Akt3稳定表达细胞株的建立及其对MDA-MB-231细胞增殖的影响

目的 构建人蛋白激酶Bγ（Akt3）基因编码区序列（cDNA）的真核表达载体、建立其稳定表达细胞株并观察其对MDA-MB-231细胞增殖的影响.方法 从流产胎儿脑组织中提取总RNA,采用RT-PCR方法扩增Akt3 cDNA的全长序列后克隆入pEGFP-N2质粒中,构建成Akt3基因真核表达载体,然后转染入MDA-MB-231细胞中,新霉素筛选稳定转染细胞克隆,通过MTT实验,研究转染Akt3基因前后细胞增殖的变化.结果 重组载体经酶切鉴定和测序证实目的 基因正确无误.Western印迹检测结果显示AKT3融合蛋白在MDA-MB-231细胞中表达良好,而转染空载体及未转染细胞对照中未见有此融合蛋白质条带;MTT结果显示AKT3表达上调的稳定克隆组,其增殖活性显著高于空载体稳定转染细胞组及未转染亲代细胞组,差异具有统计学意义（P＜0.01）,而后两者差异无统计学意义（P＞0.05）.结论 Akt3过表达可增强MDA-MB-231细胞的增殖.     

潮霉素抗性3T3细胞的建立和鉴定

目的　建立具有潮霉素 (hygromycin)抗性的 3T3细胞系 ,用于转染目的基因 (pTRE Ins human)的ES阳性细胞克隆筛选的饲养层。方法　通过脂质体转染的方法 ,将含有潮霉素B磷酸转移酶基因的质粒pHyg导入 3T3细胞中 ,利用潮霉素的药物选择特性 ,对转染细胞进行压力筛选 ,并对其进行PCR鉴定。结果　经 5 0 0 μg ml的潮霉素压力筛选后 ,获得了抗性细胞克隆。抗性 3T3细胞的形态和生长速度与正常 3T3细胞没有差异 ,特异性核苷酸引物检测抗性细胞基因组DNA ,可以扩增出对应的核苷酸片段。结论　成功地培育了潮霉素抗性的 3T3细胞 ,为进行目的基因 (pTRE Ins human)转染ES细胞的阳性细胞克隆筛选奠定了基础。     

Comparative study of the genetic effect of various tritium compounds in male mice

Comparative study of the genetic effect of some tritiated biogenic++ compounds and tritiated water was carried out. Reciprocal translocations induced in mice stem spermatogonia by 3H-glucose, 3H-glycine and 3H-lysine occur with similar frequency, whereas the difference in values of the absorbed doses of beta-irradiation in some cases was about two-fold. The highest genetic effect per unit of activity of 3H-nucleosides (3H-TdR and 3H-CdR) was revealed at lowest activities. The frequency of reciprocal translocations induced by 3H-TdR was twice as that induced by 3H-CdR.     

凋亡素蛋白多克隆抗体的制备及免疫学评价

凋亡素由鸡贫血病毒中的VP3基因编码,能诱导多种肿瘤细胞发生凋亡。以真核表达载体pcDNA3.0-VP3为模板构建原核表达载体pET8a-VP3,经NdeⅠ/BamHⅠ双酶切鉴定和基因测序无误后,在IPGT诱导下表达VP3蛋白并对其进行纯化,将纯化后的VP3蛋白与弗氏完全佐剂或不完全佐剂乳化后,分别对两只新西兰大耳白兔进行皮下多点注射,间接ELISA检测免疫后血清效价,效价达到指标后第2天以心脏穿刺的方法采全血后分离抗血清。抗血清效价高的兔子进一步采用Protein A纯化总IgG,最终纯化后的抗体效价可以达到1 ∶ 243 000。用重组腺相关病毒rAAV-VP3感染细胞后对抗体的特异性进行免疫学评价。首先利用免疫荧光技术检测VP3基因在人膀胱癌细胞株T24、EJ细胞以及Vero细胞中的表达情况,观察到凋亡素在T24、EJ细胞中主要定位于细胞核,而在Vero细胞中则定位于细胞质。其次通过Western blotting检测纯化后的抗体能与细胞内腺相关病毒介导表达的凋亡素蛋白特异性结合。实验证明了制备的凋亡素蛋白多克隆抗体的有效性和特异性,为进一步阐明凋亡素抗肿瘤效应的分子机制及生物学特性奠定了基础。     

水稻OsNCED3基因的RNAi载体构建

水稻OsNCED3基因是水稻抗逆过程中重要的基因之一.以水稻中花10号幼苗为材料,提取基因组DNA.设计引物扩增区段cDNA并引入相应的酶切位点,以基因组DNA作为模板,进行RNAi-OsNCED3顺式和反式目的片段的PCR扩增.将PCR产物连接到pMD19-T载体上,经酶切和PCR检测后进行测序.测序结果表明:RNAi-OsNCED3顺式和反式目的片段均已正确的连接到pMD19-T载体上.然后将RNAi-OsNCED3顺式和反式目的片段通过酶切和连接,连接到含有发夹结构的质粒pFGC5941上.PCR及双酶切结果显示,构建的pFGC5941-OsNCED3即RNAi-OsNCED3载体结构完整.     

臭氧胁迫下冬小麦物质生产与分配的数值模拟

利用ML9810B型臭氧监测分析仪,测定了浙江嘉兴麦田空气O3浓度,并通过改进的开顶式气室实验确定O3浓度变化对冬小麦叶片光合速率的影响函数.在此基础上,加入O3对叶片生长和穗部光合影响的模拟函数,建立反映O3对冬小麦生长和产量形成影响的作物模型.模型的检验结果表明,该模型较好地反映了O3对冬小麦生长的影响,生物量平均相对误差为10.3%.对冬小麦春后生育期(3—5月)的研究表明,水肥适宜时,由O3影响造成的该地区冬小麦干物质累积总损失量为11.4%,产量损失为17.8%.     

Biosynthesis of cholestanol: 5-alpha-cholestan-3-one reductase of rat liver

The 3-beta-hydroxysteroid dehydrogenase of rat liver which catalyzes the conversion of 5alpha-cholestan-3-one to 5alpha-cholestan-3beta-ol is localized mainly in the microsomal fraction. The enzyme required NADPH as hydrogen donor and differed from the known 3-beta-hydroxysteroid dehydrogenases of the C(19) series in being inactive in the presence of NADH. The microsomal preparations did not reduce the 3-keto groups of cholest-4-en-3-one, cholest-5-en-3-one, or 5beta-cholestan-3-one to the corresponding 3beta-hydroxy compounds. The conversion of 5alpha-cholestan-3-one to 5alpha-cholestan-3beta-ol was only slightly inhibited by the reaction product or by other monohydroxy steroids, but a strong inhibitory effect was noted with cholest-5-en-3-one, 5alpha-cholestane-3beta, 7alpha-diol and 5alpha-cholestan-7-on-3beta-ol. The microsomes, but not high speed supernatant solution, catalyzed the reverse of the cholestanone reductase reaction, namely the conversion of 5alpha-cholestan-3beta-ol to 5alpha-cholestan-3-one in the presence of oxygen and an NADP-generating system. The action of the microsomal preparations upon 5alpha-cholestan-3-one produced 5alpha-cholestan-3alpha-ol in addition to the 3beta-epimer. The 3-alpha-hydroxysteroid dehydrogenase involved functioned with either NADH or NADPH as hydrogen donor. The ratio of 5alpha-cholestan-3beta-ol to 5alpha-cholestan-3alpha-ol formed from 5alpha-cholestan-3-one was approximately 10:1 and was independent of the sex of the animal from which the microsomes were prepared.     

STK15的表达对NIH3T3细胞的影响

目的构建pcDNA3.1-STK15表达质粒,探讨STK15基因对小鼠成纤维细胞（NIH3T3）的影响。方法构建pcDNA3.1-STK15质粒,将其转染NIH3T3,应用RT-PCR、免疫细胞化学和Western印迹方法检测STK15的表达;MTT法检测细胞增殖能力;Transwell检测细胞侵袭能力。结果转染pcDNA3.1-STK15质粒的NIH3T3细胞在48 h有STK15的表达,而且该细胞的增殖速度和穿透Matrigel胶的细胞数均明显高于对照组（P〈0.05）。结论STK15基因具有增加细胞增殖和细胞侵袭力的功能,进而形成肿瘤。     

Expression of non-structural protein NS3 gene of Bombyx mori densovirus （China isolate）

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 (China isolate),named BmDNV-3,is a kind of bidensovirus.It is a new type of virus with unique replication mechanisms.To investigate the effects of the NS3 gene during viral DNA replication,a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus (China isolate).Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe,respectively.The NS3 protein was expressed in Escherichia coli BL21.The pFastBacHTe-NS3 was transformed to E.coli DH10Bac.The recombinant bacmid baculoviruses (rBacmid-EGFP-NS3)isolated from the white colonies were transfected into BraN-4 cells using a transfection reagent.BmN-4 cells were infected with recom-binant virus to express fusion proteins.The expression of fusion protein around 30 kDa in E.coli BL21 was identified by SDS-PAGE,Western blotting,and mass spectrometry.The expressed NS3 protein by B.mor/nucleopolyhedrovirus bacmid system was confirmed byWestern blotting using an anti-NS3 polyclonal antibody.And about 45 kDa protein was found.The expressed fusion protein was smalleithan the expected size of EGFP-NS3,55 kDa.Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected lar-vae with smaller molecular size.     

β-甘露聚糖酶末端残基变体对其酶学特性的影响

本研究用宇佐美曲霉Aspergillus usamii的5家族β-甘露聚糖酶AuMan5A为母本,借助同源建模、分子对接及分子动力学模拟等理性设计方法,将AuMan5A的N-末端和C-末端分别截去3个无规则的氨基酸残基,构建出截短的β-甘露聚糖酶 AuMan5A^N3C3.将AuMan5A和AuMan5A^N3C3的编码基因分别在毕赤酵母GS115中进行表达,对表达产物进行了初步纯化并分析比较了其酶学特性及各自的表达水平. 结果表明,reAuMan5A和reAuMan5A^N3C3的最适温度Topt均为70 ℃,reAuMan5A^N3C3在60 ℃的半衰期t_1/2⁶⁰为38 min,较reAuMan5A（t_1/2⁶⁰=40 min）略有降低;在相同表达条件下,reAuMan5A^N3C3上清液的β-甘露聚糖酶活性为73.4 U/mL,较reAuMan5A 的52.8 U/mL提高了39.0%;纯化的reAuMan5A^N3C3酶比活性为182.7 U/mg protein,较reAuMan5A的126.3 U/mg protein提高了44.7%. 与reAuMan5A相比,reAuMan5A^N3C3对角豆胶的Km值下降不明显,Vmax值有显著提高.     

Synthesis and in vitro biological evaluation of a carbon glycoside analogue of morphine-6-glucuronide

Attachment of a glucose moiety to 6-beta-aminomorphine afforded compound 3, where the glucose moiety was linked to the C-6 nitrogen atom by a two-carbon bridge. The synthesis of 3 was accomplished in eight steps from 3-triisopropylsilyl-6-beta-aminomorphine and 2,3,4,6-tetra-O-benzyl-D-glucose. The C-glycoside 3 was prepared with the objective of examining a metabolically stable analogue of morphine-6-glucuronide and determining the potency and selectivity of opioid receptor binding. Competition binding assays showed that 3 bound to the mu opioid receptor with a Ki value of 3.5 nM. The C-glycoside 3 exhibited delta/mu and kappa/mu selectivity ratios of 76 and 165, respectively. The synthetic intermediate (i.e., benzyl precursor, compound 11) bound to the mu opioid receptor with a Ki value of 0.5 nM, was less selective for the mu opioid receptor. The [35S]GTPgammaS assay was used to evaluate the functional properties of compounds 3 and 11. Compound 3 was determined to be a full agonist at the mu opioid receptor, whereas compound 11 was found to be a partial agonist. Compound 3 was determined to be very stable in the presence of human liver S9, and rat and monkey liver microsomes: no detectable loss of 3 was observed up to 90 min. Compound 3 was also very stable at pH 2 and pH 7.4, suggesting that 3 possessed properties for sustained duration of action.     

14-3-3σ干扰逆转录病毒载体的构建及其稳定转染HaCat细胞系的建立

目的：构建14-3-3σ干扰逆转录病毒载体,建立稳定转染的HaCat细胞系。方法：人工合成14-3-3σ基因干扰序列并定向插入到pSuper-retro-neo-EGFP质粒,并在STBL3菌内进行质粒扩增,刷选阳性克隆,酶切测序鉴定,转染293FT细胞进行病毒包装、扩增、纯化、获取逆转录病毒载体,将逆转录病毒载体感染HaCat细胞后Western免疫印迹法、Real-timePCR法检测14-3-3σ的表达情况。结果：连接重组后经酶切和测序筛选出pSuper-retro-neo-EGFP-si14-3-3σ;干扰质粒稳定转染的HaCat细胞系在倒置荧光显微镜下呈绿色荧光,Western免疫印迹法和Real-timePCR法表明14-3-3σ表达明显抑制。结论：成功构建了14-3-3σ干扰的逆转录病毒载体,并构建了其稳定转染的HaCat细胞系。     

LMO3逆转录病毒表达载体的构建及其对SK-N-AS细胞增殖的影响

目的构建包含LM03（LIM-only3,LM03）全长基因的逆转录病毒表达载体,感染人神经母细胞瘤SK-N-AS,检测LM03对SK-N-AS细胞增殖的影响。方法将质粒pEGFP-Cl-一LM03经EcoRI和BamHI双酶切后亚克隆至逆转录病毒载体pLXSN,构建重组逆转录病毒表达载体pLXSN-LMO3,导人包装细胞pA317,获得逆转录病毒颗粒,感染SK-N-AS细胞,用RT-PCR及Western印迹鉴定,检测LM03感染后细胞的增殖及细胞周期分布情况。结果获得了能正确表达LM03基因的重组逆转录病毒表达载体pLX-SN-LMO3;LM03基因被逆转录病毒成功导入SK-N-AS细胞后,与对照组细胞相比,LM03感染组G1／G1期细胞减少,S期细胞增加,感染48h后,LM03感染组细胞的增殖能力显著高于空载体对照组及SK-N．AS组（P〈0．05）。结论成功构建了LM03基因的逆转录病毒表达载体,LMO3可以通过促进SK-N-AS细胞由G0／G1期进入S期,从而促进细胞的增殖。     

The effect of some analogues of disulfiram on the aldehyde dehydrogenases of sheep liver.

1. The liver microsomal metabolism of [4-14C]cholesterol, endogenous cholesterol, 7 alpha-hydroxy-4-[6 beta-3H]cholesten-3-one, 5-beta-[7 beta-3H]cholestane-3 alpha, 7 alpha-diol and [3H]lithocholic acid was studdied in control and clofibrate (ethyl p-chlorophenoxyisobutyrate)-treated rats. 2. The extent of 7 alpha-hydroxylation of exogenous [414C]cholesterol and endogenous cholesterol, the latter determined with a mass fragmentographic technique, was the same in the two groups of rats. The extent of 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one and 5 beta-cholestane-3 alpha, 7 alpha-diol was increased by about 60 and 120% respectively by clofibrate treatment. The 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol was not significantly affected by clofibrate. The 6 beta-hydroxylation of lithocholic acid was about 80% higher in the clofibrate-treated animals than in the controls. 3. The results are discussed in the context of present knowledge about the liver microsomal hydroxylating system and bile acid formation in patients with hypercholesterolaemia, treated with clofibrate.     

Involvement of oxidoreductive reactions of intracellular haemoglobin in the metabolism of 3-hydroxyanthranilic acid in human erythrocytes.

3-Hydroxyanthranilic acid, a metabolite of tryptophan, was rapidly metabolized by human erythrocytes. The final product was determined to be cinnabarinic acid as detected by spectrophotometry, paper chromatography and t.l.c. The formation of cinnabarinic acid from 3-hydroxyanthranilic acid in the cells was markedly inhibited by CO when intracellular haemoglobin was in a ferrous state, and by cyanide when it was in a ferric state. Ferrous haemoglobin in erythrocytes was oxidized to (alpha 3+ beta 2+)2, (alpha 2+ beta 3+)2 and (alpha 3+ beta 3+)2 by 3-hydroxyanthranilic acid, and the oxidation rates were very high, like those of cinnabarinic acid formation, suggesting that the metabolism of 3-hydroxyanthranilic acid is coupled with oxidoreductive reactions of intracellular haemoglobin. This view was further confirmed by the findings that 3-hydroxyanthranilic acid was metabolized by ferrous or ferric haemoglobin and that ferrous and ferric haemoglobins were oxidized and reduced by the compound respectively. The significance of the metabolism of 3-hydroxyanthranilic acid and the oxidoreductive reactions of haemoglobin with this compound may be associated with the pathological conditions with increased 3-hydroxyanthranilic acid levels in the blood of diabetic subjects.     

人Tim-3基因真核表达载体的构建及表达

目的：为了构建可在真核细胞中高效表达人Tim-3（hTim-3）的真核表达质粒,以便用于hTim-3肿瘤免疫治疗研究。方法：取健康人外周血的单个核细胞,用高保真聚合酶扩增hTim-3基因,先进行hTim-3基因的亚克隆,用Bgl II和SalI限制性内切酶切下带有酶切位点的hTim-3基因,最终构建hTim-3基因的真核表达质粒pEGFP-N1-hTim-3。用脂质体方法转染质粒至肝癌细胞SMMC7721和巨噬细胞U937中。48h后荧光显微镜观察转染细胞绿色荧光表达情况,初步判断转染效率。结果：酶切和测序鉴定证实目的基因hTim-3正确插入到真核表达载体pEGFP—N1中,转染肝癌细胞SMMC7721和巨噬细胞U937后,在荧光显微镜下观察到绿色荧光蛋白的表达。结论：成功构建了可在真核细胞中高效表达hTim-3基因的真核表达质粒pEGFP-N1-hTim-3,为进一步研究hTim-3的肿瘤免疫治疗奠定了基础.     

人红细胞带3蛋白胞质段的克隆及其表达

人红细胞带3蛋白胞质段(cytoplasmic domain of band 3, cdb3)起着将膜与膜骨架、细胞内环境相联系的重要作用. 以带3蛋白全长基因为模板,用PCR方法扩增出cdb3片段,克隆至pRSET表达质粒上,转化大肠杆菌BL21(DE3). 转化菌经诱导表达出较高含量的cdb3蛋白,纯化后,测得对醛缩酶有抑制活性.     

In vitro effect of larval stages of Ascaris lumbricoides on human blood clotting

The effects of larval stages of Ascaris lumbricoides on human blood clotting was studied in vitro. Extracts and excretory/secretory products of third-stage larvae (L3) and late third-stage larvae (LL3) cultured from ova obtained from infected patients were analysed for anti-coagulant activity. Prothrombin time (PT) was prolonged by the addition of either whole extract of L3/LL3 or ES products of L3/LL3 as compared to controls. Partial thromboplastin time with kaolin (PTTK) was also prolonged on the addition of either extracts of ES products of L3/LL3. The prolongation of PTTK was significantly higher with extracts/ES products of L3 when compared to the extracts/ES products of LL3 (p less than 0.005). Thrombin time (TT) was prolonged by extracts of L3/LL3 and their ES products.     

Metabolism and binding in vitro of 5 alpha-androstane-3 beta, 17 beta-diol and of 5 alpha-androstane-3 alpha, 17 beta-diol in cell fractions of rat ventral prostate and liver.

Rat ventral prostate and liver were investigated for the binding in vitro to particulate fractions and for the metabolism of 5 alpha-androstane-3 beta, 17 beta-diol. Comparative investigations were carried out on the metabolism of 5 alpha-androstane-3 alpha, 17 beta-diol. Preparations of the liver were investigated in order to establish the organ specificity of the method. In the prostate, the bulk of the metabolites of 5 alpha-androstane-3 beta, 17 beta-diol was present as steroids of high polarity. Of the less polar metabolites, 17 beta-hydroxy-5 alpha-androstan-3-one, 3 beta-hydroxy-5 alpha-androstan, 17-one and 5 alpha-androstane-3 alpha, 17 beta-diol were detectable. The binding of a 5 alpha-androstane-3 beta, 17 beta-diol to mitochondria and microsomes was unspecific. In the liver, among the less polar metabolites, 3 beta-hydroxy-5 alpha-androstan-17-one was the main metabolite, and the binding was unspecific. The main metabolite in the prostate homogenate of 5 alpha-androstane-3 alpha, 17 beta-diol was 17 beta-hydroxy-5 alpha-androstan-3-one. The portion of highly polar steroids was very low. The portion of unmetabolized hormone was distributed almost equally among the different cell preparations except the nuclei, in which 17 beta-hydroxy-5 alpha-androstan-3-one was higher and 5 alpha-androstane-3 alpha, 17 beta-diol was lower than in the remaining cell fractions.