改构型酸性成纤维细胞生长因子对细胞增殖活性的影响

目的:研究aFGF和MaFGF对正常的肾小管上皮细胞及胃癌细胞增殖的影响。方法:用不同浓度的aFGF和MaFGF分别作用于肾小管上皮细胞及胃癌细胞,48h后采用WST-8法测定aFGF和MaFGF对两种细胞的促增殖活性。结果:在各浓度下,MaFGF组对肾小管上皮细胞和胃癌细胞的促增殖作用都显著低于aFGF组。结论:MaFGF对肾小管上皮细胞及胃癌细胞的促分裂活性较aFGF明显下降。     

胸腺细胞和细胞器水平的线粒体膜电势研究

以地塞米松(DEX)诱导小鼠胸腺细胞凋亡;利用PI和AnneXin V/PI流式细胞术分别检测细胞晚期和早期凋亡;利用JC-1和DiOC_6(3)/PI在细胞水平检测凋亡中线粒体膜电势(△ψm)变化：抽提线粒体,利用JC-1直接染色技术检测现存线粒体△ψm情况。实验结果显示,DEX显著诱导胸腺细胞早期和晚期凋亡,凋亡细胞主要来自G_0/G_1期;细胞水平可见DEX介导与△ψm相关的J-aggregate和DiOC_6(3)可染性降低,同时介导线粒体数量显著降低,6h细胞膜完整性无显著变化：单纯线粒体检测结果显示,多数线粒体维持正常△ψm。提示,DEX介导胸腺细胞凋亡中线粒体数量降低,现存线粒体多保持着正常△ψm以维持凋亡过程细胞能量供给。     

β-蜕皮激素对小鼠胸腺退化的保护作用

目的通过腹腔注射地塞米松(Dex)构造小鼠胸腺退化模型,探究β-蜕皮激素(Ecd)对小鼠胸腺退化的保护作用。方法小鼠随机分为空白对照组、胸腺退化模型组以及3个给药组(Ecd 50 mg/kg、75 mg/kg和100 mg/kg)。腹腔注射给药7 d,于采集胸腺前15 h给予模型组和给药组腹腔注射Dex造模。取小鼠胸腺组织,计算胸腺指数,HE染色切片观察胸腺组织变化,免疫组织化学检测胸腺组织中相关凋亡基因Bcl-2和Bax蛋白的表达。结论 Ecd可提高胸腺指数,保护胸腺组织结构,有效提高Bcl-2的表达并降低Bax的表达,从而提高Bcl-2/Bax比率,抑制胸腺细胞凋亡,提高胸腺细胞存活率。     

地塞米松对TRAIL双向诱导猪肾成纤维细胞增殖与凋亡作用的影响

采用MTT掺入法检测细胞活率变化,分别筛选出诱导细胞增殖与凋亡的药物浓度;半定量PCR法检测不同浓度下地塞米松对骨保护素(Osteoprotegerin,OPG)和核因子κB受体激活剂受体配体(Ligand of receptor activator of nuclear factor kappa B,RANKL)基因在mRNA水平上的调控作用;流式细胞术检测细胞周期分布和凋亡率变化,显微镜观察FRSs存活、增殖和凋亡变化.经MTT检测和浓度筛选,发现TRAIL在0.01-5 mg/L浓度范围内诱导FRSs增殖,浓度增加到10 mg/L出现凋亡趋势.地塞米松可协同TRAIL双向诱导FRSs增殖或凋亡,有效浓度约10-6-10-10 mol/L.细胞周期分析与凋亡率检测结果表明RAIL诱导FRSs增殖峰浓度为5 mg/L.TRAIL(5 mg/L)与地塞米松协同作用,细胞增殖指数(Prl.)较TRAIL5 mg/L组上升2.49%(P<0.05);TRAIL 10 mg/L组与空白对照组比较细胞增殖指数和凋亡率无显著变化,TRAIL 10 mg/L与地塞米松协同作用G0/G1期比例较TRAIL10 mg/L组增加2.36%(P<0.05),凋亡率较之上升6.79%(P<0.01).地塞米松作用下,OPG基因mRNA水平表现下调,RANKL基因则表现上调,二者均表现为剂量依赖型.综上所述,TRAIL对FRSs呈诱导增殖或凋亡的双向调控作用,并呈剂量依赖型.地塞米松可协同TRAIL对FRSs作用,该现象与TRAIL和地塞米松诱导FRSs细胞周期的改变以及地塞米松对OPG/RANK/RANKL信号通路的OPG和RANKL基因的表达调控相关.     

FGF-1改构体对小鼠脾细胞增殖与凋亡的影响

目的:主要探讨非促分裂改构人酸性成纤维细胞生长因子(MrhFGF-1)对balb／c小鼠脾细胞增殖和凋亡的影响。方法:采用3H-TdR掺入的方法检测MrhFGF-1同野生型hFGF-1相比对脾细胞增殖的影响,实验组分为(1)对照组;(2)FGF-1处理组;(3)ConA处理组;(4)FGF-1+ConA处理组。用流式细胞仪定量分析MrhFGF-1对脾细胞的凋亡保护作用,(1)对照组;(2)DEX处理组;(3)DEX+FGF-1(hFGF-1、rhFGF-1、MrhFGF-1)处理组。结果表明,利用DNA重组技术构建的一种非促分裂FGF-1突变体MrhFGF-1和野生型FGF-1相比,对脾细胞的促细胞增殖能力明显降低,但其对细胞凋亡保护作用的影响并无显著性变化,说明FGF-1的促细胞增殖能力和细胞凋亡保护信号通路并非由同一信号通路来实现的。     

甘青虎耳草乙酸乙酯提取物对小鼠肝癌细胞及原位移植瘤的抑制作用

目的探讨甘青虎耳草乙酸乙酯提取物(Et OAc extract from Saxifraga taugutica,EST)对小鼠肝癌细胞及原位移植瘤的抑制作用。方法体外实验用不同浓度EST处理H22细胞48 h后,AO/EB双荧光染色,激光共聚焦显微镜观察细胞形态变化;用核酸电泳和流式细胞术检测H22细胞的凋亡情况。建立小鼠肝癌原位移植瘤模型进行体内实验,对比EST低、中、高剂量组对荷瘤小鼠抑瘤率、T淋巴细胞增殖能力以及肝病理变化的影响。结果 EST处理后H22细胞出现凋亡形态学变化,且与浓度正相关;核酸电泳呈凋亡特征性梯状带;流式细胞分析,500μg/m L处理组凋亡率达31%;体内试验显示,EST低、中、高剂量的抑瘤率分别为33.0%、46.7%、64.3%;显著提高小鼠的胸腺指数、T淋巴细胞增殖能力(P 0.05);组织学观察证明EST各剂量组癌细胞有不同程度的生长抑制,形态固缩、向周围组织浸润减少。结论 EST可增强小鼠免疫功能和诱导肿瘤细胞凋亡,具明显的抗肿瘤作用,与剂量正相关。     

糖皮质激素抑制结肠癌细胞增殖和侵袭的作用机制

本研究目的是为了证实地塞米松对结肠癌LoVo细胞增殖的抑制作用,并阐明其中的分子机制。LoVo细胞经不同浓度梯度地塞米松干预,再加入TGF-β1受体抑制剂SB431542阻断TGF-β1信号传导途径,通过MTS分析各组细胞增殖情况,借助Hoechst 33342和Annexin V/PI染色法检测细胞凋亡率;结合Western blotting对TGF-β1、Smad2和caspase-3蛋白表达情况的检测结果,分析地塞米松诱导结肠癌LoVo细胞凋亡的作用机理。LoVo细胞在1.0 mmol/L和10.0 mmol/L地塞米松干预48 h后,细胞增殖率与对照组相比分别降低32%(p<0.01)和47%(p<0.001),2组细胞凋亡率分别为28%和36%(p<0.001)。Western blotting结果显示,与对照组相比,地塞米松以浓度依赖性方式显著上调LoVo细胞TGF-β1、Smad2和Cleavedcaspase-3蛋白水平(p<0.01),而TGF-β1受体抑制剂SB431542明显下调TGF-β1、Smad2和Cleaved-capase-3蛋白表达(p<0.05)。流式细胞术检测结果表明,SB431542+地塞米松干预组与地塞米松处理组LoVo细胞凋亡率分别为8%和23%(p<0.001)。地塞米松可显著诱导LoVo细胞凋亡,而SB431542能够挽救这一过程,这表明,地塞米松通过TGF-β1/Smad2通路诱导LoVo细胞凋亡。     

高原低氧免疫损伤及其干预措施的研究

目的:探讨高原低氧损伤免疫系统的特征及其可能机制,研究高原低氧免疫损伤的干预措施。方法:测定低氧暴露不同时间小鼠免疫器官指数、外周血和免疫器官T淋巴细胞亚群的变化;观察小鼠免疫器官淋巴细胞凋亡率及小鼠肺脏和肾脏病理学改变。采用预防给药方式,研究中药组方对低氧免疫损伤小鼠的干预作用。结果:①模拟海拔8000m低氧暴露8h后,小鼠胸腺CD4+CD8+细胞数显著下降,CD4+CD8-、CD4-CD8+细胞数显著增加(P0.01);低氧暴露3d后,外周血CD4+细胞明显减少(P0.05),CD4+/CD8+比值显著降低(P0.05),胸腺CD4+CD8+细胞数进一步下降,CD4+CD8-、CD4-CD8+细胞数进一步增加,小鼠脾脏、胸腺淋巴细胞晚期凋亡和坏死率均显著增加(P0.05);低氧暴露6d后,小鼠脾指数显著性增加(P0.01);胸腺指数显著性降低(P0.01),脾CD4+、CD8+细胞数显著降低(P0.01),脾脏和胸腺淋巴细胞晚期凋亡率和坏死率进一步增加(P0.01),活细胞率显著降低(P0.01),脾脏淋巴细胞早期凋亡率显著增加(P0.01)。整个低氧暴露过程中外周血CD8+无显著性变化。②新复方党参、香杞多糖、二者联合应用均能显著增加低氧免疫损伤小鼠外周血CD3+、CD4+、脾脏CD4+的细胞水平(P0.01,P0.05),对脾脏CD8+细胞水平没有显著影响。香杞多糖及其与新复方党参联合应用均能进一步降低胸腺CD4+CD8+,进一步增加CD4+CD8-的细胞水平(P0.01),未见对CD4-CD8+细胞水平的影响;新复方党参对低氧免疫损伤小鼠胸腺没有显著性影响。结论:模拟海拔8000m低氧暴露后小鼠外周发挥免疫作用的淋巴细胞数减少可能与低氧暴露早期淋巴细胞凋亡率和坏死率增加和肺脏淋巴细胞分布增多有关。新复方党参和香杞多糖作为低氧免疫损伤干预措施,具有一定发展前景。     

虎杖苷通过激活TrkA抑制芬太尼诱导的海马神经元细胞凋亡

本文旨在研究虎杖苷对芬太尼麻醉后的神经保护作用及其潜在机制。分离小鼠海马神经元进行培养,并分别使用芬太尼,虎杖苷+芬太尼,虎杖苷+芬太尼+IgG,虎杖苷+芬太尼+MNAC13(TrkA封闭抗体)进行处理,然后通过TUNNEL检测细胞凋亡,通过免疫组织化学检测Caspase-9和原肌球蛋白受体激酶(Trk)受体的表达,并通过莫里斯水迷宫试验检测芬太尼,芬太尼和虎杖苷联合处理后小鼠的学习和记忆能力。结果显示,虎杖苷预处理后可以显著增加TrkA磷酸化并抑制细胞凋亡,而MNAC13可以显著抑制TrkA磷酸化并诱导细胞凋亡;此外,与对照组小鼠相比,芬太尼组小鼠学习和记忆能力显著下降,而虎杖苷处理可以显著提高小鼠学习和记忆能力。上述结果表明,虎杖苷可以抑制芬太尼诱导的海马神经元细胞凋亡,发挥神经保护作用,改善小鼠学习和记忆能力。     

三叶青黄酮对荷Lewis肺癌小鼠免疫功能及肿瘤组织凋亡相关蛋白的影响

探索三叶青黄酮对荷Lewis肺癌小鼠免疫功能及肿瘤组织凋亡相关蛋白的影响。将60只小鼠随机分为对照组、模型组、三叶青黄酮高、中、低剂量组(100、50、25 mg/mL)各10只。连续干预14天后,检测各组胸腺指数、脾脏指数、移植瘤组织凋亡及凋亡相关蛋白表达,检测外周血调节性T细胞(Treg)比例。结果显示,与模型组比,三叶青黄酮高中剂量组胸腺指数、脾脏指数和移植瘤组织凋亡率升高(P<0.05),凋亡相关蛋白表达升高(P<0.05),外周血CD4+CD25+Foxp3+Treg细胞所占比例降低(P<0.05)。综上,三叶青黄酮可降低荷Lewis肺癌小鼠Treg细胞比例,提高免疫功能,诱导移植瘤组织凋亡。     

Heparin modulation of the neurotropic effects of acidic and basic fibroblast growth factors and nerve growth factor on PC12 cells

Nerve growth factor (NGF) and acidic or basic fibroblast growth factor (aFGF and bFGF, respectively) induce neurite outgrowth from the rat pheochromocytoma cell line, PC12. The neurites induced by these three factors are stable for up to a month in cell culture in the continued presence of any of the above growth factors. bFGF (ED50 = 30 pg/ml) is 800 fold more potent in stimulating neurite outgrowth than aFGF (ED50 = 25 ng/ml) and 260 fold more potent than NGF (ED50 = 8 ng/ml). While the neurotropic activities of aFGF and NGF are potentiated by heparin, that of bFGF is both partially inhibited or stimulated, depending upon the concentration of bFGF. Radioreceptor binding experiments show that aFGF and bFGF bind to a common binding site on the PC12 cell surface. Affinity labeling studies demonstrate a single receptor with an apparent molecular weight of 145,000 daltons, which corresponds to the high molecular weight receptor identified in BHK-21 cells. NGF does not appear to compete with aFGF or bFGF for binding to the receptor. Heparin blocked the binding of bFGF to the receptor but had only a small inhibitory effect on the binding of aFGF to the receptor. Thus, it appears that heparin inhibition of the neurotropic effects of bFGF occurs, at least in part, by impairing the interaction of bFGF with the receptor, while having little effect on that of aFGF. The stimulatory effects of heparin on the neurotropic activity of aFGF, bFGF, and NGF may occur through a site not associated with the respective cellular receptor for the growth factors.     

Stabilization by heparin of acidic fibroblast growth factor mitogenicity for human endothelial cells in vitro

The effects of heparin and other glycosaminoglycans (GAGs) on the mitogenicity and stability of acidic fibroblast growth factor (aFGF) were studied. The mitogenic activity of aFGF was assayed utilizing cultured adult human endothelial cells (AHECs) isolated from iliac arteries and veins as target cells. In most experiments, aFGF purified from bovine brain was employed; in some experiments recombinant bovine aFGF was used and qualitatively similar results were obtained. In the presence of heparin, bovine aFGF at doses between 0.5 and 1.0 ng/ml (30-60 pM) elicited half the maximum AHEC growth over a 4-day period depending on the cell line tested; in the absence of heparin, significant growth was not observed at aFGF concentrations less than 10-20 ng/ml. This effect of heparin was dose-dependent over the range 0.1-10 micrograms/ml (half-maximum dose, 2 micrograms/ml). The mitogenic activity of bovine aFGF for AHECs decreased by 50% after preincubation in culture medium without cells at 37 degrees C for 2 1/2 to 3 hours. In contrast, the mitogenic activity of bovine aFGF preincubated in the presence of heparin-containing culture medium without cells was dramatically stabilized (half-life 24-29 hours). These effects also were observed in serum-free medium. Several GAGs structurally related to heparin such as chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and hyaluronic acid neither potentiated nor stabilized aFGF mitogenic activity. However, heparan sulfate from bovine lung was found to be nearly as active as heparin in both these effects. These data suggest that the binding and stabilization of mitogens by extracellular and tissue-associated heparan sulfates might play important roles in the regulation of AHEC growth.     

Developmental Changes in Distribution of Acidic Fibroblast Growth Factor in Rat Brain Evaluated by a Sensitive Two-Site Enzyme Immunoassay

We developed a sensitive two-site enzyme immunoassay (EIA) system for acidic fibroblast growth factor (aFGF), using a polyclonal antibody raised in rats. This assay is based on the sandwiching of the antigen between anti-aFGF antibody immunoglobulin G (IgG) coated on plates and biotinylated anti-aFGF antibody IgG; the detection of biotinylated IgG was performed by enzyme reaction of streptavidin-conjugated beta-D-galactosidase (beta-D-galactoside hydrolase; EC 3.2.1.23). Our system was specific for aFGF, because basic fibroblast growth factor, which shares a 55% homology of amino acid sequence with aFGF, hardly cross-reacted at all. The sensitivity of this system (0.2 ng/ml) enabled us to quantify endogenous immunoreactive aFGF in the CNS. Using this two-site EIA system, we examined the levels of aFGF in various regions of rat brain and their developmental changes. At the early stage of neonatal development, i.e., 2 days after birth, all brain regions registered low aFGF levels (less than 10 ng/g tissue). However, at the young adult stage (21- to 49-day-old animals), an extremely high level of aFGF (75-90 ng/g tissue) was found in the ponsmedulla; relatively high levels (30-40 ng/g tissue) were found in the diencephalon and mesencephalon; and comparatively low aFGF levels (5-15 ng/g tissue) were found in various other brain regions such as the frontal cortex, piriform cortex, hippocampus, olfactory bulb, cerebellum, and striatum. This marked change in the regional distribution of aFGF in the rat brain during postnatal development from 2 to 21 days after birth suggests that this factor plays a significant role in the brain during this period.     

Basic and acidic fibroblast growth factors modulate the epidermal growth factor receptor by a protein kinase C-independent pathway

Human acidic and basic fibroblast growth factors (aFGF and bFGF) inhibit epidermal growth factor (EGF) receptor binding in mouse Swiss 3T3 cells. Scatchard analysis indicates that aFGF and bFGF cause a decrease in the high affinity EGF receptor population, similar to that observed for activators of protein kinase C such as phorbol esters, platelet-derived growth factor (PDGF) and bombesin. However, unlike phorbol esters, aFGF and bFGF inhibit EGF binding in protein kinase C-deficient cells. The time course and dose response of inhibition of EGF binding by both aFGF and bFGF are very similar, with an ID50 of approximately 0.10 ng/ml. In contrast to bombesin but like PDGF, neither aFGF nor bFGF act on the EGF receptor through a pertussis toxin-sensitive G protein. These results indicate that both acidic and basic FGF depress high affinity EGF binding in Swiss 3T3 cells with similar potency through a protein kinase C/Gi-independent pathway.     

The reversal of hyperglycemia after transplantation of mouse embryonic stem cells induced into early hepatocyte-like cells in streptozotocin-induced diabetic mice

Cellular replacement therapy is a potential therapeutic strategy for diabetes. In this study, we investigated the effect of transplantation of induced mouse embryonic stem cells (mESCs) into endoderm and early hepatocyte-like cells in streptozotocin (STZ)-diabetic mice. After embryoid body (EB) formation from mESC, the EBs were cultured in the presence of dexamethasone (DEX) and insulin for 4 days then was added acidic fibroblast growth factor (aFGF), hepatocyte growth factor (HGF) and oncostatin M (OSM) for 10 days, respectively. Blood glucose levels, intraperitoneal glucose tolerance (IGT) test and islet histology were assessed. The result revealed that transplantation of induced mESCs into early hepatocyte-like cells could repair pancreatic islets of control group. Blood glucose levels and intraperitoneal glucose tolerance test were significantly improved in test group compared to control group. Furthermore, there was significant increase in the number of islets in test group compared to control group. The findings declare that induced mESCs into endoderm and early hepatocyte-like cells, are appropriate candidate for regenerative therapy of pancreatic islets in type I diabetes.     

Acidic fibroblast growth factor stimulates adrenal chromaffin cells to proliferate and to extend neurites, but is not a long-term survival factor

Acidic fibroblast growth factor (aFGF) is a heparin-binding polypeptide that is a mitogen for endothelial cells and glial cells, as well as a differentiation factor for PC12 cells and certain neurons. We show here that aFGF is as potent as nerve growth factor (NGF) in stimulating both neuritic outgrowth and proliferation in adrenal chromaffin cells from young rats, but it fails to support long-term survival. Heparin strongly potentiates aFGF-dependent neuritic outgrowth but not aFGF-dependent proliferation. As is the case with NGF, phorbol myristate acetate depresses aFGF-induced cell division and increases the outgrowth of neurites. On the other hand, dexamethasone antagonizes neuritic outgrowth elicited by both NGF and aFGF but inhibits only proliferation induced by NGF. The effects of basic FGF (bFGF) are similar but not identical to those of aFGF. Thus the regulatory pathways controlled by aFGF, bFGF, and NGF are partially distinct.     

酸性成纤维细胞生长因子的促细胞增殖作用和对实验秃毛大鼠的疗效研究

利用大肠杆菌菌株表达出高纯度的酸性成纤维细胞生长因子,并对其促3T3细胞的增殖作用和对实验秃毛大鼠的疗效进行了研究。结果表明,在体外实验中,1.95 ng/ml~1000ng/ml的aFGF溶液可以促进balb/c 3T3细胞的分裂增殖,与PBS组比较具有显著性差异(P<0.05,P<0.01);在体内实验中,在第7天,4μg/ml aFGF组大鼠毛发长度变长,与模型对照组比较有显著性差异(P<0.05);在第14天,2μg/ml 和4μg/ml aFGF组大鼠毛发长度继续变长,与模型对照组比较有显著性差异(P<0.05)。病理检查结果显示aFGF可以促进实验秃毛大鼠的毛囊数目增多,毛囊无萎缩变小现象,血管无充血现象,基本恢复到正常水平。由此得出aFGF可以促进3T3细胞的分裂增殖,以及促进实验秃毛大鼠的毛发生长。aFGF具有很好的开发前景。