黄芪总苷对肝星状细胞增殖和合成胶原的抑制作用

目的　探讨黄芪总苷 (AST)对肝星状细胞增殖和合成胶原的影响。方法　采用枯细胞条件培养基 (KCCM )及含新生牛血清 (NBS)和健康大鼠血清 (RS)的混合血清刺激大鼠肝星状细胞 (HSC)系HSC T6 ,用 3H TdR和3H 脯氨酸参入法检测HSC增殖活性和胶原合成状况。结果　AST(1 6、32、64、1 2 8和 2 56mg·L- 1 )对KCCM 1∶4刺激HSC T6细胞增殖和胶原合成均有明显的抑制作用 ;在含 1 0 %NBS- 3 %RS混合血清刺激的HSC T6细胞 ,AST(32、64、1 2 8和 2 56mg·L- 1 )作用 48h对HSC T6细胞增殖 ,及AST(1 6、32、64、1 2 8和 2 56mg·L- 1 )作用 72h对HSC T6细胞胶原合成均有明显的抑制作用 ,呈浓度依赖型趋势。结论　AST对体外激活肝星状细胞的增殖和产生胶原有明显抑制作用 ,该作用可能是AST抗肝纤维化的机制之一     

阿米洛利对大鼠肝星状细胞增殖的影响

目的：探讨阿米洛利对大鼠肝星状细胞(HSC)增殖的影响。方法：将HSC T6细胞与不同浓度的阿米洛利共同培养24 h。MTT法观察阿米洛利对HSC T6增殖的效应，以乳酸脱氢酶(LDH)法观察阿米洛利对HSC T6的毒性作用。结果：阿米洛利浓度＞1.875 μmol·L 1时对HSC增殖有抑制作用(P＜0.01)，1.875～60 μmol·L 1未发现阿米洛利对HSC有毒性作用(P＞0.05)。结论：阿米洛利可抑制HSC的增殖，其作用不是非特异性细胞毒作用所致。     

西红花酸对乙醛诱导的肝星状细胞增殖和胶原合成的影响

目的:探讨西红花酸(Crocetin)对乙醛刺激的大鼠肝星状细胞(Hepatic stellate cell,HSC)增殖和胶原合成的影响及其作用机制.方法:培养大鼠肝星状细胞HSC-T6,建立乙醛诱导的HSC纤维化模型;用不同浓度的西红花酸(10-6、10-7、10-8 mol/L)对乙醛刺激的HSC-T6进行处理,MTT法检测细胞增殖;羟脯氨酸测定检测HSC-T6胶原含量;流式细胞分析仪测定细胞凋亡;Western blot检测细胞ERK1/2、Bax、Bcl-2蛋白的表达;RT-PCR检测Ⅰ型、Ⅲ型胶原蛋白、间质胶原酶(MMP-2)、组织金属蛋白酶抑制因子-1(TIMP-1)的基因表达.结果:在一定浓度范围里,西红花酸能抑制乙醛引起的HSC增殖和胶原合成;西红花酸诱导乙醛刺激的HSC细胞凋亡;西红花酸能增加Bax蛋白表达,降低乙醛刺激升高的ERK1/2、Bcl-2蛋白表达;西红花酸能明显降低Ⅰ型、Ⅲ型胶原、TIMP-1的表达,提高MMP-2的表达.结论:西红花酸通过抑制乙醛诱导的HSC增殖和胶原合成以及促进活化的HSC凋亡起到抗肝纤维化作用,其机制可能与ERK信号传导通路和对基质金属蛋白酶的调节有关.     

联苯双酯对大鼠肝星状细胞增殖和凋亡及PPARγ表达的影响

摘 要 目的： 探讨联苯双酯(dimethyl dicarboxylate biphenyl,DDB)对大鼠肝星状细胞HSC-T6增殖和凋亡及过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor gamma, PPARγ)表达的影响。方法: 接种HSC T6细胞于96孔板和6孔板中,分别用CCK 8法和流式细胞术测定不同浓度联苯双酯作用于细胞24 h后对细胞增殖和凋亡的影响;实时荧光定量PCR（Quantitative Real time PCR,Q-PCR）和Western blotting分别检测药物对HSC-T6细胞PPARγ mRNA和蛋白的表达的影响。结果: 相比于对照组（0 μmol·L^-1）,DDB在实验浓度（8～64 μmol·L^-1）能明显抑制HSC T6的增殖（P<0.05),促进HSC T6的凋亡（P<0.05);药物处理过的HSC T6细胞PPARγ mRNA及其蛋白表达均有显著提高。结论:DDB可通过上调HSC T6细胞中PPARγ的表达抑制细胞的增殖,促进细胞的凋亡。     

虫草多糖对离体肝星状细胞增殖及胶原基因表达的影响

目的：探讨虫草多糖(CP)抗肝纤维化的作用机理。方法：用链酶蛋白酶和胶原酶原位灌流消化正常大鼠肝脏,Nycodenz密度梯度离心分离肝星状细胞(HSC),以MTT比色法和3H-脯氨酸(3H-Pro)掺人法观察CP对HSC增殖及胶原合成的影响。结果：在(1．0～40)μg／ml剂量范围内,CP可明显抑制HSC增殖及胶原合成,呈药物浓度依赖关系。结论：CP在体外可抑制HSC的增殖和胶原合成,提示该作用可能是其抗肝纤维化的机制之一。     

高迁移率族蛋白B1与晚期糖基化终末产物受体结合对激活肝星状细胞的作用

目的探讨晚期糖基化终末产物受体(RAGE)在高迁移率族蛋白B1(HMGB1)激活肝星状细胞(HSC)中的作用。方法大鼠HSC-T6细胞系培养至对数生长期,分为5组,对照组加DMEM培养液1ml;HMGB1组加0.1μg/ml的HMGB11ml;抗RAGE组将20μg/mlRAGE抗体加入培养板2h后,再加0.1μg/mlHMGB1;sRAGE组将20μg/mlsRAGE加入培养板2h后,再加0.1μg/mlHMGB1;AGE组:将160μg/mlAGE加入培养板2h后,再加0.1μg/mlHMGB1。刺激24h后,收集各组上清培养液酶联免疫吸附试验(ELISA)方法检测透明质酸(HA)、Ⅲ型胶原(PⅢP)和Ⅳ型胶原(CⅣ)的含量;小心取出六孔板内载玻片,免疫组织化学法检测α-SMA、转化生长因子(TGF)-β1的表达。结果抗RAGE组、sRAGE组与HMGB1组相比,HSC细胞质中的α-SMA和TGF-β1表达水平和培养液中HA、PⅢP和CⅣ的含量均显著下降(P<0.05);AGE组与对照组相比,HSC细胞质中的α-SMA和TGF-β1表达水平和培养液中的HA、PIIIP和CⅣ含量均明显增高(P<0.05)。结论 HMGB1与RAGE结合,使HSC活化,合成释放细胞外基质增加。     

1-(3-氟苯基) -5-甲基-2-(1H)吡啶酮对肝星状细胞的影响

目的探讨吡啶酮类化合物1-(3-氟苯基)-5-甲基-2-(1H)吡啶酮(FMP)抗肝纤维化的机制.方法MTT法观察FMP对肝星状细胞(HSC)增殖的影响,免疫细胞化学观察FMP对HSC Ⅰ、Ⅲ型胶原表达的影响.结果FMP 200、300、400、500、600 μg/mL 在24、48 h 可抑制HSC增殖, 显著强于阳性对照组(P＜0.05).免疫细胞化学显示FMP在 200 μg/mL 可抑制HSCⅠ、Ⅲ型胶原蛋白表达.结论FMP治疗肝纤维化的原理可能与抑制HSC增殖和HSC Ⅰ、Ⅲ型胶原蛋白表达有关.     

红景天苷对乙醛刺激的大鼠肝星状细胞增殖及胶原基因表达的影响

目的探讨红景天苷对乙醛刺激的大鼠肝星状细胞(hepatic stellate cell,HSC)增殖、α1(I)型胶原mRNA合成、IκB和NF-κB表达的影响。方法用原位杂交、免疫细胞化学和凝胶电泳迁移率技术(EMSA)检测HSC的增殖和胶原基因表达。结果乙醛刺激HSC增殖、α1(I)型胶原mRNA合成 ,使IκB表达下降、NF-κB活性增加。红景天苷(0.5～2.5 mg·mL^-1)对上述影响有抑制作用,且以1.5 mg·mL^-1组效果最好。结论红景天苷明显抑制乙醛刺激的HSC增殖及胶原基因的表达。     

渥漫青霉素对大鼠肝星状细胞增殖的影响

目的探讨渥漫青霉素对大鼠肝星状细胞(HSC)增殖的影响.方法用链霉蛋白酶和胶原酶灌流大鼠肝脏,Nycodenz密度梯度离心分离大鼠HSC,进行体外培养;并以MTT比色法观察渥漫青霉素对HSC增殖的影响.结果低浓度渥漫青霉素的抑制作用不明显(P>0.05),当药物浓度上升为2.5mg/L时,抑制作用增强,呈明显药物浓度依赖关系.结论渥漫青霉素抗肝纤维化的作用是抑制了HSC的增殖,它对于防治肝纤维化可能有一定的临床意义和应用前景.     

脂多糖对肝脏Kupffer细胞增殖和分泌功能及超微结构的影响

目的 探讨脂多糖(LPS)对肝脏Kupffer细胞增殖和分泌功能及超微结构的影响.方法 将小鼠原代Kupffer细胞培养扩增后,随机分为LPS组和正常对照组.2组细胞培养24 h后,LPS组加入LPS,继续培养6 h,甲基噻唑基四唑法(MTT)测定细胞增殖情况,流式细胞仪检测细胞周期变化,透射电镜观察细胞超微结构;并收集细胞上清液,检测肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)水平.结果 LPS组吸光度值高于正常对照组,G0/G1期细胞分布低于正常对照组,S+G2/M期细胞分布高于正常对照组(P<0.01).LPS组细胞上清液中TNF-α、IL-1β和IL-6水平高于正常对照组(P<0.01).LPS组Kupffer细胞内可见大量空泡及自噬体形成,正常对照组个别细胞胞质内仅见少量空泡.结论 LPS能激活肝脏Kupffer细胞的增殖和分泌功能,并可引发肝脏Kupffer细胞自噬和超微结构改变.     

引流淋巴结树突状细胞对自身肿瘤细胞作用研究

目的　从胃癌患者引流淋巴结中分离和定向诱导培养扩增树突状细胞 (DC) ,观察其经自身肿瘤细胞抗原冲击后对自身细胞因子诱导的杀伤细胞 (CIK细胞 )杀伤活性的影响。方法　取手术切除肿瘤周围转移引流淋巴结 ,提取单个核细胞 ,将贴壁生长的细胞用细胞因子rhGM CSF、rhIL 4、rhTNF α联合诱导培养DC ,然后用自身肿瘤细胞抗原冲击DC并作用CIK细胞 ,最后检测CIK细胞杀伤三种靶细胞 (自身胃癌细胞、K5 6 2和SGC 790 1细胞 )的活性。结果　胃癌患者转移引流淋巴结中的贴壁细胞 ,经细胞因子联合诱导培养 ,DC含量可达 4 5 %。经自身肿瘤抗原冲击的引流淋巴结DC活化的CIK细胞 ,杀伤自身肿瘤细胞活性达 79 3% ,单纯CIK细胞为 33% ,两者比较差异有显著意义 (P <0 0 1) ;而对K5 6 2和SGC 790 1细胞杀伤活性无明显差异。结论　肿瘤周围转移引流淋巴结贴壁细胞在体外能定向诱导扩增出大量DC :DC经自身肿瘤抗原冲击后能明显提高CIK细胞对自身肿瘤细胞的杀伤活性。此结果为肿瘤DC的免疫治疗应用奠定了理论基础     

Comparative effect of cadmium on osteoblastic cells and osteoclastic cells

Cadmium(Cd) has been thought to disturb the bone metabolism directly. The mechanism for the bone lesion is unknown, however. To examine the effects of cadmium on bone metabolism, we compared its effects on osteoblasts and osteoclasts in vitro. We used an established cell line, MC3T3-E₁, as osteoblasts and tartrate resistant acid phosphatase (TRACP)-positive multi-nucleated cells (MNC) formed by a bone marrow culture system as osteoclasts. Alkaline phosphatase (ALP) activity was decreased by 10^–7 M Cd and DNA content and hydroxyproline content of osteoblastic cells were decreased by 10^–5 M Cd. Cadmium at 10^–7 M inhibited the osteoclastic cell formation from mouse bone marrow in the presence of 10^–8 M 1,25(OH)₂ vitamin D₃. A 100-fold higher concentration of zinc(Zn) simultaneously added to the cadmium-containing medium prevented the toxicity of cadmium to osteoclastic cells as observed in the culture of osteoblastic cells. These results indicate that both bone formation and bone resorption are inhibited by cadmium. The responses of osteoclasts and osteoblasts to cadmium in this culture system were the same and the responses of cadmium-damaged osteoblasts and osteoclasts to zinc were also similar. These results suggest that another mechanism by which cadmium could cause bone damage should be considered in addition to the specific induction of osteoclastic cells by Cd.     

静脉血管平滑肌细胞中的多倍体细胞分析

目的探讨流式细胞术分析静脉血管平滑肌细胞多倍体细胞的方法和意义。方法选取普通级健康成年日本大耳白兔2只,雄性,8月龄,大约3 kg;普通级SD大鼠2只,雄性,8周龄,大约200 g。人大隐静脉为2例心脏冠状动脉旁路移植术中修补血管所得。在麻醉日本大耳白兔和大鼠后开腹腔获取下腔静脉,采用多种酶组合的消化液消化兔、大鼠以及人的静脉血管得到适合流式细胞检测的静脉血管平滑肌单细胞悬液,用碘化丙啶标记细胞,利用细胞流式仪分析兔、大鼠以及人的静脉血管平滑肌单细胞悬液各5 000个细胞,检测细胞DNA含量,DNA含量翻倍的细胞为多倍体细胞。并利用荧光原位杂交技术（FISH）验证阳性对照HEK293细胞系中的多倍体的存在。结果流式结果显示分析的5 000个细胞中,阳性对照HEK293细胞的多倍体细胞为1 355个（27.1%）,从2例患者中取得的大隐静脉多倍体细胞为310个（6.2%）和250个（5.0%）;2只大鼠的下腔静脉平滑肌细胞中多倍体细胞为360个（7.2%）和450个（9%）;2只兔的下腔静脉平滑肌细胞中多倍体细胞分别为270个（5.4%）和305个（6.1%）。结论成年的静脉血管包括人大隐静脉的平滑肌细胞中普遍存在一定比例的多倍体细胞。     

Reprogramming of B cells into regulatory cells with engineered fusokines

B cells play a pivotal role in host adaptive immunity against pathogenic microorganisms, but may also maladaptively contribute to the pathogenesis of autoimmune diseases. In contrast, distinct B cell subsets have the capacity to regulate host immune response, and suppress inflammation. B regulatory cells are a rare population of endogenous Blymphocytes defined in part by production of the anti-inflammatory cytokine IL-10. Although "natural" B regulatory cells exist in vivo, the low frequency of B regulatory cells may be a limiting factor on their impact in autoimmune ailments. In answer to this unmet need, we have developed a novel strategy for alternate lymphoid activation: fusokines. These wholly engineered chimeric leukines fuse two functionally unrelated cytokines for the purpose of alternate immune modulation. The GM-CSF- and IL-15-derived fusokine: GIFT15, possesses entirely novel and unheralded immune modulating properties mediated through the IL15 receptor which reprograms naive B cells into B regulatory cells (Bregs). In this article, we review the current approaches to generate Bregs in vitro, and highlight gain-of-function mechanisms by which GIFT15- induced Bregs abrogate pathogenic autoimmunity in mice. We also demonstrate that the human equivalent of inducible Bregs may also serve as a new potent therapeutic tool for treatment of autoimmune disease.     

Dendritic cells and T cells in immunotherapy

Autologous cellular immunotherapies have been used experimentally in humans to treat many types of cancer. These therapies are divided into two principal types: active cellular immunotherapies that rely on autologous dendritic cells or other antigen presenting cells; and adoptive T-cell therapies, in which large numbers of antigen-specific T lymphocytes are propagated ex vivo and then infused back into the patient. With the FDA approval of the antigen presenting cell vaccine sipuleucel-T for prostate cancer, active immunization has become an accepted approach for the treatment of established cancer.     

肥大细胞对前列腺癌细胞上皮间质转化的作用

目的利用体外细胞共培养模型,探讨肥大细胞对前列腺癌细胞增殖及侵袭、转移的作用。方法选取肥大细胞瘤P815细胞系及前列腺癌LNCa P细胞,使用24孔Transwell小室构建2种细胞体外共培养模型。四甲基偶氮唑蓝(MTT)比色试验检测前列腺癌LNCa P细胞的增殖,反转录定量聚合酶链反应(q RT-PCR)方法和蛋白印迹法检测前列腺癌LNCa P细胞E-cad、N-cad、Vimentin的表达。结果前列腺癌LNCa P细胞与不同浓度肥大细胞共同培养12 h后吸光度(A)值变化与对照组差异无统计学意义(P>0.05),共同培养24 h及48h后A值显著高于对照组,差异有统计学意义(P<0.05);与对照组比较,实验组E-cad表达明显减弱;N-cad、Vimentin表达增强,差异有统计学意义(P<0.05)。结论肥大细胞可促进前列腺癌细胞的增殖并促进前列腺癌细胞的上皮间质转化,可能促进前列腺癌细胞侵袭、转移。     

无血清条件下Vero细胞流感病毒的大规模培养

目的 探索在无血清条件下Vero细胞和流感病毒在反应器中的培养条件.方法 采用5 L反应器,接种经无血清适应的不同代次Vero细胞,观察细胞生长情况.以不同感染复数接种流感病毒,观察病毒的生长情况.结果 细胞在反应器中生长繁殖成功,4 d左右达到平台期,增殖倍数为5～16.接种甲型流感病毒后72 h,病毒血凝滴度达到高峰(1/64～1/128).结论 在无血清条件下,细胞和流感病毒都能在反应器中成功培养.     

Differentiation of functional cells from iPS cells by efficient gene transfer

Induced pluripotent stem (iPS) cells, which are generated from somatic cells by transducing four genes, are expected to have broad application to regenerative medicine. Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. By using an adenovirus (Ad) vector containing the cytomegalovirus enhancer/beta-actin (CA) promoters, we have developed an efficient transduction system for mouse mesenchymal stem cells and embryonic stem (ES) cells. Also, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector could efficiently transduce transgenes into mouse iPS cells. We found that the CA promoter had potent transduction ability in iPS cells. Moreover, exogenous expression of a PPARγ gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to promote cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells.     

Apoptosis of astroglial cells

Astrocytes, the most abundant glial cell type in the brain, are considered to have physiological and pathological roles in neuronal activities. We found that reperfusion of cultured astrocytes after Ca2+ depletion causes delayed cell death and that the Na(+)-Ca2+ exchanger in the reverse mode is responsible for this Ca(2+)-mediated cell injury (Ca2+ paradox injury). The Ca2+ paradox injury of cultured astrocytes is considered to be an in vitro model of ischemia/reperfusion injury, since a similar paradoxical change in extracellular Ca2+ concentration is reported in ischemic brain tissue. Furthermore, we demonstrated that heat shock proteins, glutathione and calcineurin inhibitors protected astrocytes against Ca2+ paradox-induced cell toxicity. We also observed DNA fragmentation, a typical apoptotic ladder, 2-3 days after hydrogen peroxide exposure. In addition, laser microscopic observation showed that reperfusion after the exposure to hydrogen peroxide caused nuclear condensation of astrocytes. Hydrogen peroxide-induced cell injury and DNA fragmentation were attenuated by the NF-kappa B inhibitor ammonium pyrrolidinedithiocarbamate, 1,10-phenanthroline and a caspase 3 inhibitor. These findings suggest that astrocytes are one of the targets for ROS and the oxidative stress-induced delayed death of astrocytes is at least due to apoptosis.     

Culture of human renal tubular cells: positive selection of kallikrein-containing cells

Human renal tubular cells derived from nephrectomy specimens were maintained in culture and grown to confluence. Immunocytochemistry, using a panel of antibodies selected for their ability to bind to different cell types within the nephron, showed the cells to be predominantly of epithelial origin with no significant contamination by fibroblasts or cells of endothelial origin. Ten to thirty percent of the cells expressed the putative distal marker, epithelial membrane antigen (EMA), a finding confirmed by flow cytometry. Cells expressing EMA were positively selected from mixed cultures by magnetic activated cell sorting (MACS). Kallikrein activity, expressed as mU/10(6) cells, in the EMA +ve cells, was increased fourfold to sixfold when compared with that in the EMA -ve cells. Cultures of characterized human renal tubular cells and sub-populations enriched with distal cells should prove useful in studies of synthesis and release of parameters of the kallikrein kinin system (KKS) to physiological stimuli. Furthermore, a better understanding of the toxic effect on the KKS of clinically useful drugs, particularly those used for immunosuppression, may lead to therapeutic interventions to lessen unwanted effects.