Transwell侵袭小室技术的改良及其在人骨髓间充质干细胞诱导分化中的应用

目的：分离、培养和鉴定人骨髓间充质干细胞（Mesenchymal stem cells，MSCs），应用改良的Transwell侵袭小室技术，探讨血管内皮生长因子（VEGF）及内皮细胞条件诱导液对其体外诱导分化中的作用。方法：采用Percoll（1．073g／ml）分离液分离骨髓单个核细胞，体外培养MSCs，流式细胞术分析鉴定MSCs的纯度，Transwell侵袭小室技术结合LSCM，实时监测MSCs在Matrigel与VEGF／内皮细胞条件诱导液构成的内皮细胞生长微环境中的运动迁移情况。结果：经Percoll分离、体外培养扩增的MSCs，细胞纯度可达95％左右；VEGF组迁移的深度虽高于对照组（P〈0．05），但迁移至聚碳酸脂膜下的细胞与对照组相比，并无统计学意义（P〉0．05）。内皮细胞条件诱导液促进MSCs的迁移，在Matrigel内迁移的深度及迁移至聚碳酸脂膜下的细胞均明显多于对照组（P〈0．05）。结论：共聚焦激光扫描显微术与Transwell侵袭小室技术的结合，能够从时间和空间上对后者进行观察，使该实验得到改良；利用内皮细胞条件诱导液与Matrigel模拟体外内皮细胞生长的微环境，并从空间上观测了MSCs穿越人工基底膜的情况，为MSCs向内皮细胞体外诱导开辟了新的思路。     

Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxia-induced apoptosis of retinal ganglion cells

Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stron-ger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we ifrst isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by lfow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells signiifcantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more signiifcant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.     

miRNA-20a促进卵巢癌细胞OVCAR3的转移

目的 检测miRNA-20a对卵巢癌细胞系OVCAR3转移能力的影响.方法 通过实时定量RT-PCR验证反义寡核苷酸与小干扰RNA封闭与过表达的效果,然后利用MTT、软琼脂集落形成和transwell 侵袭实验检测封闭和过表达miRNA-20a对OVCAR3细胞增殖及转移能力的影响.结果 封闭内源性miRNA-20a后,细胞活性基本不受影响,但集落形成能力和细胞的转移能力明显降低.过表达miRNA-20a后,细胞活性基本不受影响,但集落形成能力和细胞的转移能力明显升高.结论 miRNA-20a可能参与了卵巢癌细胞OVCAR3的转移.     

富血小板血浆对牙周膜成纤维细胞的增殖、迁移和分化的影响

目的: 体外研究不同浓度的富血小板血浆(platelet-rich plasma, PRP)对牙周膜成纤维细胞(periodontal ligament fibroblasts,PDLFs)的增殖、迁移和分化的影响.方法:不同浓度PRP(10, 50, 100, 200, 300, 500 ml/L),加入到原代培养的人PDLFs,培养时间为3、 5、 7 d,MTT法测定细胞增殖效果,transwell系统测定细胞迁移效果,AKP试剂盒测定碱性磷酸酶活性.结果:PRP有明显促进增殖作用,以200 ml/L浓度PRP效果最好,更高浓度促进作用反而减低.细胞迁移效果中,各浓度组均比阴性对照组效果好,尤其是100 ml/L的效果最佳.对于碱性磷酸酶活性,也具有明显的促进作用,以300 ml/L的效果最佳.结论:PRP在一定浓度范围内对人PDLFs功能有明显促进效果,但是在更高浓度下,这种促进效果反而减低.     

Micronuclei Formation by Promutagens in Metabolism-Incompetent V79 Cells Interacting With Activation-Proficient Cells in Various Experimental Settings

The accessibility of reactive metabolites to test cells is critical for a genotoxic response. However, sulfo-conjugates formed outside may not readily enter cells, and some metabolites formed by cytochromes P450 (CYPs) may not endure transport. This topic was addressed in the present study, using V79 cells engineered for human CYPs and/or a sulfotransferase (SULT). First, 1-methylpyrene, 1-hydroxymethylpyrene, benzo[a]pyrene, and aflatoxin B1 significantly induced micronuclei in V79-hCYP1A2-hSULT1A1, V79-hSULT1A1, V79-hCYP1A1, and V79-hCYP1A2 cells, respectively. Subsequently, we used these cell lines as external activating systems in various experimental settings in combination with V79-derived target cells lacking critical enzymes. 1-Methylpyrene (activated by CYPs and SULTs sequentially) showed an activity similar to that in V79-hCYP1A2-hSULT1A1 cells, in each following model: a mixed V79-hCYP1A2:V79-hSULT1A1 (1:1) culture, exposure of V79-hCYP1A2 to 1-methylpyrene followed by transfer of medium to V79-hSULT1A1 target cells, and V79-hSULT1A1 communicating with V79-hCYP1A2 through 0.4-μm pores and over a 1-mm distance in a unique transwell system. These results suggest ready transfer of 1-hydroxymethylpyrene formed in V79-hCYP1A2 to V79-hSULT1A1 for further activation. In the last two models, with V79-hSULT1A1 for activation and V79-Mz as target, 1-hydroxymethylpyrene induced micronuclei mildly, suggesting limited intercellular transfer of the ultimate genotoxicant, 1-sulfooxymethylpyrene. Benzo[a]pyrene induced micronuclei in V79-Mz communicating with V79-hCYP1A1 via porous membranes, whereas aflatoxin B1 was inactive in V79-Mz communicating with V79-hCYP1A2. Our results suggest that the sulfo-conjugate tested may have difficulty entering cells for a genotoxic effect, and the reactive metabolite of aflatoxin B1, unlike that of benzo[a]pyrene, could not travel an adequate distance to enter cells. Environ. Mol. Mutagen. 61:224–234, 2020. © 2019 Wiley Periodicals, Inc.     

多西紫杉醇对乳腺癌细胞增殖及侵袭力影响的研究

目的探索多西紫杉醇对人类乳腺非肿瘤细胞和乳腺癌细胞增殖及侵袭力的影响。方法应用MTT法检测多西紫杉醇对人乳腺细胞HBL100、人乳腺腺癌细胞MCF7和人乳腺导管癌细胞MDA MB435增殖的影响,采用Tran swell法明确该3株细胞的侵袭能力及多西紫杉醇对其侵袭能力的影响。结果在人乳腺细胞HBL100、人乳腺腺癌细胞MCF7和人乳腺导管癌细胞MDA MB435中,MDA MB435S侵袭力最高(425.20±54.09),MCF7次之(239.00±91.39),HBL100侵袭力最低(101.00±63.88)。1.0PPC(药物血浆峰浓度)的多西紫杉醇处理24h后,MDA MB435S和MCF7细胞的侵袭力分别为18.20±4.32和58.40±50.53,显著低于未处理的对照组(P值分别为0.000和0.013)。不同浓度的多西紫杉醇(0.1、1.0和10.0PPC)分别与3株细胞共同孵育24、48、72和96h,其对3株的增殖的抑制率均随着作用时间的延长以及给药浓度的增大而升高。结论多西紫杉醇对3株细胞的增殖抑制作用呈时间及浓度依赖性;并明显抑制MCF7和MDA MB435S细胞的侵袭能力。多西紫杉醇是一个通过抑制肿瘤细胞生长和侵袭双重途径起效的、具有很好的靶向性的抗肿瘤新药。     

Importance of bicarbonate transport in pH control during amelogenesis — need for functional studies

Dental enamel, the hardest mammalian tissue, is produced by ameloblasts. Ameloblasts show many similarities to other transporting epithelia although their secretory product, the enamel matrix, is quite different. Ameloblasts direct the formation of hydroxyapatite crystals, which liberate large quantities of protons that then need to be buffered to allow mineralization to proceed. Buffering requires a tight pH regulation and secretion of bicarbonate by ameloblasts. Many investigations have used immunohistochemical and knockout studies to determine the effects of these genes on enamel formation, but up till recently very little functional data were available for mineral ion transport. To address this, we developed a novel 2D in vitro model using HAT‐7 ameloblast cells. HAT‐7 cells can be polarized and develop functional tight junctions. Furthermore, they are able to accumulate bicarbonate ions from the basolateral to the apical fluid spaces. We propose that in the future, the HAT‐7 2D system along with similar cellular models will be useful to functionally model ion transport processes during amelogenesis. Additionally, we also suggest that similar approaches will allow a better understanding of the regulation of the cycling process in maturation‐stage ameloblasts, and the pH sensory mechanisms, which are required to develop sound, healthy enamel.     

洛美利嗪对大鼠脑微血管内皮细胞上P-糖蛋白活力的影响与P-gp及mdr1基因mRNA表达无关

目的：研究洛美利嗪对原代培养的大鼠脑微血管内皮细胞（RBMECs）上P-糖蛋白（P-gP）功能和表达的影响。方法：使用流式细胞术分析洛美利嗪对P-gp底物-罗丹明123（rhodaminel23，Rh123）在RBMECs中外排的影响，使用流式细胞术分析了洛美利嗪对RBMECs上P-gp表达的影响，应用RT-PCR技术分析了洛美利嗪对RBMECs mdr1基因mRNA水平表达的影响，还使用Transwell模型研究了洛美利嗪对Rh123通透RBMECs单层细胞转运的影响。结果：洛美利嗪通过抑制了RBMECs胞内Rh123的外排；洛美利嗪对P—gP功能的影响与RBMECs上P-gp及mdr1基因mRNA表达无关；在Transwell模型中，洛美利嗪还能显著增加Rh123跨RBMECs单层细胞膜的、经上室至下室的转运，并抑制相反方向的Rh123的转运。结论：洛美利嗪能显著地抑制RBMECs上P-gp的活性，并对P-gp底物的跨细胞转运产生影响。     

MUC1 Mediates Transendothelial Migration <Emphasis Type="Italic">in vitro</Emphasis> by Ligating Endothelial Cell ICAM-1

MUC1 is a transmembrane glycoprotein expressed by normal breast epithelium and virtually all breast cancers. MUC1 is normally restricted to the apical surface of epithelia and loss of this polarized distribution in breast carcinomas is associated with lymph node metatases. Our previous work found that MUC1 can bind intercellular adhesion molecule-1 (ICAM-1), mediating adhesion of breast cancer cells to a simulated blood vessel wall, and also triggering a calcium-based signal in the MUC1-bearing cells. It is possible that the depolarized membrane distribution of MUC1 in breast carcinomas may facilitate interactions with stromal/endothelial ICAM-1 leading to adhesion and subsequent migration through the vessel wall. In the current study, we provide evidence that ICAM-1 can influence the migration of cells that express endogenous or transfected MUC1. Migration across a gelatin-coated Transwell membrane could be increased in a step-wise manner by the sequential addition of ICAM-1-expressing cells (endothelial cells and fibroblasts), and ICAM-1-inducing inflammatory cytokines (tumour necrosis factor-α and interleukin-1β). Antibodies against MUC1 or ICAM-1, but not a control antibody, could abrogate migratory increases. Cells that did not express MUC1 were unresponsive to ICAM-1. Our current findings build on our earlier work, by suggesting that the end result of the MUC1/ICAM-1-mediated cell–cell adhesion and calcium-based signal is migration. This has implications for the exit of circulating tumour cells from the vasculature, as well as tumour cell migration through fibroblast-containing stroma underlying the endothelial wall.     

人胃癌细胞高转移亚系的初步筛选及母亚两系生物学特性的比较

目的 筛选高转移胃癌细胞亚系并与其母系进行生物学特性的比较.方法 利用transwell小室筛选高转移力胃癌细胞亚系,通过HE染色比较母亚两系细胞形态改变;流式细胞仪检测母亚两系细胞周期;免疫细胞化学法检测两系细胞中MMP-9蛋白表达;transwell侵袭小室模型比较母亚两系的迁移能力.结果 利用transwell小室从母系MKN-28中成功筛选出高转移力胃癌细胞亚系MKN-28S, 母亚两系细胞形态上无明显差异,亚系增殖指数(PI)高于母系,且亚系中 MMP-9蛋白表达同母系相比显著增高,显微镜下细胞计数示亚系迁移至膜背面的数量较母系明显增多 (P＜0.05),细胞运动能力明显增强.结论 MKN-28S细胞较MKN-28细胞具有更强增殖能力和更高迁移力.