运用siRNA建立稳定低表达IGF1R基因的肝癌细胞株的实验研究

目的运用siRNA技术在肝癌细胞株中建立稳定低表达人胰岛素样生长因子1类受体(IGF1R)基因的细胞株。方法构建包含封闭IGF1R基因的真核表达载体pSUPER-IGF1R-siRNA,转染SMMC7721和Hep3B细胞,G418筛选表达稳定的细胞株。通过RT-PCR、Western-blot分析与鉴定IGF1R mRNA和蛋白及cyclin D1、cyclinB1蛋白的表达,并绘制细胞生长曲线。结果在SMMC7721和Hep3B细胞中成功建立低表达IGF1R基因的细胞株,其生长明显减慢(P<0.05),且其cyclinD1表达亦明显下降(P<0.05)。结论pSUPER-IGF1R-siRNA能抑制SMMC7721和Hep3B细胞的生长。     

小干扰RNA干扰大鼠神经元Nogo受体mRNA表达的实验研究

目的观察大鼠Nogo受体(NgR)特异性小干扰RNA(small interfering RNA,siRNA)在原代神经元干扰其mRNA表达的效果。方法体外培养大鼠原代皮层和海马细胞,应用阳离子脂质体转染试剂转染4对化学合成的大鼠NgR特异性siRNA,72h后提取细胞总RNA,实时荧光定量RT-PCR检测NgR mRNA表达情况。结果4对siRNA均能够下调靶基因NgR的mRNA表达水平,siNgR199、siNgR562、siNgR772和siNgR964等干扰后,NgR mRNA的表达分别为对照siRNA干扰组的6.5%、62.4%、15.2%和6.86%,与对照siRNA干扰组比较有统计学意义(P<0.05)。结论化学合成的NgR特异性siRNA能够有效的干扰大鼠原代皮层和海马细胞内NgR基因mRNA的表达水平。     

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。     

bFGF和硫糖铝局部应用促扩张术方法研究

目的探讨碱性成纤维细胞生长因子(bFGF)不同剂量、用药方法对扩张组织的影响.方法以白色家猪为实验动物,采用持续恒压扩张技术,观察不同剂量和不同给药时间,bFGF和硫糖铝对扩张组织的影响及组织结构变化.结果扩张术同时每日2次局部应用bFGF 9AU/cm2 硫糖铝100μg/ml,共7 d,浸于明胶海绵上缓慢持续释放对扩张组织的影响最明显,效果最佳,扩张器内实际注液量、扩张所获得的皮肤净增面积明显增加,皮瓣回缩率降低.表皮细胞层数增多,胶原纤维、弹力纤维、成纤维细胞密度和毛细血管密度显著增高.药量增加效果未随之增加.结论持续恒压扩张术时合用bF-GF 9AU/cm2 硫糖铝100μg/ml,每日2次注药浸入明胶海绵持续缓慢释放效果最佳.     

碱性成纤维细胞生长因子在实验性硬脑膜重建中的应用

[目的] 探讨碱性成纤维细胞生长因子( bFGF)在实验性自体筋膜硬脑膜重建中的应用价值.[方法]取 SD大鼠 35只,分成 5组.其中 A组 3只,直接将背部筋膜覆盖在硬膜外, B组 5只,采用自体筋膜硬脑膜重建模型,通过免疫组织化学方法观察这两组大鼠自体筋膜硬脑膜移植的愈合过程及细胞因子 bFGF在其中的表达; C组、 D组、 E组各 9只,分别进行自体筋膜硬脑膜重建, D组加用外源性 bFGF, E组仅用明胶海绵,通过脑脊液漏研究、免疫组织化学方法研究外源性 bFGF对自体筋膜重建硬脑膜愈合过程的影响并用 RT- PCR方法研究内源性 bFGF mRNA的表达情况.[结果] 大鼠自体筋膜移植硬脑膜部位 bFGF表达明显; C、 D、 E组大鼠重建硬脑膜抵抗脑脊液漏压力值( mmH2O)分别为 311± 75, 497± 153, 338± 88,Ⅰ型胶原纤维表达值分别为 4.9± 0.8, 10.9± 1.6, 5.3± 0.9, D组均好于对照组( P< 0.05);内源性 bFGF mRNA的表达没有明显变化.[结论] 在自体筋膜移植硬脑膜的愈合过程中 bFGF可能起了重要的作用;外源性 bFGF处理的大鼠其移植筋膜愈合要好于对照组.     

胰岛素样生长因子-Ⅰ和胰岛素样生长因子结合蛋白-1与妊高征的关系

目的：探讨胰岛素样生长因子 I(IGF-Ⅰ)和胰岛素样生长因子结合蛋白 1(IGFBP—1)与妊高征(PIH)的关系。方法：采用免疫放射法测定50例妊高征孕妇(PIH组)和108例正常孕妇(对照组)的血清 IGF-Ⅰ、IGFBP-1水平，并对其结果进行相关性分析。结果：①妊高征孕妇血清 IGF-Ⅰ水平低于正常孕妇(P<0.05)，且重度妊高征孕妇血清 IGF-Ⅰ水平低于中度和轻度孕妇(P＜0.05，P＜0.01)。②妊高征孕妇血清 IGFBP-1水平高于正常孕妇(P＜0.05)，且重度妊高征孕妇血清 IGFBP-1水平高于中度和轻度孕妇(P＜0.05，P＜0.01)。③两组孕妇血清 IGF-Ⅰ水平与血清 IGFBP-1水平呈负相关(r=-0.386，P＜0.05)。④妊高征孕妇血清 IGF-Ⅰ水平与舒张压呈负相关(r=-0.386，P＜0.05)，血清 IGFBP-1水平与舒张压呈正相关(r=0.632，P＜0.01)。结论：妊高征孕妇血清 IGF-Ⅰ、IGFBP-1水平的高低可反映妊高征的严重程度。     

Relaxin down-regulates renal fibroblast function and promotes matrix remodelling in vitro.

BACKGROUND: Renal fibroblasts are important effector cells in tubulointerstitial fibrosis, with experimental antifibrotic strategies focusing on the functional down-regulation of these cells. Several experimental models of fibrosis have provided evidence for the effectiveness of the polypeptide hormone relaxin as a potential antifibrotic agent. This study was conducted to further elucidate the antifibrotic mechanisms of relaxin on renal fibroblasts in vitro. METHODS: Rat cortical fibroblasts were obtained from outgrowth culture of renal tissue isolated from kidneys 3 days post-unilateral ureteric obstruction and constituted 100% of cells studied. A relaxin radio-receptor assay was used to establish binding of relaxin to renal fibroblasts in vitro. Functional studies then examined the effects of H2 relaxin (0, 1, 10 and 100 ng/ml) on fibroblast kinetics, expression of alpha-smooth muscle actin (alpha-SMA), total collagen synthesis, collagenase production and collagen-I lattice contraction. CTGF mRNA expression was also measured by northern analysis. RESULTS: H2 relaxin bound with high affinity to rat renal fibroblasts, but receptor numbers were low. Consistent with its previously reported bimodal effect, transforming growth factor (TGF-beta 1) reduced fibroblast proliferation, an effect abrogated by H2 relaxin. Fibroblasts exposed to H2 relaxin (100 ng/ml) for 24 h demonstrated decreased immunostaining for alpha-SMA and reduced alpha-SMA protein expression compared with controls. There was a trend for a relaxin-mediated reduction in total collagen synthesis and alpha 1(I) mRNA expression with large dose-related increases in collagenase protein expression being observed. TGF-beta 1-stimulated collagen-I lattice contraction was significantly inhibited following co-incubation with 100 ng/ml relaxin. Incremental doses of H2 relaxin had no significant effect on CTGF mRNA expression. CONCLUSIONS: The findings of this study suggest that the antifibrotic effects of relaxin involve down-regulation of fibroblast activity, increase in collagenase synthesis and restructuring of collagen-I lattices, which are consistent with its known physiological role of matrix remodelling. Although there appears to be an interaction between TGF-beta 1 and H2 relaxin, this does not appear to involve a reduction in CTGF mRNA expression.     

胰岛素样生长因子-1基因转染脂肪间充质干细胞向软骨细胞分化的研究

目的 观察胰岛素样生长因子(IGF)-1基因转染的脂肪间充质干细胞(ADSCs)向软骨细胞分化的效果.方法 原代培养兔ADSCs,免疫荧光法检测细胞表面抗原CD44、CIM9;脂质体介导人IGF-1基因转染兔ADSCs联合低浓度血清培养基向软骨细胞分化诱导,RT-PCR及Western blot方法检测IGF-1的表达,MMT法绘制细胞增殖曲线、甲苯胺蓝染色软骨结节、免疫组织化学检测Ⅱ型胶原的表达.结果 脂肪间充质干细胞CD44、CD109表达阳性,基因转染后细胞IGF-1表达阳性,细胞增殖速度增快,出现软骨结节,Ⅱ型胶原表达增高.结论 从脂肪组织中能够分离出增殖旺盛的ADSCs,IGF-1在ADSCs内获得稳定表达,细胞增殖能力增强,促进其向软骨细胞分化.     

Thyrotrophin-Releasing Hormone Raises Cytosolic Free Calcium Concentration in Human Adenomatous Somatotrophs and Corticotrophs; Comparison with in vivo Responsiveness to Thyrotrophin-Releasing Hormone in Patients with Acromegaly or Cushing's Disease

The effect of thyrotrophin-releasing hormone (TRH) on intracellular free Ca²⁺ concentration, [Ca²⁺)i, was investigated with the fluorescent dye fura-2 in cell suspensions obtained from 13 human growth hormone-secreting adenomas and 6 adrenocorticotrophin-secreting adenomas. Preoperatively, 9 out of 13 acromegalic patients showed a positive growth hormone response to TRH administration while none of the 6 patients with Cushing's disease had a plasma adrenocorticotrophin increase after TRH injection. In all the growth hormone-secreting adenomas the addition of TRH (100 nM) caused a significant rise in [Ca²⁺]i (from a resting level of 133±40 (±SD) to a value of 284±119 nM at 100 nM TRH, n = 42; P<0.001). The transient induced by TRH was found to have a dual origin, one due to Ca²⁺ mobilization from intracellular stores which was maintained in presence of EGTA (3mM) and verapamil (10 μM) and a plateau phase due to Ca²⁺ influx from the extracellular media. Somatostatin (0.1 μM) lowered both resting [Ca²⁺]i and TRH-induced transients. The effect of gonadotrophin-releasing hormone on [Ca²⁺]i was evaluated on cell suspensions obtained from 6 growth hormone-secreting adenomas. Gonadotrophin-releasing hormone (100 nM) caused a marked rise in [Ca²⁺]i (from 179±25 to 283±15nM) on the cell suspension obtained from the only in vivo responsive adenoma while it was ineffective in the remaining 5. Although TRH was ineffective in modifying plasma adrenocorticotrophin levels in all patients with Cushing's disease, in 5 out of 6 tumors the addition of 100 nM TRH caused a significant rise in [Ca²⁺]i (from 102.5 ± 36 to 163±66 nM, n = 22; P < 0.005). However, the effect of TRH on [Ca²⁺]i was significantly lower than that caused by arginine vasopressin, a physiological stimulator of adrenocorticotrophin release ([Ca²⁺]i values; 145±78 nM at 100 nM TRH versus 300±140 at 10 nM arginine vasopressin, n = 15; P<0.05). Moreover, the effect of arginine vasopressin on [Ca²⁺]i was detectable at concentrations as low as 0.1 nM while TRH was effective at concentrations higher than 1 nM. By contrast, gonadotrophin-releasing hormone was ineffective in increasing [Ca²]i in all the adrenocorticotrophin-secreting adenomas studied. Collectively, these data indicate that sensitivity to TRH is present in almost all the growth hormone- and adrenocorticotrophin-secreting adenomas independently of the responsiveness of the individual patients to the peptide.