白细胞介素13对肾小球系膜细胞增殖及白细胞介素1β表达的影响

目的：探讨白细胞介素13（IL－13）对体外培养的大鼠系膜细胞的增殖及其产生白细胞介素1β（IL-1β）的影响。方法：用四甲基偶氮唑（MTT）法测定系膜细胞增殖,用逆转录聚合酶链反应（RT－PCR）及酶联免疫吸附（ELISA）法测定系膜细胞IL－1β mRNA表达IL-1β蛋白水平。结果IL－13在1．0,10,100ng/ml浓度范围呈剂量依赖性地抑制系膜细胞的增殖。含5％小牛血清（FCS）的RPMI 1640培养状态下的系膜细胞检测不出IL－1β,IL－13在抑制系膜细胞的增殖的相应浓度坷显著地抑制LPS诱导的系膜细胞IL－1β mRNAg表达及L-1β分必,IL－13在10ng/ml浓度时即可几乎完全抑制LPS诱导的系膜细胞IL－1β mRNA表达,结论：IL－13对体外培养的系膜细胞增殖及炎症效应具有抑制作用。     

白细胞介素—10对鼠肾小球系膜细胞增殖和核因子κB的抑制作用

目的：研究白细胞介素－10（rhIL-10)对小鼠肾小球系膜细胞（mesangial cell,MC)增殖的影响及其细胞内作用机制。方法：采用3H－TdR掺入法观察MC的增殖反应,采用电泳适移改变法（EMSA）检测核因子κB（nuclear factorκB,NF-κB)活性。结果：rhIL-10(20ng/ml)抑制LPS刺激的小鼠MC增殖（P＜0．05）,同时rhIL-10亦显抑制LPS诱导的MC中NF－κB活化,结论：IL－10对LPS诱导的小鼠MC增殖的抑制作用至少部分与其对NF－κB活化的抑制有关。     

三七总皂苷对大鼠肾小球系膜细胞增殖及细胞周期的影响

目的：研究三七总皂苷（PNS）对大鼠肾小球系膜细胞（MC）增殖以及细胞周期的影响。方法：用不含或含有高、中、低浓度PNS的RPMI-1640细胞培养液刺激体外培养的正常大鼠MC。于实验终点,运用MTT比色法测定细胞增殖情况以及PNS的细胞毒性作用;运用流式细胞术测定细胞周期。结果：PNS能够显著抑制血清诱导的大鼠MC的增殖;通过减少MC进入S＋G2/M期,从而抑制MC的有丝分裂;且在本实验浓度内（100-400μg/ml）不具有明显的诱导凋亡作用和细胞毒性。结论：PNS能通过抑制MC的有丝分裂而抑制其活化、增殖,在慢性肾脏疾病的治疗中具有一定得应用价值。     

金水清对大鼠肾小球系膜细胞增殖及凋亡的影响

目的:通过观察中药复方金水清对大鼠肾小球系膜细胞(mesangial cells,MC)增殖及凋亡的影响,探讨其治疗小儿血尿的作用机制.方法:以噻唑蓝法检测MC的增殖,荧光显微镜下观察MC的凋亡形态,流式细胞仪检测细胞周期及凋亡.结果:在1 000～62.5 μg/L浓度范围内,金水清能抑制MC增殖,可使G0～G1期细胞增高,S、G2-M期细胞减少,且呈浓度依赖关系,并能明显诱导MC凋亡.结论:MC是金水清发挥作用的重要靶细胞,金水清可能通过:(1)减少细胞有丝分裂,干扰细胞周期,抑制系膜细胞的增殖及代谢活性;(2)诱导系膜细胞的凋亡等环节发挥抑制MC增殖的作用,从而减少炎性因子的合成与释放,减少细胞外基质的聚积,达到治疗小儿血尿,延缓慢性肾脏病理进程的目的.     

反义增殖细胞核抗原联合p16基因转染抑制膀胱癌细胞生长的研究

目的 探讨膀胱癌联合基因治疗的新策略。方法 反义增殖细胞核抗原(PCNA)联合p16转染膀胱癌细胞，观测共转染1—7d后癌细胞增殖活性、DNA合成速率、细胞周期时相、克隆形成能力、细胞凋亡和PCNA、p16基因表达情况。结果 联合转染后癌细胞PCNA表达减弱，p16表达显著增强，增殖活性抑制15．45％—68．47％(P＜0．01)，DNA合成速率减慢65．77％(P＜0．01)，细胞周期阻滞于G0／G1期，克隆形成率抑制64．49％(P＜0．01)，凋亡率为22．00％(P＜0．01)。结论 反义PCNA与p16共转染具有抑制增殖、诱导凋亡的双重作用，有望成为膀胱癌联合基因治疗的新途径。     

穿心莲内酯体外对人胃腺癌BGC823细胞增殖凋亡的影响

目的探讨穿心莲内酯（andrographolide,AD）对人胃癌细胞株BGC-823细胞增殖、细胞周期以及细胞凋亡的影响。方法分别采用MTT法、流式细胞术和流式细胞仪AnnexinV／PI双染色法检测AD对BGC-823细胞体外增殖、细胞周期和细胞凋亡的影响：应用光镜和透射电镜观察不同浓度的AD作用后BGC．823细胞形态学改变。结果各浓度组AD均对人低分化胃癌细胞株BGC-823的增殖有抑制作用。并具有时间和浓度依赖关系（均P〈0．05）。浓度7．5μg／ml以下的AD抑制效果较弱,而15．0-60．0μg／ml抑制效果显著提高（P〈0．05）,60．0μg／ml以上抑制率增高不显著（P〉0．05）。24、48和72h的IC50分别为（35．3±4．3）、（25．5±3．5）和（18．2±2．7）μg／ml。BGC-823细胞经AD作用后,G0／G1期细胞的比例增加,S期和G2/M期细胞的比例下降,细胞被阻滞在G0／G1期,呈浓度依赖关系。AD浓度为7．5、10．0和15．0μg／ml组作用24h后,早期凋亡率分别为（19．3±4．7）％、（29．4±4．1）％和（52．7±6．7）％,晚期凋亡率为（10．8±1．8）％、（10．9±4．7）％和（14．7±4．8）％,均显著高于阴性对照组的早期凋亡率[（3．4±1．0）％]和晚期凋亡率[（4．1±0．7）％],差异有统计学意义（均P〈0．05）,并呈浓度依赖关系。结论AD能抑制BGC-823细胞增殖、阻滞其细胞周期在G0／G1期和诱导其细胞凋亡．是潜在的胃癌抗肿瘤中药制剂成分。     

金雀异黄素与5-氟尿嘧啶对SGC-7901细胞的抑制作用

目的研究金雀异黄素（genistein，Gen）与5-氟尿嘧啶（5-fluorouracil，5-FU）对人胃癌细胞SGC-7901的抑制作用。方法采用MTT法检测Gen和5-FU对SGC-7901细胞的抑制作用，流式细胞术检测细胞周期分布，并在透射电镜下观察细胞超微结构改变。结果Gen和5-Fu单用或联合应用对胃癌SGC-7901细胞的生长均有显著的增殖抑制作用，且呈剂量和时间依赖性。两药联合应用时，当药物效应在0．2～0．8时，具有协同或相加作用（CI≤1）；18．8btmol／L的Gen作用后，62．97％的SGC-7901细胞阻滞在G2／M期；8．84μmol／L的5-FU作用后，63．76％的SGC-7901细胞阻滞在s期；3．06μmol／L的Gen与7．96μmol／L的5-Fu联合应用后，67．46％的SGC-7901细胞阻滞在G0／G1期。Gen和5-FU均可诱导SGC-7901细胞凋亡。结论Gen与5-FU通过阻滞细胞周期和诱导细胞凋亡而抑制胃癌细胞的生长，对胃癌细胞的增殖抑制具有协同作用。     

酸性成纤维细胞生长因子及表皮生长因子对兔韧带细胞增殖的影响

目的：观察酸性成纤维细胞生长因子(aFGF)和表皮生长因子(EGF)对内侧副韧带(MCL)和前十字韧带(ACL)细胞增殖行为的影响。方法：培养10周龄新西兰白兔内侧副韧带和前十字韧带细胞，在培养液中分别加入aFGF和EGF，以XTT方法测定细胞的增殖行为。结果：aFGF在1ng／ml时即对两种细胞具有显著的促进增殖作用，其浓度达50ng／ml时，对MCL细胞的促进作用最大，达100ng／ml时对ACL细胞的促进作用最大。EGF在0．78ng／ml时即对MCL细胞有显著的促增殖作用，在1．56ng／ml时始对ACL细胞有显著的促增殖作用，其浓度达3．125ng／ml时对2种细胞的促进作用最大。aFGF和EGF在超过其最佳浓度后，随浓度升高促进作用均下降。结论：aFGF和EGF可以促进韧带成纤维细胞增殖。     

成纤维细胞生长因子和表皮生长因子及复合因子对兔ACL、MCL体外增殖作用

目的观察酸性成纤维细胞生长因子(acid fibroblast growth factor，aFGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)和表皮生长因子(epidermal growth factor，EGF)以及复合因子对兔前交叉韧带(anterior cruciate ligament，ACL)和内侧副韧带(medial collateral ligament，MCL)的促增殖作用。方法分离传代培养10周龄新西兰大白兔骨关节韧带的ACL和MCL的成纤维细胞，接种96孔板，并加入浓度为0(对照组)、1、5、10、50、100ng／ml的aFGF或bFGF，浓度为0(对照组)、1．56、3．13、6．25、12．50、25、50ng／ml的EGF，单独或aFGF EGF两种因子联用与细胞(n=4)共同培养48h，以XTT方法测定其促细胞增殖作用。结果3种生长因子单独应用对ACL和MCL都有促进作用，aFGF对两种细胞均存在量效关系；bFGF 1ng／ml，EGF 5ng／ml对ACL作用最好，而对MCL则是bFGF和EGF均存在量效关系。浓度为5ng／ml的aFGF与50ng／ml的EGF联合1ng／ml aFGF与3．13ng／mlEGF作用于ACL或MCL均有协同作用。结论3种生长因子对ACL和MCL均有促进作用，单独应用aFGF或联用EGF优于单一因子促进兔ACL和MCL细胞的增殖，并且提示低浓度的aFGF联用EGF优于单一生长因子。     

内皮抑制素抑制内皮细胞增殖的细胞周期调控机制

目的 探讨内皮抑制素(endostatin)抑制内皮细胞增殖的细胞周期调控机制。方法 将ECV-304细胞分为对照组、碱性成纤维细胞生长因子(bFGF，10μg／L)处理组、endostatin(10mg／L)处理组。检测ECV-304体外生长情况及细胞周期分布，观察细胞周期蛋白依赖激酶4(CDK4)、cyclinD1、p21、p27表达。结果 bFGF处理后，ECV-304 G0／G1期细胞由57％减少至37％，而S期、G2／M期细胞增多。加入endostatin后，G0／G1期细胞增多至54％，S期、G2／M期细胞增多；bFGF刺激后，ECV-304细胞内cydinD1、CDK4表达增强，p21、p27表达无明显变化。加入endostatin后，cyclinD1、CDK4表达减弱，p21表达增强，p27无明显表达。结论endostatin可能通过上调p21表达，抑制cyclinD1／CDK4表达，阻止内皮细胞G1→S期进程，从而抑制内皮细胞增殖。     

FK506对许旺细胞体外增殖和分泌NGF的影响

目的 研究FK506促进许旺细胞体外增殖及对许旺细胞(SOs)分泌NGF的影响. 方法 将纯化的许旺细胞分6组:A组(空白对照组):含10%胎牛血清的DMEM/F12;B组:0.1 ng/mlFK506;C组:0.5 ng/ml FK506;D组:1.0 ng/ml FK506;E组:5.0 ng/mk FK506;F组:10 ng/ml FK506.将许旺细胞于倒置显微镜下观察并用S-100蛋白免疫组化染色鉴定;MTT法筛选FK506促SCs增殖的最佳作用浓度;流式细胞仪检测SCs周期;ELISA法检测培养72 h后SCs的NGF的分泌量. 结果 MTT法筛选:0.5 ng/ml FK506为促进SCs增殖的最佳作用浓度;当FK506浓度大于1.0 ng/ml时,SCs的生长活性逐渐下降并随着FK506浓度的逐渐增高,SCs的生长活性受抑制作用逐渐加强.流式细胞仪检测:含10%胎牛血清的DMEM/F12培养24 h、48 h、72 h,SCs S期百分比分别为:27.8%,39.3%和58.4%;0.5 ng/mlFK506培养24 h、48 h、72 h,SCs S期百分比分别为:54.2%、60.3%和94.6%.EUSA法检测FK506促SCs增殖后表达NGF的实验研究中发现:0.5 ng/ml FK506作用72 h后的SCs所分泌的NGF高达0.188 ng/ml. 结论 FK506应用于体外培养的SCs初期就能促进SCs增殖并使其保持良好的活性而高分泌NGF.     

Interleukin-1 beta stimulates human mesangial cells to synthesize and release interleukins-6 and -8.

Interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) have been reported to stimulate human mesangial cells (HMC) to proliferate and synthesize eicosanoids. We have examined whether they also induce HMC to release cytokines. In this study we show that both IL-1 and TNF stimulate HMC to release IL-6 and IL-8. Cycling and quiescent HMC were stimulated with various concentrations of either recombinant IL-1 beta or TNF for 1 to 24 hours. IL-1 beta at doses as low as 6 pg/ml stimulated mesangial cells to synthesize mRNA for both IL-6 and IL-8 as assessed by Northern analysis; mRNA for tubulin remained constant, which demonstrated a specific increase in mRNA. Secretion of IL-6 and IL-8 into the culture medium increased (4.5 to 18 ng/ml and 4 to 40 ng/ml, respectively) measured by ELISAs. TNF had similar effects but only in high concentrations (greater than 100 ng/ml). IL-1 beta did not stimulate cells to proliferate, as measured by 3H thymidine incorporation. TNF caused proliferation but only in concentrations over 100 ng/ml. We conclude that IL-1 beta is a potent stimulator of human mesangial cell production of IL-6 and IL-8, both of which may influence injury in nephritis. TNF also stimulates mesangial cells but only in pharmacological doses.     

曲古菌素A对结肠癌Lovo细胞增殖抑制的研究

目的:研究组蛋白去乙酰化酶抑制剂曲古菌素A（TSA）对结肠癌Lovo细胞增殖活性的抑制作用。方法:采用不同浓度的TSA处理结肠癌Lovo细胞。MTT法检测药物作用前后的细胞增殖情况。流式细胞仪检测TSA处理前后细胞周期的变化。结果:TSA在500ng/mL浓度以上才能明显抑制结肠癌Lovo细胞的增殖，抑制率由第2天至第5天显著增加（57.21%至82.76%）细胞周期检测发现20ng/mL TSA即可导致细胞G1期阻滞，但没有诱导明显的细胞凋亡;≥100ng/mL TSA可诱导明显的细胞凋亡。结论:TSA可以抑制体外结肠癌Lovo细胞的生长，可能通过细胞周期G1期阻滞及诱导细胞凋亡发挥抑癌作用。TSA可能是结肠癌治疗的潜在靶点。     

Human mesangial cells synthesize interleukin 1 alpha but not interleukin 1 beta, interleukin 1 receptor antagonist, or tumour necrosis factor.

There is controversy over whether mesangial cells synthesize and release IL-1 and TNF, and many of the positive experiments were performed before specific reagents and molecular probes were available. Consequently we have stimulated human mesangial cells using protocols known to stimulate the synthesis of other cytokines. No mRNA for IL-1 beta or TNF could be detected in quiescent or proliferating mesangial cells irrespective of whether they had been exposed to cytokines or not. In contrast mRNA for IL-1 alpha was detected in cells stimulated with IL-1 beta 10 ng/ml or with TNF 500 ng/ml; IL-1 alpha was also detected in cell lysates from stimulated mesangial cells. We could not detect mRNA for IL-1 receptor antagonist in any of the cell preparations. These results suggest that mesangial cells are unlikely to be a major source of IL-1 beta or TNF.     

Vascular endothelial growth factor receptors in human mesangium in vitro and in glomerular disease

Mesangial cell proliferation and growth factor over-expression are characteristic features of several glomerular diseases. Vascular endothelial growth factor (VEGF), a potent mitogen, is expressed in podocytes in the glomerulus, and VEGF receptors (flt-1, KDR, and neuropilin-1) are present on endothelial cells and other cell types. This study examined whether human mesangial cells (HMC) express VEGF receptors in vitro and ex vivo and evaluated the effect of VEGF on HMC proliferation. All receptor types were detected in HMC in vitro by immunofluorescence and Western blotting. VEGF(165) induced a dose-responsive increase in (3)H-thymidine incorporation (25 ng/ml VEGF(165) : 2.3-fold increase; 50 ng/ml : 3.8-fold; 100 ng/ml : 4. 8-fold; 200 ng/ml : 3.4-fold; P = 0.016) and in cell number (50 ng/ml VEGF(165) : 1.2-fold increase; 100 ng/ml : 1.6-fold; 200 ng/ml : 1.4-fold; P = 0.005), effects prevented by an anti-VEGF(165) polyclonal neutralizing antibody (100 microg/ml). The proliferative effect was confirmed by a tetrazolium dye-based assay (100 ng/ml VEGF(165) : 1.4-fold increase). In ex vivo experiments, VEGF receptors in biopsy material from normal and diseased kidneys were detected by immunohistochemistry. No mesangial flt-1 receptor staining was seen in normal renal cortical tissue samples, and only weak mesangial KDR staining was detected. In contrast, mesangial flt-1 and KDR receptor staining were both clearly seen in biopsy samples from proliferative renal diseases. In conclusion, flt-1, KDR, and neuropilin-1 are present on cultured HMC, and VEGF(165) induces HMC proliferation. In addition, the flt-1 and KDR receptors are expressed in the mesangium in mesangioproliferative disease.     

Increased IL-6 in supernatant of rat mesangial cell cultures treated with erythrogenic toxin type B and its precursor isolated from nephritogenic streptococci

BACKGROUND/AIMS: Previous reports have shown the presence of streptococcal erythrogenic toxin type B (ETB), IL-8, transforming growth factor-beta (TGF-beta) and glomerular proliferation in renal biopsies from patients with acute poststreptococcal glomerulonephritis (APSGN). In addition, increased levels of plasma IL-6 and tumor necrosis factor-alpha (TNFalpha) and urinary IL-6 have also been reported in this disease. To determine the effect of ETB in mesangial cell cytokine production and proliferation, the concentration of several cytokines (IL-6, IL-1beta, TNFalpha, IL-10, IL-4, RANTES), soluble TNF receptor I (STNFR-I), soluble TNF receptor II (STNFR-II) and proliferation were measured in rat mesangial cells cultures after treatment with ETB or its precursor (ETBP). METHODS: To analyze the levels of cytokines and production of soluble receptors as well as proliferation, rat mesangial cells were cultured with ETB or ETBP (50 microg/ml). After 24, 48 and 96 h of incubation, culture supernatants were assessed for cytokines and receptors by ELISA and for proliferation by incorporation of radioactive thymidine. RESULTS: A significant increase in IL-6 levels was found in mesangial cell cultures treated with either ETBP or ETB when compared with controls. Streptococcal proteins treated mesangial cells also showed elevated levels of proliferation at 96 h. Increased production of IL-6 was not correlated with proliferation. A polyclonal anti-ETB antibody abolished the IL-6 stimulatory effect of ETB on mesangial cells. ETB/ETBP failed to increase the levels of other cytokines and cytokine soluble receptors. CONCLUSION: Streptococcal ETB/ETBP is capable of inducing increased production of IL-6 and proliferation on mesangial cells. These findings could be relevant in a possible early interaction of streptococcal proteins with mesangial cells and during the course of APSGN.     

Inhibitory effects of rosmarinic acid on the proliferation of cultured murine mesangial cells.

BACKGROUND: Rosmarinic acid is a phenolic compound widely distributed in Labiatae herbs such as rosemary, sweet basil, and perilla, which are frequently used with meat and fish dishes in Western and Asian countries. In the present study we investigated the effects of rosmarinic acid on cultured murine mesangial cell proliferation. METHODS: Cultured murine mesangial cells were stimulated by growth factors with or without rosmarinic acid, and [(3)H]thymidine incorporation was measured in regard both to signal transduction and to cell cycle dependency. In other experiments, mRNA extracted from the cells was analysed by Northern blotting. RESULTS: Rosmarinic acid inhibited the cell proliferation induced by platelet-derived growth factor (PDGF) (P<0.01; IC(50) values, 1.4 microg/ml) or tumour necrosis factor-alpha (P<0.01; IC(50) values, 3. 8 microg/ml), and these effects involved both the G(0)/G(1) and G(1)/S phases of the cell cycle. Rosmarinic acid also suppressed the mRNA expressions of PDGF and c-myc in PDGF-stimulated mesangial cells. CONCLUSIONS: Rosmarinic acid inhibits cytokine-induced mesangial cell proliferation and suppresses PDGF and c-myc mRNA expression in PDGF-stimulated mesangial cells. Rosmarinic acid in Labiatae herbs might be a promising agent to prevent mesangial cell proliferation.     

Broad antiproliferative effects of benidipine on cultured human mesangial cells in cell cycle phases

BACKGROUND AND PURPOSE: Recently, in vitro studies have shown that some calcium channel blockers inhibit the proliferation of mesangial cells. In the present study, we evaluated the antiproliferative effects of benidipine, a calcium channel blocker, in comparison with other calcium channel blockers, and attempted to further clarify its mechanism of action on cultured human mesangial cells in relation to cell cycle. METHODS: Human mesangial cells were cultured in medium containing 5% fetal calf serum (FCS), with or without benidipine, or other calcium channel blockers for 20 h, and [(3)H]thymidine incorporation were measured. Analysis of cell cycle dependency was carried out, using platelet-derived growth factor as a competence factor, which transfers cells from the G0 to the G1 (G0/G1) phase, and insulin as a progression factor, which transfers cells from the G1 to the S (G1/S) phase. Cells were also analyzed by flow cytometry. RESULTS: Benidipine and nifedipine showed significant inhibitory effects on FCS-induced proliferation (p < 0.001 and p < 0.01, respectively, by ANOVA), with 3.8 and 41.9% of the control level of [(3)H]thymidine incorporation at the concentration of 10 microM of the blockers, respectively. Diltiazem had no inhibitory effect at this concentration. Benidipine was found to inhibit cells in both the boundaries of G0/G1 and G1/S phases (p < 0.001 and p < 0.0001, respectively), whereas nifedipine inhibited only cells in G1/S phase (p < 0.05). The effects of benidipine (10 microM) on cells in G1/S were stronger than those on cells in the G0/G1 phase (p < 0.0001). Furthermore, flow cytometry showed that 10 mM benidipine significantly inhibited the G1 to S phase transition of FCS-stimulated mesangial cells (p < 0.03). CONCLUSIONS: Benidipine markedly inhibited the proliferation of mesangial cells, at both the G0/G1 and G1/S phases, and it might be effective to suppress the progression of mesangioproliferative glomerular diseases.