Effects of neurotrophin-3 intervention on on bone marrow mesenchymal stem cell osteoblast differentiation as well as cell proliferation and apoptosis

Objective:To study the effects of neurotrophin-3 (NT-3) intervention on bone marrow mesenchymal stem cell osteoblast differentiation as well as cell proliferation and apoptosis. Methods: Bone marrow mesenchymal stem cells were cultured and divided into control group, 25 ng/mL NT-3 group, 50 ng/mL NT-3 group and 100 ng/mL NT-3 group, they were treated with different doses of NT-3 for 24 h, and then osteoblast marker gene, cell proliferation gene and apoptosis gene expression were determined.Results: RUNX2, Osterix, ALP, OCN, BMP-2, Bcl-2, Nrf2, ERK1/2 and PCNA mRNA expression in 25 ng/mL NT-3 group, 50 ng/mL NT-3 group and 100 ng/mL NT-3 group were significantly higher than those in control group whereas Bim, Bax, Caspase-3, CHOP and Beclin1 mRNA expression were significantly lower than those in control group, and the larger the dose of NT-3, the higher the RUNX2, Osterix, ALP, OCN, BMP-2, Bcl-2, Nrf2, ERK1/2 and PCNA mRNA expression whereas the lower the Bim, Bax, Caspase-3, CHOP and Beclin1 mRNA expression.Conclusion: NT-3 intervention in bone marrow mesenchymal stem cells can promote osteoblast differentiation and cell proliferation and inhibit apoptosis.

Propofol and remifentanil at moderate and high concentrations affect proliferation and differentiation of neural stem/progenitor cells

Propofol and remifentanil alter intracellular Ca^2＋ concentration （[Ca^2＋]i） in neural stem/progen-itor cells by activating γ-aminobutyric acid type A receptors and by reducing testosterone levels. However, whether this process affects neural stem/progenitor cell proliferation and differenti-ation remains unknown. In the present study, we applied propofol and remifentanil, alone or in combination, at low, moderate or high concentrations （1, 2–2.5 and 4–5 times the clinically effective blood drug concentration）, to neural stem/progenitor cells from the hippocampi of newborn rat pups. Low concentrations of propofol, remifentanil or both had no noticeable effect on cell proliferation or differentiation; however, moderate and high concentrations of propofol and/or remifentanil markedly suppressed neural stem/progenitor cell proliferation and differen-tiation, and induced a decrease in [Ca^2＋]i during the initial stage of neural stem/progenitor cell differentiation. We therefore propose that propofol and remifentanil interfere with the prolifer-ation and differentiation of neural stem/progenitor cells by altering [Ca^2＋]i. Our ifndings suggest that propofol and/or remifentanil should be used with caution in pediatric anesthesia.

miR-103-3p targets Ndel1 to regulate neural stem cell proliferation and differentiation

The regulation of adult neural stem cells(NSCs) is critical for lifelong neurogenesis. MicroRNAs(miRNAs) are a type of small, endogenous RNAs that regulate gene expression post-transcriptionally and influence signaling networks responsible for several cellular processes. In this study, mi R-103-3 p was transfected into neural stem cells derived from embryonic hippocampal neural stem cells. The results showed that mi R-103-3 p suppressed neural stem cell proliferation and differentiation, and promoted apoptosis. In addition, mi R-103-3 p negatively regulated Nud E neurodevelopment protein 1-like 1(Ndel1) expression by binding to the 3′ untranslated region of Ndel1. Transduction of neural stem cells with a lentiviral vector overexpressing Ndel1 significantly increased cell proliferation and differentiation, decreased neural stem cell apoptosis, and decreased protein expression levels of Wnt3 a, β-catenin, phosphor-GSK-3β, LEF1, c-myc, c-Jun, and cyclin D1, all members of the Wnt/β-catenin signaling pathway. These findings suggest that Ndel1 is a novel mi R-103-3 p target and that mi R-103-3 p acts by suppressing neural stem cell proliferation and promoting apoptosis and differentiation. This study was approved by the Animal Ethics Committee of Nantong University, China(approval No. 20200826-003) on August 26, 2020.

Cerebral dopamine neurotrophic factor promotes the proliferation and differentiation of neural stem cells in hypoxic environments

Previous research found that cerebral dopamine neurotrophic factor(CDNF)has a protective effect on brain dopaminergic neurons,and CDNF is regarded as a promising therapeutic agent for neurodegenerative diseases.However,the effects of CDNF on the proliferation,differentiation,and apoptosis of neural stem cells(NSCs),which are very sensitive to hypoxic environments,remain unknown.In this study,NSCs were extracted from the hippocampi of fetal rats and cultured with different concentrations of CDNF.The results showed that 200 nM CDNF was the optimal concentration for significantly increasing the viability of NSCs under non-hypoxic environmental conditions.Then,the cells were cultured with 200 nM CDNF under the hypoxic conditions of 90%N_2,5%CO_2,and 5%air for 6 hours.The results showed that CDNF significantly improved the viability of hypoxic NSCs and reduced apoptosis among hypoxic NSCs.The detection of markers showed that CDNF increased the differentiation of hypoxic NSCs into neurons and astrocytes.CDNF also reduced the expression level of Lin28 protein and increased the expression of Let-7 mRNA in NSCs,under hypoxic conditions.In conclusion,we determined that CDNF was able to reverse the adverse proliferation,differentiation,and apoptosis effects that normally affect NSCs in a hypoxic environment.Furthermore,the Lin28/Let-7 pathway may be involved in this regulated function of CDNF.The present study was approved by the Laboratory Animal Centre of Southeast University,China(approval No.20180924006)on September 24,2018.

Effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro

Objective To observe the effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro.Methods Iliac trabecular bone specimens were obtained from adult patients undergoing necessary surgery. After the bone pieces were digested with collagenase-trypsin, osteoblasts were released and incubated at 37℃in a relative humidity of 95% and 5% CO2. Then, the cells were purified, and their passages were given DMEM-F12 and fetal bovine serum medium. Subsequently, 10^(-8) mol/L dexamethasone was added into the culture medium to incubate the osteoblasts for three days, and the cells from control groups were incubated without any drugs. All cells were observed continually with phase contrast microscope and transmission electron microscope. Finally, apoptosis was detected by the use of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and biochemical indices, alkaline phosphatase (ALP) and osteocalcin (OCN) were used to determine the effects of dexamethasone on proliferation, differentiation and apoptosis of adult osteoblasts in vitro.Results In the adult osteoblasts obtained by collagenase-trypsin digestion, it achieved high survial, stable biochemical indices and excellent purification. Under the condition of dexamethasone 10^(-8) mol/L and osteoblasts 10 000/ml, there was significant promotion of ALP and OCN secretion without cell apoptosis.Conclusions Dexamethasone has a significant effect on the proliferation and differentiation of adult osteoblasts in vitro without apoptosis, and dexamethasone at the suggested concentration can be used as positive control in drug studies for osteoporosis treatment.

Proliferation and adipogenic differentiation of human adipose-derived stem cells isolated from middle-aged patients with prominent orbital fat in the lower eyelids

Aim:To evaluate the age-related effects on the adipogenic differentiation and proliferation potentials of human orbital adipose-derived stem cells(OASCs).Methods:Orbital adipose samples were harvested from the central fat compartment in the lower eyelids of 10 young and middle-aged patients during routine blepharoplasty surgery.After assessment of the morphological changes of adipocytes with aging,OASCs were isolated from the fat samples and expanded in vitro.Differences in the stem cell colony number(fibroblast colony-forming unit),growth rate and phenotype characterization(flow cytometry analysis)were evaluated.The ability of OASCs to differentiate into adipocytes was determined by oil red O staining and the mRNA expression level of peroxisome proliferator-activated receptorγ.Results:Fat cell size showed a decreasing trend with advancing age.Although no difference was found in the expression of cell surface markers,the colony number and proliferative rate of OASCs from middle-aged donors were significantly lower than those from the young donors.The adipogenic differentiation capacity of middle-aged OASCs was also reduced.These differences were statistically significant(P<0.001).Conclusion:The data showed that the progenitor cell number,proliferation capacity and adipogenic potential of OASCs decreased with aging,suggesting that using OASCs from elderly patients for therapeutic purposes might be restricted.

Effect of soy saponin on the growth of human colon cancer cells

AIM:To investigate the effect of extracted soybean saponins on the growth of human colon cancer cells.METHODS:WiDr human colon cancer cells were treated with 150,300,600 or 1200 ppm of soy saponin to determine the effect on cell growth,cell morphology,alkaline phosphatase(AP) and protein kinase C(PKC) activities,and P53 protein,c-Fos and c-Jun gene expression.RESULTS:Soy saponin decreased the number of viable cells in a dose-dependent manner and suppressed 12-Otetradecanol-phorbol-13-acetate-stimulated PKC activity(P < 0.05).Cells treated with saponins developed cytoplasmic vesicles and the cell membrane became rougher and more irregular in a dose-dependent manner,and eventually disassembled.At 600 and 1200 ppm,the activity of AP was increased(P < 0.05).However,the apoptosis markers such as c-Jun and c-Fos were not significantly affected by saponin.CONCLUSION:Soy saponin may be effective in preventing colon cancer by affecting cell morphology,cell proliferation enzymes,and cell growth.

Novel nanometer scaffolds regulate the biological behaviors of neural stem cells

Ideal tissue-engineered scaffold materials regulate proliferation, apoptosis and differentiation of cells seeded on them by regulating gene expression. In this study, aligned and randomly oriented collagen nanofiber scaffolds were prepared using electronic spinning technology. Their diameters and appearance reached the standards of tissue-engineered nanometer scaffolds. The nanofiber scaffolds were characterized by a high swelling ratio, high porosity and good mechanical properties. The proliferation of spinal cord-derived neural stem cells on novel nanofiber scaffolds was obviously enhanced. The proportions of cells in the S and G2/M phases noticeably increased. Moreover, the proliferation rate of neural stem cells on the aligned collagen nanofiber scaffolds was high. The expression levels of cyclin D1 and cyclin-dependent kinase 2 were increased. Bcl-2 expression was significantly increased, but Bax and caspase-3 gene expressions were obviously decreased. There was no significant difference in the differentiation of neural stem cells into neurons on aligned and randomly oriented collagen nanofiber scaffolds. These results indicate that novel nanofiber scaffolds could promote the proliferation of spinal cord-derived neural stem cells and inhibit apoptosis without inducing differentiation. Nanofiber scaffolds regulate apoptosis and proliferation in neural stem cells by altering gene expression.

Granulosa cell proliferation differentiation and its role in follicular development

Granulosa cells (GCs) are the most important cells in the ovary that undergo serious changes morphologi- cally and physiologically during the processes of follicular proliferation, differentiation, ovulation, lutenization and atresia. Oocyte (OC) directs GC proliferation and differen- tiation, while GCs influence OC maturation. Many ovarian factors are involved in the regulation of these processes via different molecular mechanisms and signal pathways. P38 MAPK can selectively regulate steroidogenesis in GCs con- trolled by FSH; Transcript factors LRH-1 and DAX-1 play an important role in this process; FSH induces GC prolifera- tion and differentiation by stimulating PCNA and StAR ex- pression and steroidogenesis. Activated ERK1/2 signal pathway may be involved in the FSH-regulated GC prolif- eration and differentiation. Therefore,GC is an ideal model for studying cell proliferation, differentiation and interaction as well as signal transduction. This review briefly summa- rizes the latest data in the literature, including the results achieved in our laboratory.

罗哌卡因对大鼠脂肪干细胞增殖、凋亡及成骨分化的影响观察

目的观察罗哌卡因对大鼠脂肪干细胞(ADSCs)增殖、凋亡及成骨分化的影响。方法分离并提取大鼠ADSCs,传代培养后,取对数生长期ADSCs分别加入含0 mg/mL、200 mg/mL、500 mg/mL罗哌卡因的培养基100µL,分别记为对照组、200 mg/mL组、500 mg/mL组。取各组细胞,继续培养24、48、72 h时,采用CCK-8实验观察细胞增殖能力。取各组细胞,继续培养24 h,使用流式细胞仪检测细胞凋亡情况。取各组细胞,加入成骨诱导培养基,观察各组细胞成骨分化能力,包括成骨潜能(以ALP活性表示)、矿化能力(以OD值表示)和成骨相关蛋白骨钙素(OCN)表达水平。结果与对照组相比,200 mg/mL组、500 mg/mL组细胞培养24、48、72 h时的增殖能力均显著下降,且500 mg/mL组低于200 mg/mL组。对照组、200 mg/mL组、500 mg/mL组细胞凋亡率分别为2.78%±0.23%、4.15%±0.62%、5.88%±0.78%,组间相比,P均<0.05。与对照组相比,200 mg/mL组、500 mg/mL组细胞ALP活性、矿化能力、OCN蛋白表达水平均显著下降,且500 mg/mL组低于200 mg/mL组。结论200 mg/mL、500 mg/mL的罗哌卡因均可抑制大鼠ADSCs增殖能力,降低其成骨能力,促进其凋亡,且500 mg/mL罗哌卡因的抑制效果高于200 mg/mL。

玉米赤霉烯酮对奶牛乳腺上皮细胞生长和乳脂合成相关基因表达的影响

旨在探索不同浓度玉米赤霉烯酮(ZEN)对奶牛乳腺上皮细胞(MAC-T)生长和乳脂合成相关基因表达的影响。首先,用不同剂量ZEN处理MAC-T细胞36 h,血细胞计数板统计细胞数量,染色并分析细胞凋亡与坏死情况,筛选ZEN添加的合适剂量;接着,试剂盒检测ZEN对MAC-T细胞中活性氧(ROS)以及线粒体膜电位的影响;最后,利用实时荧光定量PCR(RT-qPCR)技术,分析ZEN对MAC-T细胞增殖、凋亡、氧化应激和乳脂合成相关基因表达的影响。结果表明,低剂量ZEN(0.01~1.00μmol/L)有促进MAC-T生长的趋势,而高剂量ZEN(5.00~10.00μmol/L)显著降低MAC-T细胞数量;0.10μmol/L ZEN对MAC-T中ROS和线粒体数量无明显影响,但10.00μmol/L ZEN显著增加了MAC-T中ROS水平,降低了线粒体数量;RT-qPCR结果表明,0.10μmol/L ZEN显著促进增殖基因(CDK1、CCND2)、抗氧化基因(DHODH、GPX4)和抗凋亡基因(BCL-2)表达,但同时提高了凋亡基因(CAS-3、BAX)表达量;10.00μmol/L ZEN显著抑制增殖基因(PCNA、CDK1、CCND2)、抗氧化基因(DHODH、GPX4、AIFM2)和抗凋亡基因(BCL-2)表达,但显著促进凋亡基因(CAS-3、BAX)表达;值得注意的是,0.10μmol/L ZEN显著促进了乳脂合成相关基因(PPARγ、FASN、JAK-2),但10.00μmol/L ZEN显著抑制了这些基因表达。上述结果提示,不同浓度ZEN对MAC-T细胞的作用存在差异:0.10μmol/L ZEN可以促进MAC-T细胞生长和乳脂合成相关基因表达的同时,也会诱导凋亡基因表达;10.00μmol/L ZEN会诱导MAC-T细胞氧化应激、降低线粒体数量,抑制增殖、抗氧化、抗凋亡和乳脂合成相关基因表达,同时促进凋亡基因表达,导致细胞死亡。

LETM1对骨肉瘤细胞恶性生物学行为的作用及其机制

目的 探讨亮氨酸拉链-EF-hand跨膜蛋白1(LETM1)对人骨肉瘤MG63及143B细胞增殖、迁移、凋亡、成骨分化及体内成瘤的作用及其机制。方法 将骨肉瘤MG63及143B细胞分为空白对照组(未加入腺病毒感染)、阴性对照组(sh-NC组,用RNAi阴性对照病毒感染)及LETM1敲低组(sh-LETM1组,用sh-LETM1腺病毒感染)。采用Western blotting检测各组LETM1蛋白在健康人成骨细胞hFOB1.19及骨肉瘤细胞中的表达水平,以验证腺病毒敲低效果;细胞克隆形成实验及CCK-8法检测细胞增殖情况;划痕愈合实验及Transwell实验检测细胞迁移情况;DAPI染色法及Annexin V-APC/7-AAD流式细胞术双染法检测细胞凋亡情况;碱性磷酸酶(ALP)染色及茜素红S染色检测细胞的早期及晚期成骨分化情况。将10只裸鼠分为sh-NC组(n=5,以RNAi阴性对照病毒感染的143B细胞注入裸鼠皮下)与sh-LETM1组(n=5,以sh-LETM1腺病毒感染的143B细胞注入裸鼠皮下),并采用裸鼠皮下成瘤实验检测各组细胞的体内成瘤能力。结果 Western blotting检测结果显示,LETM1蛋白在骨肉瘤MG63及143B细胞中的表达明显高于健康人成骨细胞hFOB1.19(P<0.05),sh-LETM1腺病毒感染MG63及143B细胞后明显降低了LETM1蛋白的表达。细胞克隆形成实验及CCK-8法检测结果显示,在MG63及143B骨肉瘤细胞中,sh-LETM1组较sh-NC组及空白对照组的克隆形成能力及增殖能力明显降低(P<0.01)。Wound-healing实验及Transwell实验结果显示,在MG63及143B骨肉瘤细胞中,sh-LETM1组较sh-NC组及空白对照组的细胞迁移率明显降低(P<0.01)。DAPI染色及流式细胞术结果显示,在MG63及143B骨肉瘤细胞中,sh-LETM1组较sh-NC组的细胞凋亡率明显增高(P<0.01)。ALP染色及茜素红S染色实验结果显示,在MG63及143B骨肉瘤细胞中,sh-LETM1组较sh-NC组及空白对照组染色区域及钙盐结节增多。裸鼠皮下成瘤实验结果显示,143B sh-LETM1组较143B sh-NC组细胞的皮下成瘤能力降低。结论 LETM1可促进MG63和143B骨肉瘤细胞的增殖、迁移及体内成瘤,其机制可能与抑制细胞凋亡及成骨分化有关。

DDX5对白血病K562细胞生物学功能的影响及其机制

目的探讨DDX5解螺旋酶对白血病细胞系K562细胞增殖、凋亡和分化的影响。方法利用癌症基因数据库GEPIA中的数据分析DDX5 mRNA在白血病患者组织中的表达;生存曲线分析DDX5 mRNA表达水平与白血病患者预后关系;采用小干扰RNA(siRNA)瞬时转染K562细胞以敲低DDX5;使用实时荧光定量(RT-PCR)检测沉默效果;采用Western blot检测蛋白表达水平;采用CCK-8实验检查细胞增殖能力、采用Western blot和免疫荧光检测细胞增殖相关蛋白的表达水平;采用流式细胞术检测细胞凋亡;采用流式细胞术和Western blot检测细胞分化相关蛋白的表达水平。最后,通过RT-PCR和Western blot检测沉默DDX5对P21和P53蛋白表达水平的影响。结果GEPIA数据库分析结果显示DDX5在人白血病骨髓组织中的表达水平显著高于健康人群,低表达DDX5的患者具有更长的生存期。体外实验表明敲低DDX5可显著抑制K562细胞的增殖,流式细胞术检测结果表明可以促进细胞凋亡、促进CD11b和CD14蛋白的表达诱导细胞分化。沉默DDX5促进P21和P53蛋白的表达水平。结论DDX5可能通过靶向P53抑制白血病细胞系K562细胞增殖,促进凋亡诱导分化。

镍钛泡沫合金对成骨细胞生物活性的影响

目的观察镍钛泡沫合金(PNT)在体外实验对细胞的生物安全性。方法选择PNT和镍钛致密合金(NiTi)材料(中南大学材料科学工程学院提供),以及纯钛(Ti)(宁波慈北医疗器械有限公司,中国);MC3T3-E1来源于中国科学院典型培养物保藏委员会细胞库。用PNT、NiTi、Ti 3种合金的浸提液,培养小鼠前成骨细胞MC3T3-E1。用CCK-8法检测细胞的增殖活性,碱性磷酸酶法测定细胞的分化能力,流式细胞术检测细胞早期凋亡情况。结果PNT组细胞增殖能力在第1天、第3天、第7天均明显小于NiTi组(P<0.001)和Ti组(P<0.001)。PNT组细胞分化能力在第1天、第3天、第7天均明显小于NiTi组(P<0.001)和Ti组(P<0.001)。PNT组细胞的凋亡细胞百分比在第1天、第3天、第7天均明显高于NiTi组细胞(P=0.001)和Ti组细胞(P<0.001)。结论PNT对细胞的增殖和分化的影响明显大于NiTi组和Ti组,更明显地抑制了细胞的增殖与分化能力,更容易导致细胞早期凋亡。

泛素化连接酶c-Cbl对急性髓系白血病细胞增殖分化能力的影响

目的:探讨泛素化连接酶c-Cbl对急性髓系白血病细胞株THP-1细胞(人急性单核细胞白血病细胞)增殖分化能力的影响。方法:构建并筛选出稳定转染HBLV-h-CBL-ZsGreen-PURO敲低和HBLV-h-CBL-HA-BSD过表达病毒载体的THP-1细胞,分为4组:NC组(c-Cbl敲低组对照)、S3组(c-Cbl敲低组)、EV组(c-Cbl过表达组对照)、c-Cbl组(c-Cbl过表达组)。采用Cell Counting Kit-8(CCK-8试剂盒)和细胞周期与细胞凋亡检测试剂盒检测稳转细胞的增殖和凋亡。采用蛋白免疫印迹技术检测基因敲低或过表达细胞的凋亡相关蛋白(Bcl-2、caspase-9、cleaved-caspase-9)的表达情况。采用硝基四氮唑蓝(NBT)还原实验、瑞氏吉姆萨染色、流式细胞术检测表面分化抗原CD11b表达差异。采用蛋白免疫印迹技术检测PU.1蛋白表达情况来研究稳转细胞的分化能力。结果:转染慢病毒后,与NC组相比,S3组中c-Cbl基因的蛋白表达水平下降;与EV组相比,c-Cbl组中c-Cbl基因的蛋白表达水平上升。CCK8和流式细胞术结果表明c-Cbl能够促进THP-1的增殖(P<0.01)。蛋白免疫印迹结果表明c-Cbl能够抑制凋亡蛋白的表达,并且促进Bcl-2蛋白表达。NBT还原实验结果表明c-Cbl敲除后NBT阳性细胞增多,c-Cbl过表达后阳性细胞减少(P<0.01)。瑞氏吉姆萨染色结果显示,c-Cbl敲低后细胞核分化成肾型、蚕豆型。流式细胞术结果表明c-Cbl敲除后CD11b表达增多,过表达后CD11b表达减少。蛋白免疫印迹结果也显示c-Cbl敲除后PU.1蛋白表达增多,过表达后减少。结论:c-Cbl能够促进THP-1细胞的增殖,抑制其凋亡和分化。

葫芦巴碱调节CD47/SIRPα信号通路对胃癌细胞增殖、凋亡和免疫逃逸的影响

目的:研究葫芦巴碱调节分化簇47(CD47)/信号调节蛋白α(SIRPα)信号通路对胃癌MGC803细胞增殖、凋亡和免疫逃逸的影响。方法:以四甲基偶氮唑盐(MTT)法检测0、20、40、80、160、320μmol/L的葫芦巴碱处理后的人胃癌细胞株MGC803存活率,筛选其最佳作用浓度。将体外培养的MGC803细胞随机分为对照组、葫芦巴碱组(160μmol/L的葫芦巴碱处理)、CD47敲低组[转染CD47小干扰RNA(siRNA)质粒]、空载组(转染CD47空载质粒)、葫芦巴碱+CD47过表达组(160μmol/L的葫芦巴碱干预处理+转染CD47过表达质粒),培养24 h后。采用实时荧光定量PCR和免疫印迹检测各组细胞CD47/SIRPα通路表达;EdU染色和MTT法检测各组细胞增殖;流式细胞术检测各组细胞凋亡;免疫荧光染色检测各组细胞凋亡[B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)]相关蛋白表达;以不同细胞比例共培养人外周血单个核细胞与各组MGC803细胞后以MTT法检测人外周血单个核细胞对各组MGC803细胞的杀伤率;裸鼠体内移植瘤实验验证葫芦巴碱对CD47/SIRPα通路的调控作用。结果:20、40、80、160、320μmol/L的葫芦巴碱均可降低MGC803细胞存活率(P<0.05),且其降低作用随着葫芦巴碱浓度升高而增强,选择接近半抑制浓度值(168.94μmol/L)的160μmol/L葫芦巴碱进行后续实验。与对照组比较,葫芦巴碱组、CD47敲低组细胞和移植瘤组织CD47、SIRPα mRNA及蛋白表达、EdU阳性率、存活率、Bcl-2相对荧光强度、移植瘤重量、移植瘤体积降低,凋亡率、Bax相对荧光强度、人外周血单个核细胞对MGC803细胞杀伤率升高(P<0.05)。与葫芦巴碱组比较,葫芦巴碱+CD47过表达组细胞和移植瘤组织CD47、SIRPα mRNA及蛋白表达、EdU阳性率、存活率、Bcl-2相对荧光强度、移植瘤重量、移植瘤体积升高,凋亡率、Bax相对荧光强度、人外周血单个核细胞对MGC803细胞杀伤率降低(P<0.05)。结论:葫芦巴碱可通过抑制CD47/SIRPα通路表达来抑制胃癌细胞增殖并诱导其凋亡,还可减轻其免疫逃逸。

刺梨三萜对人肝癌SMMC-7721细胞增殖的影响

目的:观察刺梨三萜(Rosa roxburghii Tratt triterpene,RRTT)对人肝癌细胞SMMC-7721细胞增殖的影响,研究其可能的作用机制。方法:采用形态学观察和MTT法分析RRTT对SMMC-7721细胞增殖的影响;通过NBT还原实验和细胞培养上清液中甲胎蛋白(alpha-fetoprotein,AFP)含量的测定分析RRTT对SMMC-7721分化的影响;采用AO/EB染色法和FCM检测RRTT对肿瘤细胞周期和细胞凋亡的影响;RT-PCR检测Bad mRNA的表达。结果:RRTT对SMMC-7721细胞增殖的抑制作用呈时间和剂量依赖性。与阴性对照组比,随RRTT剂量的增加,NBT阳性细胞比率增加,AFP含量逐渐降低。RRTT对SMMC-7721细胞凋亡和增殖周期均无明显影响。RRTT作用后,Bad mRNA的表达下调。结论:RRTT具有体外抗SMMC-7721作用,其机制可能通过下调Bad mRNA的表达而诱导细胞分化,而与抑制细胞增殖和诱导细胞凋亡无关。

EPO对体外培养的神经干细胞增殖、分化和凋亡的影响

为探讨促红细胞生成素(erythropoietin,EPO)对大鼠神经干细胞增殖、分化和凋亡的影响,本研究采用显微解剖、机械吹打和无血清悬浮培养方法分离培养大鼠神经干细胞,向培养基中添加不同剂量的EPO,通过计数干细胞克隆形成率、四甲基偶氮唑蓝(MTT)检测法检测神经干细胞的增殖情况,用Annexin Ⅴ-FITC/PI染色和激光扫描共聚焦显微镜检测细胞凋亡率;向有血清分化培养基中加入不同剂量的EPO,以神经元特异性烯醇化酶(neuron-specific enolase,NSE)和胶质纤维酸性蛋白(glial fibril-lary acidic protein,GFAP)免疫荧光染色检测神经干细胞的分化情况。结果显示:与对照组相比,加入>5U/mlEPO后神经干细胞克隆形成率和MTT检测OD值明显增高,而凋亡率显著下降,分化培养后NSE阳性细胞较对照组明显增多。结果提示:EPO可促进大鼠神经干细胞的增殖,抑制其凋亡,促进其向神经元方向分化。

缺氧对离体培养神经干细胞增殖、分化及凋亡的影响

为探讨缺氧对体外培养的神经干细胞生长、分化及凋亡的影响,本研究采用显微解剖、机械吹打、无血清悬浮培养方法分离培养神经干细胞,用巢蛋白(nestin)免疫荧光染色对其进行鉴定。三气培养箱予以缺氧干预,分为5%缺氧组、10%缺氧组和正常对照组,每组又依缺氧干预时间的不同,分为6、12、24、48、72、96和120h组。通地绘制细胞生长曲线(MTT法)和计数克降形成率检测缺氧对神经干细胞增殖的影响。缺氧培养后,再用含10%胎牛血清的培养基进行分化培养,用免疫荧光技术检测缺氧对神经干细胞分化、凋亡及形态变化的影响。结果显示:缺氧干预后神经干细胞的增殖率明显下降,细包凋亡数增加,分化的神经元和胶质细胞的突起变短变粗,数量减少,但神经元和胶质细胞的分化比例未见明显变化。结果提示:缺氧可严重影响神经干细胞的存活和正常分化、且影响程序与缺氧剂量有量效关系。

青蒿素对人白血病U937细胞凋亡及分化的影响

目的:通过青蒿素对经典人白血病细胞系U937的作用研究,探索青蒿素治疗白血病的可能.方法:采用细胞培养、四唑盐比色试验(MTT)、细胞形态学、DNA电泳及NBT还原试验等方法观察不同浓度青蒿素促进U937细胞凋亡及诱导分化作用.结果:青蒿素在浓度>6μmol/L时可显著抑制U937细胞的增殖,促进其凋亡,并表现出剂量和时间依赖效应(P<0.01);在诱导分化浓度下,青蒿素可促进U937细胞向成熟粒细胞分化(P<0.01).结论:青蒿素对白血病细胞U937的选择性促凋亡作用以及诱导分化效应表明其可能是一种理想的高效低毒抗白血病药物,有望进入临床应用.