重组大鼠肝再生增强因子对肾小管上皮细胞增殖及凋亡的影响

目的探讨重组大鼠肝再生增强因子（rrALR）对体外培养的肾小管上皮细胞增殖及凋亡的影响。方法将体外培养的人肾小管上皮细胞株（HK2）分组，分别加入不同浓度的庆大霉素（GM）或（和）m～LR，^3H-胸腺嘧啶核苷（^3H-TdR）掺入法检测HK2细胞的增殖：吖啶橙／溴乙啶（AO／EB）染色和钙磷脂结合蛋白／碘化丙啶（annexin V／PI）双标记流式细胞术检测上述各组HK2细胞的凋亡。结果（1）rrALR对常规培养条件下的HK2细胞有直接的促增殖作用。并具有量效关系（在25ng／ml～50μg／ml的浓度范围内逐渐增强，P〈0．01）和时效关系（培养12h即有该作用，48h达到高峰，以后逐渐下降，P〈0．05）；（2）rrALR能够促进GM损伤后的HK2细胞增殖，有明显剂量依赖性（P〈0．05）；（3）rrALR呈剂量依赖性抑制GM诱导的HK2细胞凋亡（P〈0．01）。结论rrALR能够促进体外常规培养和GM损伤后的肾小管上皮细胞增殖．抑制GM诱导的小管上皮细胞凋亡，提示ALR可能对小管上皮细胞的中毒性损伤有改善作用。     

热打击对培养肠黏膜上皮细胞IEC-6细胞活性和增殖的影响

目的：研究梯度热打击对体外培养肠黏膜上皮细胞活性和增殖的影响。方法：肠黏膜上皮细胞株IEC-6经培养后分为正常对照组（37℃、5％CO2培养箱培养）及39℃、41℃、43℃热打击组（分别在相应温度培养箱中培养）,相差显微镜观察各组细胞培养1h的形态学改变,CCK8法比较各组细胞培养0、1、3、5、7h的细胞活性和24h增殖率,流式细胞术研究细胞周期的改变。结果：与正常对照组比较,各热打击组各个时相细胞形态变圆,伪足变短,细胞间隙增大,活力下降（P〈0．01）。24h细胞增殖实验显示,与正常对照组比较,39℃组7h及41℃和43℃组各时相增殖率显著下降（P〈0．01）,呈时间及温度依赖关系。流式细胞术检查显示,热打击组细胞呈现细胞周期G0／G1和G2／M期阻滞。结论：热打击对IEC-6细胞具有细胞毒效应,可抑制IEC-6细胞的增殖,造成细胞周期G0／G1和G2／M期阻滞。     

选择性诱生型一氧化氮合酶抑制剂对人结直肠癌Lovo细胞生长的影响

目的研究选择性诱生型一氧化氮合酶（iNOS）抑制剂氨基胍（AG）对人结直肠癌细胞株Lovo增殖与凋亡的影响，并对其作用机制进行初步探讨。方法应用四唑盐（MTT）比色法检测AG对Lovo细胞增殖的抑制作用，流式细胞术检测分析不同浓度AG作用后Lovo细胞的凋亡率和细胞周期分布变化，并用丫啶橙结合溴化乙锭染色荧光显微镜观察凋亡细胞形态学改变。结果AG 0．5 mmol／L组和AG 1．0 mmol／L组的A值与对照组比较，差异有统计学意义（P〈0．05）；各组在不同浓度AG作用24、48和72h后A值比较，差异有统计学意义（P〈0．05）。Lovo细胞生长抑制率曲线显示，AG对Lovo细胞生长的抑制率呈明显的时间和浓度依赖性。流式细胞分析显示，随AG浓度的增高，Lovo细胞G0／G1期比率增高，S期与G2/M期则相应降低（P〈0．05）；SPF值和增殖指数（PI）降低，凋亡率增加（P〈0．05）。AG作用于Lovo细胞24h后细胞体积缩小、核固缩、荧光染色增强及凋亡小体形成。结论AG可抑制Lovo细胞的增殖。促进细胞凋亡；其作用的可能机制为氨基胍阻止Lovo细胞周期的进展。     

p38蛋白激酶在小肠移植后肠黏膜上皮细胞凋亡机理中的作用

目的 探讨p38蛋白激酶（p38 MAPK）在小肠移植早期排斥反应中肠黏膜上皮细胞凋亡机理中的作用。方法 选用近交系SD和Wistar大鼠进行节段性小肠移植,实验分3组：同基因移植组（Wistar→Wistar组）、异基因移植组（SD→Wistar组）和异基因移植加环孢素A组（SD→Wistar＋CsA组）。分别于移植术后1、3、5及7d采集移植肠管行病理学检查排斥反应,TUNEL法检测凋亡细胞,并行Western—blotting测定p38MAPK表达;同时,ELIsA法测定血清TNF—α活性。结果 SD→Wistar组肠黏膜上皮细胞发生轻、中、重度排斥反应中存在细胞凋亡,凋亡细胞数随排斥反应的加重而增加（P〈0．01）;Wistar→Wistar组排斥反应轻微,凋亡细胞数无明显变化（P〉0．05）;SD→Wistar＋CsA组随着CsA的应用,排斥反应逐渐得到控制,且凋亡细胞数也随着减少。SD→Wistar组及sD→Wistar＋CsA组的血清TNF-α随移植肠管上皮细胞凋亡的轻重而发生相应的变化（P〈0．01）。p38 MAPK在SD→Wistar组随凋亡细胞数增加而表达加强（P〈0．01）,Wistar→Wistar组p38 MAPK表达无明显变化（P〉0．05）,在SD→wistar＋CsA组随凋亡细胞数而发生相应的变化（P〈0．01）。小肠移植早期排斥反应中肠黏膜上皮细胞凋亡现象与p38MAPK呈正相关（r=0．875,P〈0．01）,血清TNF-α与移植肠上皮细胞凋亡呈正相关（r=0．837,P〈0．01）,血清TNF-α与p38 MAPK亦呈正相关（r=0．826,P〈0．01）。结论 大鼠小肠移植排斥反应中存在肠黏膜上皮细胞凋亡现象,p38 MAPK参与细胞凋亡信号转导过程并起重要作用。     

谷氨酰胺缺乏诱发肠上皮细胞凋亡

谷氨酰胺是血中最丰富的氨基酸,缺乏后可导致肠粘膜萎缩,小肠粘膜是处于细胞增殖和细胞凋亡的平衡之中,已知肠上皮细胞的分裂素诱发性增殖需要谷氨酰胺,但不了解谷氨酸胺如何调节肠上皮细胞凋亡。作者以往的研究已证实谷氨酸胺缺乏导致肠粘膜细胞数减少,而因细胞凋亡引起的细胞死亡数增加;同样,蛋氨酸（Met）和半脱氨酸（Cys）的缺乏可导致肠粘膜细胞数减少,但不引起细胞凋亡。为此,作者进一步作实验研究,对谷氨酸胶、Met和CyS缺乏的结果进行比较。取鼠上皮细胞（RIE－1）与无谷氨酸胺或Met／Cys的DMEM培养基（Dubecco改良…     

Smoothened（Smo）基因在结肠腺癌Lovo细胞的表达及其作用

目的探讨Smoothened（Smo）基因在结肠腺癌Lovo细胞的表达水平及在细胞增殖与凋亡过程中的作用。方法应用Westernblot及半定量RT—PCR检测结肠腺癌Lovo细胞内Smo基因的表达水平,再应用RNAi技术降解Lovo细胞内的SmomRNA表达,然后应用MTT法、流式细胞术检测SmomRNA被降解后Lovo细胞增殖水平与凋亡水平的变化。结果结肠腺癌Lovo细胞内有Smo蛋白及SmomRNA的高表达,SmomRNA被降解后,Lovo细胞的增殖水平明显抑制（P〈0．05）,凋亡率明显升高（P〈0．001）。结论结肠腺癌Lovo细胞的发生与Smo基因的高表达有关,Smo基因参与结直肠癌的增殖与凋亡过程。     

ω-3多不饱和脂肪酸二十二碳六烯酸对胃癌细胞株AGS生长和增殖的影响

目的评价ω-3多不饱和脂肪酸（ω-3PUFA）体外抑制胃癌细胞株AGS增殖和诱导细胞凋亡的能力。方法在处于指数生长期的AGS细胞株培养剂中添加二十二碳六烯酸（DHA）,采用噻唑蓝法、DNA琼脂糖凝胶电泳和流式细胞仪等方法检测AGS细胞株的生长和增殖情况,并通过免疫组化检测经DHA处理前后细胞中COX-2的表达。结果经DHA作用后,AGS细胞增殖受抑制,随DHA浓度的递增AGS细胞增殖率逐次下降,呈现明显量效关系,同时诱导细胞凋亡;琼脂糖凝胶电泳显示DNA裂解片段出现典型的阶梯状条带;流式细胞仪检测出凋亡峰,细胞周期分析表明细胞阻滞于G0／G1期。ω-3PUFA对正常肠上皮细胞增殖无明显影响。经DHA作用后,AGS细胞中COX-2表达下调。结论DHA可能通过抑制COX-2而阻遏AGS细胞的增殖,诱发细胞凋亡。     

重组人生长激素减少人结肠癌细胞株放疗诱导的细胞凋亡

目的：探讨重组人生长激素（rhGH）对结肠直肠癌细胞株放疗敏感性的影响,并研究其与细胞凋亡的关系。方法：应用流式细胞术及免疫荧光法检测9个人结肠直肠癌细胞株表面生长激素受体（GHR）的表达水平;应用克隆形成实验检测结肠直肠癌细胞经照射后的增殖能力,从而评估其放疗敏感性;应用流式细胞术（Annexin V-FITC染色）检测放疗诱导的细胞凋亡;应用Western blot方法检测rhGH干预后Akt磷酸化水平的变化。结果：从9个细胞株中选择HCT-8为GHR阳性表达细胞,LoVo细胞为阴性表达对照。rhGH显著提高了GHR阳性表达的HCT-8细胞经放疗后的克隆形成率[在高剂量8Gy照射下尤为明显,（52.1±2.9）%比（21.0±2.7）%,P〈0.001],同时减少了细胞凋亡（P〈0.05）;而对GHR阴性表达的LoVo细胞作用不明显（P〉0.05）。rhGH能诱导HCT-8细胞Akt快速磷酸化,呈PI-3K依赖（P〈0.001）。结论：rhGH使GHR阳性表达的结肠直肠癌细胞对放疗有抵抗作用,这种作用可能与其减少细胞凋亡有关。     

皮肤血管瘤和血管畸形细胞增殖与凋亡的对比研究

目的：探讨皮肤血管瘤和血管畸形的细胞凋亡与增殖的平衡关系及与临床生物学行为的可能关系。方法：采用流式细胞术检测皮肤血管瘤和血管畸形组织中凋亡细胞比率及细胞周期分布,分析细胞凋亡/增殖水平。结果：血管瘤增生期和消退期凋亡/增殖水平出现下调和上调（P〈0.05）,而血管畸形细胞凋亡/增殖水平与正常皮肤相比无显著性差异（P〉0.05）。结论：细胞凋亡/增殖水平在皮肤血管瘤和血管畸形之间存在差异且可能与二者的病理演变过程密切相关。     

当归挥发油对人脐静脉内皮细胞增殖、凋亡及Ⅲ型胶原合成的影响

目的探讨当归挥发油对人脐静脉内皮细胞增殖、凋亡和胶原合成的影响。方法体外培养人脐静脉内皮细胞，用噻唑蓝比色法检测细胞增殖活性，用流式细胞术分析细胞周期及凋亡，用放射免疫法测定胶原合成。结果低浓度（≤4mg／L）当归挥发油促进细胞增殖（P〈0．05），降低G0/G1期细胞且增加S期细胞（P〈0．05），降低凋亡率（P〈0．05），而高浓度（≥16mg／L）时抑制增殖（P〈0．05），增加G0/G1期细胞且减少S期细胞（P〈0．05），增加凋亡率（P〈0．05）。当归挥发油呈剂量和时问依赖性抑制细胞合成胶原（P〈0．05或0．01）。结论当归挥发油对人脐静脉内皮细胞的增殖呈低浓度刺激高浓度抑制的双向调节作用，但对胶原合成呈抑制效应。     

多种化疗药物对胃癌细胞杀伤效应的研究

目的：探讨不同分化的胃癌细胞对不同化疗药物的敏感性。 方法：选择低分化的MGC-803和高分化的AGS两种胃癌细胞系,以及正常胃黏膜上皮细胞GES-1,分别加入不同浓度的5-氟尿嘧啶（5-FU）、奥沙利铂（L-OHP）、多西他赛（DXT）、顺铂（DDP）、伊立替康（CPT-11）作用48 h后,用MTT法检测药物对细胞的抑制作用,并计算药物半数抑制浓度（IC50）。用IC50的5-FU、DDP、L-OHP作用于MGC-803和AGS细胞48 h后,检测细胞凋亡及细胞周期分布情况。 结果：5种化疗药物对MGC-803、AGS细胞以及GES-1细胞均有明显的抑制作用（均P<0.05）,其中5-FU、DDP、CPT-11对AGS细胞的作用较强;L-OHP、DXT对MGC-803细胞作用较强;DDP对GES-1细胞的抑制作用较强。5-FU、DDP、L-OHP均明显诱导两种胃癌细胞凋亡,其中5-FU组与L-OHP组凋亡率高于DDP组（均P<0.05）;细胞周期分析显示,L-OHP增加两种细胞的S期阻滞,DDP增加MGC-803细胞的S期阻滞,5-FU与DDP增加AGS细胞的G1阻滞（均P<0.05）,但5-FU对MGC-803细胞周期无明显影响（P>0.05）。 结论：化疗药物对胃癌细胞的抑制作用与其病理类型有关,且对胃癌细胞凋亡的诱导和细胞周期阻滞作用均不同。     

Safe injection of cultured schwann cells into peripheral nerve allografts

The effects of cultured host Schwann cells on axonal regeneration in peripheral nerve allografts were studied. Fischer rats served as recipient animals and Buffalo rats provided nerve allografts. Animals were randomized into 9 groups. Rats receiving tibial nerve isografts were left untreated (group I), or injected with isogeneic Fischer Schwann cells (group II) or placebo suspension (group III). Allografts obtained from Buffalo rats were left untreated (group IV), or received isogeneic Fischer Schwann cells (group V), 2 mg/kg Cyclosporin A and Fischer Schwann cells (group VI), 5 mg/kg Cyclosporin A (group VII), or 5 mg/kg Cyclosporin A with Schwann cells (group VIII). No Schwann cell tumors were identified 4 or 8 weeks postoperatively. Group IX animals, harvested 3 days postoperatively, demonstrated no evidence of injection injury. Schwann cells modestly improved axonal regeneration in both isografts and allografts and may have a clinical role in the treatment of peripheral nerve allografts.     

Prostatic fluid concentrations of isoflavonoids in soy consumers are sufficient to inhibit growth of benign and malignant prostatic epithelial cells in vitro

BACKGROUND: The differential intestinal metabolism of the soy isoflavones is likely to influence the ability of soy to prevent prostate cancer. While daidzein, genistein, and equol have direct antiproliferative effects on prostatic epithelial cells in vitro, there are no such data for the isoflavone glycitein, or seven metabolites: O-desmethylangolensin (ODMA), 6-hydroxyODMA (6H-ODMA), dihydrodaidzein (DHD), cis-4-hydroxyequol (C4HE), 3'-hydroxydaidzein (3HD), 6-hydroxydaidzein (6HD), and 8-hydroxydaidzein (8HD). In the current study, the in vitro activities of these compounds were elucidated, and the active ranges of concentrations were compared to that found in Caucasian prostatic fluid (PF) and plasma samples. METHODS: The effects of isoflavonoids on cell growth, cell cycle distribution, and apoptosis (active Caspase 3) were examined on benign prostatic epithelial cells (PrEC), and the prostate cancer cell line LNCaP. RESULTS: PF concentrations of genistein, equol, and daidzein (but not ODMA or DHD) were often within the ranges that reduce PrEC growth in vitro. Profound differences in sensitivities were observed with LNCaP. The hydroxydaidzeins, C4HE, and 6H-ODMA had significant inhibitory effects at 10(-5)M on PrEC growth (but not LNCaP). Glycitein had significant effects on both. Reductions in cell growth were typically associated with both changes in cell cycle distribution and Caspase 3 activation. When five isoflavonoids were used in combination at concentrations present in PF samples, synergistic effects were observed. CONCLUSION: The profound differences in sensitivities of prostatic epithelial cells to these compounds along with their synergistic effects suggest that multiple metabolites in vivo may be optimal for preventing prostate cancer.     

Sonic Hedgehog 信号通路在肝癌细胞增殖中的作用

目的：探讨Sonic Hedgehog（SHH）信号通路在肝癌细胞增殖中的作用及抑制该通路的活性对肝癌细胞对化疗药物敏感性的影响。 方法：分别用不同浓度重组SHH N-末端肽（rSHH-N）、SHH中和抗体（anti-SHH）、SHH通路抑制剂cyclopamine作用人肝癌SMMC-7721细胞不同时间,用MTT法检测细胞增殖状态。比较 5-氟尿嘧啶（5-FU）、anti-SHH、cyclopamine、anti-SHH+5-FU、cyclopamine+5-FU对SMMC-7721细胞增殖抑制作用的差异。 结果：rSHH-N作用后,SMMC-7721细胞增殖明显增加,而anti-SHH和cyclopamine作用后,SMMC-7721细胞增殖明显降低,且均呈时间和浓度依赖性（均P<0.05）;anti-SHH或cyclopamine联合5-FU对SMMC-7721细胞增殖抑制作用明显强于各药单用（均P<0.05）。 结论：SHH信号通路在肝癌生长中起重要作用,阻断SHH信号通路能抑制肝癌细胞增殖且能增加肝癌细胞对化疗药物的敏感性。     

Destruction of rat islet cell monolayers by cytokines. Synergistic interactions of interferon-gamma, tumor necrosis factor, lymphotoxin, and interleukin 1

An assay was developed to detect the cytotoxic effects of cytokines on rat pancreatic islet cells in monolayer culture. Cell lysis was detected by a 51Cr-release assay after 4 days of incubation with various cytokines. When tested alone, murine (rat and mouse) interferon-gamma (mIFN-gamma) produced a small dose-dependent lysis of islet cells; human IFN-gamma, mouse IFN-alpha/beta, interleukins 1 and 2 (IL-1 and IL-2), tumor necrosis factor (TNF), and lymphotoxin (LT) were inactive. When added together, the following combinations of cytokines showed synergistic cytotoxic effects: TNF (or LT) plus IL-1, TNF (or LT) plus mIFN-gamma, and IL-1 plus mIFN-gamma. These results indicate that the cytokine products of mononuclear cells of the immune system, IFN-gamma, TNF, LT, and IL-1 have strong synergistic cytotoxic effects on islet cells and therefore may act as direct chemical mediators of islet beta-cell destruction in type I (insulin-dependent) diabetes.     

Paracrine effects of cell transplantation: strategies to augment the efficacy of cell therapies

Within the last few years, it has become evident that the beneficial effect of cell transplantation on ventricular function and myocardial perfusion is in large part mediated through paracrine effects on the host myocardium. Studies in which medium conditioned by cultured cells, usually mesenchymal stem cells, were injected into infarcted animal hearts have provided definitive evidence of this mechanism of action. Paracrine effects of the donor cells include but are not limited to angiogenesis, mobilization of both circulating and bone-marrow-derived stem cells, activation of cardiac-resident stem cells (CRSCs), and stabilization of the extracellular matrix (ECM). These paracrine effects can be augmented by transplantation of cells modified to express therapeutically useful transgenes, or by preconditioning through hypoxic or pharmacologic means. Strategies to enhance the paracrine effects of cell transplantation may thus be employed in the next generation of cell therapies, with greater functional benefit.     

枸杞、黄芪对大鼠睾丸支持细胞功能的影响

目的:探讨中药枸杞、黄芪对大鼠睾丸支持细胞(Sertoli细胞)功能的作用及机制。方法:对18~22 d的SD大鼠睾丸Sertoli细胞进行分离培养,用MTT法检测枸杞(50、100、200、400μg/m l枸杞多糖)、黄芪(12.5、25、50、100 g/L黄芪提取液)对Sertoli细胞增殖的影响,用一步法RT-PCR方法检测枸杞、黄芪对正常培养状态和过氧化物(H2O2)损伤状态下Sertoli细胞抑制素(INH)βB亚基转录的影响。结果:高浓度枸杞(400μg/m l)、黄芪(100 g/L)对Sertoli细胞的增殖有促进作用(P<0.05),高浓度杞芪合剂(400μg/m l枸杞+100 g/L黄芪)对Sertoli细胞增殖有明显促进作用(P<0.01);在正常培养状态下,枸杞、黄芪及杞芪合剂对Sertoli细胞INHβB亚基的转录具有明显促进作用(P<0.01);在过氧化物(H2O2)损伤状态下,Sertoli细胞INHβB亚基的转录明显降低(P<0.01),黄芪对Sertoli细胞INHβB亚基的转录水平具有上调作用(P<0.05),枸杞、杞芪合剂对其转录水平具有明显上调作用(P<0.01)。结论:枸杞、黄芪及杞芪合剂对体外培养的Sertoli细胞INHβB亚基的转录具有促进和保护作用。     

Effects of activated T cells on osteoclastogenesis depend on how they are activated

INTRODUCTION: Activated T cells are emerging as important regulators of osteoclast function in inflammatory diseases. Both pro- and anti-resorptive properties have been described. We reasoned that this reported variability of the effects of T cells on osteoclast formation depends on how T cells are activated in vitro. METHODS: We harvested T lymphocytes from 5-week-old C57BL/6 mouse spleens. Activation was performed with anti-CD3epsilon and -CD28 Ab (Abs), concanavalin A (Con A), phytohemagglutinin (PHA), or the superantigen Staphylococcal enterotoxin A (SEA). Osteoclastogenesis was induced by receptor activator of NF-kappaB ligand (RANKL) in the mouse macrophage cell line RAW 264.7 cells, or primary macrophage CD11b+ cells from mouse spleen. Cells were cultured with T cells or with their conditioned medium. RESULTS: Co-culture of activated T lymphocytes with RAW 264.7 cells inhibited osteoclastogenesis but only when activated by Abs. This effect was CD4+ -dependent. Conditioned medium from activated T lymphocytes with Abs consistently blocked osteoclastogenesis in RAW 264.7 and CD11b+ cells. T cells activated with SEA, Con A, and PHA had inconsistent effects on osteoclastogenesis. We then tested the role of interferon (IFN)-gamma, a known inhibitor of osteoclastogenesis, in the effects of T cells on osteoclast formation. IFN-gamma neutralizing antibody blocked the inhibitory effect of T-cell conditioned medium on osteoclastogenesis. Osteoclast precursors from IFN-gamma receptor-null mice treated with 0.1% medium from activated T cells formed osteoclasts. However, higher doses of medium inhibited osteoclastogenesis, so that we cannot exclude that other factors besides IFN-gamma may be involved. CONCLUSIONS: Available methods to activate T lymphocytes result in variable effects on osteoclastogenesis. IFN-gamma is the main factor responsible for the inhibitory effects of activated T cells on osteoclast formation.     

Prolactin and testosterone regulation of mitochondrial zinc in prostate epithelial cells

The prostate gland of many animals accumulates extremely high levels of zinc and citrate. Evidence currently exists in support of a concept that zinc might be an important regulator of m-aconitase and citrate oxidation of prostate epithelial cells. No information concerning the mitochondrial levels of zinc has been available. The roles of testosterone and prolactin in the regulation of zinc have not been established. In this report, we determined the levels of tissue and mitochondrial zinc of rat lateral prostate (LP), ventral prostate (VP), dorsal prostate (DP), liver, and kidney. The results demonstrate that the mitochondrial zinc levels of the prostate cells were higher than levels of nonprostatic cells. The LP contained severalfold higher zinc levels than DP and VP. The effects of testosterone and prolactin in vivo and in vitro on the zinc levels were also determined. Both hormones significantly increased cellular and mitochondrial zinc levels of LP cells; decreased the zinc levels of VP cells; and had no effect on the zinc levels of DP, liver, or kidney cells. These effects are direct and physiological effects of the hormones on the targeted prostate epithelial cells. The hormonal effects on mitochondrial zinc of LP and VP epithelial cells correlate perfectly with their effects on citrate oxidation. The results support the concept that mitochondrial zinc is an inhibitor of m-aconitase and citrate oxidation; and that prolactin and testosterone regulation of mitochondrial zinc provides a mechanism for their regulation of citrate oxidation in citrate-producing prostate epithelial cells. Prostate 30:26–32, 1997 © 1997 Wiley-Liss, Inc.