytes, which are more effective than that of EGF. This study supports KGF-2 can improve the healing of chronic wounds in adults in clinic.     

Quantitative study of endocrine cells immunoreactive for calcitonin in the normal adult human lung.

There was no significant variation in the numbers of bronchopulmonary endocrine cells immunoreactive for calcitonin in five pairs of adult human lungs either from case to case or between groups of anatomically equivalent lobes. This was the case whether their numbers were expressed in relation to epithelial length or to the total number of epithelial cells. The mean (SD) values for the frequency of occurrence of these cells in all 25 lobes studied were 4.3 (1.9) per 10 cm of epithelial length or 1.70 (0.78) per 10 000 epithelial cells. Most immunoreactive cells were single and situated in the airways; only three neuroepithelial bodies were observed, and no cells were present in the parenchyma examined. This study provides further evidence that the functional character of these cells may not be confined to early life.     

Effects of tamoxifen on normal human dermal fibroblasts

OBJECTIVE: To evaluate the effects of tamoxifen on the growth and autocrine growth factor production of human dermal fibroblasts from the face. METHODS: In vitro study of normal adult dermal fibroblast cells developed from surgical specimens in a serum-free model. Cell cultures were exposed to 5-, 8-, 12-, 16-, and 50-microg/mL concentrations of tamoxifen solution. Cell counts were performed, and the cell-free supernatants were collected at 0, 1, 3, 5, and 7 days after the initial exposure. Population doubling times were calculated, and supernatants were quantitatively assayed for basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF) beta1. RESULTS: Tamoxifen appears to delay cellular proliferation rates in a dose-dependent manner up to a concentration of 12 microg/mL. Higher concentrations, approaching 50 microg/mL, appear to have a toxic effect on cell growth. The analysis of growth factor production revealed decreased levels of bFGF and VEGF but no change in the levels of TGF-beta1. CONCLUSIONS: The in vitro findings of delayed cell proliferation and decreased production of VEGF and bFGF in cells exposed to tamoxifen are consistent with previous in vivo reports of delayed wound healing but improved scar formation. The in vitro findings of growth factor modulation by tamoxifen provide cellular and molecular evidence supporting the clinical use of tamoxifen to ultimately improve scar formation.     

不同牵拉方式对成纤维细胞增殖影响的研究

目的 建立一种模拟临床扩张术的细胞牵拉培养装置,比较不同牵拉方式对成纤维细胞增殖情况的影响,寻找能充分模拟临床扩张术的体外细胞牵拉培养方式.方法 自行设计一种可置入普通培养皿的细胞牵拉培养装置,将正常皮肤的成纤维细胞种植在弹性硅胶膜上,设计单次最大牵拉组(共牵拉延长率达30％)、渐进式牵拉组(牵拉10％→停留→牵拉10％→停留→牵拉10％→停留,共牵拉延长率达30％)、非牵拉组,MTT法和Western Blot显示细胞增殖情况.结果 细胞在牵拉模型上生长良好,细胞被拉长,并与牵拉方向平行排列,30％延长度的牵拉能刺激细胞增殖,渐进式牵拉组对细胞的促增殖作用强于单次最大牵拉组.结论 细胞在牵拉模型上生长良好,牵拉能刺激细胞增殖,模型可以用于模拟体外应力刺激细胞效应的实验研究.渐进式牵拉作用方式是模拟临床皮肤软组织扩张术较好的作用方式.     

Effect of prior bronchoconstriction on the airway response to histamine in normal subjects.

We have examined the effect of prior bronchoconstriction on the bronchial responsiveness to inhaled histamine in nine normal subjects. The airway response to increasing concentrations of histamine aerosol was assessed by measurement of specific airways conductance (sGaw) in a body plethysmograph. The threshold provocative dose of histamine needed to cause a 35% fall in starting sGaw (PD35) and the steepest slope of the response were measured from cumulative log dose response curves. Histamine challenges were performed in duplicate after premedication with 0.9% sodium chloride (control) or methacholine aerosol on separate days. The mean starting sGaw did not change significantly after inhalation of 0.9% sodium chloride but methacholine caused a mean reduction in sGaw of 42%. Mean control PD35 values did not differ significantly from mean PD35 values after methacholine. The mean steepest slope of the response after methacoline was 47% lower than the mean control value. There was a significant linear relationship between starting sGaw and the steepest slope for the control and for the methacholine premedicated challenges. The reduction in slope after methacholine was accounted for by the fall in starting sGaw. Because histamine PD35 was not altered by prior bronchoconstriction, it is concluded that the bronchial hyperresponsiveness of asthmatic subjects to non-specific bronchoconstrictor stimuli is unlikely to be a direct consequence of their low starting airway calibre.     

脂多糖对小鼠肺成纤维细胞活化的影响

目的 评价脂多糖(LPS)对小鼠肺成纤维细胞活化的影响.方法 原代培养的小鼠肺成纤维细胞,接种于96孔培养板,采用随机数字表法,将其随机分为2组,正常对照组(C组,外=6)不作任何处理,LPS组(n=24)加入LPS 1μg/ml,分别于LPS孵育3、6、24、72 h时取6孔(C组于培养72 h时),收集细胞,采用实时PCR法测定Ⅰ型前胶原mRNA、α-平滑肌肌动蛋白(α-SMA)mRNA、Toll样受体4(TLR4)mRNA和整合素β1 mRNA的表达水平.结果 与C组比较,LPS组LPS孵育3、6和24 h时Ⅰ型前胶原mRNA、α-SMA mRNA、TLR4 mRNA和整合素β1 mRNA的表达水平差异无统计学意义(P＞0.05),LPS孵育72 h时上述指标表达水平上调(P＜0.05).结论 在急性肺损伤的早期LPS一方面可直接活化小鼠肺成纤维细胞,导致肺纤维化;另一方面可上调TLR4和整合素β1的表达,增加细胞对LPS的反应性,从而加速小鼠肺成纤维细胞的活化,促进肺纤维化.

Abstract：

Objective To investigate the effect of lipopolysaccharide (LPS) on the activation of mouse lung fibroblasts. Methods Primary cultured mouse lung fibroblasts were incubated in 96 well plates and randomly divided into 2 groups: control group ( group C, n = 6) and LPS group ( n = 24). The fibroblasts were cultured for 72 h in group C. LPS 1 μg/ml was added and then the fibroblasts were incubated for 72 h in group LPS. The expression of type Ⅰ procollagen mRNA, α-smooth muscle actin (α-SMA) mRNA, Toll-like receptor 4 (TLR4)mRNA and integrin β1 mRNA was determined using real-time PCR at 3, 6, 24 and 72 h of incubation (6 wells at each time point). Results Compared with group C, there was no significant change in the expression of type Ⅰ procollagen mRNA, α-SMA mRNA, TLR4 mRNA and integrin β1 mRNA at 3, 6 and 24 h of incubation ( P ＞0.05), but the parameters mentioned above were significantly up-regulated at 72 h of incubation in group LPS ( P ＜ 0.05). Conclusion In the early acute lung injury, LPS leads to pulmonary fibrosis through activating lung fibroblasts directly, and also accelerates the activation of lung fibroblasts and promotes the process of pulmonary fibrosis through up-regulating the expression of TLR4 and integrin β1 in mice.     

巢蛋白在人真皮成纤维细胞中的表达

目的探讨巢蛋白(nestin)在人皮肤真皮组织成纤维细胞(adu lt hum an derm is fibrob lasts,HDF)中的表达。方法采用体外培养、免疫组织/细胞化学技术检测巢蛋白在6例成人皮肤真皮组织及体外培养3、5、7、10、12代龄皮肤真皮成纤维细胞中的表达,计数分析人皮肤真皮组织成纤维细胞中巢蛋白+成纤维细胞数量,权重法分析体外培养成纤维细胞各代间巢蛋白的表达差异。结果人皮肤真皮组织中,巢蛋白+成纤维细胞表达数量占成纤维细胞总数(9.5±3.0)%,其数量为(103.3±67.4)个/mm2。与人皮肤真皮组织巢蛋白+成纤维细胞的表达数量相比,体外培养的真皮成纤维细胞中巢蛋白+成纤维细胞数量明显升高,并出现代龄之间的差异(P<0.05),其中第7、10代细胞表达量高于第5、12代细胞,第3代细胞最少(P<0.05)。结论在人皮肤真皮组织成纤维细胞中存在着巢蛋白+成纤维细胞。体外培养条件下,人皮肤真皮巢蛋白+成纤维细胞数量增加,提示体外培养作为刺激因子可能使成纤维细胞反分化为其前体细胞,作为干细胞标志物的巢蛋白有可能成为用于成纤维前体细胞鉴定的标志物之一。     

Transplanted fetal fibroblasts: survival and distribution over time in normal adult dermis compared with autogenic, allogenic, and xenogenic adult fibroblasts.

Cell therapy is a widely applicable therapeutic approach using cells and cell elements, frequently from fetal or young animals, for their beneficial effects. This study evaluated the host response to and tolerance of transplanted fetal skin fibroblasts. Cultured fibroblasts from adult rabbit skin (autogenic and allogenic), 21-day fetal rabbit skin (allogenic), and adult pig skin (xenogenic) were labeled with a fluorescent vital dye CM-DiI, injected intradermally into the dorsal skin of adult rabbits at multiple sites and then biopsied over an 8-week period. Each cell type showed a biphasic distribution curve with an early phase (0 to 28 days) and a late phase (28 to 56 days). In the early phase, cells showed a rise and fall in total cell density (reflecting an increase and then a decrease in total cell number), followed by a slow decrease in cell density with cells still detectable at 56 days. Fetal cells showed the highest survival at the end of the study. None of the groups showed clinical or histologic signs of acute inflammation or rejection. This study demonstrated that (1) transplanted fibroblasts are well tolerated by an immunologically competent host, (2) CM-DiI-labeled cells are detectable in vivo for at least 8 weeks, and (3) fetal fibroblasts have a distribution and survival profile that is distinct from that of adult fibroblasts.     

消瘢醑对瘢痕疙瘩成纤维细胞增殖的影响

目的探讨复方消瘢醑对人瘢痕疙瘩成纤维细胞增殖的作用,为中药治疗瘢痕提供实验依据. 方法以氢化可的松为实验对照,在体外培养的6例瘢痕疙瘩和6例正常皮肤成纤维细胞培养液中加入不同剂量的消瘢醑,利用3H-TdR掺入法检测12 h,24 h,48 h瘢痕疙瘩成纤维细胞(KFB)和正常皮肤成纤维细胞(NFB)增殖活性.结果 10、100、1 000 μg/ml消瘢醑和1×10-7 mol/L氢化可的松在12、24、48 h均可抑制NFB和KFB的增殖,P＜0.05.氢化可的松对KFB的抑制作用与NFB相似.10 μg/ml和100 μg/ml消瘢醑对NFB和KFB增殖的抑制与氢化可的松相似. 结论消瘢醑能抑制成纤维细胞的增殖,可能起到治疗瘢痕过度增生的作用.     

Effect of histamine on human vas deferens in vitro

Histamine, 2-methylhistamine and 4-methylhistamine produced concentration-related contractions in some isolated human vas deferens preparations. The contractions produced by histamine and its analogues were reversibly and competitively antagonised by the H1-receptor blocker, mepyramine, but not the H2-receptor blockers, burimamide and cimetidine. Phentolamine, atropine and guanethidine did not affect the excitatory action of histamine. Histamine and 4-methylhistamine did not show any inhibitory effect on KCl-induced tone. The results showed that histamine receptors were present in the human vas deferens and the histamine receptors mediating the excitatory response were likely to be H1-receptors.