Effect of cycloleucine on amino acid accumulation by human diploid fibroblasts

Cycloleucine is a synthetic amino acid which produces, in vivo, biochemical abnoramlities comparable to those seen in human cystinuria-lysinuria. The effect of cycloleucine on intracellular accumulation of amino acids overlapping separate transport systems was studied using human diploid fibroblasts subcultures on glass coverslips. The data indicated that alpha-alanine, serine and proline accumulation was inhibited significantly by cycloleucine. The percentage of inhibition was approximately the same. Lysine was less affected by cycloleucine, but this amino acid accumulation proceeded at a rate slower than for neutral amino acids. In vitro, this inhibitory effect seems to be a generalized phenomenon affecting substrates. These results confirm in human fibroblasts data reported for human and rat kidney slices.     

Effects of bile and bile acids on cultured human fibroblasts

Impaired healing induced by leakage of bile has been postulated as one factor responsible for complications after reconstructive bile duct surgery. The cytotoxicity of human bile and its major bile acids on cultured human fibroblasts was therefore studied by evaluation of their effects on cell morphology and growth, on synthesis and secretion of 35SO4-mucopolysaccharides and on release of a lysosomal enzyme. Normal human fibroblasts derived from a standard culture strain (MRC-5) were grown to confluence and exposed to: (1) sterile human T-tube bile, (2) a mixture of bile acids resembling that of human bile, or (3) various concentrations of the glycine- and taurine conjugates of cholic, chenodeoxycholic or deoxycholic acid. Medium containing whole bile (total bile acid concentration 0.25, 0.75 or 1.6 mmol/l) exerted time and dose dependent cytotoxic effects on morphology and growth and release of lysosomal enzyme. Synthesis and secretion of 35SO4-mucopolysaccharides were markedly inhibited. The bile acid mixture exhibited the same time and dose dependent effects. The conjugates of deoxycholic acid were found to be the most toxic of the individual bile acids studied.     

虎杖苷对成纤维细胞生物学特性的影响

背景：虎杖多年来一直是烧伤、创伤创面愈合治疗方剂中的一味主药,因成分复杂,具体药理作用难以进一步研究,对于虎杖苷促愈合作用目前未见文献报道。目的：分析不同浓度虎杖苷对成纤维细胞生物学特性的影响。方法：取烧伤后行瘢痕切除植皮4例患者剩余小中厚皮片,原代培养人成纤维细胞。用含有10-6,10-5,10-4,10-3,10-2mol/L不同浓度虎杖苷培养液作用第2代人成纤维细胞,未加虎杖苷的培养液作为对照。MTT法检测细胞增殖情况;流式细胞仪检测细胞周期及凋亡;ELISA法检测上清中纤维结合蛋白、Ⅰ型胶原蛋白、Ⅰ型胶原蛋白的表达情况。结果与结论：10-5、10-4mol/L组促进成纤维细胞增殖最明显,10-2mol/L组吸光度值显著下降,细胞生长受抑制。10-3mol/L组G1期细胞大幅度下降,细胞有S期阻滞现象。10-2mol/L组有明确的促凋亡作用。10-2mol/L组上清中Ⅰ、Ⅰ型胶原蛋白较其他各组显著增高（P〈0.05）;纤维结合蛋白较对照组显著增高（P〈0.05）,较其他各组有所下降（P〈0.05）。说明低浓度虎杖苷有促进成纤维细胞增殖、保护细胞免于凋亡及促进纤维结合蛋白的表达与合成分泌的作用,促进成纤维细胞增殖的最适浓度应为10-5～10-4mol/L。     

多核白细胞弹性蛋白酶对人角膜基质细胞MMP-2、MMP-9表达的影响

目的观察多核白细胞弹性蛋白酶与白细胞介素-1α对人角膜基质细胞表达的MMP-2、MMP-9的影响,进而探讨角膜溃疡的发生机理。方法体外培养人角膜基质细胞,在培养液中添加或不添加多核白细胞弹性蛋白酶或白细胞介素-1α,培养5天,通过明胶酶谱分析的方法测定培养上清中的MMP-2和MMP-9的表达。结果体外培养的人角膜基质细胞无任何刺激下可释放MMP-2;白细胞介素-1α可加强并激活MMP-2的表达,同时可诱导MMP-9前体的释放;多核白细胞弹性蛋白酶在白细胞介素-1α诱导下可完全激活前体MMP-9转化为活化形式。结论多核白细胞弹性蛋白酶和白细胞介素-1α协同作用参与角膜溃疡的发生。     

抑制胆碱酯酶对精神分裂症动物模型大鼠的记忆力和游离氨基酸的影响

目的:探讨在精神分裂症动物模型大鼠中,动物模型的记忆力和脑组织游离氨基酸与乙酰胆碱含量的变化关系。方法:实验在兰州军区精神病防治中心完成。将8周龄的Wistar雄性大鼠随机分成对照组、模型对照组和治疗组,每组8只。其中对照组空白对照,模型对照组和治疗组用浓度为0.6mg/kg,注射量为5μL/g的5-甲基二氢二苯并环庚稀亚氨马来酸或地卓西平马来酸盐(简称MK801)溶液注射于大鼠左侧腹腔,建立精神分裂症的动物模型;模型对照组以等量生理盐水对照治疗,治疗组以石杉碱甲片进行治疗。采用主动回避反应,一次性被动回避反应,空间分辨记忆3种模式对各组大鼠的学习记忆过程进行系统分析,并测定游离氨基酸水平的变化。结果:3组各8只动物均进入结果分析。①各组大鼠主动回避反应的差异:对照组大鼠主动回避反应习得率明显高于模型对照组和治疗组大鼠,治疗组大鼠主动回避反应习得率明显高于模型对照组大鼠,差异均有显著性意义(P均<0.05);对照组大鼠的主动回避反应消退明显慢于模型对照组和治疗组大鼠,治疗组大鼠的主动回避反应消退明显慢于模型对照组大鼠,差异均有显著性意义(P均<0.05)。②各组大鼠一次性被动回避反应的差异:对照组步入潜伏期最长,治疗组次之,模型对照组最短,且各组间差异有显著性意义(P均<0.05)。③各组大鼠空间分辨记忆的差异:对照组最佳,治疗组次之,模型对照组最差,且各组间差异有显著性意义(P<0.05)。④各组大鼠各脑区游离氨基酸含量的差异:模型对照组大鼠的谷氨酸含量在海马、皮质和纹状体观察区明显低于正常对照组和治疗组,天冬氨酸含量在海马和纹状体观察区低于对照组和治疗组,而氨基丁酸仅在纹状体区高于正常对照组,差异有显著性意义(P均<0.05)。结论:精神分裂症的动物模型伴有学习记忆能力下降,石杉碱甲片能改善精神分裂症动物模型的学习记忆能力,主要是通过抑制乙酰胆碱酯酶的活性同时直接或间接地影响体内氨基酸的含量。     

抑制胆碱酯酶对精神分裂症动物模型大鼠的记忆力和游离氨基酸的影响

目的：探讨在精神分裂症动物模型大鼠中，动物模型的记忆力和脑组织游离氨基酸与乙酰胆碱含量的变化关系。方法：实验在兰州军区精神病防治中心完成。将8周龄的Wistar雄性大鼠随机分成对照组、模型对照组和治疗组，每组8只。其中对照组空白对照，模型对照组和治疗组用浓度为0．6mg/kg，注射量为5μL/g的5-甲基二氢二苯并环庚稀亚氨马来酸或地卓西平马来酸盐（简称MK801）镕液注射于大鼠左侧腹腔，建立精神分裂症的动物模型；模型对照组以等量生理盐水对照治疗，治疗组以石杉碱甲片进行治疗。采用主动回避反应，一次性被动回避反应，空间分辨记忆3种模式对各组大鼠的学习记忆过程进行系统分析，并测定游离氨基酸水平的变化。结果：3组各8只动物均进入结果分析。①各组大鼠主动回避反应的差异：对照组大鼠主动回避反应习得率明显高于模型对照组和治疗组大鼠，治疗组大鼠主动回避反应习得率明显高于模型对照组大鼠，差异均有显著性意义（P均〈0．05）；对照组大鼠的主动回避反应消退明显慢于模型对照组和治疗组大鼠，治疗组大鼠的主动回避反应消退明显慢于模型对照组大鼠，差异均有显著性意义（P均〈0．05）。②各组大鼠一次性被动回避反应的差异：对照组步入潜伏期最长，治疗组次之，模型对照组最短，且各组间差异有显著性意义（P均〈0．05）。③各组大鼠空间分辨记忆的差异：对照组最佳，治疗组次之，模型对照组最差，且各组间差异有显著性意义（P〈0．05）。④各组大鼠各脑区游离氨基酸含量的差异：模型对照组大鼠的谷氨酸含量在海马、皮质和纹状体观察区明显低于正常对照组和治疗组，天冬氨酸含量在海马和纹状体观察区低于对照组和治疗组，而氨基丁酸仅在纹状体区高于正常对照组，差异有显著性意义（P均〈0．05）。结论：精神分裂症的动物模型伴有学习记忆能力下降，石杉碱甲片能改善精神分裂症动物模型的学习记忆能力，主要是通过抑制乙酰胆碱酯酶的活性同时直接或间接地影响体内氨基酸的含量。     

三七总甙对人瘢痕疙瘩成纤维细胞的作用

目的:观察三七总甙对体外培养的人瘢痕疙瘩成纤维细胞增殖及胶原合成的作用,以寻找治疗人瘢痕疙瘩的有效药物。方法:体外培养人瘢痕疙瘩成纤维细胞,分别应用噻唑蓝(MTT)比色法和H3-脯氨酸掺入法检测三七总甙作用后细胞增殖及胶原合成的变化。结果:与空白对照组相比,三七总甙含量在0.5,1.0和1.5g/L时均能够显著抑制成纤维细胞增殖及胶原合成(P<0.01),但在1.5g/L时对细胞具有毒性作用。结论:三七总甙具有体外抗纤维化作用,可能成为治疗瘢痕疙瘩的有效药物。     

Effects of the antithrombotic drug suloctidil on low density lipoprotein processing and cholesterol metabolism in cultured human fibroblasts

Human foetal lung fibroblasts were pretreated for 24 h with the antithrombotic drug, suloctidil (1 to 10 mumol/l), which induced a dose-dependent increase in LDL binding, uptake and degradation. At 10 mumol/l suloctidil, the respective increases in these parameters were 40%, 80% and 50%. The same treatment also resulted in increases of 1.5 to 2-fold in the synthesis of sterols, fatty acids and triacylglycerols from sodium acetate. In contrast, the esterification of cholesterol with oleic acid was specifically decreased by 35% by 24 h pretreatment of fibroblasts with 10 mumol/l suloctidil. A similar decrease of cholesterol esterification was observed in cholesterol-laden fibroblasts. It is suggested that these effects of suloctidil on LDL processing and cholesterol metabolism are related to the amphiphilic characteristics of the drug and to its calcium-blocking properties.     

Cell density affects prolidase and prolinase activity and intracellular amino acid levels in cultured human cells

Prolidase (EC 3.4.13.9) and prolinase (EC 3.4.13.8) and intracellular amino acid levels in cultured human cells increased when cell density rose. Firstly, two normal fibroblast strains were continuously cultured for 21 days and these parameters were measured on days 3, 7, 10, 14, 17 and 21 after plating. Prolidase, prolinase and amino acid levels varied considerably depending on the duration of culture and growth rate. Secondly, we studied the action of different cadmium and cobalt concentrations on prolidase activity. These two effectors altered this enzyme activity, but secondarily to modifying cell density. Thirdly, prolidase activity was investigated in 8 control amniotic cell strains, with a view to prenatal diagnosis of inherited prolidase deficiency, and we noted the same cell density interference. Due to the large variations related to cell density, we recommend specifying the number of cells per unit surface, and avoiding the term 'cells at confluency' which is unduly vague.     

Metabolic studies on normal and pyruvate dehydrogenase deficient cultured human fibroblasts.

Secondary metabolic derangements may occur in cultured fibroblasts with a defined enzyme deficiency. The metabolism of cells deficient of pyruvate dehydrogenase (5% of normal) has been studied using radioactive labelled substrates. Compared to normal control cells the activity of glycolysis was 149% (P less than 0.001), pentose phosphate shunt 144% (P less than 0.01), citric acid cycle 80% (P less than 0.002), and oxidation of acetate was 30% (P less than 0.01). The oxidation of palmitate and octanoate were not significantly different from that of control cells. Metabolic studies on fibroblasts may serve as a useful screening procedure for the detection of enzyme defects, but the results should be cautiously interpreted with respect to the localization of the primary defect.     

Synthesis and secretion of alpha2-macroglobulin by cultured human fibroblasts

The following observations indicate that cultured human WI-38 fibroblasts synthesize and secrete alpha2-macroglobulin into serum-free medium: (a) after incubation of cultures with [35S]L-methionine, a labeled protein appeared in the medium which was precipitated by antiserum directed against alpha2-macroglobulin; (b) after incubation of cultures with [35S]L-methionine, a major band of radioactivity detected by polyacrylamide gel electrophoresis of the proteins in medium co-migrated with alpha2-macroglobulin; and (c) the amount of alpha2-macroblobulin in the medium, estimated both functionally and immunologically, increased with time in normal but not not puromycin-treated cultures.     

Effects of high doses of glucocorticoids on free amino acids,ribosomes and protein turnover in human muscle

BACKGROUND: Treatment with glucocorticosteroids causes a negative nitrogen balance, but the kinetic mechanisms responsible for this catabolic effect are controversial. We investigated the effects of 60 mg day(-1) prednisolone on protein synthesis and degradation in human skeletal muscle. MATERIALS AND METHODS: Healthy adults (n = 9) were studied in the postabsorptive state, before and after 3 days of prednisolone treatment. The L-[ring 2,6(-3)H(5)]-phenylalanine tracer technique, concentration and size distribution of the ribosomes, mRNA content of the ubiquitin-proteasome pathway components in muscle, phenylalanine flux across the leg, and the free amino acid concentrations in skeletal muscle were used to study muscle protein metabolism. RESULTS: The concentrations of most amino acids in arterial blood increased after prednisolone. There were also increased effluxes of phenylalanine, asparagine, arginine, alanine, methionine and isoleucine from the leg. The rate of protein degradation, as measured by the appearance rate (Ra) of phenylalanine, increased by 67% (P = 0.023) which, together with a doubling of the net release of phenylalanine from the leg (P = 0.007), indicated accelerated protein degradation. The pathway was not identified but there was no significant increase in mRNAs' encoding components of the ubiquitin-proteasome pathway. There was a 6% reduction in polyribosomes (P = 0.007), suggesting a decrease in the capacity for protein synthesis, although there was no measured decrease in the rate of protein synthesis. CONCLUSIONS: These findings indicate that high doses of prednisolone lead to a sharp increase in net protein catabolism, which depends more on enhanced protein breakdown, and an uncertain effect on protein synthesis. The mechanisms stimulating proteolysis and the pathway stimulated to increase muscle protein degradation should be explored.     

氧自由基对培养人脐静脉内皮细胞代谢产物的影响

目的：观察氧自由基对培养的人脐静脉内皮细胞产生的裂解不对称二甲精氨酸的酶--二甲精氨酸-二甲赖氨酸水解酶活性及表达的影响，以探讨不对称二甲精氨酸代谢机斜及卡托普利的作用。方法：实验于2003—05／2004—06在南昌大学医学分子中心进行。采用改良的Jaffe法培养人脐静脉内皮细胞，取生长良好的3～6代人脐静脉内皮细胞分为4组：①空白对照组：加DMEM培养液。②氧自由基0．01，0.1mmol／L组：分别加入氧自由基0.01，0.1mmol／L。③卡托普利组：同时加入氧自由基0.1mmol／L及卡托普利（100mg／L）共孵。孵育24h后，检测上清液中一氧化氮、一氧化氮合酶活性、内皮细胞的代谢产物不对称二甲精氨酸浓度以及反应二甲精氨酸-二甲赖氨酸水解酶酶活性的L-瓜氨酸浓度，采用Western blotting测定细胞裂解液中二甲精氨酸-二甲赖氨酸水解酶的蛋白表达。结果：①二甲精氨酸浓度：氧自由基0，01，0.1mmol／L组均高于空白对照组[（2．71&;#177;0．35），（4．78&;#177;0．67），（0．81&;#177;0．12）μmol／L，P〈0．05，0．01]，卡托普利组低于氧自由基0．1mmol／L组[（2．03&;#177;0．35）μmol／L．P〈0．01]。②L-瓜氨酸浓度：氧自由基0．01，0．1mmol／L组均低于空白对照组（P〈0．05，0．01）．卡托普利组高于氧自由基0．1mmol／L组（P〈0．01）。③一氧化氮浓度及一氧化氮合酶活性：氧自由基0．01，0．1mmol／L组均低于空白对照组（P〈0．05，0．01），卡托普利组高于氧自由基0．1mmol／L组（P〈0．01）。④二甲精氨酸-二甲赖氨酸水解酶的蛋白表达：各组间无差异（P〉0．05）。结论：氧自由基培养下，内皮损伤，不对称二甲精氨酸的增加与二甲精氨酸-二甲赖氨酸水解酶的活性减弱有关，与其蛋白的表达无关。而卡托普利则通过增加二甲精氨酸-二甲赖氨酸水解酶活性促进不对称二甲精氨酸代谢，使一氧化氮合酶活性增加，抑制氧自由基对内皮功能的损伤。     

氧自由基对培养人脐静脉内皮细胞代谢产物的影响

目的:观察氧自由基对培养的人脐静脉内皮细胞产生的裂解不对称二甲精氨酸的酶--二甲精氨酸-二甲赖氨酸水解酶活性及表达的影响,以探讨不对称二甲精氨酸代谢机制及卡托普利的作用。方法:实验于2003-05/2004-06在南昌大学医学分子中心进行。采用改良的Jaffe法培养人脐静脉内皮细胞,取生长良好的3～6代人脐静脉内皮细胞分为4组:①空白对照组:加DMEM培养液。②氧自由基0.01,0.1mmol/L组:分别加入氧自由基0.01,0.1mmol/L。③卡托普利组:同时加入氧自由基0.1mmol/L及卡托普利(100mg/L)共孵。孵育24h后,检测上清液中一氧化氮、一氧化氮合酶活性、内皮细胞的代谢产物不对称二甲精氨酸浓度以及反应二甲精氨酸-二甲赖氨酸水解酶酶活性的L-瓜氨酸浓度,采用Westernblotting测定细胞裂解液中二甲精氨酸-二甲赖氨酸水解酶的蛋白表达。结果:①二甲精氨酸浓度:氧自由基0.01,0.1mmol/L组均高于空白对照组[(2.71±0.35),(4.78±0.67),(0.81±0.12)μmol/L,P<0.05,0.01],卡托普利组低于氧自由基0.1mmol/L组[(2.03±0.35)μmol/L,P<0.01]。②L-瓜氨酸浓度:氧自由基0.01,0.1mmol/L组均低于空白对照组(P<0.05,0.01),卡托普利组高于氧自由基0.1mmol/L组(P<0.01)。③一氧化氮浓度及一氧化氮合酶活性:氧自由基0.01,0.1mmol/L组均低于空白对照组(P<0.05,0.01),卡托普利组高于氧自由基0.1mmol/L组(P<0.01)。④二甲精氨酸-二甲赖氨酸水解酶的蛋白表达:各组间无差异(P>0.05)。结论:氧自由基培养下,内皮损伤,不对称二甲精氨酸的增加与二甲精氨酸-二甲赖氨酸水解酶的活性减弱有关,与其蛋白的表达无关。而卡托普利则通过增加二甲精氨酸-二甲赖氨酸水解酶活性促进不对称二甲精氨酸代谢,使一氧化氮合酶活性增加,抑制氧自由基对内皮功能的损伤。     

Effects of propofol and taurine on intracellular free amino acid profiles and immune function markers in neutrophils in vitro.

We have examined the effects of propofol, taurine, and the combination of propofol and taurine on amino acid profiles and the immune function markers superoxide anion (O2-), hydrogen peroxide (H2O2), and released myeloperoxidase (MPO) activity in neutrophils (PMN). Propofol led to significant changes in the dynamic PMN-free amino acid pool. Exogenous taurine significantly reduced PMN neutral amino acid and alpha-aminobutyrate (alpha-aba) as intracellular taurine increased. Incubation with propofol plus taurine resulted in lower intracellular taurine levels and elevated alpha-aba and neutral amino acid concentrations compared to propofol alone. Concerning PMN immune function markers, propofol significantly decreased O2- and H2O2 formation and released MPO. Taurine led to an increased release of MPO and concomitant significantly reduced O2- and H2O2 levels. When propofol and taurine were applied together they appeared to act additively with regard to superoxide and hydrogen peroxide formation. In the case of MPO, taurine neutralized propofol's effects, supporting the idea that MPO activity may be regulated by taurine. We believe therefore that taurine is important for strengthening PMN host defense capability, although the mechanisms are not yet clear. Moreover, taurine appears to act primarily by altering the PMN osmotic balance, while propofol seems to affect PMN amino acid metabolism and/or uptake and release.     

硒化壳聚糖对体外培养成纤维细胞增殖及胶原合成的影响

目的:观察硒化壳聚糖在成纤维细胞生长过程中的作用。方法:实验于2004-01/12在郧阳医学院附属太和医院完成。选取体外培养的第6代成纤维细胞,分为对照组和药物组。药物组浓度为25,50,100,200和400mg/L,分别加入由培养基稀释成的同等体积不同浓度的硒化壳聚糖,对照组加入等体积培养基。5×108L-1接种于24孔板,培养24h后,加入不同浓度硒化壳聚糖继续培养48h,收集细胞用于电镜观察;5×106L-1接种于96孔板,培养48h后,加入不同浓度的硒化壳聚糖,继续培养24h,分别加入3H-脱氧胸腺嘧啶核苷酸或3H-脯氨酸再作用24h,液闪计数仪测定每分钟计数值来反映细胞增殖及胶原合成的情况。结果:①对细胞增殖的影响:药物组与对照组比较,细胞核均有不同程度的增大,核浆增大,核仁变大,变多,扩张的内质网增多,细胞表面突起增加,线粒体更为丰富,更易见到聚集成团的糖原。②对DNA影响:400mg/L组3H-脱氧胸腺嘧啶核苷酸掺入量高于对照组犤(18658±356.42,10564±356.47)min-1,P<0.01犦;③对细胞胶原合成的影响:100mg/L组3H-脯氨酸掺入量高于对照组犤(2867±224.09,1762±186.22)min-1,P<0.01犦。结论:创面愈合过程中,硒化壳聚糖可促进皮肤成纤维细胞分裂增殖及胶原合成,促进创面愈合。具有量效关系。     

异搏定对人皮肤成纤维细胞增殖的影响

目的：探讨钙离子通道阻滞剂异搏定对人皮肤正常成纤维细胞增殖的作用，为钙离子通道阻滞剂治疗瘢痕过度增生提供依据。方法：实验皮肤标本来源于2002—07／2003—07北京大学第三医院整形外科5例美容手术或植皮手术患者，取冗余全层皮肤组织，患者知情同意。体外培养3～8代的人皮肤正常成纤维细胞，实验分为异搏定组：包括1&;#215;10^-3, 1&;#215;10^-4, 1&;#215;10^-5, 1&;#215;10^-6, 1&;#215;10^-7, 1&;#215;10^-8,1&;#215;10^-9,1&;#215;10^-10mol／L组，分别在细胞培养孔中加入2mL相应浓度异搏定的培养液；氢化可的松组：在细胞培养孔中加入2mL浓度为1&;#215;10^-7mol／L氢化可的松的培养液；对照组：在细胞培养孔中加入2mL体积分数为0．005的新生牛血清的Dulbecco＇s改良的Eade＇s培养基。检测细胞活力采用锥虫蓝染色；细胞增殖状况测定应用^3氚标记的胸腺嘧啶掺入法；观察细胞增殖抑制率[抑制率（1％）=（阴性对照组每分钟脉冲数值-实验组每分钟脉冲数值）／阴性对照每分钟脉冲数值]，取各例样本的平均值。结果：①人皮肤正常成纤维细胞的增殖动力学结果：对照组人皮肤正常成纤维细胞在接种12h开始增殖，24h达到高峰，48h又逐渐降低。②异搏定对人皮肤正常成纤维细胞增殖抑制的浓度效应：各浓度的异搏定对人皮肤成纤维细胞的增殖均有抑制作用，其中1&;#215;10^-6mol／L异搏定与1&;#215;10^-7mol／L氢化可的松对成纤维细胞增殖的抑制率无显著性差异（t=0．135,P=0．896〉0．05）。③浓度为1&;#215;10^-6mol/异搏定对人皮肤正常成纤维细胞增殖抑制的时间效应：氢化可的松在24h时对人皮肤正常成纤维细胞的增殖抑制作用显著高于6，12,48h时[（60．67&;#177;18．94）％比（-19．69&;#177;21．26）％，（10．89&;#177;11．61）％，（37．03&;#177;12．17）％，q=10．867．6．732，3．197；P〈0．051；异搏定在24h时对人皮肤正常成纤维细胞的增殖抑制作用显著高于6，12，48h时[（51．42&;#177;15．30）％比（-16．83&;#177;16．90）％．（1．09&;#177;11．62）％，（17．41&;#177;13．41）％，q=10．566，7．791，5．265，〈0．051；在药物作用48时异搏定对人皮肤正常成纤维细胞的抑制作用低于氢化可的松（t=2．423,P=0．042〈0．05）。结论：不同浓度的异搏定对体外培养人正常皮肤成纤维细胞增殖有抑制作用，异搏定可能通过抑制成纤维细胞的增殖达到治疗瘢痕过度增生的作用。     

异搏定对人皮肤成纤维细胞增殖的影响

目的:探讨钙离子通道阻滞剂异搏定对人皮肤正常成纤维细胞增殖的作用,为钙离子通道阻滞剂治疗瘢痕过度增生提供依据。方法:实验皮肤标本来源于2002-07/2003-07北京大学第三医院整形外科5例美容手术或植皮手术患者,取冗余全层皮肤组织,患者知情同意。体外培养3～8代的人皮肤正常成纤维细胞,实验分为异搏定组:包括1×10-3,1×10-4,1×10-5,1×10-6,1×10-7,1×10-8,1×10-9,1×10-10mol/L组,分别在细胞培养孔中加入2mL相应浓度异搏定的培养液;氢化可的松组:在细胞培养孔中加入2mL浓度为1×10-7mol/L氢化可的松的培养液;对照组:在细胞培养孔中加入2mL体积分数为0.005的新生牛血清的Dulbecco’s改良的Eagle’s培养基。检测细胞活力采用锥虫蓝染色;细胞增殖状况测定应用3氚标记的胸腺嘧啶掺入法;观察细胞增殖抑制率[抑制率(I%)=(阴性对照组每分钟脉冲数值-实验组每分钟脉冲数值)/阴性对照每分钟脉冲数值],取各例样本的平均值。结果:①人皮肤正常成纤维细胞的增殖动力学结果:对照组人皮肤正常成纤维细胞在接种12h开始增殖,24h达到高峰,48h又逐渐降低。②异搏定对人皮肤正常成纤维细胞增殖抑制的浓度效应:各浓度的异搏定对人皮肤成纤维细胞的增殖均有抑制作用,其中1×10-6mol/L异搏定与1×10-7mol/L氢化可的松对成纤维细胞增殖的抑制率无显著性差异(t=0.135,P=0.896>0.05)。③浓度为1×10-6mol/L异搏定对人皮肤正常成纤维细胞增殖抑制的时间效应:氢化可的松在24h时对人皮肤正常成纤维细胞的增殖抑制作用显著高于6,12,48h时[(60.67±18.94)%比(-19.69±21.26)%,(10.89±11.61)%,(37.03±12.17)%,q=10.867,6.732,3.197;P<0.05];异搏定在24h时对人皮肤正常成纤维细胞的增殖抑制作用显著高于6,12,48h时[(51.42±15.30)%比(-16.83±16.90)%,(1.09±11.62)%,(17.41±13.41)%,q=10.566,7.791,5.265;P<0.05];在药物作用48时异搏定对人皮肤正常成纤维细胞的抑制作用低于氢化可的松(t=2.423,P=0.042<0.05)。结论:不同浓度的异搏定对体外培养人正常皮肤成纤维细胞增殖有抑制作用,异搏定可能通过抑制成纤维细胞的增殖达到治疗瘢痕过度增生的作用。