骨髓间充质干细胞共培养对肝星状细胞增殖、凋亡和RohA表达的调控

目的:观察体外大鼠骨髓间充质干细胞(BMSCs)共培养对肝星状细胞(HSCs)增殖、凋亡和RohA表达的影响,探讨BMSCs旁分泌HGF在其中的作用机制.方法:贴壁筛选法培养、纯化SD大鼠BMSCs,传代至第4代使用;大鼠肝星状细胞(HSC-T6)系及纤维原细胞系冻融后传代使用.应用6孔塑料细胞培养盒,每孔使用半透膜(transwell insert)建立上下双层细胞共培养体系,常规培养.实验分4组:空白对照组、阴性对照组、BMSCs实验组、预处理实验组(c-met多克隆抗体预处理).用四甲基偶氮唑蓝(MTT)法检测HSCs细胞增殖能力;流式细胞仪检测细胞凋亡;RT-PCR、Westernblot检测BMSCs与HSCs共培养后HSCs内RohAmRNA和蛋白的表达.酶联免疫吸附法(ELISA)检测BMSCs与HSCs共培养上清液中肝细胞生长因子(HGF)浓度.结果:BMSCs对HSCs增殖具有抑制作用,BMSCs与HSCs共培养后24h、48h的增殖抑制率分别为12.21%,35.43%,与空白对照组、实验对照组和C-met抗体预处理组比较有显著性差异(P<0.01).Annexin-V-FITC/PI双染法检测BMSCs与HSCs共培养48h后HSCs的凋亡率为25.80%,与空白对照组、实验对照组与c-met抗体预处理组比较有显著性差异(P<0.01).BMSCs与HSCs共培养48h,BMSCs组RohAmRNA的表达抑制明显,且显著低于空白对照组、实验对照组与C-met抗体预处理组,有显著性差异(P<0.01).BMSCs与HSCs共培养48hRohA蛋白的表达明显抑制,且显著低于空白对照组、实验对照组与C-met抗体预处理组,有显著性差异(P<0.01).ELISA检测BMSCs与HSCs共培养24h、48h上清液中HGF浓度分别为250ng/L与570ng/L,明显高于单独BMSCs培养和单独HSCs培养,有显著性差异(P<0.01).结论:BMSCs与HSCs共培养能抑制HSCs的增殖,促进凋亡,抑制RohA表达,其机制可能是通过BMSCs旁分泌HGF发挥抑制大鼠HSCs增殖,促进凋亡的作用.

体外培养大鼠胰岛细胞凋亡的实验研究

目的 研究体外培养条件下大鼠胰岛凋亡发生及其机制。方法 采用网状纤维染色观察大鼠胰岛分离前后网状纤维分布情况 ;Hoechst 332 5 8荧光染色法 ,透射电镜法测定培养 1,3 ,7,14,2 1d胰岛细胞凋亡率 ;放射免疫法测定胰岛素 2 4h累积分泌量 ;MTT比色法间接测定细胞存活率 ;免疫细胞化学ABC法评价Fas,Fas配体 (Fas-L)阳性蛋白表达率。结果 大鼠分离后的胰岛网状纤维消失。培养 3d后 ,电镜观察可见胰岛细胞核皱缩 ,染色质边集于核膜处等典型的凋亡表现。培养 3d ,胰岛凋亡率为 (7.6± 5 .8) % ;随培养时间延长 ,胰岛凋亡率逐渐增加 ,培养 14,2 1d ,胰岛凋亡率分别为 (6 3.0± 2 .6 ) % ,(4 7.2± 8.1) %。免疫细胞化学检测Cateninβ表达培养 7d消失 ;Fas阳性蛋白表达率从培养 3d时 (8.1± 1.8) %上调至 14d ,2 1d时 (38.5± 4.7) % ,(35 .6±6 .5 ) % ;Fas -L在培养 3d前无表达 ,培养 7d为 (6 .8± 3 .2 ) % ,14d ,2 1d显著上调 ,分别达到 (19.6± 4.8) % ,(12 .4± 7.1) %。同时 ,胰岛素 2 4h累积分泌量 ,细胞存活率逐渐下降。结论 大鼠胰岛在体外培养条件下易发生凋亡 ;Fas,Fas-L可能参与胰岛凋亡事件的发生和发展。

人牙周膜成纤维细胞的培养及牵张应变诱导凋亡研究

目的:探讨人牙周膜成纤维细胞的分离、培养及在牵张应变作用下发生早期凋亡的情况。方法:刮取健康人前磨牙牙周膜,进行牙周膜成纤维细胞的分离、培养及生长曲线描绘。经3～4代传代培养后,对细胞加载1%～20%的牵张应变,加载时间分别为30min,1h、6h、12h然后通过流式细胞仪检测Annexin V-FITC(异硫氰酸荧光素)及激光扫描共聚焦显微镜进行细胞的早期凋亡鉴定。结果:人牙周膜成纤维细胞传代到12代以后,细胞逐渐呈衰老生状,胞体变大,细胞生长缓慢。人牙周膜成纤维细胞的凋亡率在30min组低于对照组,但两者间无统计学差异。在6h内,人牙周膜成纤维细胞的凋亡情况随加载时间和加载应力的增加而增加(P<0.05)。在12h时所有组的细胞凋亡均减少。结论:人牙周膜成纤维细胞的使用最好在12代以内,尤其是3～4代细胞的活力、增殖能力较好。牵张应变可以诱导细胞发生早期凋亡。

2-甲氧雌二醇诱导人子宫内膜癌细胞株凋亡的实验研究

目的:探讨2-甲氧雌二醇(2-ME)对人子宫内膜癌细胞株KLE细胞凋亡的影响。方法:选用人子宫内膜癌细胞株KLE进行体外培养,实验组加入不同浓度2-ME,对照组不含2-ME。用电子显微镜(电镜)观察细胞形态变化;流式细胞仪(FCM)观察细胞的凋亡率及细胞周期的变化,以及免疫组织化学方法检测P53和Bcl-2的表达强度。结果:2-ME作用后G0/G1期细胞增加,并伴随G0/G1期细胞的增加,出现细胞凋亡峰和凋亡率的升高(P<0.05)。电镜下观察到KLE细胞染色体边集、核固缩、凋亡小体,P53和bc1-2阳性表达率明显下降。结论:2-ME对人子宫内膜癌KLE细胞株有诱导凋亡作用。

大气细颗粒物PM_(2.5)对人微血管内皮细胞坏死性凋亡的影响

[目的]观察大气细颗粒物PM_(2.5)对体外培养血管内皮细胞坏死性凋亡的影响。[方法]以人微血管内皮细胞株(HMEC-1)为研究对象,应用系列浓度PM_(2.5)处理24 h后,CCK-8检测HMEC-1细胞活力,试剂盒法检测培养上清液乳酸脱氢酶(LDH)的活性,Western blot法检测坏死性凋亡相关蛋白RIPK3和MLKL的表达及磷酸化水平,Transwell法检测HMEC-1细胞通透性改变。采用坏死性凋亡抑制剂(Ne-crostatin-1)处理后,观察PM_(2.5)对HMEC-1坏死性凋亡、细胞损伤和通透性的影响。[结果]PM_(2.5)可呈剂量依赖性抑制HMEC-1的增殖,并增加培养上清液中LDH活性、RIPK3和MLKL的蛋白表达以及其磷酸化水平。Transwell检测结果表明PM_(2.5)可增加单层血管内皮细胞通透性,而坏死性凋亡抑制剂Necrostatin-1预处理可显著性抑制PM_(2.5)应激下血管内皮细胞RIPK3和MLKL的蛋白表达及磷酸化水平,降低上清液LDH活性和单层血管内皮细胞通透性。[结论]PM_(2.5)可诱导HMEC-1坏死性凋亡,损伤血管内皮完整性。

Cell Apoptosis Induced by Oligosaccharide in Suspension Cultures of Taxus chinensis

Taxol production of Taxus chinensis (Pilger) Rehd. var. mairei (Lemee et Lévl.) Cheng et L. K. Fu induced by oligosaccharide from Fusarium oxysporum f. vasinfectum (Atkinson) Snyder et Hansen was studied in suspension cultures, and it was found that oligosaccharide triggered cell apoptosis. Under transmission electron microscope the following morphological changes were observed: cell shrinkage, condensation of cytoplasm, nuclear fragmentation, and the increase of high electron density bodies in vacuole in great quantity. In oligosaccharide_treated cells, agarose gel electrophoresis revealed that DNA was digested into oligonucleosomal fragments that were times of 200 bp appearing as DNA ladders. Control cells were in normal physiological state, they were intact, abundant in organelle and with integral nucleus DNA, and the rate of taxol biosynthesis in these cells was very low. After the oligosaccharide to the culture system, the defense system of cells was elicited and the secondary metabolism was strengthened, i.e. phenolics were accumulated in the medium, the activity of polyphenol oxidase (PPO) was increased quickly and secondary wall of cells was thickened. The activity of L _phenylalanine ammonia lyase (PAL), the critical enzyme of the phenylpropanoid pathway, was increased promptly 1 h after elicitation. The rate of taxol production was improved sharply and the maximal taxol concentration at 72 h was six times that of control. Appearance of cell apoptosis was accompanied with the highest concentration of taxol in suspension cultures.

Effect of Coriolus Versicolor Polysaccharide-B on the Biological Characteristics of Human Esophageal Carcinoma Cell Line Eca109

Objective To investigate the effect of Coriolus versicolor polysaccharide-B （CVPs-B） on the biological characteristics of human esophageal carcinoma cell line Ecal09 in vitro. Methods The cells of experimental group （EG） were cultured in DMEM with 10% FCS and 150μg/mL CVPs-B, the cells of control group （CG） were cultured in DMEM with 10% FCS without CVPs-B. MTT reduction assay was performed to detect the effect of CVPs-B on the proliferation of Ecal09 cells after the compound was administrated in varying concentrations. The living conditions of the Ecal09 cells were determined using trypan blue exclusion. Then, cell growth curves were drawn. Flow cytometry was performed to detect the effect of CVPs-B on the apoptosis and cell cycle of Ecal09. Results In comparison with the CG, a marked decrease in the proliferation of Eca09 cells was observed in the EG, after incubation with CVPs-B. The survival rate of Eca09 cells decreased as the time of CVPs-B incubation prolonged. Comparing the cell cycles and apoptotic rates between the two groups, the proportions of cells in the G0/G1, S, and G2/M phases in the EG were found to be （68.4±3.7）%, （13.9±2.1）%, and （17.7±1.4）%, respectively, after 24 h incubation with CVPs-B. The cells had an apoptotic rate of （9.7±0.7）%. On the other hand, the proportions of the G0/G1, S, and G2/M cells of the CG were found to be （53.9±3.6）%, （26.6±2.8）%, and （19.5±2.3）%, respectively, with an apoptotic rate of （5.7±1.4）%. In comparison with the CG cells, significant cell growth in the G0/G1 phase was observed in the EG （P〈0.05）. Furthermore, a significant decrease in the number of cells in the S phase was observed （P〈0.05） in the EG. Conclusions CVPs-B can inhibit proliferation and enhance apoptosis of Ecal09 cells and may be useful in the treatment of esophageal carcinoma.

Mechanism of induction apoptosis of Onychin in ovarian cells in vitro

Objective： The aim of the study was to investigate the possible mechanism of induction apoptosis of Onychin （ONY） in ovarian cancer HO-8910 cells in vitro. Methods： Human ovarian cancer HO-8910 cells were cultured in vitro. Inhibi- tory effect of ONY on the viability of HO-8910 cells was evaluated by the MTT assay. Apoptosis of HO-8910 cells treated with different concentrations of ONY for 48 h was detected by FCM. Expression of proteins related to apoptosis was analyzed by Western blot. Results： ONY significantly inhibited the viability of human ovarian cancer HO-8910 cells in a dose-dependent and time-dependent manner, and the ICso was 10.48 pg/mL for 48 h. The cells treated with ONY showed typical morphological change of apoptosis and increased cells of sub-G1 population by FCM in a dose-dependent. Western blot showed that ex- pression of Bax, cytochrome C, caspase-9 and caspase-3 proteins were upregulated and protein level of Bcl-2 was depressed after treatment with ONY in a concentration dependent. Conclusion： Apoptosis of ovarian cancer HO-8910 cells was induced by ONY through mitochondrial apoptosis pathway in vitro.

Induction of apoptosis of human ovarian cancer cells by SGI-1776 combination with DDP in sub-toxic concentration in vitro

Objective： The aim of the study was to investigate the effect of SG1-1776 combination with DDP in sub-toxic concentration on induction of apoptosis of human ovarian cancer HO-8910 ceiis in vitro and to unravei the associated mechanisms. Methods： Human ovarian cancer HO-8910 cells were cultured in vitro. The inhibitory effect of SG1-1776 combination with DDP in sub-toxic concentration on induction on viability of human ovarian cancer HO-8910 cells was evaiuated by the MTT assay. Cell apoptosis rate was analyzed by flow cytometry. The proteins expression level related to apoptosis were analyzed by Western blot. Results： SG1-1776 combination with DDP in sub-toxic concentration significantiy inhibited the proliferation of human ovarian cancer HO-8910 cells, and proliferation inhibition rate was increased drastically compared with normai saline （NS） group or DDP group in sub-toxic concentration or SG1-1776 group in sub-toxic concentration （P 〈 0.01）. Apoptosis rate markedly increased after the treatment of SG1-1776 combination with DDP in sub-toxic concentration for 48 h. Western blot showed that the expression of bcl-2 protein was down-regulated and protein level of Bax and Cyto-c were depressed by SG1-1776 combination with DDP in sub-toxic concentration. Cenclusion： SG1-1776 combination with DDP in sub-toxic concentration could inhibit the cell proliferation and lead to cell apoptosis inhuman ovarian cancer HO-8910 cells, and its mechanism may be related to through mitochondrial apoptotic pathway.