miR-199a-3p 通过靶向 CABLES-1 调控流体剪切力介导的成骨细胞增殖

目的 探讨miR-199a-3p在流体剪切力(fluid shear stress, FSS)诱导成骨细胞增殖中的作用及其可能的分子机制。方法 对成骨细胞MC3T3-E1加载1.2 Pa FSS,时间分别为0、15、30、45、60、75、90 min。使用miR-199a-3p模拟物或miR-199a-3p抑制物转染MC3T3-E1细胞。使用将过表达的miR-199a-3p以及其阴性对照分别转染MC3T3-E1细胞,并以1.2 Pa FSS处理45 min。将pcDNA NC、pcDNA-CABLES-1、si RNA NC、si RNA CABLES-1转染至MC3T3-E1细胞中。分别共转染pc DNA-CABLES-1与miR-199a-3p mimic以及si RNA-CABLES-1与miR-199a-3p inhibitor。CCK-8实验检测细胞活性;RT-qPCR检测CABLES-1、 miR-199a-3p、CDK 6、Cyclin D1、PCNA表达水平;荧光素酶报告实验检测CABLES-1和miR-199a-3p的靶向关系。免疫荧光检测CABLES-1蛋白表达。...     

cyclinD1过表达对子宫颈鳞状细胞癌SiHa细胞增殖及上皮间质转化的影响北大核心CSCD

目的 探讨cyclin D1过表达对子宫颈鳞癌SiHa细胞增殖、分化的影响及其他信号分子的变化情况.方法 采用PCR法扩增cyclin D1基因全长,构建cyclin D1-pcDNA3.1质粒转染人乳头状瘤病毒16阳性的SiHa细胞,筛选cyclin D1稳定过表达细胞株.采用逆转录聚合酶链反应（RT-PCR）法和Western blot分别检测转染细胞cyclin D1 mRNA和蛋白表达.采用四甲基偶氮唑盐（MTT）比色法绘制细胞生长曲线,采用RT-PCR法和Western blot检测转染细胞CK7、E-cadherin、波形蛋白、Snail基因的mRNA和蛋白表达;采用RT-PCR法检测增殖分化相关基因CDK4、CDK2、p21、p27、cyclin E、Rb、E2F、E6/E7、Ki-67基因的mRNA表达水平.对细胞同步化处理,用RT-PCR法检测细胞周期不同时间点cyclin D1及p21基因的mRNA表达情况.结果 成功构建cyclin D1稳定过表达的G-3细胞株.生长曲线及Ki-67 mRNA升高（P〈0.05）提示G-3细胞增殖速度加快,G-3细胞中波形蛋白、Snail基因和蛋白表达明显增加（均P〈0.05）,E-cadherin、CK7基因和蛋白表达明显降低,提示转染细胞发生了上皮间质转化.cyclin D1过表达后,CDK4、CDK2、p21、p27、cyclin E表达增加,Rb、E2F、E6/E7、p16表达无明显改变,p21与cyclin D1表达趋势基本一致,在细胞周期不同时间点表达存在波动性.结论 转染诱导cyclin D1过表达可促进SiHa细胞增殖和上皮间质转化,这一过程中伴随着CDK4、CDK2、p21、p27、cyclin E基因表达的上调.cyclin D1过表达时,p21表达量也增高,可能通过影响波形蛋白表达参与了对SiHa细胞上皮间质转化的调控.     

K-ras反义核酸抑制肺腺癌细胞A549的生长

目的: 探讨K-ras反义核酸抑制肺癌生长的机制。方法: 应用Western blotting检测K-ras基因在6例肺腺癌标本和A549细胞中的表达;构建K-ras基因的反义表达载体(命名为antisense-K-ras-pcDNA3.1),转染后,MTT法测细胞的生长曲线,Annexin V检测细胞凋亡;Western blotting检测cyclin A、cyclinD1、cyclinE、CDK2、CDK4、P53、Rb和caspase-3的表达。结果: 在A549细胞和6例肺腺癌标本中,A549细胞和4例标本中K-ras基因高表达;MTT结果显示从第4 d开始,转染K-ras反义核酸的细胞生长明显受到抑制,而且细胞凋亡现象较明显;Western blotting显示转染反义K-ras基因的细胞中cyclin A、cyclin D1、cyclin E、CDK2和CDK4的表达降低,而caspase-3、P53和Rb的表达升高。结论: K-ras基因反义核酸能够抑制细胞生长,可能与cyclin A、cyclin D1、cyclin E、CDK2和CDK4的表达降低和caspase-3、P53、Rb的表达升高有关。     

RUNX3小干扰RNA对SH-SY5Y细胞生长和药物敏感性的作用

目的：探讨RUNX3基因对人神经母细胞瘤细胞生长和药物敏感性的调节作用。方法:构建RUNX3基因的小干扰RNA载体并将其转导入SH-SY5Y细胞，G418筛选后获得稳定转染的阳性克隆后，应用RT-PCR和Western blotitng进行鉴定；MTT法和流式细胞仪检测细胞转染前后生长速度和细胞周期的变化；MTT法和流式细胞仪检测细胞转染前后对阿霉素敏感性的变化；Western blotting检测细胞转染前后细胞周期相关蛋白cyclin D1、CDK4、CDK6、p21、 p27和凋亡相关蛋白Bcl-2、Bax以及耐药相关蛋白P-gp、MRP的表达变化。结果:成功构建了RUNX3的小干扰RNA载体并将其转染SH-SY5Y细胞；筛选到稳定的RUNX3低表达的神经母细胞瘤细胞模型；MTT和流式细胞仪检测结果显示，转染RUNX3小干扰RNA后的细胞的生长速度显著快于对照组（P<0.05），且G1期的细胞比率显著低于对照组（P<0.05）；MTT法和流式细胞仪结果显示, 转染RUNX3小干扰RNA后的细胞对化疗药物的敏感性降低，细胞内的阿霉素蓄积量显著减少（P<0.05）；Western blotting显示，转染RUNX3小干扰RNA后的细胞中Bcl-2、P-gp和cyclin D1的表达明显增高，p21的表达明显降低。结论:下调RUNX3基因能促进神经母细胞瘤细胞生长，降低细胞对化疗药物的敏感性，提示RUNX3在神经母细胞瘤的发生和发展中可能扮演重要角色。     

重组人内抑素对佐剂性关节炎大鼠滑膜组织p21、cyclin D1及CDK4表达的影响

目的 观察重组人内抑素(rhEndostatin)对佐剂性关节炎(AA)大鼠滑膜组织p21,细胞周期素D1(cyclin D1)及细胞周期素依赖性激酶4(CDK4)基因表达的影响,探讨rhEndostatin抑制AA大鼠成纤维样滑膜细胞(FLS)增殖的分子机制. 方法 雄性SD大鼠36只,随机分为正常组(n=12)、AA模型组(n=12)和rhEndostatin(2.5mg/kg)治疗组(n=12).制备AA大鼠模型,分别采用实时荧光定量PCR和Western blotting方法,定量分析rhEndostatin对AA大鼠滑膜组织p21、cyclin D1、CDK4 mRNA及cyclin D1蛋白表达的影响. 结果 rhEndostatin治疗组大鼠滑膜组织p21、cyclin D1 mRNA和cyclin D1蛋白表达水平降低,CDK4 mRNA表达水平增加,与AA模型组比较,差异均有统计学意义(P< 0.01). 结论 rhEndostatin降低AA大鼠滑膜组织 cyclin D1表达水平可能是其抑制AA FLS增殖的分子机制之一.     

三丁基过氧化氢诱导WI-38细胞衰老的细胞周期调控机制

目的：探讨三丁基过氧化氢(t-BHP)诱导WI-38细胞衰老的细胞周期调控机制。方法： 从30代开始，隔代用t-BHP作用WI-38细胞4次，每次1 h，诱导细胞衰老，从细胞超微结构、细胞周期分析和β-半乳糖苷酶细胞化学染色观察衰老细胞的特点，同时用Western blotting方法检测细胞周期调控蛋白CDK4、CDK2、cyclin D1、cyclin E 、p21和p16的表达程度。 结果：100 μmol/L t-BHP作用4次后，WI-38细胞出现衰老的特征，细胞增殖分裂停止，细胞体积增大、胞体变平、次级溶酶体增多，同时G1期细胞比例增加，β-半乳糖苷酶染色阳性细胞数增加，提示t-BHP能有效地诱导细胞衰老。t-BHP作用后CDK4、CDK2、cyclin E 表达下降，cyclin D1、p21和p16表达增加。 结论： t-BHP有诱导细胞衰老的作用，其机制可能与通过调节细胞周期调控分子的表达有关。     

基于CRISPR/Cas9技术构建SETD2基因敲除鼻咽癌细胞株并分析其增殖特性

目的:基于CRISPR/Cas9技术构建SETD2基因敲除鼻咽癌(NPC)细胞株,并对该细胞株的增殖特性进行分析。方法:采用RT-PCR及Western blot检测永生化鼻咽黏膜细胞系NP-69及不同分化NPC细胞系CNE1、CNE2Z和C666-1中SETD2的表达情况,筛选出SETD2高表达细胞系CNE1。应用CRISPR/Cas9技术敲除CNE1细胞中的SETD2基因,筛选SETD2稳定敲除细胞株。采用CCK-8和平板集落形成实验分析SETD2基因敲除前后CNE1细胞的增殖能力,流式细胞术检测细胞周期的分布,Western blot检测细胞周期相关蛋白的表达。结果:与NP-69细胞相比,随着细胞分化程度的降低,SETD2在CNE1、CNE2Z和C666-1细胞中的表达逐渐下降(P 0. 01)。基于CRISPR/Cas9方法成功地从15个转染了小向导RNA(small guide RNA,sgRNA)的CNE1细胞单克隆中筛选出2个SETD2稳定敲除的细胞株CNE1-SETD2-KO-#5和#9。CCK-8及平板集落形成实验结果证实,相对于CNE1-WT细胞,CNE1-SETD2-KO-#5和#9细胞的增殖能力增强(P 0. 05);流式细胞术分析表明,CNE1-SETD2-KO-#5和#9细胞G1期减少而G2/M和S期均增加(P 0. 05); Western blot证实,SETD2敲除后增殖细胞核抗原(PCNA)、细胞周期素D1(cyclin D1)、cyclin B1、cyclin A2、cyclin E1、细胞周期素依赖性激酶2(CDK2)和CDK4表达增加,p21表达减少(P 0. 05)。结论:基于CRISPR/Ca9技术成功构建SETD2基因敲除NPC细胞株。NPC细胞中SETD2表达与细胞分化程度有关; SETD2表达缺失通过上调cyclin D1、cyclin B1、cyclin A2、cyclin E1、CDK2和CDK4并下调p21表达而促进细胞增殖。     

miR-3619-5p对乳腺癌细胞MCF-7和T47D增殖、凋亡的影响及分子机制

目的探讨miR-3619-5p转染对乳腺癌细胞系MCF-7和T47D增殖和凋亡的影响及可能的分子机制。方法乳腺癌培养至对数生长期,根据对其不同处理分为2组:对照组(转染ds Control)和实验组(转染miR-3619-5p)。5-乙炔基-2'脱氧尿嘧啶核苷(Ed U)增殖实验和集落形成实验检测乳腺癌细胞增殖能力变化;流式细胞术(flow cytometry,FCM)检测乳腺癌细胞周期分布和凋亡情况;实时定量聚合酶链反应(qRT-PCR)检测p21、CDK6和Cyclin D1 mRNA的表达;Western blot法检测p21、CDK6和Cyclin D1蛋白的表达变化。结果与dsControl组相比,转染miR-3619-5p的乳腺癌细胞增殖能力显著降低(P0.05)。转染miR-3619-5p后,位于G_0/G_1期的细胞比例明显升高,位于S期和G_2/M期的细胞比例明显降低,细胞凋亡率显著上升。与ds Control组相比,转染miR-3619-5p后两种细胞系中p21 mRNA均明显上调(P0.01),而CDK6和Cyclin D1 mRNA的表达均明显下调(P0.01)。Western blot检测结果与qRT-PCR结果一致。结论 miR-3619-5p可显著抑制乳腺癌细胞的增殖能力,促进细胞凋亡,其分子机制可能是通过激活乳腺癌细胞中抑癌基因p21的表达。     

Ikaros的3种亚型对人卵巢癌SKOV3细胞增殖的影响

目的:探讨Ikaros的3种亚型对人卵巢癌SKOV3细胞增殖的影响。方法:利用逆转录病毒转染人卵巢癌SKOV3细胞,分别表达Ikaros的3种亚型(IK1、IK2和IK6);采用CCK-8法分析表达不同亚型后SKOV3细胞的增殖能力;流式细胞术检测细胞周期的改变;Western blot检测细胞周期相关蛋白的表达。结果:CCK-8结果显示IK1和IK2能明显抑制SKOV3细胞的增殖;细胞周期结果表明IK1和IK2能诱导SKOV3细胞发生G1期阻滞,IK6则对SKOV3细胞的增殖能力和细胞周期则无明显影响;Western blot检测结果显示,IK1和IK2明显降低cyclin D1和cyclin D2蛋白的表达水平,同时升高p21蛋白的表达水平。IK6则对SKOV3细胞的cyclin D1、cyclin D2及p21蛋白表达水平无明显改变。结论:IK1和IK2这2种亚型能明显抑制卵巢癌SKOV3细胞的增殖,其机制可能是由于IK1和IK2能降低细胞周期促进蛋白cyclin D1和cyclin D2的表达及增加细胞周期抑制蛋白p21的表达,从而诱导细胞周期发生G1期阻滞。而IK6亚型对SKOV3细胞增殖能力及细胞周期无明显影响。     

AEG-1表达下调对人宫颈癌细胞细胞周期和侵袭能力的影响及其机制

 目的：研究星形细胞上调基因1（astrocyte elevated gene-1,AEG-1）在人宫颈鳞癌细胞和组织中的表达,探讨AEG-1表达下调对宫颈癌SiHa细胞细胞周期和侵袭能力的影响,并分析其可能的分子机制。方法：采用Western blotting检测正常宫颈组织、宫颈鳞癌组织、HeLa、SiHa和CaSki细胞中AEG-1蛋白的表达。将对照siRNA和AEG-1 siRNA分别转染SiHa细胞,利用Western blotting检测SiHa细胞中AEG-1蛋白的表达,采用流式细胞术检测细胞周期分布的变化,采用Boyden小室检测细胞侵袭能力的变化,最后采用Western blotting检测cyclin D1、细胞周期素依赖性激酶 2（CDK2）和基质金属蛋白酶9(MMP-9)蛋白表达的变化。结果：宫颈鳞癌组织中AEG-1蛋白表达显著高于正常宫颈组织（P<0.05）,同时3株宫颈癌细胞中AEG-1蛋白表达均显著高于正常宫颈组织,其中SiHa细胞中AEG-1蛋白表达最高（P<0.05）。此外,AEG-1 siRNA能显著下调SiHa细胞中AEG-1蛋白的表达（P<0.05）,其表达下调能明显促使SiHa细胞在G0/G1期的比例增加和降低其侵袭能力。Western blotting结果表明,AEG-1 siRNA组中cyclin D1、CDK2和MMP-9蛋白的表达均显著低于未处理组和对照siRNA组（P<0.05）。结论：AEG-1在宫颈癌中的高表达可能与宫颈癌的发生发展密切相关,其表达下调介导的细胞周期静止和侵袭能力降低可能与cyclin D1、CDK2和MMP-9蛋白表达下调密切相关。     

3-Hydroxy-3-Methylglutaric Aciduria

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglecemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.     

3-Hydroxy-3-methylglutaric aciduria

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglycemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.     

Fetal thymocyte potential for T cell receptor Vγ3-Jγ1 junctional modification

Junctional modifications of T cell receptor (TcR) and immunoglobulin (Ig) gene joining regions provide great diversity to respective protein repertoires. The addition of non-germ-line-encoded nucleotides (N-regions) in the V-Jγ junction is one such modification which is developmentally regulated, rarely evident in the fetal animal, but common in the adult. A question has recently arisen as to whether developmentally patterned N-region additions in V-Jγ joins are a reflection of T cell progenitors which are committed to particular types of rearrangement prior to the event, or of changing environmental influences on uncommitted cell populations. To address this question with regard to theVγ3-Jγ1 join, T cells were examined in the fetal thymic organ culture (FTOC), a system with which the environment of early progenitor cells could be deliberately altered. At various times following FTOC initiation, cells were isolated for examination by the polymerase chain reaction, cloning and sequencing. Vγ3-Jγl sequences within genomic DNA as well as cDNA were evaluated. Data from these studies revealed frequent N-region additions within V-Jγ joins among day 14 fetal thymocyte populations, a situation dissimilar from that in vivo. Also dissimilar from the in vivo situation was the degree of exonuclease activity evident in FTOC. The canonical Vγ3-Jγl join (a frequent junction lacking N-region addition) was recognized in all experiments, but was least common among DNA versus cDNA sequences. Results illustrate that early progenitor cell populations are not programmed to exclude junctional modifications from Vγ3-Jγ1 joins.     

γδ cells involved in contact sensitivity preferentially rearrange the Vγ3 region and require interleukin-7

Ptak and Askenase showed that both αβ and γδ cells are required for transfer of contact sensitivity (CS). This study confirms that day 4 immune cells depleted of γδ cells fail to transfer CS to trinitrochlorobenzene (TNP-Cl) systemically and demonstrates that administration of anti-γδ monoclonal antibodies (mAb) in vivo abolishes the CS reaction. Moreover, γδ cells accumulate at the antigen challenge site: these cells have the unusual phenotype CD8α⁺, CD8β^-, IL-4 R⁺ which we suggest is due to their state of activation. Following immunization with contact sensitizer on the skin, the absolute number of γδ cells increases in the regional lymph nodes with a peak at 4 days. Of the γδ cells, 80%, both in the lymph nodes of TNP-Cl-immune mice and accumulating at the antigen challenge site are Vγ3⁺. The γδ cells expressing Vγ3, which is characteristic of dendritic epithelial T cells (DETC), obtained 4 days after sensitization, proliferate in response to interleukin (IL)-7, but only poorly to IL-2 and IL-4. They also respond to concanavalin A and immobilized anti-γδ mAb, but not to haptens or heat-shocked syngeneic spleen cells. Furthermore, injection of mice with mAb to IL-7 inhibits accumulation of Vγ3⁺ cells both in the lymph nodes after skin sensitization and at the antigen-challenge site. Altogether, these results strongly support the view that DETC are related to, or the original source of, the γδ cells found in the lymph node after skin sensitization and at the site of challenge, and that IL-7 is implicated in these phenomena.     

Hypoxia Increases Serum Amyloid A3 (SAA3) in Differentiated 3T3-L1 Adipocytes

Hypoxia has been implicated as a possible cause of adipose tissue inflammation. Furthermore, the acute phase protein serum amyloid A (SAA) has been associated with the modulation of the adipogenic process, and it is well-known that obese individuals have increased levels of SAA. The effect of hypoxia in the expression and production of SAA was examined in murine 3T3-L1 adipocytes. Hypoxia leads to a substantial increase in SAA3 mRNA and protein level, apparently in a time-dependent manner (threefold in 48 h), in fully differentiated 3T3-L1, followed by reestablishment of gene expression to basal levels after 24 h of reoxygenation. Hypoxia-induced SAA may be one of the key molecules to the development of the inflammatory response in adipose tissue.     

γδ cells regulate autoimmunity

γδ cells are attractive candidates for mediators of autoimmune disease. They can expand in germ-free mice, probably through recognition of autoantigens, and γδ-cell-deficient mice, unlike mice deficient in αβ T cells or B cells, show no severe defects in the immune response to foreign antigen challenge. A capacity of γδ cells to effect or regulate tissue damage is also plausible, given their ready localization to tissues, and their myriad of effector functions. Added to this, attempts to reconstruct the physiological course of autoimmune diseases with only autoreactive αβ T cells seem invariably to fall short for lack of other unidentified players. γδ cells and their putative ligands have been linked to autoimmune conditions, and recent experiments confirm that γδ cells play a significant role in autoimmune disease in vivo.     

Assembly of the TCR/CD3 complex: CD3ϵ/δ and CD3ϵ/γ dimers associate indistinctly with both TCRα and TCRβ chains. Evidence for a double TCR heterodimer model

The TCR/CD3 complex is composed of six subunits which are expressed on the cell surface in a coordinate fashion after assembly in the endoplasmic reticulum (ER). The TCR/CD3 complex is assembled after a series of pairwise interactions involving the formation of dimers of CD3ϵ with either CD3γ or CD3δ. These dimers assemble with TCRα and TCRβ chains, and finally, the CD3ζ homodimer is added to allow export of the full complex from the ER. A model has been proposed suggesting that during assembly the CD3ϵ/CD3γ dimer interacts exclusively with TCRβ and the CD3ϵ/CD3δ dimer with TCRα to form a complex with a single TCRα/β heterodimer. We show in this study, by immunoprecipitation and two-dimensional gel electrophoresis, that in the human T cell line Jurkat as well as in total human thymocytes, this preferential interaction does not occur and instead, the CD3ϵ/CD3γ and CD3ϵ/CD3δ dimers associate with both TCR chains simultaneously and indistinctly. These data are confirmed by the analysis of the TCRα-negative T cell line MOLT-4 in which TCRβ is found separately associated with CD3ϵ/CD3γ and with CD3ϵ/CD3δ dimers. Indirectly, our results support a model of stoichiometry in which two TCRα/β heterodimers are present in a TCR/CD3 complex. Furthermore, immunoprecipitation with anti-CD3γ and anti-CD3δ antibodies from 1 % NP40 and 1 % Brij96 cell lysates showed that these subunits form independent partial complexes which are cross-linked through the CD3ζ homodimer. This suggests that CD3ζ mediates the interaction between both TCRα/β heterodimers contained in the double TCR complex. Further proof for this hypothesis is obtained after analysis of a Jurkat cell transfectant containing a point mutation in the transmembrane domain of TCRβ that impairs the association of CD3ζ. In this mutant cell line, unlike a control line with wild-type TCRβ, the CD3γ- and CD3δ-containing complexes were found completely independent. Altogether, these results support a model of TCR/CD3 assembly and stoichiometry in which two TCR-α/β heterodimers form two hemicomplexes containing either CD3ϵ/γ or CD3ϵ/δ dimers which become associated via the CD3ζ homodimer.