Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

The use of plants as bioreactors for the expression oftherapeutic proteins has developed into a new field in biotechnology research[1]. Plant systems provide simple genetic manipulation, low production costs, and low risk of contamination by animal viruse…     

The molecular mechanism of different sensitivity of breast cancer cell lines to TRAIL

Although Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of various cancer cells, some caner cell lines are resistant to TRAIL-induced cell death. To investigate the molecular mechanisms underlying TRAIL-resistance, two human breast cancer cell lines, MCF-7 (resistant to TRAIL) and MDA-MB-231 (sensitive to TRAIL), were used as a model system to analyze the different sensitivities to TRAIL cytotoxicity. PKCδ inhibitor rottlerin, but not MEK and ERK1/2 inhibitor U0126 nor PI3K inhibitor LY294002, was shown to enhance TRAIL-induced apoptosis in MCF-7 cells significantly, suggesting that PKCδ might play an important role in the resistance of MCF-7 cells to TRAIL. In contrast, rottlerin, U0126, and Ly294002 had no effect on MDA-MB-231 apoptosis induced by TRAIL under the same conditions. Further experiment showed that the combination of rottlerin and TRAIL cleaved PARP in the MCF-7 cells synergistically, but not in the MDA-MB-231 cells. The role of PKCδ in TRAIL-resistant MCF-7 cells was confirmed by knocking down the endogenous PKCδ expression using RNAi technology. Furthermore, caspase-3 reconstitution in MCF-7 cells was unable to alter PKCδ expression, suggesting that innate caspase-3 deficient in the cells does not cause PKCδ high expression. These data provide evidence for the first time that PKCδ plays a critical role in breast cancer cell lines to TRAIL cytotoxicity.     

可溶性人TRAIL蛋白诱导肝癌细胞的凋亡

为研究可溶性人TRAIL蛋白（sTRAIL）对肝癌细胞株SMMC7721的生长抑制效应及凋亡诱导作用．采用显微镜、台盼蓝排斥试验、MTT比色试验、TUNEL法和DNA断裂实验等方法检测细胞增殖和细胞凋亡．通过显微镜观察到核染色质凝集等凋亡的形态学变化,台盼蓝排斥试验、MTT比色试验结果显示,sTRAIL蛋白可显著抑制SMMC7721细胞的生长和增殖,并且TUNEL法检测到经sTRAIL处理后的细胞凋亡指数与对照比较有显著差异,DNA断裂实验亦观察到典型的DNA梯形条带,这些结果提示sTRAIL可诱导肝癌细胞株SMMC7721发生凋亡,具有抗肝癌的作用．     

XIAP在TRAIL抗性细胞HCT116 bax-/-中的作用机制研究

为了探索结直肠癌细胞HCT116抗TRAIL诱导凋亡的分子机制,本课题以其抗性细胞HCT116 bax~(-/-)为实验对象进行了研究.通过利用目前最热的基因定点编辑技术Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)9系统将HCT116bax~(-/-)的XIAP(X-linked inhibitor of apoptosis protein)基因彻底敲除后,用TRAIL处理,发现其恢复了对TRAIL的敏感,形态学发生了明显凋亡,而且western blot检测显示PARP蛋白发生了完全剪切.由此证明敲除XIAP基因能克服HCT116 bax~(-/-)对TRAIL的抗性.这些发现对肿瘤细胞抗TRAIL的分子机理研究以及肿瘤的个性化治疗有非常重要的意义.     

Recombinant sTRAIL induces cell death of tumor cell lines as well as primary cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a new member of TNF family. It was reported that TRAIL could induce apoptosis of tumor cells but not normal cells in tissue culture system. To further study the biological activity and potential clinical significance, a recombinant soluble TRAIL (rsTRAIL) has been expressed stably in E. coli after transformation of pET28b vector containing the extracellular domain of TRAIL. The yield of rsTRAIL is approximately as high as 60% of whole bacterial proteins. The rsTRAIL, purified by Ni+ -agarose affinity chromatography, could remarkably trigger apoptosis at the concentrations of 0.1-1 μg/mL in all 7 tumor cell lines tested in vitro. However, this killing activity has not been observed in mouse fibroblast cell line (NIH3T3) as normal control. Further investigation shows that the rsTRAIL could also kill primary tumor cells isolated freshly from patients with cardiac cancer, breast cancer and malignant thymoma, while the normal human peripheral blood lymphocytes are not killed under the same conditions. These results provide new evidence that rsTRAIL could induce apoptosis of tumor cells specifically and it could be a new promising medicine for tumor therapy.     

碳纳米管电极对水溶液中苯酚的电化学氧化处理

制作了多壁碳纳米管电极,并将其应用于苯酚的氧化处理上．结果发现有很好的氧化峰出现在电位窗口内,峰电流在一定范围内与苯酚的浓度成良好的线性关系．长时间恒电位氧化实验表明,能克服传统碳电极的缺点,电极表面没有积垢,电极的重现性较好,可以逐渐将苯酚氧化．     

Human Soluble TRAIL Protein Inducing Apoptosis in Osteosarcoma Cell

This study is to examine the effect of human recombinant soluble TRAIL （TNF-related apoptosis-inducing ligand） protein inducing apoptosis in MG-63 human osteosarcoma cells. The inhibitive rates of TRAIL to MG-63 cells were detected by MTT assay. The apoptosis induced by TRAIL in MG-63 human osteosarcoma cells was analyzed with FACS and TUNEL and the apoptotic bodies were observed by transmission electron microscope. MTT assay showed that the inhibitive rates of 500, 1 000, 2 000 and 4 000 ng/mL TRAIL for 24 h were 10.1%, 24.3%, 50.6% and 97.7% respectively. Flow cytometric analysis showed that after MG-63 cells were treated with 2 gg/mL TRAIL for 6 h, obvious apoptotic peak would immediately appear before diploid peak. Human soluble TRAIL protein can quickly kill MG-63 osteosarcoma cells selectively, and may have potential value for clinical treatment of osteosarcoma.     

重组质粒pEgr-1-TRAIL辐射诱导乳腺癌细胞效应的实验研究

目的将具有辐射诱导表达特性的重组质粒p Egr-1-TRAIL转入乳腺癌MCF-7细胞中,诱导肿瘤杀伤基因TRAIL的表达,实现辐射和基因对MCF-7细胞的双重杀伤效应.方法用ELISA法检测不同剂量X射线诱导TRAIL表达的量效关系及2 Gy X射线照射后不同时间TRAIL的表达;用流式细胞仪检测不同处理组MCF-7细胞早期凋亡情况.结果不同剂量X射线照射转染p Egr-1-TRAIL重组质粒的乳腺癌细胞MCF-7,TRAIL表达量明显高于假照组(P0.001~0.05),其中5 Gy X射线照后表达量最高,TRAIL表达量为假照组的5.1倍.2 Gy X射线照射后4 h,TRAIL表达量明显升高(P0.05),并随时间延长逐渐增加,照射后32 h达峰值,表达量为照射前的4.6倍.MCF-7-p Egr-1-TRAIL/0 Gy组早期凋亡细胞百分数较MCF-7/0 Gy和MCF-7-p3.1Egr/0 Gy组明显增加(P0.01),随着照射剂量的增加,MCF-7-p Egr-1-TRAIL/5 Gy组细胞早期凋亡率与其他处理组比较均存在统计学意义(P0.001~0.05),MCF-7/0 Gy组细胞生长最快,而MCF-7-p Egr-1-TRAIL/8 Gy组细胞生长最慢.结论乳腺癌细胞转染p Egr-1-TRAIL重组表达质粒联合X射线照射能够增加MCF-7细胞凋亡,抑制其生长.     

TRAIL与kallistatin联合表达载体的构建及抗肿瘤活性分析

为研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)与组织激肽释放酶结合蛋白(kallistatin)联合用药的抗肿瘤作用,构建TRAIL与kallistatin双表达的重组质粒pAM-CAG-Kal-IRES-TRAIL,将重组质粒转染A549,LO-2,NCI-H446和Hela细胞,考察其抗肿瘤活性.实验结果表明:构建的双表达载体能同时表达TRAIL与kallistatin,且均能分泌至培养基中;TRAIL与kallistatin联合表达对肿瘤细胞活力的抑制作用明显增强,诱导肿瘤凋亡的作用也明显增强,说明联合表达TRAIL与kallistatin能够增强抗肿瘤活性.     

人可溶性TRAIL原核分泌表达载体的构建

为了克隆人可溶性TRAIL基因片段,构建其新型原核分泌表达载体,从HL-60细胞中提取总RNA,根据GeneBank提供的人TRAIL基因序列,设计扩增人可溶性TRAIL基因114～281片段的特异性引物,同时引入NcoI、BamHI、TEV酶的酶切位点及His标签,以便纯化及纯化后切去His标签,并将目的基因克隆至原核表达载体PhoA,经测序分析鉴定,于大肠杆菌MM294中进行表达.结果表明:克隆到人sTRAIL基因序列,经DNA测序结果与GeneBank基因库报道的一致,成功构建了可分泌表达的人源可溶性TRAIL原核表达载体PhoA-sTRAIL,并在大肠杆菌MM294中成功表达.     

伤寒沙门菌激活TRAIL信号通路促进巨噬细胞凋亡

为探究伤寒沙门菌对巨噬细胞凋亡及肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis inducing ligand,TRAIL)信号通路的影响,对伤寒沙门菌感染THP-1细胞早期和后期提取细胞RNA进行转录组测序(RNA-seq).通过定量PCR(qPCR)技术检测凋亡相关基因的转录水平,对RNA-seq结果进行验证,流式细胞术和蛋白质免疫印迹法检测THP-1细胞的凋亡和凋亡相关蛋白;加入TRAIL抗体结合TRAIL蛋白后,流式细胞术和蛋白质免疫印迹法检测感染早期THP-1细胞凋亡的变化.结果显示,伤寒沙门菌感染THP-1细胞后,凋亡相关基因caspase-3、caspase-8、caspase-9在感染后期转录水平显著增加,Tnfsf10、Tnfrsf10b、Tnf、Fas在感染早期和后期转录均增加,细胞凋亡率明显增加,caspase-3、caspase-8、caspase-9蛋白活化增加,TRAIL、DR5蛋白表达增加;与PBS对照组相比,加入TRAIL抗体后caspase-3蛋白活化明显减弱,细胞凋亡率明显下降.综上,伤寒沙门菌通过部分激活TRAIL信号通路来诱导巨噬细胞凋亡.     

异硫氰酸异丁酯抑制肝癌细胞HepG2生长的效应与机理

为了探究异硫氰酸异丁酯(isobutyl isothiocyanate)对肝癌细胞HepG2的生长抑制效应及其分子机制，该研究通过MTT实验检测化合物对细胞的生长抑制率，计算出IC50值为3.5 μg·mL－1；通过流式细胞技术检测发现0.437 μg·mL－1以上浓度的化合物能诱导HepG2细胞凋亡；通过划痕实验检测发现0.875 μg·mL－1以上浓度的化合物能抑制细胞迁移；进一步通过生物信息学分析表明异硫氰酸异丁酯的靶蛋白可能为巨噬细胞迁移抑制因子(MIF)，且HepG2细胞中MIF表达量高于低敏感株.免疫印迹实验也进一步发现化合物不仅能够抑制蛋白酪氨酸激酶2/信号转导子和转录激活子3(JAK2/STAT3)信号通路的激活，还能下调p53蛋白表达.研究结果表明，异硫氰酸异丁酯可能通过靶向HepG2细胞中的MIF蛋白，影响MIF与其受体CD74相互作用，从而下调p53蛋白表达，阻滞细胞周期的正常进行，诱导细胞凋亡，对肝癌具有特异性的抑癌潜力.     

Effect of Quercetin on Breeding and Apoptosis of Cervical Cancer HeLa Cell and on Growth of Transplanted Tumor in Nude Mice

Effect of quercetin on HeLa cell system of cervical cancer was studied by methods of MTT and Annexin V-FITC/PI. The results show that quercetin has functions of inhibiting breeding of HeLa cells and inducing apoptosis of the cells. The total apoptosis rate is positively proportional to reaction duration and concentration of quercetin used. The maximum apoptosis rate being （88.76±2.35）% was obtained when the concentration was 50.0 μmol/L and the cells were treated with quercetin for 72 hours. Based on establishing a model of tumor of cervical cancer transplanted into nude mice, quercetin of different concentrations was injected into abdominal cavity of nude mice and situation of tumor growth was reviewed. The result showed that with quercetin concent＇ration increasing from 0 to 100.0 μmol/L, the transplantation volume and weight of the tumors decreased from （279.59±70.58） mm^3 and （0.145±0.019） g to （128.72±36.12） mm^3 and （0.089± 0.019） g respectively, while apoptosis rate of the transplanted tumor increased from （9.63±1.85）% to （34,98±0.47）%, which proved that quercetin inhibited increment of volume and weight of transplanted tumor in nude mice bodies.     

Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency

Apoptosis is a form of programmed cell death that is controlled by aspartate-specific cysteine proteases called caspases. In the immune system, apoptosis counters the proliferation of lymphocytes to achieve a homeostatic balance, which allows potent responses to pathogens but avoids autoimmunity. The CD95 (Fas, Apo-1) receptor triggers lymphocyte apoptosis by recruiting Fas-associated death domain (FADD), caspase-8 and caspase-10 proteins into a death-inducing signalling complex. Heterozygous mutations in CD95, CD95 ligand or caspase-10 underlie most cases of autoimmune lymphoproliferative syndrome (ALPS), a human disorder that is characterized by defective lymphocyte apoptosis, lymphadenopathy, splenomegaly and autoimmunity. Mutations in caspase-8 have not been described in ALPS, and homozygous caspase-8 deficiency causes embryonic lethality in mice. Here we describe a human kindred with an inherited genetic deficiency of caspase-8. Homozygous individuals manifest defective lymphocyte apoptosis and homeostasis but, unlike individuals affected with ALPS, also have defects in their activation of T lymphocytes, B lymphocytes and natural killer cells, which leads to immunodeficiency. Thus, caspase-8 deficiency in humans is compatible with normal development and shows that caspase-8 has a postnatal role in immune activation of naive lymphocytes.     

突变型P53和肿瘤易感基因101在肺癌组织中的表达及意义

探讨了P53基因突变对肺癌组织中TSG101基因的影响,结果是:TSG101在正常组织中呈强阳性表达100%(30/30),在高分化肺癌(包括肺鳞癌和肺腺癌)组织、低分化肺癌组织、淋巴结转移组织的表达分别为77.97%(92/118)、25.37%(17/67)、18.95%(18/95);P53的表达则相反,在正常组织、高分化肺癌组织、低分化肺癌组织、淋巴结转移组织的表达分别为6.67%(2/30)、67.80%(80/118)、86.57%(58/67)、94.63%(90/95);P53基因PCR-SSCP分析谱检测P53第5~8外显子阴性56例占30.27%,阳性129例占69.73%,总突变率为69.73%(127/180);P53与TSG101呈现负相关.这些结果提示P53和TSG101可能共同参与了肺癌的发生、发展和转移.     

The application of Tau protein testing to gastric cancer patients treated with paclitaxel

Paclitaxel is one of the main drugs used to treat gastric cancer, but many tumors develop drug resistance, resulting in treatment failure. The levels of expression of Tan protein in breast tumors have been found to be related to paclitaxel resistance, suggesting that Tau protein expression may predict breast cancer sensitivity to paclitaxel treatment. To determine whether Tan protein ex- pression can predict gastric cancer sensitivity to paclitaxel, we assayed Tan protein expression levels in gastric cancer specimens from 70 patients. We observed Tan protein expression in 54 of 70 （77.1%） specimens. Assays in gastric cancer cell lines showed that Tan protein expression was significantly lower in BGC823 than in MKN45 cells （P -- 0.0147）. MTT assays showed that dif- ferent concentrations of paclitaxel inhibited the growth of MKN45 and BGC823 cells, but inhibition and apoptosis were more obvious in cells expressing low levels of Tau protein. Paclitaxel chemotherapy was effective in 34 of the 70 patients （48.6%） and was significantly correlated with low expression of Tau protein （P 〈 0.01）. These findings indicate that Tau protein is expressed in a high percentage of gastric cancers, with paclitaxel being more effective in tumors with low Tau expression.     

Determination of global mean sea surface WHU2000 using multi-satellite altimetric data

In this study, the overall editing criteria for altimetric data are considered and the geophysical correction models is improved. The datum for various altimetric data is also unified and the method of a full-combined crossover adjustment for different altimetric tracks is used to improve the radial orbits of Geosat, ERS-1 and ERS-2 data. In addition, the method for determining mean sea surface (MSS) by using multi-altimetric data is developed. The data used to compute WHU2000 MSS include 7-year Topex/Poseidon data (cycles 11-249), 2-year Geosat ERM data (cycles 1-44), 5- year ERS2 data (cycles 1-52) and all ERS-1 168-day data. The WHU2000 MSS is determined with a grid resolution of 2′× 2′ within the ± 82° latitude and its precision is better than 0.05 m. Comparing WHU 2000MSS with 3.75′ 3.75 CLS_SHOM98.2 MSS, 3′×3′ GFZ MSS95A and 3.75′×3.75′ OSU MSS95, as external checks, the corresponding standard deviation (STD) of their differences is 0.090 m, 0.211 m and 0.079 m respectively.     

PI3K/Akt signaling pathway involved in regulation of T lymphocyte activation and apoptosis mediated by CD3ε

To study the expression and kinase activity of phosphatidylinositol 3′ -kinase (PI3K) and protein kinase B (PKB or Akt) during activation and apoptosis of human Jurkat T lymphocytes (TJK) with stable expression of CD8ε chimera fused human CD8α extracellular and transmembrane domains to intracellular domain of mouse CD3ε, Western blot, kinase activities detection and immunoprecipitation were carried out. It was shown that Jurkat cells with expression of wild type chimera CD8ε died by apoptosis after continuous stimulation of anti-CD8 monoclonal antibody. The expressions of PI3K and Akt, and the kinase activity of Akt remarkably increased during the process. However, this phenomenon did not occur in the Jurkat cells (T1JK) with expression of the mutant of CD8ε chimera (Y170F), suggesting that PI3K/Akt signaling pathway is involved in activation and apoptosis of T lymphocyte mediated by CD3ε.     

Inhibiting mechanism of baculovirus p35 gene to apoptosis

We transfected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 gene and neomycin-resistant gene (as a selection marker). By G418 screening, we got transformed cells that appeared resistant to G418 and picked one clone named Sf9-35. By hybridization in situ, it was found that p35 gene had integrated into the chromosome of Sf9-35 cells; By using actinomycin D treatment and cellular DNA electrophoresis, Sf9-35 cells were found to resist apoptosis induced by infection of vAcΔp35 deleting p35 gene and actinomycin D treatment; And it was also found that apoptosis induced by viral infection and actinomycin D treatment can only be delayed, but can not be stopped in Sf9-35. Supported by the National Natural Science Fundation of China Li Xiaofeng: born in 1965, Lecturer     

Apoptosis in suspension culture of tobacco cells induced by heat shock

The induction of apoptosis in suspension culture of tobacco cells by heat shock is reported for the first time. Heat treatment (48℃ for 4 h) of tobacco cells led to the appearance of typical hallmarks of apoptosis. It was demonstrated by DNA laddering analysis that the cells treated with heat shock at 48℃ for 4 h had a serious degradation of nuclear DNA into multi-nu-cleosomal sizes, suggesting that heat shock activated endogenous nuclease which led to DNA cleavage at the linkage sites between the nucleosomes, but ladders were very faint for DNA from 2 and 9 h heat-treated cells. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) detection also showed that most of these treated cells (48℃ for 4 h) displayed positive reactions, indicating a serious DNA 3'-OH cleavage in their nuclei. Moreover, some other cytological changes in apoptotic cells, such as cell shrinkage, chromatin aggregation, nucleus collapse, have also been observed by 4', 6'-diamidino-2-phenyl-indole (DAPI) staining.