siRNA沉默DNA甲基化转移酶1基因干扰胎牛成纤维细胞增殖和凋亡（英文）

为阐明Dnmt1-siRNA对胎牛成纤维细胞(FBFCs)的影响,设计了3种针对Dnmt1的siRNA来转染FBFCs.结果显示:Dnmt1-siRNA3转染FBFCs 24,48,72 h后,Dnmt1 mRNA表达水平显著降低(P0.01).在转染后48 h,siRNA3对Dnmt1抑制效率达到近80%,Dnmt1-siRNA3处理的FBFCs增殖也受到显著影响,FBFC细胞活力下降、处于G0/G1过渡期细胞数量增多(P0.05).但Dnmt1-siRNA3组FBFC细胞的凋亡率也显著增加(P0.05).说明通过Dnmt1特异性siRNA能够有效地敲低FBFC中的Dnmt1 mRNA表达量,由于细胞周期分布变化,siRNA处理后FBFC作为核供体能提高SCNT的效率.     

人类T型钙通道α1G和α1H亚单位基因在细胞增殖中的功能

已经发现T型钙通道的表达与细胞增殖关系密切，然而T型钙通道的表达是细胞增殖的原因还是结果有待进一步研究．利用过量表达人类T型钙通道α_1G和α_1H亚单位基因 （CACNA1G和CACNA1H） 的HEK-293细胞研究了这两种基因在细胞增殖中的直接作用．通过RT-PCR和标准全细胞膜片钳记录分别从mRNA转录水平和T型钙通道蛋白功能水平验证了α_1G和α_1H亚单位基因的过量表达；从生长曲线分析得到了细胞群体倍增时间，HEK_α1G ⁺细胞为（13.7±0.3）h，HEK_α1H ⁺细胞为（14.0±0.4）h，都短于对照HEK-293细胞的（22.1±1.1）h.流式细胞分析结果表明，在稳定转染细胞处于S期的细胞百分率比对照HEK-293细胞高，相反地处于G₁期的百分率比对照HEK-293细胞低．结果表明，T型钙通道α_1G和α_1H亚单位基因的过量表达都能显著促进细胞增殖，这一作用可以被T型钙通道特异性阻断剂mibefradil抑制．Western杂交结果提示了T型钙通道的过量表达是通过提高与细胞周期有关的蛋白质（CDK2，cyclin A和 cyclin E）的表达水平刺激了细胞周期的进程从而促进细胞增殖．     

一个构造等能量图族的方法

一个图的能量定义为图的邻接矩阵的特征值的绝对值之和,是一类重要的图指标. 利用矩阵性质给出了一类联并图的谱刻划：正则图G₁,G₂,…,G_n的联并图G［G₁,G₂,…,G_n］的谱是由正则图G₁,G₂,…,G_n的谱（去掉每个正则图的第一个最大特征值）和一个由图G决定的辅助矩阵的特征值组成. 这个刻划能够给出一个构造等能量图的方法. 作为方法的应用,给出一些等能量图的例子.     

乙型肝炎病毒HBx基因的shRNA重组表达载体的构建和鉴定

目的: 构建 pGenesil-3.1-HBx-shRNA 重组表达载体, 探讨其作用于 HepG2.2.15 细胞的生物活性． 方法: 构建了针对HBx基因的siRNA 表达载体,通过电穿孔法转染 HepG2.2.15 细胞,以荧光显微镜观察细胞的转染效率,QRT-PCR检测细胞中HBx基因的相对表达量,利用CCK-8细胞增殖检测试剂盒检测了细胞增殖活力,流式细胞术检测细胞凋亡．结果: 酶切和测序鉴定证实构建质粒含有HBx基因,QRT-PCR检测和荧光显微镜观察到构建的pGenesil-3.1-HBx-shRNA 可以在 HepG2.2.15 细胞中表达,HBx基因的表达下降了 28%(P＜0.05),细胞增殖水平下降了47%(P＜0.05),质粒 pGenesil-3.1-HBx-shRNA 转染后的HepG2.2.15 细胞凋亡率显著升高．结论: 乙型肝炎病毒HBx基因的shRNA重组表达载体构建成功,沉默HBx基因的shRNA能够抑制HBx基因的表达,且对HepG2.2.15细胞的增殖水平起明显的抑制作用．     

P53亚细胞定位变化对POLD1基因启动子活性的影响

POLD1基因编码DNA聚合酶δ（pol δ）的催化亚基．体外实验表明p53能够抑制POLD1基因启动子活性．文中探讨p53是否在细胞内直接与POLD1启动子结合调节POLD1基因表达. 在瞬时转染pEGFP-p53重组质粒的人乳腺癌MCF7细胞中，p53表达增强; RT-PCR结果显示POLD1基因mRNA表达受到抑制． 染色体免疫共沉淀和荧光素酶报告基因实验证明p53在细胞内与POLD1启动子直接结合抑制其启动子活性．荧光显微镜观察发现EGFPp53在G1期进入细胞核而在S期和G2期则被转运至细胞质．在稳定表达的pEGFP-p53细胞系中，细胞周期同步化后POLD1启动子活性在细胞周期各时相均处于较低水平．证明了细胞内p53高表达后通过直接与POLD1启动子结合而抑制POLD1基因表达，且这种抑制作用不受细胞周期进程的影响．     

岗松总黄酮对脂多糖诱导RAW264.7细胞炎症的影响

为探讨岗松总黄酮（GSH）的抗炎活性机制,通过NF-κB信号通路,研究脂多糖（LPS）诱导RAW264.7细胞炎症的保护作用。实验采用噻唑蓝（MTT）法检测GSH对RAW264.7细胞增殖活性的影响,采用酶联免疫法（ELISA）检测GSH对白介素（IL-1β、IL-8）及转换生长因子β （TGF-β）的影响,采用蛋白印迹法（WB）检测细胞中IκBα蛋白相对表达,采用实时定量基因扩增荧光检测法（QPCR）检测细胞中NF-κB p65的mRNA基因表达。研究结果表明, GSH处理细胞后,可促进RAW264.7细胞增殖（P<0.05）,抑制IL-1β、IL-8、TGF-β含量（P<0.01）,上调IκBα蛋白的相对表达（P<0.05或P<0.01）,抑制NF-κB p65的mRNA基因表达（P<0.01）。推测岗松总黄酮抑制NF-κB信号通路可能是其抗炎活性作用机制之一。     

广义Petersen图P(n,1)和P(n,2)的意大利控制数

在图G=（V, E）中,f为从顶点集合V到｛0,1,2｝的映射,如果满足所有 f（v）=0的顶点v其邻域中至少有一个被赋值为2的顶点或者至少有两个被赋值为1的顶点,则 f 称为图G的意大利控制函数。图G中所有顶点的函数值之和为f 的权重。权重的最小值为图G的意大利控制数。确定图的意大利控制数是NP （non?deterministic polynomial） 困难的。通过构造可递推的意大利控制函数,计算出广义Petersen图P（n,1）和P（n,2）意大利控制数的上界。利用袋装法和控制代价函数法分别证明出P（n,1）和P（n,2）意大利控制数的下界。最终确定了P（n,1）和P（n,2）意大利控制数的精确值。     

宿州市城区河流沱河与新汴河水质评价及污染来源对比研究

煤炭型城市河流污染评价与溯源是国内外学者关注的热点问题。以典型的煤炭型城市宿州市城区河流新汴河与沱河为研究对象,测定水体中污染物指标化学需氧量（COD）、总磷（TP）、总氮（TN）,氨氮（NH₃-N）和高锰酸盐指数（COD_Mn）等指标,利用单因子水质标识指数法、相关性分析等方法对比2条河流的水质与污染来源。结果表明：（1）新汴河与沱河水体中TN质量浓度较高,NH₃-N与TP所对应的水质类别全部符合安徽省水功能区划对应的标准,2条河流的COD与COD_Mn质量浓度均存在不同程度的超标;（2）新汴河中NH₃-N与TP质量浓度存在极显著正相关（r = 0.701,P＜0.01）,有着相似的污染源,主要受河流周边农田肥料使用的影响;沱河中TN均与COD_Mn、NH₃-N质量浓度之间都存在极显著正相关（r分别为0.637与0.555,P＜0.01）,NH₃-N与COD_Mn质量浓度之间存在显著正相关（r = 0.418,P＜0.05）,而NH₃-N与TP质量浓度之间存在显著负相关（r = -0.469,P＜0.05）,说明氮污染物与磷污染物存在着不同的污染来源,氮污染分布可能是受煤炭开采矿井废水排放影响较大,磷污染分布可能是受居民生活洗涤污水排放的影响较大。研究结果可为人类活动对煤炭型城市城区河流污染分布的影响机制提供理论参考。     

遮光对红鳞蒲桃幼苗光合特性的影响

为了解红鳞蒲桃（Syzygium hancei）对光能的需求及适应性,以红鳞蒲桃幼苗为材料,设置透光率分别为100.0%、72.3%、48.6%、24.9%的4种光照处理,测定其光合作用参数、光响应参数、叶绿素含量等数据,研究不同遮光处理对其光合特性的影响。结果表明：（1）红鳞蒲桃是喜光树种,红鳞蒲桃幼苗净光合速率（Net Photosynthetic Rate,P_n）、气孔导度（Stomatal Conductance,G_s）日均值随着遮光程度的增加而减小,胞间CO₂浓度（Intercellular Carbon Dioxide Concentration,C_i）日均值随之增大,过度遮光下（透光率为48.6%和24.9%）,蒸腾速率（Transpiration Rate,T_r）日均值显著低于全光照;（2）遮光条件下,红鳞蒲桃幼苗净光合速率下降可能是非气孔限制引起的;（3）红鳞蒲桃幼苗的最大净光合速率（Maximum Net Photosynthetic Rate,P_max）、光补偿点（Light Compensation Point,LCP）、光饱和点（Light Saturation Point,LSP）、暗呼吸速率（Respiration Rate,R_d）均随着光照强度的增加而显著增加;（4）红鳞蒲桃幼苗叶绿素a、叶绿素b及叶绿素（a+b）的含量均随着遮光程度的增加而显著增加,叶绿素a/b的值随之减小。红鳞蒲桃在幼苗期具有较明显的喜光性,因此,在红鳞蒲桃幼苗的培育及种群的恢复过程中,可通过疏伐、修剪枝条等措施来增加林内的透光量,为处于幼苗时期的红鳞蒲桃创造适宜的光照环境,为其自然更新提供良好条件。     

白肉灵芝水提物对新生大鼠胆红素脑病的影响

为了研究白肉灵芝水提物（ganoderma leucocontextum aqueous extracts, GLAE）对新生大鼠胆红素脑病的改善作用。腹腔注射胆红素建立高胆红素血症大鼠模型，将新生大鼠分成对照组、模型组、GLAE低剂量组、GLAE中剂量组和GLAE高剂量组，分别给予不同剂量的（0、50、100、200 mg/kg）GLAE处理。利用试剂盒检测血液样本和大脑组织中总胆红素水平，比色法检测大脑组织ATP酶、超氧化物歧化酶（superoxide dismutase, SOD）、过氧化氢酶（catalase, CAT）、谷胱甘肽过氧化物酶（glutathione peroxidase, GSH-Px）活性和丙二醛（malondialdehyde, MDA）含量，酶联免疫吸附（ELISA）法检测血清肿瘤坏死因子-α（tumornecrosisfactor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）、神经特异性烯醇化酶（neuron specific enolase, NSE）和中枢神经特异性蛋白（central nerve specific protein, S100β）的含量，实时荧光定量PCR和western blot检测大脑组织B淋巴细胞瘤-2（B-cell lymphoma-2, Bcl-2）、半胱氨酸天冬氨酸蛋白酶-3 （cysteine aspartate protease 3, Caspase-3）、B淋巴细胞瘤-2 相关X（Bcl-2 assaciated X, Bax）、脑源性神经生长因子（brain derived neurotrophic factor, BDNF）、神经生长因子（nerve growth factor, NGF）mRNA水平和蛋白含量。结果显示，与模型组相比，经GLAE干预后，大鼠血清及大脑组织胆红素浓度明显降低（P < 0.05，P < 0.01）；大脑组织SOD、CAT、GSH-Px、Na⁺-K⁺-ATP和Ca²⁺-ATP酶活性显著升高（P < 0.05，P < 0.01），MDA含量明显降低（P < 0.05，P < 0.01）；血清TNF-α、IL-1β、NSE、S100β含量显著降低（P < 0.05，P < 0.01）；大脑组织Caspase-3、Bax mRNA水平和蛋白含量明显降低（P< 0.05，P < 0.01），Bcl-2、BDNF、NGF mRNA水平和蛋白含量明显升高（P < 0.05，P < 0.01）。以上结果表明GLAE能减轻过量胆红素对新生大鼠的大脑损伤，其作用机制可能通过改善新生大鼠大脑能量代谢，提高大脑抗氧化能力，增加神经营养因子含量，抑制炎症反应及凋亡基因的表达来发挥作用。     

Effect of PKC pathway on G1/S progression control in HeLa cells

The effect of PKC activity on G₁/S progression in HeLa cells has been studied. The result shows that (ⅰ ) PKC activity alteration in G₁ phase affects G₁/S progression in HeLa cells. It has been observed that G₁/S progression is stimulated by PKC agonist TPA and inhibited by PKC inhibitor GF-109203X. ( ⅱ) The expression of c-myc and c-jun is stimulated by TPA and inhibited by GF-109203X treatment in early G₁ phase. (ⅲ ) During G₁/S progression, the expression of CyclinD₁ is stimulated by TPA treatment and inhibited by GF-109203X treatment. There is no effect on the expression of CDK4. It is likely that PKC pathway regulates G₁/S progression through regulating the expression of some early response genes and engine molecules in HeLa cells.     

Effects of antisense CENP-B on the proliferation of HeLa (Tet-off) cells

In order to study the change of the expression of centromere protein B (CENP-B) caused by antisense transfection, proto-eukaryotically expressed fused protein GST-CENP-B (65 ku) was injected into mouse, and a peculiar anti-CENP-B serum MaCenpB was collected. A strain of transfected HeLa Tet-off cell HaCb, which contains antisense CENP-B expressing vector pBI-EGFP-as-CenpB, was prepared. Northern blot and Western blot were used to analyze the repression of internal CENP-B in transfected cells. According to the growth curve, the proliferation of HeLa (Tet-off) is repressed by antisense CENP-B, and the multiplication time is prolonged for 32.81 h. The analysis of flow cytometry revealed that, compared with HeLa (Tet-off), the G₁ cell population of HaCb is increased (ΔG₁= 9%) while S fraction is decreased (ΔS = 11%), but the G₂/M phase is nearly unchanged (ΔG₂/M=3%). In the meanwhile, the mitotic index of HaCb declines greatly compared with that of HeLa (Tet-off). Immunofluorescence showed that the assembling of centromeres in HaCb cell is arrested. These results suggest that a normal expression of CENP-B may be necessary for cell proliferation.     

Rapid induction of mRNAs forPC3 by partial hepatectomy and epidermal growth factor

The expression of immediate early gene plays a pivotal role in rat hepatocyte proliferation from G₀ to G₁ phases and the progression through G₁ phase of the cell cycle within several hours after 2/3 hepatectomy. We investigated the different gene expressions within 1 h after 2/3 hepatectomy by representational difference analysis. Sequence analysis indicated thatPC3 induced by NGF was a kind of immediate early gene and might be correlated with liver regeneration. Moreover, we found that 2/3 hepatectomy could induce the expressing ofPC3 mRNA by Northern blot with a peak 1–2 h after surgery. In primary cultures of rat hepatocytes, addition of EGF resulted in rapid and transient induction ofPC3 mRNA. It was first reported thatPC3 gene belongs to immediate early gene associated with liver regeneration.     

Parthenolide inhibits proliferation of vascular smooth muscle cells through induction of G0/G1 phase cell cycle arrest

Objective: This study is to determine the effect of the natural product parthenolide, a sesquiterpene lactone isolated from extracts of the herb Tanacetum parthenium, on the proliferation of vascular smooth muscle cells (VSMCs). Methods: Rat aortic VSMCs were isolated and cultured in vitro, and treated with different concentrations of parthenolide (10, 20 and 30 μmol/L). [³H]thymidine incorporation was used as an index of cell proliferation. Cell cycle progression and distribution were determined by flow cytometric analysis. Furthermore, the expression of several regulatory proteins relevant to VSMC proliferation including IκBα, cyclooxygenase-2 (Cox-2), p21, and p27 was examined to investigate the potential molecular mechanism. Results: Treatment with parthenolide significantly decreased the [³H]thymidine incorporation into DNA by 30%~56% relative to control values in a dose-dependent manner (P<0.05). Addition of parthenolide also increased cell population at G₀/G₁ phase by 19.2%~65.7% (P<0.05) and decreased cell population at S phase by 50.7%~84.8% (P<0.05), which is consistent with its stimulatory effects on p21 and p27. In addition, parthenolide also increased IκBα expression and reduced Cox-2 expression in a time-dependent manner. Conclusion: Our results show that parthenolide significantly inhibits the VSMC proliferation by inducing G₀/G₁ cell cycle arrest. IκBα and Cox-2 are likely involved in such inhibitory effect of parthenolide on VSMC proliferation. These findings warrant further investigation on potential therapeutic implications of parthenolide on VSMC proliferation in vivo.     

Function of resveratrol derived from transgenic plant expressing resveratrol synthase gene

Two genes from grapevine coding for resveratrol synthase, named RS1 and RS2, were cloned by RT-PCR. AnEscherichia coli expression vector was constructed by insertion of RS1 into pBV221. A specific protein with the same molecular weight (42 ku) as the resveratrol synthase was expressed and used to prepare the rabbit antiserum. A plant expression vector was constructed by inserting the RS1 gene into pBin438 downstream of the doubled CaMV 35S promoter and TMV-Ω fragment. PCR-positive transgenic tobacco plants were obtained after transformation withAgrobacterium tumefaciens LBA4404 harboring the plant expression vector. Southern blot analysis demonstrated that the foreign gene was integrated into the tobacco genome. The results of RT-PCR and Western blot indicated that the RS1 gene was transcribed and expressed. Formation of resveratrol in transgenic tobacco was further determined by thin-layer chromatography of silica gel and HPLC. Increased accumulation of human breast adenocarcinoma cells in G₀ and G₁ phases of cell cycle was observed in cells treated with resveratrol purified from transgenic tobacco as compared to the untreated cells.     

microRNA-16 expression in HeLa cells is upregulated in cell-cycle and drug dependent manner

microRNAs are single-stranded, non-coding RNAs that regulate gene expression. The microRNA-16 family has been reported to be involved in cell-cycle regulation, which could also downregulate expression of multiple pro-proliferation genes. The present results demonstrated that miR-16 expression in HeLa cells increased when the cells were arrested during S-phase under methyl methanesulfate (MMS) treatment. This further resulted in downregulation of a target protein CDC25A, whereas miR-16 expression did not increase when HeLa cells were arrested during the MMS-treated G₀/G₁ or G₂/M phase. Furthermore, when HeLa cells were arrested during S-phase with hydroxyurea treatment, miR-16 expression did not increase. These results suggest that expression levels of microRNAs in mammalian cells are delicately regulated under variable cellular conditions.     

肺泡上皮细胞在高氧急性肺损伤中的作用研究

目的:观察新生小鼠肺组织甲状腺转录因子[TTF-1]及肺表面活性物质蛋白B[SP-B]的表达,探讨高氧急性肺损伤[HALI]可能存在的损伤机制.方法:新生1d的C57BL/6小鼠30只,随机分为:高氧暴露组及空气对照组,每组15只.高浓度氧暴露72h建立ALI小鼠模型,各组于24,48及72h随机取5只小鼠肺组织标本,测定肺湿/干重(W/D)比值、观察肺组织形态学变化并进行病理评分及肺泡计数(RAC),荧光定量PCR法及免疫荧光标记法检测各组各实验点TTF-1,SP-BmRNA及蛋白的表达情况.结果:与空气对照组比较,高氧暴露48h时,肺组织出现肺泡间质细胞数量增多及肺水肿等HALI的表现;W/D比值及病理评分显著升高(P0.05);RAC显著减少(P0.05).高氧暴露72h时,TTF-1,SP-B mRNA及蛋白表达较空气对照组下调(P0.05).结论:高浓度氧暴露能导致ALI,肺泡上皮细胞的损伤在HALI发生进程中可能发挥重要作用.