槐定碱通过调节MCP-1/CCR2信号轴抑制膀胱癌恶性进展的实验研究

目的:探究槐定碱对膀胱癌恶性进展的影响以及该过程中对MCP-1/CCR2信号轴的调控机制。方法:通过CCK-8检测槐定碱对膀胱癌细胞5637的半数抑制浓度,随后将细胞分为对照组、槐定碱低浓度组、槐定碱高浓度组、槐定碱高+pcDNA组、槐定碱高+pcDNA-MCP-1组。CCK-8检测各组细胞的增殖活性;流式细胞术检测各组细胞凋亡情况和细胞周期;Transwell实验检测各组细胞迁移和侵袭能力;Western blot检测各组细胞中E-cadherin、Vimentin、N-cadherin、MCP-1、CCR2蛋白的表达;qRT-PCR检测各组细胞中MCP-1、CCR2 mRNA的水平;建立移植瘤裸鼠模型,观察肿瘤生长情况并检测肿瘤组织中MCP-1、CCR2 mRNA及蛋白水平。结果:与对照组相比,槐定碱低、高浓度组细胞的存活率、克隆形成数量、S期细胞比例、迁移和侵袭细胞数量、Vimentin和N-cadherin表达、MCP-1和CCR2 mRNA及蛋白的水平均降低(P<0.05),细胞凋亡率、G₂/M期细胞比例、E-cadherin表达升高(P<0...     

人脐带来源间充质干细胞向HepG2肝癌细胞原位移植瘤的归巢及分化

目的:观察人脐带来源的间充质干细胞(human umbilical cord derived mesenchymal stem cell,HUMSC)向HepG2肝癌细胞原位移植瘤的归巢及分化.方法:二步法体外诱导HUMSC向肝细胞分化,通过Real-time PCR和Western blotting检测不同时间点HUMSC中肝细胞特异性基因甲胎蛋白(alpha-fetoprotein,AFP)及白蛋白(albumin,ALB)mRNA和蛋白水平的变化;Transwell实验观察HUMSC在HepG2肝癌细胞条件培养基作用下的趋化现象;采用皮下-原位移植的方法建立BALB/c裸鼠的HepG2肝癌细胞原位移植瘤模型,利用慢病毒感染的方法将HUMSC标记CMV或AFP启动子驱动的fLuc,制备成MSC.CMVLuc或MSC.AFPLuc,并将其注射入荷瘤肝组织,IVIS成像系统观察MSC.CMVLuc向肿瘤部位的迁移归巢及MSC.AF-PLuc在肝癌微环境中向肝细胞分化.结果:HUMSC在体外诱导培养过程中出现细胞形态学变化证明其向肝细胞分化,同时诱导培养后AFP、ALB mRNA和蛋白的表达明显升高(均P＜0.05);HUMSC向HepG2肝癌细胞的趋化呈瘤细胞密度依赖性(P ＜0.01);MSC.CMVLuc注射后2d迁移并聚集于肝脏,注射后5d肝脏内发光信号进一步增强;MSC.AFPLuc肝内注射后24h已向肝细胞分化,注射第7天AFP启动子活性最强,注射第9天活性消失.结论:在小鼠体内外证明了HUMSC具有向肝脏归巢及分化的能力,为今后MSC应用于肝癌基因靶向治疗提供了一种新的策略.     

HPA通过上调MCP-1/CCR2促进胃癌发生侵袭和转移

目的 检测乙酰肝素酶(HPA)、单核细胞趋化因子-1(MCP-1)和趋化因子受体2(CCR2)在人胃癌中的表达,探讨HPA诱导人胃癌细胞发生侵袭和转移的分子机制。方法 免疫组织化学方法检测74例人胃癌组织中HPA、MCP-1和CCR2蛋白的表达。培养人胃癌MGC803细胞、MGC803-HPA细胞(过表达HPA蛋白细胞株)、SGC7901细胞和SGC7901-HPA(-)细胞(敲低HPA蛋白细胞株)。MGC803细胞和SGC7901细胞作为实验组,MGC803-HPA细胞和SGC7901-HPA(-)作为对照组,MTT实验检测CCR2抑制剂(RS504393)作用于人胃癌MGC803-HPA细胞的最适抑制浓度,Western blot实验检测人胃癌细胞中各种蛋白的表达,划痕实验和Transwell侵袭实验检测细胞迁移和侵袭能力。结果 人胃癌组织中HPA、MCP-1/CCR2蛋白阳性表达与人胃癌发病部位、分化程度、侵袭深度以及淋巴结转移相关(P<0.05)。人胃癌组织中HPA蛋白表达与MCP-1/CCR2蛋白表达呈正相关(P<0.05)。HPA过表达可促进细胞迁移和侵袭(P&...     

化疗提高肿瘤细胞表面趋化因子受体表达促进间充质干细胞的归巢

目的 研究化疗造成的肿瘤细胞损伤促进间充质干细胞迁移的能力及机制。方法 CCK-8法检测常用化疗药在HepG2和MCF-7细胞中的IC50值,IC20和IC50浓度的药物短暂处理细胞后用Transwell检测肿瘤细胞培养上清对间充质干细胞的趋化作用;Real-time PCR和Western blot方法验证相关趋化因子受体的表达水平变化,免疫组织化学法研究间充质干细胞在肿瘤部位的分布情况。结果 CCK-8法检测ADR与5-Fu对不同来源的肿瘤细胞有明显杀伤作用。Transwell检测显示仅IC20浓度的5-Fu或ADR处理肿瘤细胞后可促进HUMSCs的迁移。Real-time PCR和Western blot结果显示体外化疗处理后HepG2细胞表面CCR6分子和MCF-7细胞表面CXCR4分子的基因和蛋白表达水平明显升高。免疫组织化学结果证明体内化疗后间充质干细胞向受损部位迁移明显增多。结论 化疗造成的肿瘤细胞损伤能提高肿瘤细胞表面趋化因子受体CCR6、CXCR4的表达,促进间充质干细胞向肿瘤部位归巢。     

间充质干细胞对VX2肺癌模型化疗所致心肌损害的干预研究

目的:探讨间充质干细胞(MSCs)对VX2肺癌模型化疗所致心肌损害的干预研究,为临床应用提供实验依据.方法:选用2.5～3.5kg的新西兰大白兔随机分为对照组、化疗组及干预组.在CT定位下建立VX2肺癌模型,造模成功后给予化疗组顺铂4.4mg/kg,干预化疗组顺铂4.4mg/kg和MSCs(1×106/只),1次/周,并分时段观察实验兔心脏肌钙蛋白I(TnI)及B型尿钠肽(B-type natriuretic peptide,BNP)的变化.经过3个周期治疗,处死全部实验兔取出心脏做病理学检查.结果:MSCs干预组和化疗组与对照组相比较,TnI和BNP的含量均升高,然而MSCs干预组TnI和BNP的含量趋近于正常值与对照组相比较无统计学差异;病理检查发现化疗组实验兔的心肌细胞排列紊乱,横纹消失,胞浆不均匀,疏松相间,空泡化,节段性胞浆凝聚及溶解.而MSCs干预组的心肌细胞排列尚整齐,横纹清晰,胞浆空泡化及溶解较单药化疗组明显减轻,差异具有统计学意义(P＜0.05).结论:MSCs具有减轻化疗所致心肌损害以及改善心功能的作用.     

血清MCP-1在结直肠癌肝脏转移诊断及预后判断中的作用

[目的]检测单核细胞趋化蛋白1(MCP-1)在结直肠癌患者血清中的含量,分析其在结直肠癌患者肝脏转移诊断和预后判断中的应用潜力和价值.[方法]采用ELISA方法定量检测60例健康人员及155例有随访资料的结直肠癌患者血清MCP-1水平,采用SPSS 17.0软件进行数据检测分析.[结果]结直肠癌患者血清MCP-1平均水平为260.7±111.4pg/ml,明显高于正常人血清中的188.4±80.4 pg/ml(P＜0.001).在结直肠癌组中发生肝脏转移的患者血清MCP-1平均水平为347.7±118.7 pg/ml,显著高于无肝脏转移患者平均水平228.4±89.6pg/ml (P＜0.001).Kaplan-Meier生存分析显示,血清MCP-1低表达患者(＜251.9pg/ml)的中位生存期为53个月,而血清MCP-1高表达患者(＞251.9pg/ml)的中位生存期仅为46个月;前者的3年总生存率为87.9％,而后者仅为54.7％ (P＜0.001).Cox比例风险回归模型分析显示,血清MCP-1水平可作为判断结直肠癌患者预后的独立因素(HR=1.528,95％CI:1.093 ～2.135; P=0.013).[结论]结直肠癌患者血清MCP-1水平明显高于正常人.发生肝脏转移的结直肠癌患者血清MCP-1水平明显高于无肝脏转移患者.血清MCP-1在结直肠癌及其肝脏转移诊断中具有作为辅助诊断指标的应用潜力.同时血清MCP-1可作为结直肠癌患者预后判断的独立因素,具有重要的临床应用价值.     

PD-1/PD-L1拮抗剂在小细胞肺癌治疗中的研究进展

小细胞肺癌是以广泛转移和预后不良为特征的一种恶性肿瘤。虽然34%~85%的小细胞肺癌对化疗有效,但疾病进展迅速,后续的二线治疗效果很不理想,并且近几十年的治疗方案没有突破性进展。近几年生物治疗迅速发展,免疫治疗成为继靶向治疗后的又一次飞跃。程序性死亡蛋白-1(PD-1)与程序性死亡受体-配体1(PD-L1)结合,抑制了CD4^+和CD8^+T细胞的增殖和活化,负性调控机体免疫机制应答过程,从而介导肿瘤细胞的免疫逃逸,促进肿瘤生长。PD-1/PD-L1拮抗剂在SCLC中的临床前及临床研究中均获得了良好效果,让更多的小细胞肺癌患者受益,尽可能的延长生存时间。本文就PD-1/PD-L1拮抗剂在小细胞肺癌中的研究进展作一综述。     

过表达TRAIL的间充质干细胞对肺癌A549细胞系生物学功能的影响

目的:探究过表达肿瘤坏死因子相关凋亡诱导配体（TRAIL）的小鼠骨髓间充质干细胞（BMSCs）对肺癌A549细胞系生物学功能的影响。方法:通过慢病毒载体携带TRAIL感染小鼠BMSCs,构建过表达TRAIL的小鼠BMSCs,RT-PCR法检测BMSCs中TRAIL基因表达。根据是否感染TRAIL基因,将细胞分为BMSCs组、NC-BMSCs组和TRAIL-BMSCs组,收集各组BMSCs的培养基,作为A549细胞条件培养基。根据条件培养基的不同,将A549细胞分为A549组、A549-BMSCs组、A549-NC-BMSCs组和A549-TRAIL-BMSCs组。MTT、Transwell和平板克隆形成实验检测各组A549细胞的增殖、迁移、侵袭和克隆能力,Western blot法检测细胞中E-cadherin、N-cadherin和Vimentin的表达情况。结果:RT-PCR法检测结果显示,与BMSCs组比较,TRAIL-BMSCs组细胞中TRAIL mRNA的表达水平显著升高,过表达TRAIL小鼠BMSCs构建成功。MTT、Transwell和平板克隆形成实验检测结果显示,与A549组比较,A549-BMSCs组和A549-NC-BMSCs组细胞的增殖、迁移、侵袭和克隆能力显著增加,而A549-TRAIL-BMSCs组细胞在不同时间点的增殖能力显著降低,发生侵袭、迁移的细胞数量和克隆形成数量均显著降低;Western blot法检测结果显示,A549组、A549-BMSCs组、A549-NC-BMSCs组和A549-TRAIL-BMSCs组A549细胞中E-cadherin的表达水平分别为（0.45±0.03）、（0.23±0.03）、（0.25±0.02）、（0.95±0.09）;N-cadherin的表达水平分别为（0.57±0.07）、（0.97±0.08）、（0.99±0.12）、（0.19±0.03）;Vimentin的表达水平分别为（0.52±0.05）、（1.02±0.09）、（0.99±0.11）、（0.20±0.02）,与A549组比较,A549-BMSCs组和A549-NC-BMSCs组细胞中E-cadherin的表达水平显著降低,N-cadherin和Vimentin的表达水平显著增加,A549-TRAIL-BMSCs组细胞中E-cadherin的表达水平显著增加,N-cadherin和Vimentin的表达水平显著降低,差异均有统计学意义（P＜0.05）。结论:BMSCs可促进A549细胞的增殖、迁移、侵袭、克隆和EMT转化,BMSCs过表达TRAIL基因则对A549细胞的生物学功能具有明显抑制作用。     

口腔鳞癌中单核细胞趋化蛋白-1在巨噬细胞浸润、聚集中的意义

目的:探讨口腔鳞癌中单核细胞趋化蛋白-1(monocyte chemoattractant protein-1 MCP-1)表达与肿瘤中浸润巨噬细胞的关系.方法:应用免疫组化方法检测口腔鳞癌中MCP-1的表达和巨噬细胞的浸润,光镜进行高倍视野下巨噬细胞记数和MCP-1表达的分级.结果:在口腔鳞癌中MCP-1的表达与肿瘤分化有关,并与肿瘤内浸润的巨噬细胞有关(P＜0.05).结论:巨噬细胞可能受MCP-1趋化作用的影响,参与肿瘤的生长和转移.     

上调结肠癌间充质干细胞中miR-93-5p表达抑制结肠癌细胞增殖及促进凋亡的机制探讨

目的:观察miR-93-5p上调的结肠癌间充质干细胞(colon cancer mesenchymal stem cell,CCMSC)对结肠癌细胞增殖、凋亡的影响,并初步探讨其机制.方法:以贴壁细胞培养法抽提癌组织CCMSC,从正常结肠组织抽提结肠间充质干细胞(colon mesenchymal stem cell,...     

Zoledronic acid prevents the tumor-promoting effects of mesenchymal stem cells via MCP-1 dependent recruitment of macrophages

Zoledronic acid (ZA) has been tested in clinical trials as an additive therapy for early-stage breast cancer. However, the mechanism by which ZA exerts its antitumor activity is still unclear. The aim of this study is to investigate whether the prevention of tumor growth by ZA is through regulating the mesenchymal stem cells (MSC)-monocyte chemotactic protein 1 (MCP-1)-macrophages axis in the tumor microenvironment.To address this issue, MDA-MB-231-FLUC human breast cancer cells were cultured and injected either alone, or coupled with MSC into the mammary fat pads of nude mice. MSC were treated with either ZA or untreated. Tumor growth was determined by using an in vivo bioluminescence imaging (BLI) and the tumor-associated macrophages (TAMs) in tumor tissues were immunohistochemically analyzed by using CD206 antibody. The effects of ZA on the cytokine related gene expression of MSC were assessed by using real-time PCR.In this study, we found that ZA-treated mice showed a significant delay in tumor growth. In addition, our data revealed that ZA weakened the ability of MSC to promote tumor growth by impairing TAMs recruitment and tumor vascularization. Furthermore, it was found that ZA decreased MCP-1 expression of MSC, and therefore reduced the recruitment of TAMs to the tumor sites and hence inhibited the tumor growth.Altogether, our study demonstrated ZA can prevent the tumor-promoting effects of MSC. The antitumor effects of ZA were caused by decreasing the MCP-1 expression of MSC, which further decreased the infiltration of TAMs into tumor sites, and therefore inhibited the tumor growth.     

长期传代人脐带间充质干细胞的特性及功能

目的：探讨传至10 代（P10）的人脐带来源间充质干细胞（P10-hUC-MSC）的生物学特性及功能。方法：人脐带来源于厦门弘爱医院（伦理批号：HAXM-MEC-20201012-037-01）,分离、收集、培养hUC-MSC 并传代培养,收集P1-、P10-hUC-MSC, FCM检测hUC-MSC 表型,细胞衰老β-半乳糖苷酶染色法及FCM法检测终末期细胞衰老与凋亡情况,秋水仙碱处理检测细胞染色体稳定性,体外成脂、成骨诱导实验检测其多向分化能力,以不同比例与外周血单个核细胞（PBMC）混合培养后FCM检测T细胞亚群及表型变化。结果：成功分离和培养的P10-hUC-MSC与P1-hUC-MSC 的表型相似,表现为CD45、CD34、HLA-DR表达阴性而CD105、CD90 阳性率 95%。终末期的P1-hUC-MSC 和P10-hUC-MSC 均表现出β-半乳糖苷酶表达阳性和早期凋亡特征,细胞染色体核型一致且保持稳定,未发生转化现象。P1-、P10-hUC-MSC在体外都可被诱导分化成脂肪、成骨细胞。P10-hUC-MSC与PBMC 以1∶1 混合培养7 d 后,可显著上调CD4+/CD8+ T细胞比值、CD4+ Treg 细胞比例和PD-1表达（均P<0.01）。结论：长期传代的P10-hUC-MSC仍然保持其生物学特性和安全性,并具备多向分化能力及免疫调节能力,这为最大限度发挥hUC-MSC的临床放疗损伤修复与预防作用提供了前期实验依据和指导。     

TRAIL-expressing mesenchymal stem cells kill the putative cancer stem cell population

Background:

Tumours contain stem-like, side population (SP) cells, which have increased tumorigenic potential, resistance to traditional therapies and may be responsible for treatment failures and relapse in patients.

Methods:

Mesenchymal stem cells (MSCs) were engineered to express the apoptotic ligand, TNF-related apoptosis-inducing ligand (TRAIL). Squamous (H357) and lung (A549) cancer cell lines were sorted into side and non-side populations (non-SP) by Hoechst flow cytometry. The survival and growth of both SP and non-SP cancer populations, in conjunction with TRAIL-expressing MSCs and mitoxantrone chemotherapy, were assessed by flow cytometry and colony forming ability.

Results:

Mesenchymal stem cells expressing TRAIL migrate to tumours and reduce the growth of primary cancers and metastases. This report demonstrates that these cells cause apoptosis, death and reduced colony formation of the SP of squamous and adenocarcinoma lung cancer cells and are synergistic when combined with traditional chemotherapy in apoptosis induction.

Conclusions:

The sensitivity of putative cancer stem cells to TRAIL-expressing MSCs, suggests their possible role in the prevention of cancer relapse.     

Sphk1基因对间充质干细胞诱导结肠癌RKO细胞增殖和迁移的影响

目的： 探讨鞘氨醇激酶1 （sphigosine kinase 1, SphK1）基因下调对间充质干细胞（mesenchymal stem cell, MSC）诱导的 结肠癌RKO细胞增殖和迁移的影响。 方法： 采用MSC条件培养液（MSC-CM）和对照培养液（Control-CM）分别干预RKO细胞, CCK-8法检测细胞的增殖能力,Transwell实验检测细胞的迁移能力,Western blotting法检测Ki-67抗原（Ki-67）、MMP-2/9、肿瘤干 细胞标志物CD44和CD133蛋白的表达。用慢病毒shRNA载体转染RKO细胞抑制SphK1的表达,观察抑制SphK1的表达对 MSC-CM诱导的RKO细胞增殖、迁移以及MMP-2/9、CD44和CD133蛋白表达的影响。 结果： MSC-CM可时间依赖性促进RKO 细胞的增殖(P<0.05),并明显增强细胞的迁移能力(P<0.01)和细胞中Ki-67、MMP-2/9、CD44和CD133蛋白的表达(均P<0.05或P< 0.01)。慢病毒shRNA转染明显抑制RKO细胞SphK1的表达,抑制SphK1的表达可显著抑制MSC-CM对RKO细胞增殖和迁移的 促进作用(均P<0.05或P<0.01),且明显抑制MSC-CM诱导的Ki-67、MMP-2/9、CD44和CD133蛋白的表达。 结论： MSC-CM可 促进RKO细胞的增殖和迁移,下调SphK1可通过抑制MMP-2/9、CD44和CD133的表达逆转MSC-CM诱导的细胞增殖和迁移。     

抗ENO1抗体联合二甲双胍通过靶向肿瘤干细胞逆转人非小细胞肺癌A549细胞对西妥昔单抗的抵抗

目的:探究抗烯醇化酶1 (enolase 1,ENO1)抗体、二甲双胍(metformin,MET)通过靶向肿瘤干细胞对西妥昔单抗(cetuximab,CTX)耐药型非小细胞肺癌A549细胞增殖、迁移、侵袭和干性的影响及其可能的作用机制.方法:10 mmol/L MET联合40μg/ml抗ENO1抗体处理CTX(35 ...     

Oxidized high mobility group B‐1 enhances metastability of colorectal cancer via modification of mesenchymal stem/stromal cells

High mobility group box‐1 (HMGB1) is known to be a chemotactic factor for mesenchymal stem/stromal cells (MSCs), but the effect of post‐translational modification on its function is not clear. In this study, we hypothesized that differences in the oxidation state of HMGB1 would lead to differences in the function of MSCs in cancer. In human colorectal cancer, MSCs infiltrating into the stroma were correlated with liver metastasis and serum HMGB1. In animal models, oxidized HMGB1 mobilized three‐fold fewer MSCs to subcutaneous tumors compared with reduced HMGB1. Reduced HMGB1 inhibited the proliferation of mouse bone marrow MSCs (BM‐MSCs) and induced differentiation into osteoblasts and vascular pericytes, whereas oxidized HMGB1 promoted proliferation and increased stemness, and no differentiation was observed. When BM‐MSCs pretreated with oxidized HMGB1 were co‐cultured with syngeneic cancer cells, cell proliferation and stemness of cancer cells were increased, and tumorigenesis and drug resistance were promoted. In contrast, co‐culture with reduced HMGB1‐pretreated BM‐MSCs did not enhance stemness. In an animal orthotopic transplantation colorectal cancer model, oxidized HMGB1, but not reduced HMGB1, promoted liver metastasis with intratumoral MSC chemotaxis. Therefore, oxidized HMGB1 reprograms MSCs and promotes cancer malignancy. The oxidized HMGB1–MSC axis may be an important target for cancer therapy.     

Treatment of advanced gastrointestinal cancer with genetically modified autologous mesenchymal stem cells: Results from the phase 1/2 TREAT-ME-1 trial

TREAT-ME-1, a Phase 1/2 open-label multicenter, first-in-human, first-in-class trial, evaluated the safety, tolerability and efficacy of treatment with genetically modified autologous mesenchymal stromal cells (MSC), MSC_ apceth_101, in combination with ganciclovir in patients with advanced gastrointestinal adenocarcinoma. Immunological and inflammatory markers were also assessed. All patients (3 in Phase 1; 7 in Phase 2) received three treatment cycles of MSC_apceth_101 at one dose level on Day 0, 7, and 14 followed by ganciclovir administration according to the manufacturer's instructions for 48─72 h after MSC_apceth_101 injection. Ten patients were treated with a total dose of 3.0 x 10⁶ cells/kg MSC_apceth_101. 36 adverse events and six serious adverse events were reported. Five patients achieved stable disease (change in target lesions of −2 to +28%). For all patients, the median time to progression was 1.8 months (95% CI: 0.5, 3.9 months). Median overall survival could not be estimated as 8/10 patients were still alive at the end of the study (1 year) and therefore censored. Post-study observation of patients showed a median overall survival of 15.6 months (ranging from 2.2─27.0 months). Treatment with MSC_apceth_101 and ganciclovir did not induce a consistent increase or decrease in levels of any of the tumor markers analyzed. No clear trends in the immunological markers assessed were observed. MSC_apceth_101 in combination with ganciclovir was safe and tolerable in patients with advanced gastrointestinal adenocarcinoma, with preliminary signs of efficacy in terms of clinical stabilization of disease.