大鼠脊髓缺血损伤后β管蛋白及其mRNA的表达变化

目的：研究大鼠脊髓缺血损伤后β管蛋白及其ｍＲＮＡ的表达变化。方法：利用β管蛋白的单抗及地高辛标记ｃＤＮＡ探针，借助于免疫组化、原位杂交、图像分析等方法，对腹主动脉阻断（肾动脉上）３０ｍｉｎ后再灌流不同时间点大鼠缺血损伤脊髓神经元β管蛋白及其ｍＲＮＡ的变化进行了定性定量研究。结果：ＳＣＩ后β管蛋白及ｍＲＮＡ均明显降低，３ｄ达最低水平，７ｄ有所恢复，到１４ｄβ管蛋白ｍＲＮＡ恢复到伤前，而β管蛋白仍维持低水平。结论：β管蛋白破坏，可能与Ｃａ２＋激活的中性蛋白酶有关；β管蛋白与其ｍＲＮＡ的分离，提示β管蛋白的减少，除转录水平减少所致翻译产物降低外，破坏增多亦是可能的原因。

神经再生过程中脊髓运动神经元β管蛋白基因表达的变化

损伤大鼠右侧坐骨神经后,SDS—PAGE电泳分析双侧腹角细胞中蛋白含量变化,发现损伤侧的管蛋白相对含量较对照侧增加了17％—121％。损伤后3—14d损伤侧腹角运动神经元中β管蛋白mRNA的原位杂交信号增强了27％—70％,β管蛋白mRNA的Northern杂交分析也显示基因表达的增强。

Determination of Internal Controls for Quantitative Gene Expression of Isochrysis zhangjiangensis at Nitrogen Stress Condition

lsochrysis zhangfiangensis is a potential marine microalga for biodiesel production, which accumulates lipid under ni- trogen limitation conditions, but the mechanism on molecular level is veiled. Quantitative real-time polymerase chain reaction （qPCR） provides the possibility to investigate the gene expression levels, and a valid reference for data normalization is an essential prerequisite for firing up the analysis. In this study, five housekeeping genes, actin （ACT）, α-tubulin （TUA）, β-tubulin （TUB）, ubiquitin （UBI）, 18S rRNA （18S） and one target gene, diacylglycerol acyltransferase （DGAT）, were used for determining the reference. By analyzing the stabilities based on calculation of the stability index and on operating the two types of software, geNorm and bestkeeper, it showed that the reference genes widely used in higher plant and microalgae, such as UBI, TUA and 18S, were not the most stable ones in nitrogen-stressed 1. zhangjiangensis, and thus are not suitable for exploring the mRNA expression levels under these experi- mental conditions. Our results show that ACT together with TUB is the most feasible internal control for investigating gene expres- sion under nitrogen-stressed conditions. Our findings will contribute not only to future qPCR studies of/. zhangfiangensis, but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.

The impact of beta-elemene on beta-tubulin of human hepatoma hepg2 cells

Objective: The aim of this study was to investigate the impact of beta-elemene injection on the growth and beta-tubulin of human hepatocarcinoma HepG2 cells. Methods: Cell proliferation was assessed by MTT assay. Cell cycle distribution was detected by flow cytometry(FCM). The mRNA expression of beta-tubulin was measured by RT-PCR. Western blot analysis was used to determine protein expression of beta-tubulin and the polymerization of beta-tubulin. Results: Beta-elemene injection inhibited HepG2 cells proliferation in a dose- and time-dependent manner; FCM analysis indicated beta-elemene injection induced cell cycle arrested at S phase. RT-PCR and western-blot analysis showed that beta-elemene injection down-regulated beta-tubulin expression at both mRNA and protein levels, presenting a dose-dependent manner. Moreover, beta-elemene injection reduced the polymerization of microtubules in a dose-dependent manner. Conclusion: Beta-elemene injection can inhibit the proliferation of hepatoma HepG2 cells, the mechanism might be partly related to the down-regulation of beta-tubulin and inhibition of microtubular polymerization.

腺病毒介导脑源性神经营养因子基因转移对神经根损伤后再生相关基因的影响

目的观察腺病毒介导的脑源性神经营养因子(BDNF)基因脊髓内转移对神经根损伤后再生相关基因表达的影响。方法在大鼠神经根切断吻合模型基础上,通过显微注射法在立体定位仪上将2μl复制缺陷型重组腺病毒载体(AxCA-BDNF)直接注入大鼠神经根损伤部相应的脊髓腹角。损伤组注射病毒缓冲液2μl。术后1、3、7、14、28d,利用免疫组化、原位杂交,观察β管蛋白和GAP-43mRNA表达的改变。结果与损伤组比较,注射AxCA-BDNF组动物β微管蛋白和GAP-43 mRNA表达在术后3、7、14、28d间差异有统计学意义(P＜0．05)。结论AxCA-BDNF治疗能促进神经根损伤后神经元β管蛋白的合成,促进轴突的延伸；同时发现AxCA-BDNF治疗,能上调GAP-43表达,增加损伤神经元发芽的潜能。