谷胱甘肽过氧化物酶模拟物6-ySeCD对过氧化氢诱导小鼠成纤维细胞损伤的保护作用

考察不同剂量的H₂O₂作用于小鼠成纤维细胞（NIH3T3）后, 细胞脂质过氧化、 存活率、 DNA片段化的变化情况, 并研究谷胱甘肽过氧化物酶（GPX）的模拟物6A,6B-环己胺-6A′,6B′-硒桥联-β-CD（6-CySeCD）抗H₂O₂诱导小鼠成纤维细胞损伤的能力. 结果表明: H₂O₂对NIH3T3细胞有严重损伤; 6-CySeCD能有效保护细胞, 防止紫外线引起的损伤.     

人endostatin基因重组逆转录病毒载体的构建及其在NIH3T3细胞中表达

为探讨转人血管内皮抑制基因（endostatin）在转染细胞中的表达，利用逆转录病毒载体构建人endostatin基因的重组质粒，通过脂质体（lipofectamine）将重组质粒导入包装细胞PA317,制备重组病毒液。用重组病毒液感染NIH3T3细胞，经G418筛选获得转入endostatin基因细胞株NIH3T3-endo。同法制备对照细胞株NIH3T3-pLncx.PCR检测NIH3T3-endo细胞基因组，在扩增产物中一份550bp人endostatin基因特异性片段，对照组为阴性。免疫组化测定示仅NIH3T3-endo细胞中有外源性endostatin蛋白的表达，说明人endostatin基因已被成功导入NIH3T3细胞，并获得稳定表达。     

谷胱甘肽过氧化物酶模拟物6-CySeCD对过氧化氢诱导小鼠成纤维细胞损伤的保护作用

考察不同剂量的H2O2作用于小鼠成纤维细胞(NIH3T3)后,细胞脂质过氧化、存活率、DNA片段化的变化情况,并研究谷胱甘肽过氧化物酶(GPX)的模拟物6A,6B-环己胺-6A′,6B′-硒桥联-β-CD(6-CySeCD)抗H2O2诱导小鼠成纤维细胞损伤的能力.结果表明:H2O2对NIH3T3细胞有严重损伤;6-CySeCD能有效保护细胞,防止紫外线引起的损伤.     

稀土Yb对NIH3T3细胞生长的影响

应用MIT方法,研究了稀土元素Yb对小鼠成纤维细胞生长的影响.以体外传代培养的细胞株NIH3T3为研究对象,给予Yb3+(0.001 mmoL/L、0.01 mmol/L、0.1和1.00 mmol/L)培养1d后,观察NIH3T3细胞的生长情况,同时,应用MTT法检测稀土元素Yb对NIH3T3细胞生长的作用.结果发现稀土Yb对NIH3T3细胞的生长具有明显的抑制作用(P<0.05),且随着Yb3+剂量的增加,对该细胞生长的抑制作用明显增强(P<0.05).这些结果提示稀土Yb具有体外抗纤维化的作用,有望应用于组织纤维化疾病的治疗上.     

重组白细胞介素-13及其受体对小鼠胚胎成纤维细胞增殖及表型转化的影响

将常规培养的NIH3T3细胞分为实验组和对照组,实验组加入80μg/L IL-13,作用24h后进行以下实验:使用RT-PCR技术检测NIH3T3细胞中IL-13Rα1、IL-13Rα2及α-SMA基因的表达;使用MTT法检测NIH3T3细胞的增殖率.研究结果表明:实验组NIH3T3细胞,胞体呈梭型,突起生长迅速,相互交织成网状,与对照组相比形态发生了明显的改变;MTT法测定显示,实验组NIH3T3细胞的增殖率与对照组相比显著增加,差异显著(P0.01);RT-PCR检测IL-13Rα2及α-SMA基因的表达量均显著增加,而IL-13Rα1表达量变化不大.本研究表明IL-13Rα2在成纤维细胞上的表达与其增殖和转型具有直接的相关性,IL-13可直接作用于成纤维细胞并可能以IL-13Rα2为起点发挥致肺纤维化作用.     

芦荟凝胶对成纤维细胞紫外辐射损伤的保护作用

为了探讨芦荟凝胶对紫外辐射所致皮肤成纤维细胞氧化损伤的保护作用和机制,利用长波紫外线(UVA)辐射体外培养小鼠成纤维细胞株(NIH 3T3),建立紫外线损伤成纤维细胞模型。UVA辐射NIH 3T3细胞后,分别采用CCK-8法以及生化比色法检测不同质量浓度的芦荟凝胶对其增殖及抗氧化酶活性的影响。结果显示,UVA辐射对NIH 3T3细胞造成明显损伤,100、200、400μg·m L~(-1)芦荟凝胶均可以提高受UVA辐射损伤的NIH 3T3细胞的增殖活性,提高细胞中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的含量,降低脂质过氧化物酶的活性,且呈剂量依赖性。说明UVA对NIH 3T3细胞有损伤作用,100~400μg·m L~(-1)的芦荟凝胶可以拮抗UVA所致的NIH 3T3细胞损伤,具有光保护作用。     

Spindlin 1编码的蛋白质定位于细胞核并使NIH3T3细胞发生恶性转化

研究了卵巢癌中高表达的新基因spindlin1的亚细胞定位及其对小鼠NIH3T3细胞生物学行为的影响, 并探讨了人spindlin1基因的生物学功能. 采用脂质体转染法将构建的融合蛋白表达载体pEGFP-N1/ pEGFP-N1-spindlin1瞬时转染COS-7细胞,观察spindlin1基因的亚细胞定位;同时稳定转染NIH3T3细胞,经G418抗性筛选后,用RT-PCR筛选并鉴定表达spindlin1的稳定细胞株;通过细胞增殖活性、流式细胞检测、软琼脂克隆形成、迁移实验及裸鼠成瘤等对转染细胞的生物学行为进行检测. 结果表明,spindlin1定位于胞核;并促进NIH3T3细胞的增殖,使其处于G2/M期的细胞比例显著增加;同时证实过表达spindlin1的NIH3T3细胞不仅具明显的克隆形成及迁移能力,而且能使裸鼠成瘤. 由此我们得出结论:spindlin1基因过表达可促使NIH3T3细胞发生恶性转化,提示spindlin1可能是一种与肿瘤形成相关的原癌基因.     

稀土镧对成纤维NIH3T3细胞生长的作用

研究了稀土镧对培养的成纤维NIH3T3细胞增殖,周期和凋亡的影响,并探讨了其相关机理。用MTT比色法检测了细胞的增殖,用流式细胞分析仪测定了细胞周期和凋亡。结果表明:LaCl3对NIH3T3细胞的凋亡没有明显的影响,但可明显促进成纤维NIH3T3细胞的增殖,且具有一定的时间和浓度依赖性。且随着LaCl3浓度的升高,处于G1期的细胞数量明显减少,而S期细胞数量显著增多,表明LaCl3可促进成纤维NIH3T3细胞G1/S期转换,加快细胞周期进程,从而提高成纤维NIH3T3细胞的增殖活性。此研究为进一步明确镧的生物学性质提供一定的实验依据。     

PI3K等5条信号通路对NIH3T3细胞周期进程的调节作用研究

为从基因转录水平解析信号通路对NIH3T3细胞周期进程的调控作用,用小鼠基因表达谱芯片Mouse Genome 4 302.0检测信号通路相关基因表达丰度发现,PI3K,STAT3,钙蛋白酶,Rho家族鸟苷酸激酶和VEGF等5条信号通路的105个基因在该细胞的细胞周期中发生有意义的表达变化.分析基因表达变化预示的信号通路作用表明,上述5条信号通路依次促进G1期、G1/S转换期、S期、G2/M转换期和M期进程.结论:上述5条信号通路促进NIH3T3细胞的细胞周期进程.     

用于电转染NIH3T3细胞的两种电击介质的比较

分别采用不含钙镁离子的PBS与电转缓冲液,利用电穿孔转染法将带有增强型绿色荧光蛋白(e GFP)的pe GFP-N1质粒转染到NIH3T3细胞中.所用的电转参数为:电压6 V,脉冲数2,脉冲时间1ms.结果表明,无钙镁的PBS的电转效率高达(21.2±2.1)%,而电转缓冲液的电转效率只有(3.8±1.4)%.在电转后加入G418筛选,用不含钙镁离子的PBS为电击介质的细胞状态正常且大量增殖.因此,通过对转染效率以及对细胞损伤的对比,无钙镁的PBS更适合作为NIH3T3细胞的电击介质.     

重组PAI—2基因在NIH3T3细胞中的活性表达

构建了人ＰＡＩ—２ｃＤＮＡ睥达质粒，以ＮＩＨ３Ｔ３为转染宿主细胞，ＮｏｒｔｈｅｒｎＢｌｏｔ，ＷｅｓｔｅｒｎＢｌｏｔ和ＰＡＩ活性扩散试验结果表明人ＰＡＩ—２ｃＤＮＡ可以在ＮＩＨ３Ｔ３细胞中正确表达出ＰＡＩ—２蛋白，且具有抑制ＰＡ的活性，同时未能检测到ＮＩＨ３Ｔ３细胞表达ＰＡＩ—２蛋白质．为探讨ＰＡ—ＰＡＩ—２系统的调控机制打下基础．     

A novel human gene spindlin1, encoding a protein localized in the cell nucleus and inducing NIH3T3 cell''s transformation

A novel human gene, spindlin1, recently cloned in our laboratory, is highly expressed in the tissue of ovary cancer. To study its biological function, a vector expressing green fluorescent-spindlin1 fusion protein was constructed and transfected into COS-7 and NIH3T3 cells by lipofectamine methods. The results showed that the fusion protein pEGFP-N1-spindlin1 was localized in the nucleus of COS-7 and NIH3T3 cells. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the parental NIH3T3 cells displayed a complete morphological change, improved the cell growth and increased the percentage of cells in G2/M phase (12.6% vs control cells at 3.4%). Furthermore, overexpressed spindlin1 cells formed colonies in soft agar, more motile in migration assay in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may be a prooncogene which is associated with tumorigenesis.     

Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells

Human tumours often contain DNA sequences not found in normal tissues which are able to transform cultured NIH 3T3 cells. In some tumours the gene responsible for this transformation belongs to the cellular ras gene family. A specific type of mutation is responsible for converting the cellular proto-oncogene into a ras oncogene capable of inducing transformation. In a study of the function of a cellular ras gene, its protein product (produced in a bacterial cell) was microinjected into NIH 3T3 cells; the recipient cells became morphologically transformed and were induced to initiate DNA synthesis in the absence of added serum, but only when cellular ras protein was injected at much higher concentrations than required with protein of the transforming ras gene. To further analyse the function of the cellular ras gene, we have now injected monoclonal antibodies against ras proteins into NIH 3T3 cells. We report here that NIH 3T3 cells induced to divide by adding serum to the culture medium are unable to enter the S phase of the cell cycle after microinjection of anti-ras antibody, showing that the protein product of the ras proto-oncogene is required for initiation of the S-phase in NIH 3T3 cells.     

Raf-1 protein kinase is required for growth of induced NIH/3T3 cells

Many growth factors regulate the cytoplasmic Raf-1 protein kinase, consistent with its having a central role in transduction of growth signals. The kinase is ubiquitously expressed and can promote proliferation, presumably in a manner dependent on growth-factor receptors and membrane-associated oncogenes. We have now examined the dependence of serum- and TPA (12-O-tetradecanoylphorbol-13-acetate)-regulated NIH/3T3 cell growth on RAF-1 kinase to determine whether Raf-1 is essential for receptor signalling. We inhibited Raf-1 function by expressing c-raf-1 antisense RNA or kinase-defective c-raf-1 mutants. Antisense RNA for c-raf-1 interferes with proliferation of normal NIH/3T3 cells and reverts raf-transformed cells. In revertant cells, DNA replication induced by serum or TPA was eliminated or reduced proportionately to the reduction in Raf protein levels. Expression of a kinase-defective Raf-1 mutant (craf301) or a regulatory domain fragment (HCR) inhibited serum-induced NIH/3T3-cell proliferation and raf transformation even more efficiently. Inhibition by antisense RNA or craf301 blocked proliferation and transformation by Ki- and Ha-ras oncogenes. We conclude that raf functions as an essential signal transducer downstream of serum growth factor receptors, protein kinase C and ras.     

体外培养人角膜内皮细胞的UVB损伤及抗坏血酸的抗氧化保护作用研究

以非转染人角膜内皮(HCE)细胞系为体外实验模型系统研究了UVB氧化损伤、Asc抗氧化保护及其分子机理。体外培养的HCE细胞经UVB和/或Asc处理后,利用MTT和光镜对细胞的活力和形态进行了检测,利用8-羟基脱氧鸟苷免疫荧光染色对DNA的氧化损伤进行了检测,并利用二氢乙啶染色对胞内活性氧(ROS)的水平进行了检测。结果显示,100~800 mj/cm²的UVB辐射能剂量和时间依赖性地损伤HCE细胞的活力;200 mj/cm² UVB(自然太阳光中的平均辐射剂量)能引起HCE细胞发生皱缩,并显著增加细胞的DNA氧化损伤程度及胞内ROS水平;而1 mmol/L Asc不仅能显著增强HCE细胞的活力、促进细胞分裂,而且还能显著降低200 mj/cm² UVB所引起的DNA氧化损伤及胞内ROS水平。综上所述,UVB通过诱导ROS的产生进而引起DNA氧化损伤,对HCE细胞具有显著的氧化损伤作用;而Asc能够通过降低UVB诱导的ROS水平进而保护DNA免受氧化损伤,对HCE细胞的UVB损伤具有一定的抗氧化保护作用。本文研究结果对于利用Asc等抗氧化保护剂保护HCE细胞免受UVB氧化损伤具有一定的理论指导价值。     

Prevention of chemical carcinogenesis using glutathine S-transferase-pi (GST-pi)

In order to explore whether the protective function of GST-pi can prevent transformation in vitro, NIH3T3 cells and carcinogen glycidyl methacrylate (GMA) have been used in cell transformation study. NIH3T3 cells have been transfected with GST-pi cDNA inserted retrovirus vector, pXT1, and then G418 resistant clones have been analyzed by Southern and Northern analyses. NIH3T3/pXGST clones that stably express GST-pi and control cells, untransfected NIH3T3 and NIH3T3/pXT1, have been treated three times discontinuously with GMA. 1.287% of untransfected NIH3T3 and 1.197% of NIH3T3/pXT, cells obtained a transformation pheno-type, forming type Ⅲ transformed clones, which could grow in soft agar and form fibrosarcoma in nude mice. In comparison, the transformation rate is only 0.007% in NIH3T3/pXGST cells, which could not grow in soft agar and formed no tumor in vivo. The results showed that expression of exogenous GST-pi in NIH3T3 do protect NIH3T3 cells from GMA induced transformation in vitro, which provides an evidence that GST-pi may play a role in preventing chemical car-cinogenesis.     

点突变ras癌基因全cDNA真核表达重组体的构建及诱导NIH3T3细胞转化的研究

将活化癌基因Ｔ２４－ｒａｓ的全ｃＤＮＡ序列正向插入真核载体ｐＭＡＭｎｅｏ，构建成重组质粒ｐＭＡＭｎｅｏ－Ｔ２４－ｒａｓ．将该质粒转染ＮＩＨ３Ｔ３细胞，通过药物筛选，建成细胞系３Ｔ３（Ｔ２４）．Ｓｏｕｔｈｅｒｎ杂交证明外源Ｔ２４－ｒａｓ基因已整合于受体细胞染色体中．３Ｔ３（Ｔ２４）细胞表现出形态学方面的明显变化：具失去接触抑制能力，且在裸鼠体内致瘤等恶性行为．本文构建的Ｔ２４－ｒａｓ基因真核表达重组体和建立的转化细胞系可用于肿瘤的诊断、预防、治疗及抗肿瘤药物的筛选、评估等研究．     

Rapid senescence induced by overexpression of p53 in NIH3T3 cells

An NIH3T3 cell line which overexpresses temperature-sensitive p53Val135 was constructed by introduction of p53Val135 gene. It exhibited rapidly characteristic morphological and biochemical alterations related to repli-cative senescence when being cultured in 32℃. We suggested that the overexpression of p53 activated probably the onset of senescence in NIH3T3 cells, which induced a rapid cellular senescence.