间充质干细胞释放 Dkk-1 抑制乳腺癌细胞 Wnt/β-catenin 途径的实验研究

研究表明,间充质干细胞具有向肿瘤细胞定向迁移并且抑制肿瘤细胞的特性,然而其分子机理目前尚不清楚.为了探讨间充质干细胞抑制肿瘤细胞作用的分子机制,应用BMMS-03人间充质干细胞的条件培养液作用于MCF-7乳腺癌细胞,通过软琼脂克隆形成实验、MTT实验、免疫印迹和免疫荧光染色等技术观察细胞克隆形成、增殖和基因表达的变化.结果显示:在BMMS-03细胞条件培养液作用下,MCF-7细胞的克隆形成和增殖受到了明显的抑制,β-catenin及其下游靶蛋白c-Myc、Bcl-2、PCNA和survivin的表达被明显下调,MCF-7细胞浆和细胞核内β-catenin的表达被明显抑制.BMMS-03细胞中Dkk-1的表达水平与MCF-7细胞相比较高.利用抗Dkk-1的抗体中和BMMS-03细胞条件培养液中的Dkk-1后,可明显拮抗BMMS-03细胞条件培养液对MCF-7细胞中β-catenin及c-Myc表达的抑制作用,基因转染使MCF-7细胞过表达Dkk-1后,MCF-7细胞的β-catenin及c-Myc的表达明显下调.同样经基因转染使BMMS-03细胞过表达Dkk-1后,其条件培养液可进一步下调MCF-7细胞β-catenin及c-Myc的表达.上述结果表明,间充质干细胞BMMS-03对乳腺癌MCF-7细胞的恶性表型具有明显抑制作用,其分子机制与间充质干细胞释放Dkk-1抑制乳腺癌细胞Wnt/β-catenin信号途径有关.     

COX-2在乙肝病毒X蛋白（HBx）促进肝癌细胞和肝细胞增殖中的作用

乙型肝炎病毒（hepatitis B virus, HBV）X蛋白（HBx）对肝癌的发生发展具有十分重要的作用．大量研究结果表明,与花生四烯酸代谢相关的磷脂酶COX-2与肿瘤细胞的增殖密切相关．为了阐明COX-2在HBx促进肝细胞增殖中的作用,在前期应用基因芯片方法检测发现稳定转染HBx 基因的肝癌细胞H7402-X中COX-2基因表达明显上调的基础上,本研究应用免疫印迹技术在蛋白表达水平上获得了相同的结果．进而,应用COX-2的特异性抑制剂Indo分别作用H7402-X细胞和L-O2-X细胞（稳定转染HBx 基因的人永生化L-O2肝细胞）,观察HBx蛋白是否通过COX-2促进肝癌细胞或肝细胞增殖．BrdU掺入实验和流式细胞仪检测结果显示,50 μmol/L的Indo可明显抑制H7402-X细胞和L-O2-X细胞的增殖．本研究结果提示,HBx可通过COX-2所介导的花生四烯酸代谢促进肝癌细胞和肝细胞增殖．     

IFN-β基因修饰的人脐带源性间充质干细胞抑制A549和H226肺癌细胞的克隆、迁移和增殖能力

目的探讨干扰素-β(interferon-β,IFN-β)基因修饰的人脐带源性间充质干细胞(human umbilical cord mesenchymal stem cells,huc MSCs)对非小细胞肺癌细胞株A549和H226克隆形成、迁移和增殖能力的影响。方法按照慢病毒IFN-β感染huc MSCs条件分为以下5组:慢病毒组、阴性对照病毒组、间充质干细胞组、干扰素组、空白对照组。将IFN-β慢病毒感染huc MSC,荧光显微镜观察荧光表达,确定最佳感染复数(multiplicities of infection,MOI)值,以最佳MOI值感染huc MSCs细胞。达到稳定感染后收集慢病毒组、阴性对照病毒组和间充质干细胞组的上清,ELISA法和Western blot检测以上3组的IFN-β表达的水平。克隆形成实验、Transwell小室迁移实验、细胞增殖-毒性检测法(CKK-8)检测A549、H226细胞克隆形成、迁移和增殖能力。结果慢病毒组和阴性对照病毒组的慢病毒感染效率均在85%以上,慢病毒组的IFN-β表达水平明显高于阴性对照病毒组和间充质干细胞组,慢病毒组A549细胞和H226细胞的克隆形成、迁移能力和增值能力明显降低。结论 IFN-β修饰的huc MSCs能显著抑制非小细胞肺癌细胞株A549和H226的克隆形成、迁移及增值能力,有望成为治疗非小细胞肺癌的一种新途径。     

具有间充质干细胞特征的CD105+细胞在胎儿多种组织器官的分布

CD105是骨髓间充质干细胞的特征性表型之一。为了研究机体各组织器官也存留有间充质干细胞,首先检测胎儿各组织CD105^ 细胞的分布,进而分离胎儿各组织CD105^ 细胞。将CD105^ 细胞向脂肪和成骨细胞诱导分化。结果表明胎儿心、肝、肺、血管、肌肉、皮肤等组织含有CD105^ 间充质干细胞。在间充质干细胞分化为脂肪细胞时,CD105表达明显下降。地塞米松可以促进脂肪细胞形成并提高了培养液中甘油三酯的含量。而向成骨细胞分化时,诱导的成骨细胞胞浆内外有电子密度高的钙盐沉积。以上结果提示,分布于多种组织的间充质干细胞异常分化可能与疾病的发生有关。     

黄芩甙对人肝癌BEL-7402细胞增殖和侵袭转移的影响及机制

目的探讨黄芩甙对人肝癌BEL-7402细胞系增殖、侵袭转移的影响及其机制。方法应用细胞培养技术培养人肝癌BEL-7402细胞,MTT实验、软琼脂克隆形成实验检测黄芩甙对肝癌细胞增殖的影响。通过Boyaen小室模型测定其侵袭力,细胞迁移实验测定细胞运动能力,同时观察细胞形态。流式细胞术测定肝癌细胞MMP2、TIMP2表达,免疫组化测定VEGF表达。结果黄芩甙能明显抑制肝癌细胞增殖,细胞侵袭力及运动能力明显下降,且呈量效关系（P〈0．05）。形态学观察发现,黄芩甙处理组细胞形态较圆,伪足数目较少;MMP2阳性表达细胞减少,TIMP2阳性表达细胞增多,MMP2／TIMP2比值下降;VEGF表达减少。结论黄芩甙能抑制肝癌BEL-7402增殖、侵袭与转移,其机制可能与直接抑制细胞迁移运动,抑制细胞基质溶解相关基因蛋白MMP2表达,促进TIMP2表达;VEGF表达减少有关。     

比格犬会厌黏膜来源干细胞的分离培养与生物学特性鉴定

目的:探讨建立喉部黏膜间充质干细胞的分离培养方法,并对其生物学特性进行鉴定,为进一步研究其在喉部瘢痕中形成的作用及喉部组织工程提供参考.方法:以比格犬喉部会厌背侧(舌面)黏膜为研究对象,采用消化培养的方法分离具有间充质干细胞样细胞.选取第3代细胞对其进行生物学特性鉴定,首先利用MTT法检测其增殖活性及克隆形成情况,然后通过流式细胞术检测细胞表面分子标记物CD29及CD34的表达情况,最后应用第3代细胞对其进行成脂肪细胞和成骨细胞分化培养,观察其分化能力.结果:分离培养细胞形态较为一致,绝大多数呈梭形,排列不规则.MTT增殖活性实验及克隆形成试验结果显示,所分离的细胞具有良好的增殖活性和克隆形成率;流式细胞术结果显示,该细胞表达CD29间充质干细胞细胞表面标记物,低表达造血干细胞细胞表面标记物CD34;同时,该细胞诱导分化成脂肪细胞和成骨细胞实验表明,其具有多向分化潜能.结论:从比格犬会厌背侧黏膜分离的细胞具有间充质干细胞样特性,为进一步研究喉部瘢痕形成机制及喉部组织工程提供了技术基础.     

枸杞子水提取物对Bel-7402人肝癌细胞增殖的影响

目的:探讨枸杞水提取物对Bel-7402人肝癌细胞增殖的影响。方法:用RPMI-1640完全培养液温育Bel-7402人肝癌细胞,分为空白对照组和药物组,空白对照组给予培养液处理,药物组分为低剂量组(100μg/m L)和高剂量组(200μg/mL),实验过程加入相应剂量的枸杞子水提取物。于倒置显微镜下观察三组细胞形态学变化,采用流式细胞仪观察细胞的凋亡状况及活性氧簇(ROS)含量,采用MTT法检测细胞增殖的抑制作用,采用反转录-聚合酶链式反应(RT-PCR)检测Bel-7402细胞中bax m RNA和bcl-2 m RNA的表达。结果:在倒置显微镜下观察,枸杞子水提取物使Bel-7402细胞生长受到抑制,表现为细胞贴壁减少、脱落增加,脱落的细胞浮悬于培养液中,呈圆形,体积缩小。枸杞子水提取物可促进Bel-7402细胞凋亡,药物组Bel-7402细胞的凋亡率比空白对照组明显增加(P0.01),G0/G1期细胞比率减少(P0.05),G2/M2期细胞比率增加(P0.05),且ROS含量增加(P0.05)。枸杞子水提取物对Bel-7402细胞的生长有明显抑制作用,且抑制率随着培养时间的延长而增加,且高剂量组在96小时时抑制率最高。药物组Bel-7402细胞中bax基因灰度比值显著高于空白对照组(P0.01),bcl-2基因灰度比值显著低于空白对照组(P0.01)。结论:枸杞子水提取物可抑制Bel-7402人肝癌细胞的增殖、阻滞细胞周期、诱导细胞凋亡,增加ROS含量,促进bax基因表达,抑制bcl-2基因表达,该实验结果可为枸杞子抗肝癌作用提供实验依据。     

离子通道对间充质干细胞增殖、骨向分化的作用

间充质干细胞(mesenchymal stem cells,MSCs)是一类具有向中胚层多向分化的干细胞,其细胞表面的离子通道表达多样,功能复杂。近年来,离子通道对间充质干细胞的功能调节备受关注。越来越多研究发现离子通道参与各种信号传递,调控细胞功能,如增殖、分化等基因的表达等。本文主要从离子通道表达的角度介绍离子通道在间充质干细胞的增殖、骨向分化中的作用。     

人肿瘤浸润淋巴细胞的克隆化

肿瘤组织经机械剪切和酶消化分离肿瘤浸润淋巴细胞(TIL)。TIL在含1000U/ml重组人白细胞介素-2(rhIL-2)的培养液中培养6d后,植入含1000U/ml rhIL-2的软琼脂半固体培养基中培养,7d后将形成的克隆转移到含rhIL-2的培养液中培养。~(51)Cr释放法测定结果表明,约30％的克隆对NK敏感的K562细胞和对NK不敏感的H7402肝癌细胞有细胞毒性,为具有杀瘤活性的TIL杀伤克隆(TIL-K)。60％以上TIL-K克隆在含IL-2的培养液中可持续地增殖2～3个月,其细胞数可扩增至10~8～10~9,并始终保持对肿瘤细胞的细胞毒性。TIL-K克隆的表型为CD3~ 、CD4~-、CD8~ 、CD16~-,有T细胞抗原受体β链基因的表达,说明其属T细胞系统。采用半固体-液体两步培养法可获取大量高纯度具有广谱杀瘤活性的TIL,本研究有助于TIL的深入研究和临床应用。     

DDR2基因缺失对小鼠骨髓间充质干细胞生物学特性的影响

目的:骨髓间充质干细胞(Bone Mesenchymal Stem Cells,BMSCs)是骨再生工程中重要的种子细胞,它对骨组织缺损的修复有着良好的效果。但是BMSCs向成骨细胞分化并修复骨组织缺损是是由细胞外因子共同作用产生的结果。DDR2(Discoidin Domain Receptor 2)作为I型胶原的特异性受体在成骨细胞的分化中发挥重要的调节作用。而对于其在BMSCs向成骨细胞的分化过程中的所起到的作用还鲜有研究,对其作用机理尚不明确。因此我们希望通过分离、培养并鉴定比较DDR2基因缺失小鼠与野生型小鼠来源的骨髓间充质干细胞了解其生物学特性,为后续的实验奠定理论基础。方法:采用改良型的全骨髓贴壁细胞分离方法分离培养两种小鼠来源的骨髓间充质干细胞,采用流式细胞技术鉴定其表面标记物的表达,并利用诱导培养液诱导骨髓间充质干细胞向成骨细胞和成脂肪细胞分化。结果:分离培养的两种骨髓间充质干细胞形态一致,增殖能力和自我更新能力强,流式细胞术检测其表面标记物CD29,Sca-1均表达阳性,CD105,CD45表达为阴性,分离得到的两种细胞均有向成骨细胞和成脂肪细胞分化的能力,但可以明显观察到DDR2基因缺失小鼠的骨髓间充质干细胞的成骨分化能力减弱。结论:本实验通过对于DDR2基因缺失小鼠BMSCs分离、培养和鉴定,初步探索DDR2基因缺失在在成骨过程中的作用结果,为进一步研究提高BMSCs的成骨分化能力奠定理论基础。经实验证明,DDR2基因缺失小鼠来源的骨髓间充质干细胞虽然仍具备干细胞的生物学特性,但其向成骨细胞的分化能力明显减弱,说明DDR2基因缺失对其骨髓间充质干细胞的成骨分化等有着重要的影响。     

Suppression of tumorigenesis by human mesenchymal stem cells in a hepatoma model

Human mesenchymal stem cells (hMSCs) can home to tumor sites and inhibit the growth of tumor cells. Little is known about the underlying molecular mechanisms that link hMSCs to the targeted inhibition of tumor cells. In this study, we investigated the effects of hMSCs on two human hepatoma cell lines (H7402 and HepG2) using an animal transplantation model, a co-culture system and conditioned media from hMSCs. Animal transplantation studies showed that the latent time for tumor formation was prolonged and that the tumor size was smaller when SCID mice were injected with H7402 cells and an equal number of Z3 hMSCs. When co-cultured with Z3 cells, H7402 cell proliferation decreased, apoptosis increased, and the expression of Bcl-2, c-Myc, proliferating cell nuclear antigen (PCNA) and survivin was downregulated. After treatment with conditioned media derived from Z3 hMSC cultures, H4702 cells showed decreased colony-forming ability and decreased proliferation. Immunoblot analysis showed that beta-catenin, Bcl-2, c-Myc, PCNA and survivin expression was downregulated in H7402 and HepG2 cells. Taken together, our findings demonstrate that hMSCs inhibit the malignant phenotypes of the H7402 and HepG2 human liver cancer cell lines, which include proliferation, colony-forming ability and oncogene expression both in vitro and in vivo. Furthermore, our studies provide evidence that the Wnt signaling pathway may have a role in hMSC-mediated targeting and tumor cell inhibition.     

Chlorogenic acid inhibits proliferation in human hepatoma cells by suppressing noncanonical NF-κB signaling pathway and triggering mitochondrial apoptosis

Chlorogenic acid (CGA), a phenylpropanoid derived from Eucommia ulmoides Oliver, has been shown to exhibit potent cytotoxic and anti-proliferative activities against several human cancers. However, the effects of CGA on hepatocellular carcinoma (HCC) and the underlying mechanisms have not been intensively studied. In this study, the CGA treatment effects on the viability of human hepatoma cells were investigated by MTT assay. Our data showed that CGA could dose-dependently inhibit the activity of human hepatoma cells Hep-G2 and Huh-7, but did not affect the activity and growth of normal human hepatocyte QSG-7701. The genes and pathways influenced by CGA treatment were explored by RNA sequencing and bioinformatics analysis, which identified 323 differentially expressed genes (DEGs) involved in multiple pharmacological signaling pathways such as MAPK, NF-κB, apoptosis and TGF-β signaling pathways. Further analyses by real-time quantitative PCR, Western blot and flow cytometry revealed that CGA effectually suppressed the noncanonical NF-κB signaling pathway, meanwhile it activated the mitochondrial apoptosis of HCC by upregulation of the BH3-only protein Bcl-2 binding component 3 (BBC3). Our findings demonstrated the potential of CGA in suppressing human hepatoma cells and provided a new insight into the anti-cancer mechanism of CGA.

     

Hepatitis B virus X protein drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration

Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). However, the mechanism remains unclear. Recently, we have reported that HBx promotes hepatoma cell migration through the upregulation of calpain small subunit 1 (Capn4). In addition, several reports have revealed that osteopontin (OPN) plays important roles in tumor cell migration. In this study, we investigated the signaling pathways involving the promotion of cell migration mediated by HBx. We report that HBx stimulates several factors in a network manner to promote hepatoma cell migration. We showed that HBx was able to upregulate the expression of osteopontin (OPN) through 5-lipoxygenase (5-LOX) in HepG2-X/H7402-X (stable HBx-transfected cells) cells. Furthermore, we identified that HBx could increase the expression of 5-LOX through nuclear factor-κB (NF-κB). We also found that OPN could upregulate Capn4 through NF-κB. Interestingly, we showed that Capn4 was able to upregulate OPN through NF-κB in a positive feedback manner, suggesting that the OPN and Capn4 proteins involving cell migration affect each other in a network through NF-κB. Importantly, NF-κB plays a crucial role in the regulation of 5-LOX, OPN and Capn4. Thus, we conclude that HBx drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration. This finding provides new insight into the mechanism involving the promotion of cell migration by HBx.     

Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells

The objective of the study was to evaluate differentiation of human bone marrow mesenchymal stem cells into true or pseudo neurons after treating with chemical induction medium in vitro. The morphological changes were assessed using interference contrast microscopy. Immunocytochemistry and Western blotting were performed using neuronal markers. Further evaluation was conducted with proteomic profiling, DNA microarray analysis and the whole-cell patch clamp test. After three hours of treatment with chemical induction medium, nearly three-fourths of the hMSCs changed to cells with a neuronal phenotype. The results of immunocytochemistry and Western blotting showed a high expression of neuronal markers in these cells at 3 h which decreased at 24 h. The proteomics analysis showed no change of proteins related to neuronal differentiation. DNA microarray showed downregulation of neuron related genes. The patch clamp test was unable to demonstrate any similarity to true neurons. Our findings suggest that neuron-like cells derived from chemical induction of hMSCs are not the genuine neurons as they resemble true neurons phenotypically but are different in genotypic and electrophysiological characteristics.     

HBXIP基因对乙肝病毒X蛋白诱导细胞凋亡的影响

探讨乙型肝炎病毒X蛋白结合蛋白(hepatitisBXinteractingprotein ,HBXIP)基因在乙型肝炎病毒X蛋白(HBX)诱导肝癌细胞凋亡时对细胞周期的影响.构建HBXIP基因真核表达载体pcDNA3 hbxip ,进行瞬时基因转染,将克隆有HBx基因的pCMV X (分别为1μg、2 μg和3μg)和pcDNA3 hbxip质粒分别和共转染至人H74 0 2肝癌细胞中(总体积分别为5 0 μl) .发现瞬时转染3μgpCMV X质粒后,肝癌细胞凋亡发生率为34 4 % ,肝癌细胞的细胞周期相关蛋白p2 7表达水平发生明显上调;与对照组相比,瞬时转染1μg、2 μg和3μg时,细胞周期蛋白D和细胞周期蛋白E的表达水平均发生明显上调,但随着HBX水平的增加细胞周期蛋白D和细胞周期蛋白E的表达水平发生明显下降;在稳定转染pCMV X质粒的H74 0 2 X肝癌细胞中无明显的细胞凋亡发生,研究发现p2 7的表达水平发生了明显下调,而细胞周期蛋白D和细胞周期蛋白E的表达水平发生了明显上调;当pcDNA3 hbxip质粒与pCMV X质粒进行共瞬时转染时,细胞凋亡发生率由pcDNA3质粒与pCMV X质粒共转染时的2 9 2 %下降为13 3% ,p2 7的表达水平发生了下调,但细胞周期蛋白D和细胞周期蛋白E的表达水平无明显变化.研究结果表明,瞬时转染一定剂量的x基因可导致肝癌细胞发生凋亡,细胞周期相关蛋白p2 7、细胞周期蛋白D和     

Effects of basic fibroblast growth factor on angiogenin expression and cell proliferation in H7402 human hepatoma cells

Hepatocellular carcinoma （HCC） is one of the most common cancers worldwide. Basic fibroblast growth factor （bFGF）, which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Its expression is essential for the progression and metastasis of HCC. This study aims to investigate the effects of bFGF on the expression of angiogenin, another growth factor, which plays an important role in tumor angiogenesis, and on cell proliferation in H7402 human hepatoma cells. The bFGF sense cDNA or antisense cDNA was stably transfected into H7402 cells. Genomic DNA PCR analysis demonstrated that human bFGF sense cDNA or antisense cDNA was inserted into the genome. Furthermore, the expression of bFGF and angiogenin was examined by RT-PCR and Western blot assays. MTT and colony formation assays were employed to determine cell proliferation. Stable bFGF over-expressing and under-expressing transfectants were successfully established. Expression of angiogenin was decreased in the over-expressing bFGF cells （sense transfectants） and was increased in the under-expressing bFGF cells （antisense transfectants）. Cell proliferation increased in the bFGF sense transfectants and decreased in the bFGF antisense transfectants. These results demonstrated that the endogenous bFGF may not only negatively regulate the angiogenin expression but also contribute to the overall cell proliferation in H7402 human hepatoma cells. This study may be helpful in finding a potential therapeutic approach to HCC.