川芎嗪对成纤维细胞增殖及TGF-β_1和bFGF表达的影响

目的:研究川芎嗪对体外培养大鼠成纤维细胞增殖及碱性成纤维细胞因子(Basicfibroblastgrowthfactor,bFGF)、转化生长因子β_1(Transforminggrowthfactorβ_1,TGF-β_1)表达的影响,从而探讨川芎嗪抑制瘢痕形成的作用机制。方法:体外分离培养大鼠皮肤成纤维细胞,采取MTT法及荧光染色检测不同浓度的川芎嗪对大鼠成纤维细胞增殖能力的影响,ELISA法测定大鼠皮肤瘢痕成纤维细胞TGF-β_1和bFGF的表达量。结果:在三种川芎嗪浓度条件下(1mg/ml,10mg/ml,50mg/ml),大鼠皮肤成纤维细胞增殖能力有不同程度的抑制作用,抑制作用与川芎嗪浓度成正比,在50mg/ml浓度时,抑制作用最为明显。采用ELISA法测定培养上清TGF-β_1和bFGF的分泌量,发现实验组两者含量均明显低于对照组,其中浓度为50mg/ml时,含量最低(P0.05)。结论:川芎嗪能够明显抑制体外培养的大鼠皮肤成纤维细胞的增殖能力,降低TGF-β_1和bFGF的表达量,从而在一定程度上抑制大鼠皮肤成纤维细胞的活性。     

黄芪甲苷对人增生性瘢痕成纤维细胞生长的影响

目的探讨黄芪甲苷对人增生性瘢痕成纤维细胞生长的作用。方法获取患者经手术切除的增生性瘢痕组织,从中提取成纤维细胞进行培养。采用MTT试验检测不同浓度的黄芪甲苷对成纤维细胞增殖的影响,并应用流式细胞技术对黄芪甲苷作用后的成纤维细胞的周期进行分析。通过Annexin V-FITC/PI双染色法,观察经黄芪甲苷处理的成纤维细胞的凋亡情况。采用Western blot和RT-PCR验证黄芪甲苷对细胞周期蛋白Cyclin D1和凋亡调节蛋白Bcl-2的作用。结果不同浓度的黄芪甲苷对成纤维细胞增殖均有一定的抑制作用,且随着药物浓度增加和作用时间的延长,其抑制作用愈发明显。大多数的成纤维细胞阻滞于G1期,而进入S期的细胞比例呈下降趋势,且细胞周期受到阻断;通过黄芪甲苷对抑制细胞周期蛋白Cyclin D1和抗凋亡蛋白Bcl-2的表达来影响细胞周期的进程并诱导成纤维细胞的凋亡,从而实现对细胞生长的抑制作用。结论黄芪甲苷能够抑制人增生性瘢痕成纤维细胞的增殖,并诱导其凋亡,适于对增生性瘢痕的预防和治疗。     

大黄素对人增生性瘢痕成纤维细胞的增殖及细胞周期作用的实验研究

目的观察大黄素对体外培养的人增生性瘢痕成纤维细胞增殖及细胞周期的作用,以寻找治疗人增生性瘢痕的有效药物。方法将切取于增生性瘢痕患者的瘢痕组织标本分成A（对照组）、B、C、D四组,每组6个标本,行体外成纤维细胞培养,采用0、50、100、200μg/ml的大黄素依次对A、B、C、D组的成纤维细胞进行干预,应用MTT比色法和流式细胞仪检测细胞增殖和细胞周期的变化。结果B、C、D组标本中成纤维细胞的增殖受到抑制,抑制于G0/G1期（P〈0.05）,与A组比较,差异有统计学意义（P〈0.05）。而且大黄素在50～200μg/ml的范围内,对成纤维细胞增殖的抑制有浓度的依赖性。结论大黄素具有体外抗纤维化作用,可能成为治疗人增生性瘢痕的有效药物。     

利多卡因通过增加细胞周期素依赖的蛋白激酶抑制剂1A（p21）表达抑制NIH-3T3细胞的增殖

背景研究利多卡因抑制大鼠胚胎成纤维细胞株NIH-3T3增殖的分子机制。体外实验中，局麻药可抑制细胞增殖，但它们对伤口愈合的影响目前尚存在争议。我们观察了利多卡因对体外大鼠成纤维细胞株NIH一3T3增殖的作用及机制。方法将NIH-3T3细胞置于含利多卡因（O、0．05、0．5、1、2、5rIM）的培养液中生长。细胞计数反映细胞增殖水平，并测定溴脱氧尿苷的摄取来反映利多卡因的抑制作用，基因表达和蛋白增殖水平分别采用PcR和westem测定。结果利多卡因呈浓度依赖性地抑制NIH—313细胞增殖，其抑制程度从无（Q05和Q5mM）到轻微（1mM）到很强（2mM和5mM）（P=0．006）。第3．5天，2mM利多卡因组明显抑制溴脱氧尿苷的摄取（与对照组相比P=0．02，与lmM利多卡因组相比P=Q0495）。第1．5天，利多卡因可上调细胞周期蛋白D1和细胞周期素依赖性蛋白激酶抑制剂1A（p21）的表达。第2．5天，利多卡因增加了p21蛋白水平。结论在脊髓麻醉、硬膜外麻醉和神经阻滞时，血浆中利多卡因浓度较低，其对成纤维细胞的增殖无明显影响，而高浓度的利多卡因可升高p21蛋白水平，使细胞增殖停止在细胞周期的S期，从而抑制细胞增殖。组织渗透后可能达到的高浓度利多卡因可能通过抑制成纤维细胞增殖而阻碍伤口的愈合。     

当归挥发油对增生性瘢痕成纤维细胞增殖、凋亡及胶原合成的影响

目的:探讨当归挥发油对增生性瘢痕成纤维细胞增殖、凋亡和胶原合成的影响。方法:体外培养人增生性瘢痕成纤维细胞,用噻唑蓝比色法检测细胞增殖活性,用流式细胞术分析细胞周期及凋亡,用放射免疫法测定胶原合成。结果:1mg/L、4mg/L和16mg/L当归挥发油组与对照组相比G0/G1期细胞、G2/M期减少,显著增加了S期细胞(P<0.05)。凋亡率随当归挥发油浓度增大逐渐增大(P<0.05)。当归挥发油呈剂量和时间依赖性抑制细胞合成胶原(P<0.05或0.01)。结论:当归挥发油抑制成纤维细胞的增殖和胶原合成。     

当归挥发油对增生性瘢痕成纤维细胞增殖、凋亡及胶原合成的影响

目的:探讨当归挥发油对增生性瘢痕成纤维细胞增殖、凋亡和胶原合成的影响。方法:体外培养人增生性瘢痕成纤维细胞,用噻唑蓝比色法检测细胞增殖活性,用流式细胞术分析细胞周期及凋亡,用放射免疫法测定胶原合成。结果:1mg/L、4mg/L和16mg/L当归挥发油组与对照组相比G0/G1期细胞、G2/M期减少,显著增加了S期细胞(P<0.05)。凋亡率随当归挥发油浓度增大逐渐增大(P<0.05)。当归挥发油呈剂量和时间依赖性抑制细胞合成胶原(P<0.05或0.01)。结论:当归挥发油抑制成纤维细胞的增殖和胶原合成。     

几丁糖抑制人成纤维细胞增殖的实验研究

目的通过研究几丁糖对人成纤维细胞的细胞周期和Ki-67蛋白表达的影响,探讨几丁糖预防手术后组织粘连的机制。方法分别用含0、0.01、0.1、1.0、10.0mg/ml几丁糖的培养液作用于人成纤维细胞48h后,用流式细胞仪检测细胞周期。制作成纤维细胞的细胞爬片,用以上各浓度的含几丁糖培养液作用24h后,行细胞核的Ki-67蛋白免疫组织化学染色,观察细胞核中Ki-67蛋白的表达情况。结果在几丁糖作用下,成纤维细胞的生长在形态上受到明显抑制。几丁糖浓度为1.0、10.0mg/ml时,处于增殖期的成纤维细胞百分率分别为32.3%±5.2%、14.7%±2.9%,均显著低于几丁糖浓度为0时的百分率41.9%±5.8%,具有统计学意义(P<0.05);几丁糖浓度为0.01、0.1mg/ml时,处于增殖期的成纤维细胞百分率分别为39.0%±6.0%、35.5%±3.4%,虽低于几丁糖浓度为0时,但差异无统计学意义(P>0.05)。几丁糖浓度为0.1、1.0、10.0mg/ml时,Ki-67阳性细胞百分率分别为37.3%±3.4%、30.5%±6.2%、17.8%±3.0%,均显著低于几丁糖浓度为0时的57.6%±8.9%,具有统计学意义(P<0.05);几丁糖浓度为0.01mg/ml时,Ki-67阳性细胞百分率为54.1%±8.0%,低于几丁糖浓度为0时,无统计学意义(P>0.05)。结论几丁糖能抑制人成纤维细胞增殖,使处于静止期细胞数量增多,这可能是其预防手术后组织粘连的机制之一。     

P物质抑制剂辣椒素和bFGF抗体对增生性瘢痕成纤维细胞增殖协同抑制作用的实验研究

目的：探讨P物质抑制剂-辣椒素及bFGF抗体单独或联合使用对体外培养的人体增生性瘢痕成纤维细胞增殖的影响。方法：体外培养12例增生性瘢痕组织的成纤维细胞,分别加入不同浓度的辣椒素或/和bFGF抗体,培养24h后观察细胞形态学变化,并用MTT法检测细胞增殖抑制率。结果：所有不同浓度的辣椒素（2.5,5,20,40,60,80mg/L）均能使细胞皱缩、坏死,抑制细胞增殖,其抑制作用随剂量增大而增加。中高浓度bFGF抗体（40,80mg/L）也可促使细胞皱缩、坏死,抑制细胞增殖,其抑制作用也随剂量增大而增加。辣椒素与bFGF抗体联合应用对成纤维细胞抑制作用较单独使用效果更显著（P﹤0.05）。结论：辣椒素及bFGF抗体可抑制增生性瘢痕成纤维细胞增殖,其抑制作用随浓度增加而增加,两者联合使用有协调作用,本研究结果可能在增生性瘢痕的治疗提供新的思路和手段。     

苦参碱对人增生性瘢痕成纤维细胞增殖的影响

目的：研究苦参碱(Matrine，简称Mat)对人增生性瘢痕成纤维细胞增殖的影响。方法：将Mat以不同浓度梯度作用于体外培养的增生性瘢痕成纤维细胞，测MTT反应的A^0值及LDH值，用免疫组织化学检测Mat用药前后的细胞核内增殖性抗原(proliferatmg cell nuclear antigen，PCNA)的表达。结果：增生性瘢痕成纤维细胞在Mat0．25～100mg／ml范围内呈剂量依赖性增殖抑制，但LDH值无明显改变；PCNA的表达，在Mat作用后明显减弱。结论：Mat在体外能明显抑制增生性瘢痕成纤维细胞的增殖而不引起细胞坏死性改变。     

苦参碱对瘢痕疙瘩成纤维细胞生长的抑制作用实验研究

目的:研究苦参碱对瘢痕疙瘩成纤维细胞增殖、凋亡和自噬的影响及其相关作用机制。方法:通过收集手术切除的瘢痕疙瘩进行体外培养成纤维细胞,将细胞分为3组,分别加入10μmol/L,20μmol/L的苦参碱以及对照组(0.9%NaCl)进行实验。MTT实验检测细胞增殖情况;流式细胞技术进行细胞周期分析;Hoechest33258染色检测细胞凋亡情况;Westernblot实验检测苦参碱对瘢痕疙瘩成纤维细胞增殖、凋亡和自噬相关调控蛋白表达的影响。结果:通过不同浓度的苦参碱处理细胞后,MTT结果发现成纤维细胞的增殖受到抑制,抑制作用具有剂量依赖性;细胞周期分析发现,苦参碱作用后细胞的G1-S期转化受到了不同程度的抑制;Hoechest 33258染色发现苦参碱可以促进凋亡细胞的增加;Western blot实验结果发现苦参碱可以抑制CDK4、CDK6、Bcl-2和LC3-I蛋白的表达,促进Bax、LC3-Ⅱ和Beclin-1的表达。结论:苦参碱对瘢痕疙瘩成纤维细胞具有抑制增殖、促进凋亡的作用,能够影响细胞增殖、凋亡和自噬相关调控蛋白的表达。     

Sodium hyaluronate enhances colorectal tumour cell metastatic potential in vitro and in vivo

BACKGROUND: Sodium hyaluronate has been used intraperitoneally to prevent postoperative adhesions. However, the effect of sodium hyaluronate on tumour growth and metastasis in vitro and in vivo is still unknown. METHODS: Human colorectal tumour cell lines SW480, SW620 and SW707 were treated with sodium hyaluronate (10-500 microg/ml) and carboxymethylcellulose (0.125-1 per cent), and tumour cell proliferation and motility were determined in vitro. For the in vivo experiments male BD IX rats were randomized to a sodium hyaluronate group (n = 11; intraperitoneal administration of 0.5 x 10(6) DHD/K12 tumour cells and 5 ml 0.4 per cent sodium hyaluronate) or a phosphate-buffered saline group (n = 11; 0.5 x 10(6) DHD/K12 tumour cells and 5 ml phosphate-buffered saline intraperitoneally). Four weeks later the intraperitoneal tumour load was visualized directly. RESULTS: In vitro sodium hyaluronate increased tumour cell proliferation and motility significantly. Sodium hyaluronate-induced tumour cell motility appeared to be CD44 receptor dependent, whereas sodium hyaluronate-induced tumour cell proliferation was CD44 receptor independent. In vivo there was a significantly higher total tumour nodule count in the peritoneal cavity of the sodium hyaluronate-treated group compared with the control (P = 0.016). CONCLUSION: Sodium hyaluronate enhances tumour metastatic potential in vitro and in vivo, which suggests that use of sodium hyaluronate to prevent adhesions in colorectal cancer surgery may also potentiate intraperitoneal tumour growth. Presented to the Patey Prize Session of the Surgical Research Society and the annual scientific meeting of the Association of Surgeons of Great Britain and Ireland, Brighton, UK, 4-7 May 1999     

透明质酸钠预防屈肌腱粘连的临床研究

目的　评价透明质酸钠预防术后屈肌腱粘连的临床效果。方法　 1998年～ 1999年对 47例屈肌腱手术者 ,于肌腱损伤修复部鞘内或局部分别注入两种透明质酸钠凝胶制剂。 A组注入透明质酸钠 I号 ,2 0 m g/ 2 ml,17例 ;B组注入透明质酸钠 号 ,2 0 mg/ 2 ml,16例 ;C组除不用透明质酸钠外 ,其它治疗与 A、B组相同 ,14例。于术后 1、2和 3个月测定相关部位的功能、疼痛和肿胀等情况 ,按关节功能和握拳功能评价透明质酸钠预防粘连的效果。结果　 47例经 1～ 3个月随访 ,A组优良率为 6 4.71% ,B组为 6 8.75 % ,C组为 42 .86 % ,A及 B组与 C组比较有统计学意义 (P<0 .0 5 ) ;各组均未见明显毒副作用。结论　两种透明质酸钠凝胶均有明显抑制术后屈肌腱粘连形成的作用 ,且使用安全方便     

Hyaluronic acid in bronchoalveolar lavage fluid in patients with sarcoidosis: relationship to lavage mast cells.

Hyaluronate (hyaluronic acid), a potential marker for activated pulmonary fibroblasts, appears in increased concentrations in bronchoalveolar lavage fluid from patients with sarcoidosis. The mechanisms underlying fibroblast proliferation are largely unknown but activated alveolar T lymphocytes and macrophages probably play a part; the mast cell is also important for fibroblast proliferation. This study was designed to determine whether there is any association between pulmonary mast cells in lavage fluid, which are known to be increased in patients with sarcoidosis, and signs of pulmonary fibroblast activation. A strong correlation was found between lavage fluid hyaluronate and recovered mast cells (r = 0.72, p less than 0.001). Moreover, mast cell and hyaluronate estimations correlated inversely with lung volume and transfer factor for carbon monoxide, and both indices increased with advancing radiological sarcoid stage. Macrophage and granulocyte counts were normal in lavage fluid from patients with sarcoidosis and were not related to lavage fluid hyaluronate or laboratory signs of the disease in the lungs. Lymphocytes were recovered in increased numbers (p less than 0.001) and were related to the lavage fluid mast cells and hyaluronate. It is concluded that in sarcoidosis release of hyaluronate into the airways is related to the degree of lung disease and to the local inflammatory reaction in the lung as defined by increased numbers of mast cells and lymphocytes in lavage fluid. The findings may reflect a link between the immune system, activation of mast cells, and a pulmonary fibroblast proliferation.     

臭氧联合玻璃酸钠治疗膝骨性关节炎的疗效观察

[目的]观察医用臭氧(O3)联合玻璃酸钠(SH)治疗膝骨性关节炎的临床疗效。[方法]选择120例膝骨性关节炎患者,随机分为A、B、C三组,每组40例。其中A组第1周关节腔内注射15ml(35ug/ml)的医用臭氧,第2周注射玻璃酸钠25mg,第3周注射15ml(35ug/ml)的医用臭氧;B组关节腔内注射15ml/周的医用臭氧,每周1次,连续3次为1疗程;C组关节腔内注射玻璃酸钠25mg/周,每周1次,连续5次为1疗程,比较三组病例治疗前及治疗后1、2、3个月VAS值和治疗效果。[结果]三组患者治疗后VAS评分与治疗前相比均有降低(P0.01),在治疗后的第1、2、3个月同一时点,A组疼痛缓解最明显,VAS评分三组相比差异有统计学意义(P0.05,P0.01)。A组膝关节功能的优良率要高于对照组,差异有统计学意义(P0.05和P0.01)。全部患者未见明显的不良反应和副作用。[结论]医用臭氧联合玻璃酸钠治疗膝骨性关节炎能有效解除关节疼痛,改善关节活动度,临床疗效优于臭氧、玻璃酸钠单独应用。是一种治疗膝骨性关节炎的优势方法。     

Effect of heparin on pulmonary fibroblasts and vascular cells.

BACKGROUND: There is a large increase in mast cell numbers in fibrotic lung tissue, suggesting that mast cells may play a part in the pathogenesis of pulmonary fibrosis. Glycosaminoglycans, such as heparan sulphate, that are structurally related to heparin (a mast cell product) are part of the extracellular matrix and known to regulate cell growth. Basic fibroblast growth factor is a heparin binding growth factor produced by endothelial cells. METHODS: A study was carried out to examine the effect of heparin, basic fibroblast growth factor, and mast cell products on the proliferation of normal human lung fibroblasts and the effect of adding heparin on the proliferation of lung fibroblasts and pulmonary vascular cells incubated with basic fibroblast growth factor. RESULTS: Heparin at low concentration (0.03, 0.3-1.0 micrograms/ml) stimulated the proliferation of normal human lung fibroblasts in culture whereas a higher concentration (100 micrograms/ml) had an inhibitory effect. Mast cell products also stimulated the proliferation of fibroblasts, and the effect was decreased by pretreatment with heparinase or protamine. Heparin enhanced the growth of both fibroblasts and pulmonary vascular cells induced by low concentrations of basic fibroblast growth factor. CONCLUSIONS: Mast cells in fibrotic lung tissue may regulate fibroblast proliferation by releasing heparin. These results suggest that endothelial cells may interact with mast cells and modulate fibroblast growth by release of basic fibroblast growth factor.     

几丁聚糖和透明质酸钠抑菌作用的研究

目的　比较几丁聚糖和透明质酸钠的抑菌作用及抑菌范围。方法　对奇异变形杆菌、大肠埃希氏菌、白色念珠菌、铜绿假单胞菌及金黄色葡萄球菌进行培养。每种细菌培养 33管 ,其中 30管分为 A、B及 C组 ,按倍比稀释法在 A组加入高相对分子量 (Mr)几丁聚糖 ,B组加入低 Mr几丁聚糖 ,C组加入透明质酸钠 ;另 3管为对照组 (D组 )。37℃恒温下培养 18小时 ,观察细菌的生长情况。结果　 A组最小抑菌浓度 (MIC)为 :奇异变形杆菌 0 .0 31% ,大肠埃希氏菌 0 .0 6 3% ,白色念珠菌 0 .0 6 3% ,铜绿假单胞菌 0 .0 6 3% ,金黄色葡萄球菌 0 .0 6 3% ;B组 MIC为 :奇异变形杆菌0 .12 5 % ,大肠埃希氏菌 0 .2 5 % ,白色念珠菌 0 .2 5 % ,铜绿假单胞菌 0 .2 5 % ,金黄色葡萄球菌 0 .12 5 %。 C组和 D组各管都有细菌生长。结论　透明质酸钠无抑菌作用 ;几丁聚糖有广谱抑菌作用 ,而高相对分子量几丁聚糖抑菌能力较强。     

Inhibition of Peyronie's plaque fibroblast proliferation by biologic agents

Peyronie's disease is a fibromatosis of the tunica albuginea which affects up to 2% of men. Plaque development is believed to result, at least in part, from fibroblast proliferation and excess collagen deposition. Numerous oral and intralesional therapies have been used, including verapamil, colchicine and steroids. The purpose of this study was to investigate the in vitro effects of prostaglandin-E1 (PGE1), verapamil and colchicine on the proliferation rates of fibroblasts derived from Peyronie's disease tissue. Using tissue culture, multiple cell lines comprising fibroblasts from Peyronie's plaque, normal tunica and foreskin were established. Cells of low passage were removed from the parent culture and incubated with varying concentrations of PGE1 (0.1-10 mg/ml), verapamil (10-1000 mg/ml), and colchine (2.5 mg/ml). Proliferation was assessed at 48, 72 and 96 hours using the Vybrant MTT cell proliferation and then compared to control cells. Six plaque lines and 5 normal tunical cell lines were established. These cell lines exhibited excellent linear growth in culture media alone. Co-culture wih PGE1 resulted in no significant inhibition at 0.1 and 1 mg/ml, but a mean inhibition of 60.6+/-11.5% at a concenrtation of 10 mg/ml was noted. Similar inhibition was noted with verapamil at 100 and 1000 mg/ml with a mean inhibition of 65.2+/-10.6%. Colchicine resulted in a mean inhibition of 28% at a concentration of 2.5 mg/ml. Maximum inhibition occurred at 96 hours in all cases. There was no statisitically significant difference in proliferation rates between plaque and normal tunical cell lines. We have developed an in vitro model to assess the effects of biologically active agents on the growth of fibroblasts derived from Peyronie's disease tissue. Our data suggests that PGE1, verapamil, and colchicine inhibit in vitro proliferation of fibroblasts at specific concentrations. Refinement and application of this knowledge may allow the development of useful pharmacologic strategies for men with PD.