胰岛素和IGF-I对离体犊牛小肠上皮细胞增殖和吸收功能的影响

本试验选择离体犊牛小肠上皮细胞,以细胞增殖率和葡萄糖吸收率作为细胞生长发育与功能成熟的指标,研究了胰岛素与胰岛素样生长因子-I(IGF-)对细胞生长发育的影响。结果表明,胰岛素浓度为10μg/ml时,明显促进小肠上皮细胞的增殖和对葡萄糖的吸收,浓度达到50μg/ml时则抑制细胞的增殖和吸收(P<0.01)。IGF-I浓度为100ng/ml时,对促进小肠上皮细胞增殖和吸收葡萄糖的作用最强(P<0.01),但100ng/ml、500ng/ml和1000ng/ml三种不同浓度的IGF-I对刺激细胞增殖和提高吸收功能无显著差异(P>0.05)。     

黄芪、何首乌、女贞子、菟丝子混合提取物对体外培养毛囊生长的影响及药理作用研究

目的:通过体内外实验探讨黄芪、何首乌、女贞子、菟丝子混合中药提取物对毛囊增殖的影响作用以及其作用机理。方法:通过体外培养的C57BL/6小鼠毛囊器官模型观察不同浓度中药提取物对毛囊生长的影响;采用MTT法测定不同浓度中药提取物对毛乳头增殖的影响;蛋白免疫印迹法(Western Blot)和ELISA检测中药提取物对毛乳头细胞分泌肝细胞生长因子(HGF)的影响。结果:中药提取物能够刺激体外培养的小鼠毛囊的生长,800μg/mL浓度的促进作用最强;160μg/mL中药提取物对毛乳头细胞的增殖作用最强,与米诺地尔、齐墩果酸阳性对照存在显著性差异(P0.05)。而且,药提取物促进了毛乳头细胞分泌HGF。结论:黄芪、何首乌、女贞子、菟丝子混合中药提取物在促进毛发生长中起到重要作用,促进毛乳头细胞增殖和分泌HGF是促进毛囊生长的可能性药理机制。     

牛脑成纤维细胞生长因子的分离纯化与鉴定

新鲜牛脑组织匀浆液经两步硫酸铵沉淀、CM-Sephadex C50 离子交换层析以及肝素-Sepharose 亲和层析,可得到纯化的酸性和碱性成纤维细胞生长因子(aFGF 和bFGF),分子量分别为13.2kD 和15.2—15.8kD.两种因子均可有效促进3T3细胞的 DNA 合成,ED₅₀分别为15.8ng/ml 和 0.32ng/ml.进一步对 aFGF 的等电点及氨基酸组成做了分析.     

丙磺舒对血小板衍生生长因子-BB诱发大鼠肺动脉平滑肌细胞迁移和增殖的干预作用

目的:研究丙磺舒(PROB)对血小板衍生生长因子-BB(PDGF-BB)诱发大鼠肺动脉平滑肌细胞的迁移与增殖的影响。方法:体外原代培养SD大鼠肺动脉平滑肌细胞(PASMCs),随机分为正常对照组(CON组)、造模组(10 ng/ml PDGF-BB 24 h)、丙磺舒处理组(10 ng/ml PDGF-BB和200μmol/L PROB孵育24 h, PROB为Pannexin-1的特异性阻断剂)。通过CCK-8法选取丙磺舒和PDGF-BB干预浓度,并检测各组细胞的增殖能力;通过细胞划痕实验和Transwell^TM实验检测各组PASMCs的迁移能力;通过细胞免疫荧光技术检测各组PASMCs中骨桥蛋白(OPN)和增殖细胞核抗原(PCNA)的分布和表达;通过Western blot法检测各组PASMCs中OPN和PCNA的蛋白水平差异。结果:与CON组相比,PDGF-BB组的PASMCs迁移和增殖能力均有所增强(P<0.05),经PROB干预后,细胞迁移和增殖能力降低(P<0.05);与CON组相比,PDGF-BB组OPN和PCNA的表达和蛋白水平均显著升...     

不同剂量LPS诱导肝Kupffer细胞释放TNF的体外研究

本实验观察了不同剂量LPS诱导大鼠肝Kupffer细胞释放TNF的作用。加入LPS后1小时,三种剂量LPS组Kupffer细胞培养上清中均可测到TNF活性,3小时达到峰值。100和150ng/ml LPS组TNF活性高于50ng/ml组(P<0.01),而100和150ng/ml两组之间无明显差异(P>0.05),再次加入LPS(终浓度100ng/ml),只有50ng/ml LPS组培养上清液中有TNF活性检出,但幅度明显下降(P<0.01)。上述结果提示LPS在体外诱导肝Kupffer细胞释放TNF在一定范围内具有剂量依赖关系,且呈一定的时间反应性。     

TAT-aFGF融合蛋白在大肠杆菌中的表达及其生物活性测定

构建表达Tat与人酸性成纤维细胞生长因子（acidic fibroblast growth factor, aFGF）融合蛋白的重组质粒pET3c–Tat-aFGF14-154和pET3c-Tat-aFGF27-154,并转化大肠杆菌BL21（DE3）中。37℃,l mmol/L的IPTG诱导4 hrs后,获得分子量约为18 kDa的融合蛋白,表达量分别占菌体总蛋白的42%和24%。利用阳离子交换（CM-Sepharose FF）、肝素亲和层析（Heparin-Sepharose CL-6B）以及分子筛（Sephadex G-25）联用的方法可获得纯度大于95%的目的蛋白,得率分别为93和78 mg/L。Tat-aFGF14-154及其对照蛋白aFGF14-154对Balb/c 3T3细胞有明显促细胞增殖的作用,最佳作用浓度分别为1280 ng/ml和160 ng/ml。而Tat-aFGF27-154 及其对照蛋白aFGF27-154则几乎没有促细胞增殖活性。免疫荧光分析结果表明,Tat-aFGF14-154及Tat-aFGF27-154均能穿过PC12、Balbc/3T3、HaCaT和海马神经元细胞的细胞膜,并主要定位于细胞浆中。此外,四种重组蛋白均能降低Aβ25-35对大鼠海马神经元细胞的毒性,在剂量为1000 ng/ml时,Tat-aFGF14-154、Tat-aFGF27-154、aFGF14-154及aFGF27-154细胞存活率分别为90.66%、81.87%、85.71%和78.02%,而Aβ25-35模型组的存活率仅为56.87%。     

不同浓度尼古丁对牙周膜成纤维细胞增殖及纤维结合蛋白合成的作用

目的:探讨不同浓度尼古丁对人牙周膜成纤维细胞(PDLFs)增殖及纤维结合蛋白(Fn)合成的作用。方法:不同浓度的尼古丁(50 ng/ml,250 ng/ml,500 ng/ml,1μg/ml,2μg/ml,3μg/ml)作用于PDLFs,MTT比色法检测细胞增殖活性,流式细胞仪检测细胞周期,酶联免疫吸附测定法(ELISA)检测Fn合成含量。结果:不同浓度尼古丁作用下:人PDLFs的增殖均被抑制,且呈浓度依赖性。浓度为250 ng/ml~3 ug/ml的尼古丁有明显抑制人PDLFs增殖的作用(P0.05),3 ug/ml尼古丁显示出最强的抑制增殖作用(P0.01);人PDLFs的G0-G1期、S期、G2-M期与对照组相比,G0-G1期细胞周期分布比例逐渐增高,S期和G2-M期逐渐降低,差异均有统计学意义,呈浓度依赖性;人PDLFs合成Fn逐渐减少,呈浓度依赖性。浓度为50 ng/ml~3μg/ml的尼古丁均有明显抑制人PDLFs合成Fn的作用(P0.05),其中3μg/ml抑制作用最强。结论:尼古丁抑制牙周膜细胞的增殖及Fn的合成,呈浓度依赖性,并影响其细胞周期的进程,进而影响牙周新附着的形成,加重牙周病的病情。     

脑源性神经营养因子促进海马神经干细胞的存活、增殖及向神经元分化

探讨脑源性神经营养因子(brain derived neurotrophic factor, BDNF)对海马神经干细胞(neural progenitor/stem cells, NPCs)的存活、增殖及分化的影响.采用无血清培养基体外分离、纯化、扩增胎鼠海马NPCs.通过细胞形态观察、nestin免疫荧光染色及血清促分化检测NPCs的干细胞特性; 采用神经球计数及神经球直径测定观察BDNF对NPCs的促增殖作用, 筛选出在适当细胞密度下, 促进NPCs增殖的有效浓度; 采用Tunel染色及全自动生化分析仪测定细胞培养上清液乳酸脱氢酶(lactic dehydrogenase, LDH)的含量探讨BDNF对海马NPCs存活的影响; 采用抗-b-微管蛋白(tubulin) III (Tuj-1)染色检测NPCs分化成神经元的百分率, 同时测定分化神经元突起的长度.分离的海马NPCs表现为nestin 免疫染色阳性, 具有自我增殖能力、且能分化为神经元和星形胶质细胞; 当细胞密度为5×105个/ ml 时, 10～200 ng/ml BDNF能显著促进NPCs的增殖, 其中40 ng/ml BDNF促增殖作用最强, 40 ng/ml BDNF能显著增大神经球直径; 40 ng/ml BDNF 显著减少NPCs的凋亡率(Tunel /DAPI ), 抑制LDH漏出; 40 ng/ml BDNF能显著促进NPCs分化为Tuj-1免疫染色阳性神经元, 且分化后神经元的突起长度显著大于对照组.上述结果提示: BDNF促进海马NPCs的存活、增殖及向神经元方向分化.     

三碘甲腺原氨酸对大鼠成骨细胞增殖的影响

目的:探讨三碘甲腺原氨酸(T3)对大鼠成骨细胞增殖的影响。方法:采用骨组织块法原代分离培养新生SD大鼠颅骨成骨细胞,然后配置含不同浓度(10-7mol/L、10-8mol/L、10-9mol/L)T3的培养基,与大鼠成骨细胞共培养4d,采用噻唑蓝(MTT)法检测细胞增殖能力,采用细胞化学染色法测定成骨细胞ALP活性。结果:T3可促进大鼠成骨细胞增殖,并呈剂量依赖性;随T3浓度的增高,成骨细胞表达ALP活性增强(P<0.05)。结论:10-7mol/L～10-9mol/L浓度的T3可通过刺激成骨细胞的增殖,对预防和治疗骨质疏松发挥一定作用。     

MOC-31和CD44v6在良恶性腹水鉴别诊断中的应用

目的探讨检测肿瘤标志物MOC-31和CD44v6对鉴别良恶性腹水的诊断价值。方法利用液基薄层细胞学自动涂片技术方法筛查出查到肿瘤细胞的恶性腹水标本390例以及良性腹水标本100例,分别采用酶联免疫吸附法(ELISA)和免疫细胞化学染色检测MOC-31和CD44v6的含量和表达情况。结果 ELISA结果显示MOC-31和CD44v6在良性腹水中的含量分别为21±4ng/ml和291±32ng/ml,在恶性腹水中的含量分别为98.1±19.3ng/ml和891±116ng/ml,差异均具有显著性(P<0.05);免疫细胞化学染色显示MOC-31在恶性腹水细胞中阳性表达250例,良性腹水细胞中阳性表达5例;CD44v6在恶性腹水细胞中阳性表达266例,良性腹水细胞中阳性表达3例,差异均具有显著性(P<0.05)。结论 MOC-31和CD44v6可以做为良恶性腹水鉴别诊断的重要指标,值得在临床病理工作中推广应用。     

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.     

Effects of acidic fibroblast growth factor and epidermal growth factor on subconfluent fetal rat calvaria cell cultures: DNA synthesis and alkaline phosphatase activity

The effects of acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) were examined in subconfluent fetal rat calvaria cell cultures, in the presence of 2% serum. Maximal effect of aFGF and EGF on DNA synthesis measured by [³H]thymidine incorporation was observed after 18 h. aFGF stimulated DNA synthesis by 3.5-fold with an ED₅₀ of 0.75 ng/ml while a 2.3-fold EGF stimulation was recorded with an ED₅₀ of 0.067 ng/ml. 5-Bromo-2-deoxyuridine staining showed a higher stimulation of proliferation in the scattered cells than in the cell clusters. An 18 h aFGF or EGF treatment decreased alkaline phosphatase (ALP) activity by 40 and 23%, respectively, as compared with control cultures. This inhibition was more pronounced after 48 h in the presence of the effectors but no modification of the ALP electrophoretic mobility was observed. These data suggest that aFGF is a less potent mitogen than EGF and a higher inhibitor of ALP activity in fetal rat calvaria cell culture.     

法舒地尔对单纯急性心肌梗死大鼠心肌形态学、心肌酶学及血液流变学的影响

目的:探讨法舒地尔对单纯急性心肌梗死模型大鼠心肌形态学、心肌酶学及血液流变学变化的影响。方法:将40只Wistar大鼠分为空白组、假手术组、治疗组和模型组。治疗组给予法舒地尔0.01 m L/g,股静脉注射,其余各组给予生理盐水。治疗结束后检测各组大鼠心肌形态学、心肌酶学及血液流变学各指标。结果:与其余各组相比,治疗组大鼠各指标均显著改善,空白组与假手术组各指标水平无显著差异(P0.05),治疗组具体表现为:CK、LDH、ASH水平明显降低(P0.05);PAR、PAG水平均明显改善,与模型组相比明显降低;HCT、ESR水平明显降低(P0.05);MIS水平明显下降(P0.05)。结论:法舒地尔能够明显改善单纯急性心肌梗死模型大鼠的心肌形态学、心肌酶学及血液流变学各指标,对临床有指导意义。     

Bifunctional effects of heparin-binding protein HBp17 on DNA synthesis in cells.

A 17 kD heparin-binding protein (HBp17) has a biphasic dose-dependent effect on DNA synthesis in 3T3 cells. Maximal stimulation of DNA synthesis occurs at 8 ng/ml HBp17, but a half-maximal inhibition occurs at approximately 500 ng/ml. This inhibition can easily be reversed by addition of 400 pg/ml aFGF or 100 pg/ml bFGF, whereas EGF had no effect. This biphasic action of HBp17 was also seen in human umbilical vein endothelial cells (HUVEC), whereas it was not found in the malignant cell line, A431-AJC. The functional relationship between HBp17 and FGF is discussed.     

Relationships between COL2A1 gene polymorphisms and knee osteoarthritis in Han Chinese women

To investigate the relationships between two COL2A1 single nucleotide polymorphisms (SNPs; T2088C and G4006A) and osteoarthritis (OA) in Han Chinese women. One hundred and twenty OA women and 120 control women were recruited. Genomic DNA was extracted from the whole blood. The COL2A1 polymorphisms T2088C and G4006A were analyzed by TaqMan assay. The levels of plasma N-propetide of type IIA collagen (PIIANP) and urinary C-telopeptide of type IIA collagen (CTX-II) were determined by ELISA. The level of plasma PIIANP significantly decreased in the OA group, compared with that in the control group (P < 0.05), with 15.6 ± 4.2 ng/ml (Mean ± SD) in the OA group and 30.2 ± 7.8 ng/ml in the control group. The level of urinary CTX-II significantly increased in the OA group, compared with that in the control group (P < 0.05), with 201.4 ± 10.2 ng/ml in the control group and 250.8 ± 15.6 ng/ml in the OA group. There was no difference in the T2088C genotypes between the OA and control groups. The G4006A AA homozygous genotype significantly increased in the OA patients, when compared with that in the control women (P < 0.05, χ²), with 24.2% (29/120) in the OA group and 10.0% (12/120) in the control group; The A allele accounted for 49.2% (118/240) in the OA group and 35.8% (86/240) in the control group. Among the G4006A genotypes, the plasma PIIANP level of the AA genotype (16.4 ± 6.6 ng/ml) was significantly lower than those of the GG genotype (28.6 ± 4.2 ng/ml) and GA genotype (21.5 ± 8.0 ng/ml) while the urinary CTX-II level of the AA genotype (255.2 ± 18.4 ng/ml) significantly increased, compared with those of the GG genotype (218.4 ± 13.2 ng/ml) and GA genotype (221.2 ± 15.6 ng/ml). The haplotype analysis shows that T-G was a protective factor for OA and that T-A was a risk factor. The AA genotype, A allele and T-A may increase the risk of OA in the Han Chinese women while T-G may protect these women from OA.     

山萘酚通过增强Treg细胞免疫抑制功能延长移植物生存时间

目的:探讨应用山萘酚增强Treg细胞免疫抑制功能,从而抑制大鼠移植物排斥反应并改善移植物生存的作用和机制。方法:以Wister大鼠和SD大鼠分别为供、受体,建立同种异体皮肤移植排斥反应动物模型。观察受体老鼠皮肤移植物的情况,记录移植物失功时间(移植物皮片80%面积发生排斥)。RT-PCR检测移植7天后脾细胞、淋巴细胞FOXP3、CTLA-4和IL-10的mRNA水平,用HE染色组织病理学观察术后7天移植皮片的淋巴细胞浸润程度。体外实验T细胞增殖抑制试验加入山萘酚作为对照,观察Treg功能情况。结果:1.山萘酚能增强移植后同种异体移植物的生存时间(DMSO组6.3±0.3天,山萘酚组13.7±0.39天,P<0.01);2.RT-PCR显示山萘酚可增强细胞CTLA-4(对照组9.24±0.17,山萘酚组12.48±0.145,P<0.05)、FOXP3(对照组0.96±0.07,山萘酚组1.41±0.07,P<0.01)和IL-10(对照组0.95±0.12,山萘酚组1.50±0.16,P<0.05)的mRNA水平;3.体外T细胞增殖抑制实验中,山萘酚可增强Treg细胞的免疫抑制功能。结论:在大鼠皮肤移植模型中,山萘酚可延长皮肤移植物的生存时间,提高Treg细胞相关IL-10、FOXP3和CTLA-4的mRNA水平;体外实验中,能抑制效应T细胞的增殖,表明山萘酚在提高移植物生存方面存在一定的价值。     

Direct and indirect effects of leptin on preadipocyte proliferation and differentiation

Leptin has been shown to reduce body fat in vivo. Adipocytes express the leptin receptor; therefore, it is realistic to expect a direct effect of leptin on adipocyte growth and metabolism. In vitro studies examining the effect of leptin on adipocyte metabolism require supraphysiological doses of the protein to see a decrease in lipogenesis or stimulation of lipolysis, implying an indirect action of leptin. It also is possible that leptin reduces adipose mass by inhibiting preadipocyte proliferation (increase in cell number) and/or differentiation (lipid filling). Thus we determined direct and indirect effects of leptin on preadipocyte proliferation and differentiation in vitro. We tested the effect of leptin (0-500 ng/ml), serum from leptin-infused rats (0.25% by volume), and adipose tissue-conditioned medium from leptin-infused rats (0-30% by volume) on preadipocyte proliferation and differentiation in a primary culture of cells from male Sprague-Dawley rat adipose tissue. Leptin (50 ng/ml) stimulated proliferation of preadipocytes (P<0.05), but 250 and 500 ng leptin/ml inhibited proliferation of both preadipocyte and stromal vascular cell fractions (P<0.01), as measured by [3H]thymidine incorporation. Serum from leptin-infused rats inhibited proliferation of the adipose and stromal vascular fractions (P=0.01), but adipose tissue-conditioned medium had no effect on proliferation of either cell fraction. None of the treatments changed preadipocyte differentiation as measured by sn-glycerophosphate dehydrogenase activity. These results suggest that leptin could inhibit preadipocyte proliferation by modifying release of a factor from tissue other than adipose tissue.     

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。