汉防己甲素对成纤维细胞Ⅰ型胶原蛋白合成的影响

目的　探讨汉防己甲素预防和治疗增生性瘢痕的可行性。方法　对增生性瘢痕中的成纤维细胞进行培养 ,然后加入不同浓度的汉防己甲素 ,培养后继续观察成纤维细胞合成胶原蛋白的能力的变化。结果　汉防己甲素可以抑制成纤维细胞合成胶原蛋白。结论　汉防己甲素对成纤维细胞胶原蛋白的合成具有抑制作用 ,因此它可能对增生性瘢痕具有预防和治疗作用     

汉防己甲素对增生性瘢痕基质胶原酶活性的影响

目的观察汉防己甲素对瘢痕胶原酶活性的影响，进一步阐明其抗瘢痕作用的机理。方法将汉防己甲素的实验浓度控制在低于细胞增殖抑制范围之内，采用加胶原蛋白底物的SDS—PAGE电泳法分离胶原酶，胰蛋白酶激活后利用湿胶密度扫描仪分析胶原酶的活性。同时采用改良的氯胺T法测量细胞外胶原蛋白量，并分析两者的相关关系。结果在不抑制细胞增殖的情况下，汉防己甲素可显著提高胶原酶的活性，使细胞外胶原蛋白减少，两者呈显著的负相关，此作用强于激素。结论增强胶原酶降解胶原蛋白的活性，减少细胞外胶原沉积是汉防己甲素抗瘢痕作用机理之一。     

汉防己甲素对增生性瘢痕成纤维细胞MMP-1 mRNA表达的影响

目的观察汉防己甲素对增生性瘢痕来源的成纤维细胞的MMP-1 mRNA表达的影响.方法培养增生性瘢痕来源的成纤维细胞,然后分别按终浓度为1、5、10 mg/L加入汉防己甲素,继续培养24 h,提取细胞内的RNA,经RT-PCR获得cDNA,再用MMP-1的特异性引物,通过PCR扩增,得到MMP-1的mRNA,并以β-actin为内参照物,进行琼脂糖凝胶电泳,最后用Band-scan扫描系统测定各产物密度值,用凝胶自动成像仪拍照.结果在汉防己甲素的作用下,成纤维细胞的MMP-1 mRNA表达增加.结论汉防己甲素可使增生性瘢痕来源的成纤维细胞的MMP-1的mRNA转录增加,从而增加了MMP-1的合成,因而可能对增生性瘢痕具有预防和治疗作用.     

汉防己甲素对增生性瘢痕成纤维细胞MMP-1 mRNA表达的影响

目的观察汉防己甲素对增生性瘢痕来源的成纤维细胞的MMP-1 mRNA表达的影响。方法培养增生性瘢痕来源的成纤维细胞，然后分别按终浓度为1、5、10mg／L加入汉防己甲素，继续培养24h，提取细胞内的RNA，经RT—PCR获得cDNA，再用MMP-1的特异性引物，通过PCR扩增，得到MMP-1的mRNA，并以β—actin为内参照物，进行琼脂糖凝胶电泳，最后用Band—scan扫描系统测定各产物密度值，用凝胶自动成像仪拍照。结果在汉防己甲素的作用下，成纤维细胞的MMP-1 mRNA表达增加。结论汉防己甲素可使增生性瘢痕来源的成纤维细胞的MMP-1的mRNA转录增加。从而增加了MMP-1的合成，因而可能对增生性瘢痕具有预防和治疗作用。     

汉防己甲素、川芎嗪和苦杏仁甙对人肾成纤维细胞的影响

目的 观察汉防己甲素、川芎嗪和苦杏仁甙３种中药成分对人肾成纤维细胞（ＫＦＢ）的影响。方法 采用ＥＬＩＳＡ法、四甲基偶氮唑蓝（ＭＴＴ）法、流式细胞仪、免疫组织化学法分别检测汉防己甲素、川芎嗪、苦杏仁甙对人ＫＦＢ分泌的Ｉ型胶原酶活性、人ＫＦＢ增殖、凋亡、Ｉ型胶原表达的影响。结果 汉防己甲素、川芎嗪、苦杏仁甙３种中药提取成分在各自的最佳浓度范围和作用时间内均能提高人胚ＫＦＢ分泌的Ｉ型胶原酶活性、抑制人胎     

汉防己甲素防护环孢素A肾毒性的实验研究

本文观察了中药钙拮抗剂汉防己甲素对大鼠环孢素A肾毒性（CsA－NT）的防护作用。雄性Wistar大鼠用CsA50mg·kg－1／d灌胃7天导致急性CsA－NT，表现为血清肌酐（Cr）升高34．8％，内生肌酐清除率（Ccr）下降39．4％，组织学检查发现近曲小管大片空泡变性。同时，肾皮质丙二醛（MDA）含量显著升高，超氧化物歧化酶（SOD）活性显著下降。大鼠每日用CsA灌胃前30分钟，给予汉防己甲素10mg／kg腹腔注射，能使上述指标明显改善，组织学损伤也明显减轻。实验结果提示汉防己甲素能从功能和形态上防护大鼠急性CsA－NT。     

汉防己甲素对肾小球硬化大鼠肾单位的影响

目的 :探讨中药汉防己甲素 (Tetrandrine)对单侧肾切除加两次注射阿霉素肾小球硬化大鼠肾单位的作用。方法 :将实验动物分为 :正常对照组、汉防己甲素治疗组、氨氯地平治疗对照组和模型组 ,于第 12周留取尿标本。肾组织病理学检查、图像分析肾单位变化 ,用Northernblot杂交检测TGF - βmRNA表达。 结果 :汉防己甲素治疗组和氨氯地平治疗对照组肾小球ECM明显减少 (肾小球ECM /GA为 0 .2 4± 0 .0 2、0 .2 9± 0 .0 1,比模型组 0 .39± 0 .0 2 ,(P <0 0 5 ) ,肾病理损伤减轻 ,肾皮质TGF - βmRNA表达下调 ,汉防己甲组和模型组肾小管上皮细胞高度无明显差异 (P <0 0 5 )。结论 :汉防己甲素治疗机理为通过下调肾皮质TGF - βmRNA表达参与了延缓肾小球硬化的机理。     

汉防己甲素对大鼠急性脊髓损伤的作用及意义

目的：探讨汉防己甲素（Tet）对急性脊髓损伤（ASCI）的保护作用及作用机制。方法：81只大鼠随机分为三组：生盐水对照组（NS组）、汉防己甲素治疗组（Tet组）和甲基强松龙治疗组（MP组），每组27只，用加速型Allen′s打击法制成脊髓急性损伤模型，监测大鼠ASCI后及用药后的血压变化，测定损伤区脊髓Ca^2 、MDA含量；采用氢清除法测定脊髓伤区血流量（SCBF）的变化；连续观测6周ASCI后运动功能评分情况，ASCI后4h,8h、6周取伤区标本进行组织病理检查，结果：ASCI后10s各组动物的MABP显著升高，1min内迅速降至正常水平，之后Tet组舒张压，平均动脉明显降低（P＜0．05），而收缩压降低不明显（P＞0．05）；ASCI后SCBF下降，但Tet组和MP组的SCBF较NS组高（P＜0．05）；损伤区Ca^2 及MDA含量Tet组和MP组均较NS组低，神经功能评分均较NS组高。结论：Tet能改善微循环，防止SCBF的减少；抗脂质过氧化损伤，减少脂质过氧化物MDA的生成；减轻Ca^2 的局部积聚，防止钙超载，阻断继发性损伤的链式反应，减轻组织继发性损伤。对实验性急性脊髓损伤有保护作用。     

汉防己甲素对肾病综合征大鼠肾小球细胞外基质的作用

目的探讨中药汉防己甲素(Tetrandrine)对单侧肾切除加两次注射阿霉素肾病综合征大鼠肾小球细胞外基质(ECM)的作用.方法将实验动物分为(1)正常对照组、(2)汉防己甲素治疗组、(3)氨氯地平治疗对照组和(4)模型组,于第12周留取尿标本,肾组织病理学检查和图像分析肾小球细胞外基质变化,计算ECM/肾小球面积(GA).结果汉防己甲素治疗组和氨氯地平组肾小球ECM均明显减少(ECM/GA为0.24±0.02、0.29±0.01比(4)组0.39±0.02)(P＜0.05)、球囊粘连减轻.结论汉防己甲素能减少肾病综合征大鼠肾小球ECM沉积,延缓肾小球硬化.     

Effect of laser irradiation on collagen production by fibroblasts in vitro

The effects of the low power laser irradiation (Space Mix 5 Mid Laser) on the 3H-proline incorporation in the collagenic proteins produced by two lines of normal human fibroblasts in vitro were studied. The 3H-thymidine incorporation in cultures of control and irradiated fibroblasts of the same lines was also evaluated. The obtained results show that in the experimental conditions considered, laser irradiation may have a positive effect on the production of collagen by the fibroblasts in vitro. This effect is dependent on the time of exposure, the frequency of infra-red impulses and the age of subjects from which fibroblasts were obtained. The laser irradiation in the same experimental conditions does not affect the incorporation of 3H-thymidine in the fibroblasts in vitro. Therefore, the observed increase of collagen production is not dependent on an increase of cellular population.     

MMP9 production by human monocyte-derived macrophages is decreased on polymerized type I collagen

The production of matrix metalloproteinases (MMPs), such as MMP9, by macrophages may be a critical factor in the rupture of unstable atherosclerotic plaques and aortic aneurysms. Therefore, we studied the role of matrix and soluble cytokines in the regulation of monocyte/macrophage expression of MMP9. Although freshly isolated monocytes synthesize little MMP9, cells cultured on tissue-culture plastic differentiate into macrophages and synthesize maximal amounts of MMP9. Differentiated macrophages cultured on plastic are unresponsive to further stimulation by interleukin 1beta, tumor necrosis factor alpha, or platelet-derived growth factor BB. In contrast, monocytes cultured on polymerized collagen synthesize much less MMP9 than cells cultured on plastic and demonstrate a more than three-fold increase in MMP9 synthesis in response to interleukin 1beta, tumor necrosis factor alpha, and platelet-derived growth factor BB. To determine whether the physical state of the collagen was critical for the decrease in basal synthesis of MMP9, monocytes were cultured in suspension for 5 days to allow differentiation and then seeded onto monomer or polymerized collagen. Synthesis of MMP9 was significantly decreased in cells on polymerized collagen and modestly increased in macrophages seeded on monomer collagen. These results suggest that MMP9 synthesis by macrophages in the vessel wall may be under negative control by native, polymerized collagen and that disruption of this native conformation could increase MMP9 production. In addition, cells in contact with the collagen matrix are potentially more responsive to soluble mediators such as platelet-derived growth factor, interleukin 1beta, and tumor necrosis factor alpha.     

粉防己碱对瘢痕成纤维细胞DNA和胶原合成的影响

目的探讨粉防己碱对瘢痕成纤维细胞DNA和胶原合成的影响.方法以体外培养的人瘢痕成纤维细胞为实验模型,将粉防己碱(5～80mg/L)加入细胞培养液中进行干预并与对照组比较,用3H-TdR掺入法和3H-脯氨酸掺入法分别测定各组瘢痕成纤维细胞3H-TdR掺入值与3H-脯氨酸掺入值,以反映其DNA和胶原合成水平的变化.结果粉防己碱预处理浓度由5mg/L升至80mg/L时(1)瘢痕成纤维细胞3H-TdR掺入值(min-1)由对照组的1740±165分别降至1162±226、412±82,抑制率达76.32%,组间差异有非常显著意义(P＜0.01);(2)瘢痕成纤维细胞3H-脯氨酸掺入值(min-1)由对照组的1126±193分别降至535±141、341±89,抑制率达69.71%,组间差异有非常显著意义(P＜0.01).结论粉防己碱对体外培养的瘢痕成纤维细胞DNA和胶原合成具有抑制作用,呈剂量依赖关系,具有防治增生性瘢痕的应用前景.     

Normal human fibroblasts enable melanoma cells to induce angiogenesis in type I collagen

BACKGROUND: We previously reported that fibroblasts induce human microvascular endothelial cells (HMVECs) to differentiate from monolayer to capillarylike morphology. We now test the hypothesis that fibroblasts modulate angiogenesis in melanoma cells. METHODS: We tested 12 human melanoma lines (2 radial growth phase (RGP), 3 vertical growth phase (VGP), and 7 metastatic (MM)) for ability to induce HMVECs to invade/migrate into collagen and form capillarylike networks. HMVEC monolayers were overlaid with 3-dimensional collagen gels embedded with melanoma cells alone (M), fibroblasts alone (F), or a 1:1 mixture of the 2 cells (M+F). After 5 days, gels were removed, fixed, and HMVEC networks were quantified by von Willebrand's factor (vWF) immunofluorescence. The influence of soluble factors on HMVEC invasion/migration into collagen was assessed with the use of acellular 3-D collagen gels overlaid on HMVEC monolayers, cultured with conditioned media (CM) derived from monolayers of M, F, or M+F. Angiogenic growth factors involved in the observed invasion/migration were identified with the use of a RayBio Cytokine Antibody Array (RayBiotech, Norcross, Ga). RESULTS: Cell line-specific variability in melanoma-supported angiogenesis was observed only when in combination with fibroblasts (analysis of variance [ANOVA], P < .01). Melanoma plus fibroblasts uniformly resulted in a significantly higher angiogenic response than melanoma alone (P < .05). One vertical growth phase and one metastatic melanoma line, while weakly angiogenic alone, induced significantly higher angiogenesis than either fibroblast or melanoma alone (P < .05) when combined with fibroblasts. CM from M or M+F induced significantly less HMVEC invasion/migration into collagen than CM from fibroblasts alone. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 were identified as significantly elevated in the media derived from M+F cultures, compared with either cell type alone. CONCLUSION: To our knowledge, this is the first report demonstrating that melanoma-supported angiogenesis in collagen is more significantly influenced by normal skin-derived fibroblasts than by the intrinsic biology of the melanoma cell type. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 are implicated as potential paracrine factors regulating this observed effect.     

Effects of different setting of diode laser on the mRNA expression of growth factors and type I collagen of human gingival fibroblasts

The aim of this study was to analyze the influence of non-surgical applications of diode laser (940 nm) on the cell proliferation and mRNA expressions of type I collagen and growth factors in human gingival fibroblasts (GF). Gingival fibroblasts were isolated from human gingival connective tissue of systemically healthy individuals. Cells were treated with different laser parameters as follows; (1) Infected pocket setting (power: 2 W, pulse interval: 1 ms, pulse length: 1 ms, 20 s/cm²); (2) Perio-pocket setting (power: 1.5 W, pulse interval: 20 ms, pulse length: 20 ms, 20 s/cm²); and (3) Biostimulation setting (power: 0.3 W in continuous wave, 20 s/cm²). Proliferation of GF was evaluated after different laser applications using a real-time cell analyzer. Total RNA was isolated on day 2 and cDNA synthesis was performed. Type I collagen, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β) mRNA expressions were determined with quantitative RT-PCR. In a proliferation experiment, no significant differences were observed in the different laser applications when compared to the control group. Statistically significant increases in IGF, VEGF, and TGF-β mRNA expressions were noted in the laser groups when compared to the untreated control group (p < 0.05). A significant increase in collagen type I mRNA expression was noted in only biostimulation set-up of diode laser (p < 0.05). The results of this study demonstrate that non-surgical laser applications modulate behavior of gingival fibroblasts inducing growth factors mRNA expressions and these applications can be used to improve periodontal wound healing.     

Mechanical loading regulates protease production by fibroblasts in three-dimensional collagen substrates

Mechanical loading is important in tissue formation and remodelling, notably in wound repair. The aim of this study was to measure the effects of controlled loading on the release of extracellular matrix protease activities by fibroblasts. Fibroblast populated collagen lattices were subjected to external cyclical loads through a computer controlled unit incorporated into a culture system, a tensioning-Culture Force Monitor. Cyclical loading was compared to untensioned and statically loaded gels (tethered endogenous contraction). Overall changes in a range of protease activities were monitored (chiefly by zymography) as measures of the cyto-mechanical response to these loads. Under static load, 2.5- and 13-fold more matrix metalloproteinase-2 was produced than matrix metalloproteinase-9, at 24 and 48 hours. Total matrix metalloproteinase-9 increased 37 fold on cyclical loading. Total matrix metalloproteinase-3 and urokinase plasminogen activator activities were dramatically reduced on cyclical loading while tissue type plasminogen activator activity was increased. Comparison with cell responses on stiffer substrates (collagen sponges) identified similar matrix metalloproteinase responses to load, but at much reduced levels (4-6 fold matrix metalloproteinase-9 stimulation on loading), showing the importance of matrix compliance to this mechano-response. In conclusion, physiological mechanical loading of fibroblasts in three dimensional collagen lattices elicited complex and substantial changes in matrix modifying proteases. These changes suggest that cells switch between expression of comparable protease activities mainly influencing cell-matrix interactions associated with migration or more generalized extracellular matrix remodelling.     

Interaction of ascorbic acid and glucose on production of collagen and proteoglycan by fibroblasts

Collagen and proteoglycans are two major constituents of the extracellular matrix, and their abnormalities have been incriminated in the pathogenesis of diabetic complications. A decrease of plasma ascorbic acid has been reported in diabetes and thus may play a role in the collagen and proteoglycan abnormalities in diabetes. Ascorbic acid and glucose share structural similarity, and their metabolism may interact at the level of membrane transport and cellular action. In this study, we used a fibroblast culture system to explore this possibility. Ascorbic acid increased collagen and proteoglycan both in the culture medium and the cell layer. This stimulatory action of ascorbic acid was inhibited by the presence of glucose at a concentration of 25 mM. The effect of high glucose concentration was not mediated by inhibition of ascorbic acid uptake by fibroblasts. Insulin is able to abolish this inhibitory action of glucose on collagen production, but the precise mechanism is unclear. These results show that the high glucose concentration in diabetes can impair the action of ascorbic acid at the cellular level. This may further accentuate the problem of decreased availability of this vitamin as a result of its low plasma concentration.