甲基硝基亚硝基胍对NIH3T3细胞转化作用

以ＭＮＮＧ为受试物，旨在探讨ＮＩＨ３Ｔ３细胞能否作为转化实验的细胞株。研究结果显示，ＭＮＮＧ在所设浓度下均能诱导ＮＩＨ３Ｔ３细胞出现典型的细胞转化灶，其转化率呈剂量－反应关系；对转化细胞的恶性鉴定结果表明，与正常细胞不同，转化细胞失去了接触性抑制，能在软琼脂内生长。因此，初步认为ＮＩＨ３Ｔ３细胞有可能作为评定化学物质转化作用的细胞株。     

互隔交链孢霉素对NIH/3T3细胞毒性的作用

目的 研究互隔交链孢霉素(altenuene,ALT)对NIH/3T3细胞的毒性作用,为互隔交链孢霉的致癌机制提供理论依据.方法 以互隔交链孢酚(alternariol,AOH)为阳性对照,采用形态学观察,噻唑蓝法,生长曲线,流式细胞术(FCM)等方法检测不同浓度的ALT对NIH/3T3细胞生长的影响及细胞周期的改变.结果 ALT对NIH/3T3细胞的半数抑制率为76.4、50和100 μmol/L的ALT染毒24 h可使NIH/3T3细胞生长曲线明显下降,并有明显的形态学改变;以10.0、20.0和50.0 μmol/L的ALT染毒24 h后,与对照组比较,G2/M期细胞比例增加,且差异有统计学意义(P<0.05).结论 ALT对NIH/3T3细胞有毒性作用,可抑制细胞增殖并诱导G2/M期细胞阻滞,其细胞抑制作用可能与细胞周期阻滞有关.     

携带LMO3基因逆转录病毒载体的构建及其在NIH/3T3细胞中的表达

目的构建单纯LIM蛋白3(LMO3)的逆转录表达载体,并观察其在NIH/3T3细胞中的表达情况。方法重组载体pLXSN-LMO3经酶切及测序鉴定后,脂质体法转染到pA317包装细胞,G418筛选稳定的病毒产生细胞株。重组逆转录病毒体外感染NIH/3T3,并对其进行病毒滴度检测,NIH/3T3细胞分为3组:以pLXSN-LMO3转染为实验组,以pLXSN转染为阴性对照组,正常细胞为空白对照组。采用免疫荧光组化染色和蛋白印迹(Western blot)法鉴定NIH/3T3细胞中LMO3的表达。结果重组逆转录病毒载体pLXSN-LMO3经酶切及测序鉴定构建正确,其病毒滴度平均可达4.04×106cfu/ml,各组NIH/3T3细胞免疫荧光组化染色及Western blot检测均有LMO3蛋白表达,其中实验组高表达LMO3,与阴性对照组、空白对照组比较差异具有统计学意义(P<0.05)。结论成功构建了携带LMO3基因逆转录病毒载体,并能在NIH/3T3细胞中高表达LMO3蛋白,为下一步开展基因治疗奠定了基础。     

蚯蚓蛋白对NIH3T3细胞的促进增殖作用研究

目的探讨蚯蚓蛋白(EFP)对NIH3T3细胞的促进增殖作用。方法 MTT法测定EFP对大鼠成纤维细胞(NIH3T3)的促进增殖作用;细胞划痕法测定EFP促进NIH3T3细胞划痕创面愈合作用;测定NIH3T3细胞培养液中胶原降解产物羟脯氨酸的含量。结果 EFP具有促进NIH3T3细胞的增殖作用,促进划痕创面愈合作用,增加了NIH3T3细胞培养液中羟脯氨酸的含量。结论 EFP具有促进NIH3T3细胞增殖和划痕创面愈合作用。     

替米沙坦对2型糖尿病大鼠肾脏内氧化立激及NIH3T3细胞中促成熟因子活性的影响

目的探讨替米沙坦对2型糖尿病大鼠肾脏氧化应激和NIH3T3细胞中促成熟因子（MPF）活性的影响。方法将动物分为正常对照组、糖尿病组及替米沙坦治疗组，检测各组给药4，5，11，17周后的血糖、血胰岛素、血脂（TG，TC），11，17周的血肌酐（Scr）、血尿素氮（BUN）、尿微白蛋白排泄率（UAE）、肾组织中丙二醛（MDA）含量、铜，锌-超氧化物歧化酶（Cu，Zn—SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化酶（GSH—Px）及MPF活性。结果与糖尿病组相比，替米沙坦治疗组血糖、血胰岛素及血脂无明显变化，而Scr，BUN，UAE、肾脏MDA含量、肾细胞膜MPF均明显下降，肾脏抗氧化酶活性（Cu，Zn—SOD，CAT，GSH—Px）则明显上升。肾脏内MDA含量的变化及抗氧化酶活性的变化与细胞膜、细胞浆MPF活性的变化相关。结论替米沙坦可以抑制2型糖尿病大鼠肾脏内的氧化应激，并且可能与其下调NIH3T3细胞中MPF活性有关。     

携带LMO3基因逆转录病毒载体的构建及其在NIH/3 T3细胞中的表达

目的：构建单纯LIM蛋白3（LMO3）的逆转录表达载体,并观察其在 NIH/3T3细胞中的表达情况。方法重组载体pLXSN-LMO3经酶切及测序鉴定后,脂质体法转染到pA317包装细胞,G418筛选稳定的病毒产生细胞株。重组逆转录病毒体外感染NIH/3T3,并对其进行病毒滴度检测,NIH/3T3细胞分为3组：以pLXSN-LMO3转染为实验组,以pLXSN转染为阴性对照组,正常细胞为空白对照组。采用免疫荧光组化染色和蛋白印迹（ Western blot）法鉴定NIH/3T3细胞中LMO3的表达。结果重组逆转录病毒载体pLXSN-LMO3经酶切及测序鉴定构建正确,其病毒滴度平均可达4.04×106 cfu/ml,各组NIH/3T3细胞免疫荧光组化染色及Western blot检测均有LMO3蛋白表达,其中实验组高表达LMO3,与阴性对照组、空白对照组比较差异具有统计学意义（P〈0.05）。结论成功构建了携带LMO3基因逆转录病毒载体,并能在NIH/3T3细胞中高表达LMO3蛋白,为下一步开展基因治疗奠定了基础。     

吴茱萸碱调控人结肠癌HCT-116细胞中JAK2/STAT3信号通路的机制研究

目的探讨吴茱萸碱(evodiamine,EVO)对人结肠癌HCT-116细胞中JAK2/STAT3信号通路的影响。方法EVO诱导人结肠癌HCT-116细胞2、4、6 h后,Hoechst法检测细胞核凋亡的形态学改变;6μmol·L-1的EVO作用于HCT-116细胞2、4和6 h,不同浓度的AG490作用于HCT-116细胞48 h,6μmol·L-1的EVO和50μmol·L-1的AG490分别诱导HCT-116细胞以及两药联合诱导HCT-116细胞6 h后,运用蛋白质印迹法检测各组细胞中JAK2、pJAK2、STAT3和p-STAT3的蛋白表达量。结果镜下观察EVO诱导后的细胞核浓缩聚集,染色质边缘化,并出现染色质碎裂成块,形成凋亡小体等形态学改变。Western blot结果显示:EVO可以明显下调HCT-116细胞内p-STAT3的表达;AG490可以抑制JAK2/STAT3信号通路的激活;EVO对JAK2/STAT3信号通路中p-STAT3蛋白的表达抑制效果较AG490更明显,且两药联合应用后抑制效果进一步增强。结论EVO对人结肠癌HCT-116细胞的抗肿瘤活性有可能是通过抑制JAK2/STAT3信号通路的激活来发挥的。     

槲皮素对PDGF诱导的NIH 3T3细胞增殖的影响

目的研究酪氨酸蛋白激酶抑制剂槲皮素对ＰＤＧＦ诱导的ＮＩＨ３Ｔ３细胞增殖的影响。方法采用ＭＴＴ比色法、Ｇｉｅｍｓａ染色、流式细胞术（测定ＤＮＡ含量的变化及增殖细胞核抗原的表达）、及ＷｅｓｔｅｒｎＢｌｏｔ分析技术对ＰＤＧＦ诱导的ＮＩＨ３Ｔ３细胞增殖进行分析。结果与对照组相比，槲皮素处理可显著地抑制ＰＤＧＦ诱导的ＮＩＨ３Ｔ３细胞增殖，而且主要是细胞周期Ｓ期抑制，ＷｅｓｅｔｅｒｎＢｌｏｔ分析提示槲皮素可明显地抑制ＰＤＧＦ诱导的ＮＩＨ３Ｔ３细胞酪氨酸磷酸化程度。结论酪氨酸蛋白激酶抑制剂可显著地抑制ＰＤＧＦ诱导的ＮＩＨ３Ｔ３细胞增殖，可能是通过抑制酪氨酸蛋白激酶活性来完成。     

逆转录病毒介导的牛生长激素基因转染NIH3T3细胞和人胚肺成纤维细胞

目的：以牛生长激素(bGH)为模型，建立逆转录病毒载体转移系统，体外转染鼠成纤维细胞株NIH3T3和人胚肺成纤维细胞2BS，以获得bGH表达。方法：将PCR扩增的bGH基因片段插入逆转录病毒载体pSXLC-MDR相应酶切位点，获得的重组逆转录病毒质粒。应用脂质体方法转洒重组质粒进入包装细胞PA317，筛选长春新碱抗性克隆株，收集上清液感染NIH3T3细胞和人胚肺成纤维细胞2BS，筛选携带bGH基因的成纤维细胞的克隆株3T3／bGH和2BS／bGH。结果：RT－PCR分析表明基因转染组的3T3／bGH细胞和2BS／bGH细胞在相当于680bp处有一清晰的条带，而空质粒对照组无相应条带出现。RIA分析表明转染基因细胞的培养液及胞浆蛋白有bGH蛋白表达，而作为对照的3T3／mdr细胞和NIH／3T3细胞均无此蛋白表达。结论：应用逆转录病毒IRBS载体系统转染bGH基因进入成纤维细胞，该系统易筛选，转染效率较高。     

稳定表达EGFR胞外区的NIH3T3细胞株的建立及鉴定

目的构建稳定表达表皮生长因子受体(EGFR)胞外区(EGFRex)的NIH3T3细胞株。方法构建带有EGFRex基因的重组真核表达质粒PLNCX2-EGFRex,经脂质体法转染NIH3T3细胞后,用G418筛选阳性克隆,用免疫组化和蛋白质印迹(Western blot)检测EGFRex蛋白的表达,用EGF-EGFP蛋白检测其活性。结果成功构建了真核表达载体PLNCX2-EGFRex。以其转染NIH3T3细胞后,经免疫组化和Western blot检测到EGFRex的表达。在荧光显微镜下见细胞膜上有绿色荧光。结论人EGFRex在NIH3T3细胞内以其天然活性形式稳定表达,为抗体制备奠定了基础。     

Thermotolerance inhibits various stress-induced apoptosis in NIH3T3 cells

When NIH3T3 cells were exposed to mild heat and recovered at 37°C for various time intervals, they were thermotolerant and resistant to subsequent stresses including heat, oxidative stresses, and antitumor drug methotrexate which are apoptotic inducers. The induction kinetics of apoptosis by stresses were determined by DNA fragmentation and protein synthesis using [³⁵S]methionine pulse labeling. We investigated the hypothesis that thermotolerant cells were resistant to apoptotic cell death compared to control cells when both cells were exposed to various stresses inducing apoptosis. The cellular changes in thermotolerant cells were examined to determine which components are involved in this resistance. At first, the degree of resistance correlates with the extent of heat shock protein synthesis which were varied depending on the heating times at 45°C and recovery times at 37°C after heat shock. Secondly, membrane permeability change was observed in thermotolerant cells. When cells prelabeled with [³H]thymidine were exposed to various amounts of heat and recovered at 37°C for 1/2 to 24 h, the permeability of cytosolic [³H]thymidine in thermotolerant cells was 4 fold higher than that in control cells. Thirdly, the protein synthesis rates in thermotolerant and control cells were measured after exposing the cells to the same extent of stress. It turned out that thermotolerant cells were less damaged to same amount of stress than control cells, although the recovery rates are very similar to each other. These results demonstrate that an increase of heat shock proteins and membrane changes in thermotolerant cells may protect the cells from the stresses and increase the resistance to apoptotic cell death, even though the exact mechanism should be further studied.     

Sinomenine hydrochloride inhibits the progression of plasma cell mastitis by regulating IL-6/JAK2/STAT3 pathway

Plasma cell mastitis (PCM) is a special form of mastitis characterized by periductal inflammation and large-scale plasma cell infiltration. At present, the recurrence rate of PCM after excision is quite high, making PCM a major problem for mammary surgeons. However, no effective drug exists for the treatment of PCM. Numerous studies have demonstrated that Sinomenine hydrochloride (SH) has potent anti-inflammatory and immunoregulatory properties. However, the efficacy and the underlying mechanisms of SH in the treatment of PCM remain unclear. In the present study, we first investigated the therapeutic effects of SH in the PCM mouse model and clarified the possible mechanisms. We found that the levels of plasmocytes and lymphocytes infiltration were alleviated significantly in the 100 mg/kg SH group compared to the control group. In addition, few CD138+ plasma cells were found in the mammary glands of the 100 mg/kg SH group. The levels of Bcl-2 in the 100 mg/kg SH group were dramatically decreased compared with those in the saline group. Mechanistically, we demonstrated that SH inhibited the progression of PCM mainly through downregulating IL-6/JAK2/STAT3 levels. Collectively, our results suggested that SH could inhibit the progression of PCM by suppressing IL-6/JAK2/STAT3 cascades and ultimately achieve a therapeutic effect in PCM. This study provides theoretical support for the clinical application of SH in the treatment of PCM.     

Adenosine inhibits epileptiform activity arising in hippocampal area CA3.

The ability of adenosine and structurally-related compounds to inhibit epileptiform activity induced by bicuculline in the CA3 region of the hippocampal slice of the rat was examined. Bath application of all purinoceptor agonists tested reduced the frequency of generation of burst potentials. Analysis of dose-response curves yielded the following IC50 values: adenosine, 1.5 microM; 2-chloroadenosine, 0.144 microM; 5'-(N-ethyl)carboxamidoadenosine, 30.2 nM; L-phenylisopropyladenosine, 12.1 nM; cyclohexyladenosine, 7.9 nM. Theophylline (30 microM) increased the rate of bursting and antagonized the effect of exogenous adenosine. Dipyridamole (0.03-1 microM) reduced the occurrence of burst firing. In slices untreated with bicuculline, theophylline (30 microM) and adenosine deaminase (10 micrograms ml-1) induced bursting activity. These results demonstrate that purinoceptor agonists can suppress epileptiform activity in the hippocampus and suggest that adenosine may act as an endogenous anticonvulsant.     

Glycoprotein isolated from Ulmus davidiana Nakai inhibits TPA-induced apoptosis through nuclear factor-kappa B in NIH/3T3 cells

Glycoprotein of Ulmus davidiana Nakai (UDN glycoprotein) was isolated and identified using SDS-PAGE. UDN glycoprotein was shown to have strong scavenging activities against oxygen free radicals, as detected by different oxygen-radical formation assays. To investigate the anti-apoptotic effects of UDN glycoprotein, we investigated the activity of protein kinase C alpha (PKCalpha), the DNA-binding activation of nuclear factor-kappa B (NF-kappaB), the production of nitric oxide (NO) and apoptosis in 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated NIH/3T3 cells using a western blot analysis, electrophoretic mobility shift assays (EMSA) and NO assays. Results in this experiment showed that 100 microg/ml of UDN glycoprotein has inhibitory effects on PKCalpha translocation, NF-kappaB DNA binding activity, NO production, and apoptosis in TPA (61.68 ng/ml)-stimulated NIH/3T3 cells. Interestingly however, it could not regulate the DNA binding activity of AP-1. Therefore, UDN glycoprotein, a natural anti-oxidant, is a potential modulator of apoptotic signal pathways in NIH/3T3 cells.     

BD750, a benzothiazole derivative,inhibits T cell proliferation by affecting the JAK3/STAT5 signalling pathway

Background and Purpose

A series of benzothiazole derivatives were screened for immunosuppressive activity; of these compounds BD750 was found to be the most effective immunosuppressant. The purpose of the current study was to determine the immunosuppressive activity of BD750 on T cell proliferation and its potential mode of action.

Experimental Approach

T cell proliferation, CD25 and CD69 expression and cell cycle distribution were measured in vitro by flow cytometry. Cell viability was determined by CCK-8 assay. Cytokine levels were measured by elisa. The activation of signal-regulated molecules was assessed by Western blot analysis. The effects of BD750 were evaluated in vivo in a mouse model of delayed-type hypersensitivity.

Key Results

BD750 significantly inhibited mouse and human T cell proliferation, stimulated either by anti-CD3/anti-CD28 monoclonal antibodies or by an alloantigen, in a dose-dependent manner in vitro. No obvious cytotoxic effects of BD750 were observed in our experimental conditions. Furthermore, BD750 did not inhibit CD25 and CD69 expression or IL-2 and IL-4 secretion, but induced cell cycle arrest at the G₀/G₁ phase in activated T cells. In IL-2-stimulated CTLL-2 cells and primary activated T cells, BD750 inhibited cell proliferation and STAT5 phosphorylation, but not Akt or p70S6K phosphorylation. BD750 also reduced the T cell-mediated delayed-type hypersensitivity response in mice in a dose-dependent manner.

Conclusion and Implications

These data indicate that BD750 inhibits IL-2-induced JAK3/STAT5-dependent T cell proliferation. BD750 has the potential to be used as a lead compound for the design and development of new immunosuppressants for preventing graft rejection and treating autoimmune diseases.     

利用CRISPR/Cas9n系统构建Asxl2基因敲除的NIH3T3稳定细胞系

摘要： 目的 利用 CRISPR/Cas9n 系统在 NIH3T3 小鼠胚胎成纤维细胞系中敲除 Asxl2 基因。方法 设计一对靶 向小鼠 Asxl2 基因第 5 个外显子的小向导 RNA （sgRNA）, 分别克隆进 pX462 载体。将测序鉴定正确的重组质粒转染 至 NIH3T3 细胞中, 利用有限稀释法得到单细胞, 通过培养获得单克隆细胞系。提取单克隆细胞系基因组 DNA, ge⁃ notyping PCR 扩增出靶位点附近的 DNA 片段并测序。利用 Western blot 方法检测细胞株中 Asxl2 的敲除效果。结 果 成功构建靶向 Asxl2 的 CRISPR/Cas9n 重组质粒。将 2 个重组质粒共转染 NIH3T3 细胞, 嘌呤霉素筛选后得到亚 克隆细胞系, 并且经 genotyping PCR 测序验证得到一株正确的单克隆细胞系。Western blot 证实敲除 Asxl2 后, 该 NIH3T3 细胞系中 Asxl2 蛋白表达缺失。结论 通过这个系统得到了靶向 Asxl2 的 CRISPR/Cas9n 重组质粒及稳定 敲除 Asxl2 的NIH3T3 细胞系。     

熊果酸通过ROS／AMPK／STAT3信号通路抑制胃癌细胞COX-2表达

目的：探讨胃癌细胞中活性氧（ROS ）／单磷酸腺苷激活的蛋白激酶（AM PK ）／信号转导与转录活化因子3（STAT 3）信号通路在熊果酸抑制环氧化酶2（COX‐2）表达中的作用。方法在人胃腺癌细胞株SGC‐7901中加入不同浓度熊果酸（0、10、20、30μmol／L ）培养24 h ,或用抗氧化剂N‐乙酰‐L半胱氨酸（NAC 2.5 mmol／L）预处理30 min后熊果酸再干预培养24 h。采用荧光探针2′,7′‐二氯荧光素（DCFH‐DA）检测细胞内ROS水平,Western blot检测AMPK、STAT 3磷酸化水平和COX‐2蛋白表达。结果熊果酸明显增加SGC‐7901细胞内ROS生成和AMPK磷酸化水平,抑制STAT3磷酸化和 COX‐2蛋白表达（P＜0．01或P＜0．05）;而 NAC 能有效逆转上述各指标的变化过程（P＜0．01或P＜0．05）。结论熊果酸可能通过 ROS／AMPK／STAT3信号转导通路抑制胃癌细胞COX‐2表达。