抗RIP3抗体的制备及其亚细胞定位

目的:制备兔抗受体相互作用蛋白3(RIP3)的抗体,并探讨其在鼠源NIH3T3细胞株中的定位。方法:用RTPCR技术从NIH3T3细胞株中克隆鼠rip3基因,进行原核表达及电洗脱纯化。以高纯度的RIP3抗原免疫新西兰兔制备抗血清并纯化。用Westernblot检测该抗体的特异性,以免疫荧光法确定RIP3在NIH3T3细胞株中的定位,以及TNFα和zVAD.fmk对其定位的影响。结果:获得特异性的兔抗RIP3蛋白的抗体。免疫荧光的结果显示,RIP3定位于细胞质中。单用TNFα或zVAD.fmk处理NIH3T3细胞后,RIP3的定位均无明显变化;而用zVAD.fmk和TNFα共同处理细胞后,在核内外均有RIP3分布。结论:成功地克隆rip3基因并制备高特异性的兔抗RIP3抗体。RIP3在NIH3T3细胞株中定位于细胞质中,为进一步研究RIP3在TNFα诱导的caspase非依赖的细胞凋亡途径中的作用奠定了基础。     

互隔交链孢酚通过PKA-CREB信号通路激活NIH3T3细胞中DNA聚合酶β表达

目的： 探讨互隔交链孢酚（AOH）诱导小鼠胚胎成纤维细胞NIH3T3中DNA聚合酶β（DNA polβ）表达增高的分子信号通路。方法: ①15 μmol/L AOH作用NIH3T3细胞16 h,利用Western blotting 方法检测细胞中DNA polβ的表达,并利用磷酸化CREB抗体检测AOH作用后NIH3T3细胞中CREB信号分子是否被激活。②利用PKA-CREB信号通路特异性抑制剂H89预处理NIH3T3细胞1 h,再加入AOH作用,分别检测磷酸化CREB和DNA polβ表达的变化。结果: ①与溶剂对照组相比,AOH诱导NIH3T3细胞中DNA polβ表达明显增高,而且磷酸化CREB蛋白含量也明显增加,差异均显著。②用PKA特异性抑制剂H89预处理,可部分抑制NIH3T3细胞中CREB蛋白的磷酸化,并且降低了AOH引起的NIH3T3细胞中DNA polβ的表达增加。结论: PKA-CREB信号通路在AOH诱导的 NIH3T3细胞中DNA polβ表达起重要作用。     

核内M-CSF对NIH3T3细胞内微丝的影响

目的建立M-CSF核内稳定表达的NIH3T3细胞系,探讨核内M-CSF对细胞骨架的影响。方法经脂质体介导核内定位表达重组体pCMV/M-CSF转染NIH3T3细胞,G418筛选后,用Rt-PCR、免疫细胞化学鉴定其在真核细胞中的表达及定位分布,用考马斯亮蓝染色细胞微丝测定M-CSF进入细胞核后对细胞骨架的影响。结果RT-PCR结果显示转染pCMV/M-CSF的NIH3T3细胞能稳定表达M-CSFmRNA;免疫细胞化学结果显示表达的M-CSF定位于NIH3T3细胞核。考马斯亮蓝染色结果显示转染pCMV/M-CSF的NIH3T3细胞内微丝数量减少,排列紊乱。结论核内M-CSF可引起NIH3T3细胞内微丝数量减少,排列紊乱。     

重组人骨形态发生蛋白-7对NIH3T3细胞生物学行为的影响

目的:探讨重组人骨形态发生蛋白-7(rhBMP-7)对NIH3T3增殖和骨向分化的影响。方法:向培养的成纤维细胞NIH3T3中加入不同浓度的rhBMP-7,观察NIH3T3增殖、碱性磷酸酶(ALP)活性和骨钙素(OCN)含量的变化。结果:rh-BMP-7在一定浓度范围内可以明显促进NIH3T3细胞增殖、提高ALP活性与OCN水平。结论:rhBMP-7可以刺激NIH3T3细胞增殖,诱导NIH3T3细胞向成骨细胞表型分化。     

ErbB-3 结合蛋白 Ebpl 对 NIH3T3 细胞生长增殖的影响

目的:探讨 erbB-3 结合蛋白 Ebpl 对 NIH3T3 细胞生长增殖的影响.方法:将真核表达质粒 pEGFP-C1 和 Ebpl 基因经双酶切后用 T4 连接酶连接,并经鉴定序列正确后,用脂质体转染剂 Lipofectin Reagent 稳定转染 NIH3T3 细胞,免疫细胞化学显色,在激光共聚焦扫描显微镜下观察 Ebpl 蛋白的表达和定位;通过平板集落形成率观察细胞生长增殖能力的改变.结果:重组质粒鉴定正确,成功构建 Ebpl 稳定表达的 NIH3T3 细胞系,并在显微镜下可见绿色荧光,免疫细胞化学显色可见 Ebpl 蛋白表达;转染目的基因的细胞克隆形成率明显升高.结论:成功构建 Ebpl 稳定表达细胞系,Ebpl 融合蛋白表达于胞质,呈不均匀颗粒状、环状分布;Ebpl 在体外能增强 NIH3T3 细胞生长增殖.     

NIH3T3细胞核内过表达的M-CSF对细胞运动的影响

目的构建M-CSF细胞核内定位表达载体pCMV/M-CSF,探讨核内M-CSF对NIH3T3细胞运动的影响。方法采用PCR的方法扩增人M-CSF活性片段,将其插入核内真核表达载体pCMV/myc/nuc,构建重组体pCMV/M-CSF,经脂质体介导转染NIH3T3细胞,G418筛选后,用免疫细胞化学及Western blot鉴定其在真核细胞中的表达及定位分布,用细胞划痕实验测定M-CSF进入细胞核后对细胞运动能力的影响。结果限制性双酶切及DNA测序分析结果显示插入pCMV/M-CSF的片段为1400bp左右,与预期M-CSF分子大小相当。Western blot结果显示转染pCMV/M-CSF的NIH3T3细胞能稳定表达M-CSF蛋白;免疫细胞化学结果显示表达的M-CSF定位于NIH3T3细胞核。细胞划痕实验显示转染pCMV/M-CSF的NIH3T3细胞有较强的运动能力。结论成功构建M-CSF核内定位表达载体pCMV/M-CSF,核内M-CSF可加强NIH3T3细胞运动。     

JNK信号通路对NaAsO2诱导NIH3T3细胞中hnRNP K蛋白聚集体形成的影响

 目的：观察核不均一核糖核蛋白(hnRNP)K的表达与定位以及亚砷酸钠（NaAsO2）刺激NIH3T3细胞的反应情况及氧化应激变化,探讨JNK信号通路在hnRNP K蛋白聚集体形成中的作用。方法：构建重组载体pcDNA3-hnRNP K-HA,转染NIH3T3细胞,荧光显微镜观察内源性hnRNP K在细胞中的表达与定位,以及在NaAsO2不同刺激时间该蛋白聚集体的形成情况,并利用活性氧（ROS）检测试剂盒测定胞内ROS水平;给予JNK、MEK、PI3K/Akt、NF-κB和核转运等5种信号通路的抑制剂预处理后,观察聚集体的变化情况。结果：重组质粒构建正确,荧光显微镜观察显示hnRNP K主要定位于胞核中,而重组质粒转染NIH3T3细胞后,可见胞内有蛋白聚集体形成并随NaAsO2刺激时间的延长而递增,且过表达hnRNP K可抑制NaAsO2刺激细胞生成ROS;JNK信号通路抑制剂SP600125明显抑制聚集体的形成。结论：hnRNP K主要定位在NIH3T3细胞的胞核中,胞浆少量分布;NaAsO2可诱导NIH3T3细胞形成hnRNP K蛋白聚集体,此聚集体可抑制胞内ROS生成,且该聚集体的形成有赖于JNK介导的信号通路。     

ErbB-3结合蛋白Ebp1对NIH3T3细胞生长增殖的影响

目的：探讨erbB-3结合蛋白Ebp1对NIH3T3细胞生长增殖的影响。方法：将真核表达质粒pEGFP—C1和Ebpl基因经双酶切后用T4连接酶连接，并经鉴定序列正确后，用脂质体转染剂Lipofectin Reagent稳定转染NIH3T3细胞，免疫细胞化学显色，在激光共聚焦扫描显微镜下观察Ebp1蛋白的表达和定位；通过平板集落形成率观察细胞生长增殖能力的改变。结果：重组质粒鉴定正确，成功构建Ebp1稳定表达的NIH3T3细胞系，并存显微镜下可见绿色荧光，免疫细胞化学显色可见Ebp1蛋白表达；转染目的基因的细胞克隆形成率明显升高。结论：成功构建Ebp1稳定表达细胞系，Ebp1融合蛋白表达于胞质，呈不均匀颗粒状、环状分布；Ebp1在体外能增强NIH3T3细胞生长增殖。     

长期900MHz射频电磁辐射对NIH/3T3细胞凋亡与周期的影响

目的探讨长期900 MHz电磁辐射对NIH/3T3细胞凋亡与周期的影响,以为进一步深入研究电磁辐射的细胞生物学效应提供一定理论依据。方法将NIH/3T3细胞分为对照组和辐射组,对照组NIH/3T3细胞为正常条件培养,电磁辐射组用SAR值为4.0 W/kg的900 MHz电磁辐射辐照,辐照时间为2 h/d。分别取正常培养和电磁辐射辐照的4 d、7 d、10 d和12 d的对照组和辐射组NIH/3T3细胞,用流式细胞法进行细胞凋亡和细胞周期检测和分析。结果长期900 MHz电磁辐射引起NIH/3T3细胞出现显著的早期和晚期凋亡。细胞辐照12 d时产生S期阻滞。结论在12 d的辐照周期里,900 MHz电磁辐射持续地诱导NIH/3T3细胞凋亡,在辐照超过10 d后开始对细胞周期产生一定影响。     

PRRSV M 蛋白细胞系的培育及其在细胞毒性T淋巴细胞检测方法中的应用

目的 探讨PRRSV M基因在哺乳动物细胞NIH/3T3细胞中的表达,建立一种非放射性、简便易行的检测特异性细胞毒T淋巴细胞的方法.方法 应用RT-PCR扩增得到M基因并克隆入真核表达载体pcDNA3,构建成重组表达载体pcDNA3-M;将重组质粒转染至NIH/3T3细胞,G418筛选克隆;IFA检测M蛋白在转基因NIH/3T3细胞中的表达.用表达M蛋白的重组腺病毒rAd-M免疫小鼠,用表达M蛋白的NIH/3T3细胞和乳酸脱氢酶法检测PRRSV特异性细胞毒T淋巴细胞的杀伤效应. 结果 获得有G418抗性的NIH/3T3/M细胞克隆;IFA检测到有PRRSV M蛋白表达.重组腺病毒rAd-M免疫组可以诱发CTL应答,并明显高于wtAd和PBS对照组. 结论 培育成功一株能表达PRRSV M蛋白的细胞株NIH/3T3/M,可作为PRRSV CTL检测方法中的靶细胞,证明PRRSV M基因能够诱发特异性的细胞免疫.     

Monoclonal antibodies against NIH 3T3 cells transformed by human thyroid carcinoma DNA

First cycle transformants of NIH 3T3 cells transfected with metastatic human thyroid carcinoma DNA were used as immunogen to obtain monoclonal antibodies (MAbs) against normal and transformation-related antigens. The transformed cell line (M33) was shown to contain Alu sequences. Two MAbs were selected on the basis of their differential reactivity toward untreated NIH 3T3 cells or the transformed M33 cell line. By immunofluorescence, immunoelectronmicroscopy and biochemical analysis, the first MAb (MTr1) was demonstrated to recognize an epitope on cytoskeletal filaments of proliferating murine fibroblasts. Similar MTr1-labelled filaments were also found to accumulate into cytoplast-like structures spontaneously produced by M33 cells. The characterization by immunofluorescence of MTr2, the second MAb, indicates that it recognizes a specific human antigen associated with normal thyroid epithelial cells and differentiated thyroid tumors.     

CELL RECOGNITION AND PHAGOCYTOSIS OF SV40 TRANSFORMED CELL

In the course of cell transformation by SV40 the cell membrane undergoes changes, which in turn cause its surface negatively charged, and decrease markedly the phagocytosis of negatively charged substances. The study of the relationship between such transformed cells and allo-genous hamster red cells as well as lymphocytes has revealed that the rates of adhesion to and ingestion of native cells by the transformed cell, which recognizes the former as self, are low. However, when such native cells are previously treated with half saturated ammonium sulfate and 2.5% glutaraldehyde there occurs a change in the charge of cell membrane and a marked phagocytosis is triggered. In such an instance, when L cells are coated with non-immune albumin or immune sera (anti-RBC-rabbit sera) the phagocytosis is inhibited, but the phagocytosis of transformed cell remains unchanged. The phagocytosis of transformed cells can be inhibited by Concanavalin A treatment of transformed cells and fixed RBC, while be blocking the surface antigen the phagocytosis is enhanced. Therefore, it can be said that the cell recognition of self or non-self by cancer cell depends upon changes in the cell surface of both cell groups, in other words, upon the changes in cell surface charge due to molecular architecture.     

Junctional Rab13‐binding protein (JRAB) regulates cell spreading via filamins

We previously showed that Rab13 and its effector protein, junctional Rab13‐binding protein (JRAB)/molecules interacting with CasL‐like 2 (MICAL‐L2), regulate junctional development by modulating cell adhesion molecule transport and actin cytoskeletal reorganization in epithelial cells. Here, we investigated how JRAB regulates reorganization of the actin cytoskeleton in NIH3T3 fibroblasts, in an attempt to obtain novel insights into the mechanism of JRAB action. To this end, we expressed mutant proteins that adopt a constitutively open or closed state and then examined effect on cellular morphology of the resulting actin cytoskeletal reorganization. Expression of the JRABΔCT mutant (constitutively ‘closed’ state) induced stress fibers, whereas expression of the JRABΔCC mutant (constitutively ‘open’ state) caused cell spreading with membrane ruffles. Next, we identified the proteins involved in JRAB‐induced rearrangement of actin cytoskeleton leading to morphological changes. In NIH3T3 cells expressing HA‐JRABΔCC, filamin, an actin cross‐linking protein, coimmunoprecipitated with HA‐JRABΔCC. Expression of ASB2 induced degradation of all three filamin isoforms and inhibited the JRABΔCC‐induced cell spreading. Consistent with our previous results, actinin‐1/‐4 were also immunoprecipitated with HA‐JRABΔCC. However, actinin‐1/‐4 have no effect on the cell spreading regulated by JRABΔCC. These data suggest that JRAB contributes to the rearrangement of the actin cytoskeleton during cell spreading via filamins.     

While E1A can facilitate epithelial cell transformation by several dominant oncogenes, the C-terminus seems only to regulate rac and cdc42 function, but in both epithelial and fibroblastic cells

Epithelial and fibroblast cells were differentially susceptible to transformation by oncogenic src, ras, mos, raf, rac, and cdc42 and the influence of adenovirus E1A. In contrast to NIH 3T3 cells, which are easily transformed by all the oncogenes tested, epithelial cells were more resistant to transformation by the same oncogenes. Transformation efficiency of both primary and immortal epithelial cells by E1B, V12ras, v-src, v-raf, and v-mos was increased by cotransfection of E1A 12S, which enables these cells to overcome the M1/M2 mortality blocks, which are not present in NIH 3T3 cells. NIH 3T3 cell transformation by these oncogenes was not altered by E1A. Although V12cdc42 or V12rac1 alone could produce foci on NIH 3T3 cells, morphological conversion was observed only in the presence of a hypertransforming E1A mutant and not WT E1A. Epithelial cells were not transformed by V12cdc42 or V12rac1, even in the presence of WT or mutant E1A, but could be transformed by coexpression of mos/raf and rac/cdc42, and the resultant phenotype was affected by the E1A C-terminus. Hypertransformation, which has previously been reported with ras and E1A C-terminal mutants, turns out to be due to a synergy with rac/cdc42, but not ERK/MAPK or PI3K ras effectors. Like V12rac, expression of the E1A hypertransforming mutant resulted in the upregulation of vinculin and VASP, concomitant with the altered organization of the actin cytoskeleton in these cells. The results show that in addition to requiring abrogation of M1/M2 mortality blocks, primary epithelial cells require activation of the ERK MAPK cascade and rearrangement of the actin CSK to achieve transformation. In addition, the E1A C-terminus regulates rac/cdc42 function in both epithelial and fibroblast cells to affect the extent of transformation progression.     

Colcemid but not taxol modulates the migratory behavior of human T lymphocytes within 3-D collagen lattices.

T cell migration within tissue requires engagement of the cytoskeleton, however, little is known about the functional role of both actin- and tubulin-based cytoskeleton in this process. We investigated the direct effect of microtubule disruption and stabilization using colcemid and taxol, respectively, on the locomotion of peripheral human T cells within three-dimensional (3-D) collagen lattices. Microtubules network disassembly very potently enhanced T cell migration, nearly doubling the fraction of locomoting cells. Both a recruitment of previously sessile cells as well as an increase in the mean duration of active locomotion contributed to the promigratory effect. The stimulatory effect was correlated with the loss of the integrity of the tubulin cytoskeleton. Reassembly of microtubules, subsequent to the removal of colcemid from the cells, resulted in the successive return of the migratory activity to baseline levels. On the contrary, taxol failed to modulate T cell migration in our in vitro assay despite its potency to assemble tubulin into compact clots. Our observations underscore the view that tubulin-dependent cellular deformability is not the rate-limiting factor for locomotion and provide evidence that the increase in migratory activity subsequent to colcemid-treatment is due to a secondary phenomenon, most likely the activation of the actin cytoskeleton.     

HNF transfection with chondrosarcoma DNA results in the development of a sarcoma cell surface-associated epitope.

High molecular weight DNA, which was isolated from a chondrosarcoma cell line, was transfected into human neonatal foreskin fibroblasts and NIH/3T3 cells. Both types of transfected cells expressed anchorage-independent growth in soft agar and produced tumors in nude mice. The tumors which developed in nude mice following injection of transfected human fibroblasts grew to approximately 0.8 cm in diameter in four weeks. The tumors which developed from transfected NIH/3T3 cells grew to greater than 2.0 cm in diameter in 6 weeks. After growth in soft agar the transfected human fibroblasts expressed a cell surface sarcoma-associated epitope recognized by the monoclonal antibody 345.134S. In addition to the transfected human fibroblasts, the original human chondrosarcoma tumor, the chondrosarcoma cell line derived from the tumor, and the nude mouse tumor which developed from transfected human fibroblasts all exhibited positive reactivity with the monoclonal antibody 345.134S. Transfected NIH/3T3 cells that exhibited anchorage-independent growth and tumorigenicity did not exhibit detectable reactivity with the monoclonal antibody. These results suggest that the expression of the tumor-associated cell surface antigen appears to be an early event correlated with transformation of the transfected human cells but not directly related to the tumorigenic potential of the DNA. The transfected cells which expressed anchorage independent growth exhibited the sarcoma cell surface antigen prior to attaining the potential for tumorigenicity.     

EXPRESSION OF HUMAN CD59 ANTIGEN ON MOUSE NIH3T3 AND EL-4 CELLS CONFERS PROTECTION AGAINST HUMAN COMPLEMENT ATTACK

CD59分子是广泛分布于各组织细胞表面的18kDa糖蛋白,通过肌醇磷脂酰聚糖(GPI)锚固于细胞膜,具有抑制同源补体攻膜复合物(MAC)形成和参与介导T细胞活化等多种功能.本文应用RT-PCR方法,从Jurkat细胞的总RNA中扩增得到396bp的cDNA片段,经测序证实该片段包括25aa信号肽在内的全部的CD59编码序列.进一步将此CD59的cDNA重组于逆转录病毒载体pLXSN,电穿孔转染PA317细胞,并用病毒上清感染小鼠成纤维母细胞NIH3T3及小鼠胸腺瘤细胞EL-4.经G418加压筛选,FACS检测获得表达CD59的阳性细胞克隆.补体杀伤实验结果表明:表达GPI-型CD59分子的NIH3T3和EL-4细胞对人血清补体溶破的抵抗作用较空载体转染的非表达细胞明显增强.证实了用逆转录病毒载体可成功地将人CD59基因导入异源细胞,使表达CD59的异源细胞获得抑制人补体溶破的功能.本研究为探讨CD59分子与细胞活化的关系及其信号转导机制建立了良好的细胞模型,并为进一步应用于异种器官移植,或对由于CD59遗传缺损所致PNH进行基因治疗奠定了基础.     

Release of adsorbed fibronectin from temperature-responsive culture surfaces requires cellular activity

We have previously developed a temperature-responsive cell culture surface by grafting poly(N-isopropylacrylamide) that changes its surface hydrophobicity in response to temperature. While this surface shows similar hydrophobicity to that of commercial polystyrene cell culture surfaces and facilitates cell adhesion and proliferation at 37 degrees C, grafted polymer becomes hydrophilic below 32 degrees C and releases spread cultured cells without trypsin. Temperature-regulated cell detachment requires cell metabolic activity requiring ATP consumption, signal transduction, and cytoskeleton reorganziation. Precoating these surfaces with fibronectin (FN) improves spreading of less adhesive cultured hepatocytes and reducing culture temperature releases cultured cells from FN-adsorbed grafted surfaces. Immunostaining with anti-FN antibody revealed that only FN located beneath cultured cells is removed from culture surfaces after reducing temperature. FN adsorbed to surface areas lacking direct cell attachment remained surface-bound after reducing temperature. A novel concept of active cell detachment is also discussed.