hsa＿circ＿001988抑制A549细胞增殖、迁移、侵袭及凋亡影响NSCLC发生发展

目的：本研究探究hsa＿circ＿001988在肺癌发生发展中的作用以及机制。方法：利用circbase生信系统，选取hsa＿circ＿001988作为研究对象；原位杂交FISH和qRT-PCR检测hsa＿circ＿001988在肺癌组织和细胞系中的表达；Transwell、细胞划痕、CCK-8及TUNEL细胞凋亡检测hsa＿circ＿001988过表达和基因敲减对A549、NCI-H460细胞侵袭、迁移、增殖及凋亡的影响；检测皮下瘤体体积和重量；PCNA免疫组化实验检测hsa＿circ＿001988过表达和基因敲减对体内细胞增殖的调控。结果：hsa＿circ＿001988在肺癌组织和各细胞系中呈低表达；hsa＿circ＿001988抑制A549细胞侵袭、迁移、增殖及促进凋亡，circ＿001988敲减后则促进NCI-H460细胞侵袭、迁移、增殖及抑制凋亡；hsa＿circ＿001988抑制皮下瘤体生长及抑制体内细胞增殖，hsa＿circ＿001988敲减后则促进肿瘤生长及细胞增殖。结论：hsa＿circ＿001988可能通过调控肺癌细胞生物学功能从而参与NSCLC发生发展过程。     

肺癌中Cyr61的表达及其对肺癌细胞增殖、迁移和凋亡的影响

目的探讨Cyr61在非小细胞肺癌(non-small cell lung cancer, NSCLC)中的表达,以及其对NSCLC细胞增殖、迁移和凋亡的影响。方法收集63例NSCLC组织及对应癌旁组织,采用免疫组化法检测组织中Cyr61的表达,分析Cyr61表达与NSCLC临床病理特征的关系。体外培养A549、NCI-H460细胞,转染Cyr61 mimics分为空白对照组、阴性转染组、Cyr61转染组,MTT法检测细胞增殖情况;Transwell小室检测细胞侵袭、迁移能力;流式细胞仪检测细胞凋亡情况;Western blot法检测转染后细胞中Cyr61、BCL-2、Bax、Caspase-3蛋白表达。结果与癌旁组织相比,NSCLC组织中Cyr61蛋白阳性率降低,差异有统计学意义(P0.05)。Cyr61蛋白表达与吸烟、淋巴结转移、临床分期有关(P0.05)。转染Cyr61 mimics后,Cyr61蛋白表达、细胞迁移、侵袭数量、BCL-2蛋白表达降低(P0.05),细胞抑制率、凋亡率、Bax、Caspase-3蛋白表达升高(P0.05)。结论 Cyr61在肺癌中呈低表达,体外过表达后能够抑制细胞增殖、迁移,诱导凋亡,其机制可能与Caspase-3凋亡通路激活有关。     

罂粟碱通过HMGB1/NLRP3炎症小体影响非小细胞肺癌A549和H460细胞生物学行为的作用机制

目的 探讨罂粟碱对非小细胞肺癌A549和H460细胞增殖、凋亡和迁移能力的影响,并初步探讨其分子机制.方法 利用罂粟碱处理非小细胞肺癌A549和H460细胞,CCK-8细胞增殖检测试剂盒和平板克隆实验分析罂粟碱对A549和H460细胞增殖能力的影响,流式细胞术检测罂粟碱对A549和H460细胞凋亡的影响,Transwell小室实验检测罂粟碱对A549和H460细胞迁移能力的影响;Western blot分析罂粟碱对A549和H460细胞HMGB1、NLRP3、caspase-1、IL-1β和IL-18蛋白表达的影响.结果 与对照组相比,经罂粟碱处理后,A549和H460细胞增殖和迁移能力显著下降,凋亡率明显上升,HMGB1、NLRP3、caspase-1、IL-1β和IL-18蛋白表达水平均显著降低.结论 罂粟碱下调HMGB1蛋白表达,影响炎症小体活性,抑制A549和H460细胞增殖和迁移,并诱导凋亡.     

抑制BCAR1降低肺癌细胞系A549 p-P38表达

目的探讨抑制乳腺癌抗雌激素药物耐药蛋白(BCAR1)表达对肺癌细胞系A549 P38和p-P38表达的影响。方法培养正常A549细胞(对照组)、慢病毒BCAR1基因干扰A549细胞(干扰组)和慢病毒阴性对照A549细胞(阴性对照组),用Western blot检测细胞P38和p-P38表达水平,用克隆形成实验、流式细胞计量术、Transwell实验和划痕实验检测细胞的细胞克隆、细胞周期、细胞侵袭和迁移能力。结果干扰组p-P38表达显著低于其他两组(P0.05);干扰组细胞G1期比例显著高于其他两组(P0.05);干扰组细胞增殖、侵袭和迁移能力低于其他两组(P0.05)。结论抑制BCAR1表达可下调p-P38表达,减弱肺癌A549细胞增殖、迁移和侵袭能力。     

集缩素NCAPG对非小细胞肺癌增殖和凋亡的影响及其可能机制

目的 探讨非小细胞肺癌细胞中集缩素NCAPG表达对A549与H1299细胞增殖、凋亡的影响及其机制.方法 构建shRNA-NCAPG慢病毒载体转染A549及NCI-H1299细胞,实时定量PCR法(qRT-PCR)及免疫印迹法(Western blot)验证两种细胞中NCAPG表达情况.应用CCK-8方法检测对照组及下调组的细胞增殖差异,明确NCAPG表达对细胞增殖的抑制作用.Annexin V/PI双染法流式细胞术检测两组细胞中早期凋亡及晚期凋亡细胞比例.Western blot法比较两组细胞中Caspase-3、Bax及Bcl-2的表达水平.结果 shRNA-NCAPG转染组细胞的NCAPG表达量低于对照组.下调NCAPG表达抑制A549及NCI-H1299细胞增殖,增加早期凋亡及晚期细胞凋亡比例,增加肺癌细胞促凋亡相关蛋白Caspase-3及Bax的表达、抑制Bcl-2蛋白表达.结论 下调NCAPG表达诱导非小细胞肺癌A549及H1299细胞增殖,通过线粒体通路抑制其细胞凋亡.     

慢病毒介导的SEMA3G过表达对人胰腺癌细胞系PANC-1的作用

目的通过体外实验探讨过表达SEMA3G对人胰腺癌细胞PANC-1细胞增殖、侵袭和迁移能力的影响。方法将携带SEMA3G的慢病毒载体转染人胰腺癌PANC-1细胞系,利用real-time PCR和Western blot分别检测mRNA和蛋白质水平。CCK-8法检测细胞增殖,transwell实验检测细胞侵袭和细胞划痕试验检测细胞的迁移。结果成功建立稳定转染SEMA3G胰腺癌PANC-1细胞系。SEMA3G病毒感染组与空白对照组和阴性对照组相比细胞的增殖能力显著降低(P0.05),SEMA3G病毒感染组的细胞侵袭和迁移能力明显低于两组对照组(P0.05)。结论 SEMA3G慢病毒载体能有效过表达人胰腺癌PANC-1细胞中内源性SEMA3G蛋白,进而抑制细胞增殖、侵袭和迁移。     

鞘氨醇激酶1通过ERK1/2信号通路促进非小细胞肺癌细胞的侵袭和迁移

目的:探讨鞘氨醇激酶1(SphK1)对非小细胞肺癌(NSCLC)细胞迁移和侵袭的影响及其作用机制。方法:选取31例外科手术切除并经常规组织学检查确诊为NSCLC的肿瘤组织标本和配对癌旁肺组织标本,应用免疫组织化学染色和RT-qPCR检测SphK1的表达。将pcDNA3. 1-SphK1载体(SphK1组)、空白pcDNA3. 1载体对照(NC组)、SphK1 siRNA(siSphK1组)和对照siRNA(siNC组)分别转染人肺腺癌A549细胞,Western blot法检测SphK1、E-cadherin、fibronectin和p-ERK1/2的表达;利用Transwell实验评估过表达SphK1和抑制ERK1/2对A549细胞迁移和侵袭的影响。结果:SphK1在NSCLC组织中高表达,并与肿瘤分期相关。SphK1过表达可显著促进A549细胞的迁移和侵袭,提高p-ERK1/2和fibronectin蛋白水平,减少E-cadherin蛋白表达(P0. 05),而干扰SphK1则呈现相反的结果。ERK1/2抑制剂U0126可显著抑制SphK1过表达诱导的p-ERK1/2和fibronectin上调及E-cadherin下调,也抑制了SphK1过表达增强的A549细胞侵袭和迁移能力(P0. 05)。结论:SphK1可能通过ERK1/2通路降低E-cadherin蛋白水平、提高fibronectin蛋白水平,并促进NSCLC细胞的侵袭和迁移。     

RNA干扰抑制乙酰肝素酶对肺腺癌细胞A549增殖、侵袭和凋亡的影响

目的 探讨抑制乙酰肝素酶表达对人肺腺癌细胞A549增殖、侵袭和凋亡的影响.方法 构建靶向人乙酰肝素酶基因短发夹状双链RNA(shRNA)真核表达质粒(pshRNA-Hpa),用脂质体将其转入A549细胞,建立稳定转染细胞株系,经逆转录.PCR和Western blot法检测转染效率后,分别用四甲基偶氮唑蓝(MTT)、Transwell侵袭实验和流式细胞术(AnnexinV-FITC/PI双染法)检测其对A549细胞增殖、侵袭和凋亡的影响.采用Western blot法检测转染后磷酸化蛋白激酶B(PKB/Akt-Ser473)的表达.结果 转染pshRNA-Hpa的A549细胞乙酰肝素酶mRNA及蛋白表达明显低于未处理A549细胞,抑制率分别为54.09%和48.26%,细胞增殖速度减缓,侵袭能力降低,流式细胞术显示早期细胞凋亡率达(12.53±0.34)%,较空白对照组、空载体转染细胞组和阴性对照组明显增加(P＜0.01).同阴性对照质粒相比,转染pshRNA-Hpa的A549细胞Akt-Ser473的磷酸化水平降低52.15%.结论 靶向乙酰肝素酶重组shRNA质粒pshRNA-Hpa能有效抑制A549细胞乙酰肝素酶的表达,并可能通过下调磷酸化蛋白激酶B途径抑制细胞的增殖侵袭、促进细胞的凋亡.     

SFRP5基因沉默促进人类胰腺癌细胞系PANC-1增殖与迁移

目的探讨慢病毒介导短发夹RNA(shRNA)沉默SFRP5基因对人类胰腺癌细胞系PANC-1细胞增殖、侵袭和迁移能力的影响。方法构建靶向SFRP5基因特异性shRNA慢病毒载体并转染人胰腺癌PANC-1细胞系,以空白质粒转染阴性对照组,未处理细胞做为空白对照组。用real-time PCR及Western blot检测转染前后SFRP5 RNA以及蛋白的表达;CCK-8实验检测细胞体外增殖能力;使用Transwell小室实验分析细胞侵袭能力;细胞划痕实验分析细胞迁移能力。结果成功建立稳定转染shRNA-SFRP5胰腺癌PANC-1细胞株。SFRP5病毒转染组与阴性对照组及空白对照组相比细胞的增殖能力明显增加(P0.01);SFRP5病毒转染组的细胞侵袭、迁移能力明显高于阴性对照组及空白对照组(P0.01)。结论 SFRP5慢病毒干扰载体能有效抑制SFRP5基因在人胰腺癌PANC-1细胞中的表达,进而促进细胞的增殖、侵袭和迁移。     

miR-520c-3p靶向MEX3A调控肺癌细胞增殖、凋亡的分子机制

为研究miR-520c-3p对肺癌细胞增殖和凋亡的影响及其潜在的作用机制,用实时定量RT-PCR检测肺癌细胞H1299、NCI-H460、A549和正常肺上皮细胞BEAS-2B中miR-520c-3p和MEX3同源物A(MEX3 homolog A,MEX3A)的表达水平。将miR-NC、miR-520c-3p、si-NC、si-MEX3A、anti-miR-NC、anti-miR-520c-3p转染至H1299细胞后,用细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测细胞增殖,流式细胞术检测细胞凋亡,Western blotting检测蛋白表达,双荧光素酶报告基因检测实验检测细胞的荧光活性。结果显示,与正常肺上皮细胞BEAS-2B比较,肺癌细胞H1299、NCI-H460、A549中miR-520c-3p的表达水平均显著降低(P0.05),MEX3A mRNA和蛋白的表达水平显著升高(P0.05)。miR-520c-3p过表达、MEX3A抑制表达均可抑制H1299细胞的增殖活力,促进细胞凋亡;miR-520c-3p过表达抑制CyclinD1、Bcl-2蛋白的表达,促进p21、Cleaved Caspase-3、Bax蛋白的表达;MEX3A抑制表达抑制CyclinD1、Bcl-2蛋白的表达,促进p21、Bax蛋白的表达。miR-520c-3p可靶向调控MEX3A的表达;MEX3A过表达逆转了miR-520c-3p过表达对肺癌细胞H1299的增殖抑制和凋亡促进作用。提示miR-520c-3p可抑制肺癌细胞H1299的增殖,促进其凋亡,其机制可能与靶向MEX3A有关,这将为肺癌的预防和治疗提供新靶点。     

Signet-Ring Cell Variant of Large Cell Lymphoma

A diffuse, large cell lymphoma of palatine tonsil was found to contain a considerable number of enlarged tumor cells with prominent, hyaline, Russell body-type cytoplasmic inclusions displacing the nucleus peripherally and, thus, the morphologic features of signet-ring cell lymphoma. Immunoperoxidase staining revealed that the contents of the signet-ring cells were strongly positive for μ heavy chains and κ light chains. Ultrastructurally, Russell body-type inclusions consisted of multiple, angulated, electron-dense crystalloids enclosed within expanded segments of rough endoplasmic reticulum.     

Signet-Ring Cell Variant of Large Cell Lymphoma

A diffuse, large cell lymphoma of palatine tonsil was found to contain a considerable number of enlarged tumor cells with prominent, hyaline, Russell body-type cytoplasmic inclusions displacing the nucleus peripherally and, thus, the morphologic features of signet-ring cell lymphoma. Immunoperoxidase staining revealed that the contents of the signet-ring cells were strongly positive for μ heavy chains and κ light chains. Ultrastructurally, Russell body-type inclusions consisted of multiple, angulated, electron-dense crystalloids enclosed within expanded segments of rough endoplasmic reticulum.     

基于细胞三维受控组装技术的细胞芯片构建

如何将细胞准确装配于细胞芯片的设计位置,是制约细胞芯片向组织和生理系统细胞芯片方向发展的关键问题之一。本研究用细胞组装技术将细胞装配到细胞芯片的设计位置并检测的理论和方法。首先设计构建了含多组传感器阵列的细胞芯片;选取输送和固定细胞的明胶-海藻酸钠复合水凝胶材料,探讨用细胞组装技术在芯片上精确装配细胞的方法;测试复合材料和多种电解溶液对电阻抗的影响,并用芯片对细胞增殖进行检测。结果显示:复合材料能输送并将细胞固定在芯片上超过4周;细胞组装技术可精确将细胞装配到指定传感器,组装的细胞/材料微丝直径100～120μm。复合材料对芯片基础电阻抗影响小,在大于103Hz的高频段,基础阻抗为小于102.6Ω;PBS和DMEM溶液在103.5～105Hz高频段可替代氰铁化钾溶液作为电解液;芯片上的传感器在103.5Hz可准确测出Hela细胞增殖引起的电阻抗变化。研究证明了用细胞组装技术构建复杂细胞芯片的可行性。     

人牙髓细胞向成牙本质细胞诱导分化中细胞周期变化

目的建立成牙本质细胞样细胞体外培养体系，观察体外培养的牙髓细胞向成牙本质细胞分化过程中细胞周期的变化。方法利用组织块酶解法培养人牙髓细胞并进行鉴定，使用矿化诱导液诱导其向成牙本质细胞样细胞分化。对细胞增殖情况使用流式细胞仪进行细胞周期测定。结果①获得的牙髓细胞均为波形丝蛋白阳性，角蛋白阴性，证明为中胚层来源的细胞。②诱导分化的牙髓细胞在2周左右进入复层生长期；3周左右开始有细胞结节形成．周围细胞呈放射状排列，部分细胞出现细长突起并呈极性排列：4周左右细胞团中央Von Kossa染色为阳性。③在诱导开始后1周，细胞处于活跃的增殖期，以后细胞增殖变慢．3周后多数细胞处于G0G1期。结论实验成功建立了成牙本质细胞样细胞体外培养体系，所获得的成牙本质细胞样细胞具有成牙本质细胞的部分形态和生物学功能，诱导分化后细胞增殖变慢．是研究成牙本质细胞的较好模型。     

Squamous Cell Carcinoma with Anemone Cell Features

A tumor from a 52-year-old Albino male had the ultrastructural features of an anemone cell tumor. Evidence of squamous differentiation was seen by electron microscopy and immunohistochemistry. “Anemone cell” tumors have been shown to be classifiable as either carcinomas or lymphomas; the term therefore does not describe a discrete entity or constitute a definitive diagnosis. Treatment with radiotherapy was successful in this case, while chemotherapy appeared to have little effect.     

Cell Tracing Techniques in Stem Cell Transplantation

Pluripotent stem cells have shown great therapeutic promise because of their natural capacity to regenerate damaged tissue. Likewise, autologous stem cells or genetically modified stem cells have already been successfully applied in animal or clinical experimental studies including cardiopathy, diabetic disease, system lupus erythema, pancreatic disease, and liver disease. In these studies regarding stem cell transplants in different diseases, identifying the location of implanted cells and distinguishing them from endogenous cells is the first and most important step. Moreover, different tracing techniques were applied in different studies for their different sensitivity, dynamic range, convenience and reliability of their assays. Therefore, we will here review different tracing techniques and their applications in stem cell transplants, including both experiment studies and preclinical trials. Li Yan, Ying Han and Yuanlong He contributed equally to this work.     

Regulation of B Cell:T Cell Interactions: Potential Involvement of an Endogenous B Cell Sialidase

In light of the ability of B cells treated with neuraminidase to interact more effectively with T cells, the increased capacity of activated, but not small resting B cells, to interact with T cells could be associated with the level of sialylation on certain B cell surface molecules which influences the effectiveness of the physical interaction between B and T cells. The purpose of this study was to determine if activation of B cells altered sialylation via an endogenous sialidase which affected both the initial interaction between T and B cells and subsequent B cell-induced T cell proliferation. The competitive neuraminidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), inhibited LPS-mediated enhancement of B cell conjugate formation with Ia-specific T cell clones as well as enhancement of their capacity to stimulate a mixed lymphocyte reaction. The addition of NeuAc2en during LPS stimulation did not affect the surface expression of Ia, LFA-1, ICAM-1 or mB7, suggesting that inhibition of LPS-mediated enhancement by the sialidase inhibitor was not due to changes in the level of expression of the major B cell adhesion or co-stimulatory molecules. Short term stimulation with phorbol myristate acetate (PMA) and ionomycin also enhanced the ability of resting B cells to form antigen specific T:B conjugates. However, activation of B cells with PMA and ionomycin or with LPS did not change the capacity of a sialic acid specific lectin to bind to the B cells, suggesting that activation was not associated with global changes in surface sialic acid content. B cell stimulation did not appear to increase the activity of the most prevalent B cell sialidase activity as measured in an in vitro assay system, suggesting that the major B cell sialidase may not be responsible for the alteration of B cell sialylation levels or the ability of activated B cells to interact more effectively with T cells. The possibility of intracellular compartmentalization of sialidase activity or that a minor B cell sialidase may play a role in the regulation of a B cells ability to interact with T cells are discussed.     

Regulation of B Cell:T Cell Interactions: Potential Involvement of an Endogenous B Cell Sialidase

In light of the ability of B cells treated with neuraminidase to interact more effectively with T cells, the increased capacity of activated, but not small resting B cells, to interact with T cells could be associated with the level of sialylation on certain B cell surface molecules which influences the effectiveness of the physical interaction between B and T cells. The purpose of this study was to determine if activation of B cells altered sialylation via an endogenous sialidase which affected both the initial interaction between T and B cells and subsequent B cell-induced T cell proliferation. The competitive neuraminidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), inhibited LPS-mediated enhancement of B cell conjugate formation with Ia-specific T cell clones as well as enhancement of their capacity to stimulate a mixed lymphocyte reaction. The addition of NeuAc2en during LPS stimulation did not affect the surface expression of Ia, LFA-1, ICAM-1 or mB7, suggesting that inhibition of LPS-mediated enhancement by the sialidase inhibitor was not due to changes in the level of expression of the major B cell adhesion or co-stimulatory molecules. Short term stimulation with phorbol myristate acetate (PMA) and ionomycin also enhanced the ability of resting B cells to form antigen specific T:B conjugates. However, activation of B cells with PMA and ionomycin or with LPS did not change the capacity of a sialic acid specific lectin to bind to the B cells, suggesting that activation was not associated with global changes in surface sialic acid content. B cell stimulation did not appear to increase the activity of the most prevalent B cell sialidase activity as measured in an in vitro assay system, suggesting that the major B cell sialidase may not be responsible for the alteration of B cell sialylation levels or the ability of activated B cells to interact more effectively with T cells. The possibility of intracellular compartmentalization of sialidase activity or that a minor B cell sialidase may play a role in the regulation of a B cells ability to interact with T cells are discussed.     

Immunodetection of Cell Adhesion Molecules in Rat Sertoli Cell Cultures

PROBLEM: The presence of cell adhesion molecules (CAMs) in Sertoli cells has not been explored extensively. The expression of CAMs involved in cell-matrix and cell-to-cell interactions in Sertoli cell cultures was examined. METHOD OF STUDY: Immunohistochemical and Western blot techniques were applied to rat Sertoli cell cultures using specific antibodies to α₃, α₅, and α₆ integrin subunits; NCAM; and Cadherins. RESULTS: Expression of α₃ and α₆ integrin subunits (mainly laminin receptors) and lack of expression of α₅ integrin subunit (fibronectin receptor) was observed in Sertoli cells by immunohistochemistry. These cells also expressed neural CAM (NCAM) and N-cadherin. By Western blot analysis, Sertoli cell extracts reacted with antibodies to α₃ integrin subunit revealed a band approximately 130 kDa, whereas no expression of α5 integrin subunit was detected. Cell extracts incubated with antibodies to pan Cadherin exhibited a band approximately 120 kDa, whereas bands of 180, 140, and 120 kDa were observed with antibodies to NCAM. CONCLUSION: New data about the expression of receptors for extracellular matrix proteins (α₃ and α₆ integrin subunits) as well as cell-to-cell adhesion molecules (NCAM and Cadherins) are reported in rat Sertoli cell cultures.