Inhibition of HSP70 Gene Expression by Modified Antisense and Its Effects on Embryonic Sensitivity to Heat Shock

Experiments were performed to evaluate the efficiency of inhibition of HSP70 gene expressionby antisense oligonucleotides complementary to the mRNA of HSP70 and to test the effects ofinhibition of HSP70 gene expression on subsequent embryonic sensitivity to heat shock. Theresults showed that transfection of pre-implantation embryos at 4-cell stage with 5 Mantisense oligo had no effect on in vitro blastocyst development. However, transfection with10 to 40 M antisense oligo had reduced in vitro blastocyst development to 15, 10% and 0; Forthe embryos which exposed to 40 M As arrested at the 16-cell stage, there was no blastocystformation within the heat shock groups. In contrast, transfection had no effect on embryonicsensitivity to heat shock, above 25% of embryos developed to blastocyst stage in controlgroups.

EFFECT OF TNF-a AND IFN-g ON THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE AND PROLIFERATION INHIBITION OF HUMAN COLON CANCER CELL LINE

Objective: To study the expression of the inducible nitric oxide synthase (iNOS) gene and the effects of tumor necrosis factor-α(TNF-a) and interferon-γ(IFN-g)on proliferation of the continuous cultured human colon cancer cell line CCL229. Methods: Using the molecular and biochemical techniques and electron microscopy to analyze the expression of iNOS, production of NO and growth characteristics of human colon cancer cells. Results: cytokine treatment can induce expression of the iNOS gene and production of nitric oxide was significantly higher after treatment of CCL229 cells with TNF-αor IFN-γ. Treatment with either cytokine or a combination of both significantly increased levels of Malondialdehyde (MDA) over control. Furthermore, cytokine treatment increased the proliferation inhibition rate as assessed in vitro and decreased the cell proliferation index on flow cytometry. Electron microscopy showed that cells treated with cytokines had fewer pseudopodia or cell processes than control cells and that cytokine treated cells had dilatation of the mitochondria and endoplasmic reticulum and dilated vesicular or tubular cisternae. Conclusion: Our findings indicate that TNF-α and IFN-γ induce the expression of iNOS gene in CCL229 cells, which increases the production of nitric oxide, inhibits proliferation, causes lipid peroxidation, and results in ultrastructural changes.

Effective inhibition of expression of hepatitis B virus genes by DNAzymes

AIM: To evaluate the inhibitory effects of DNAzymes on the expressions of hepatitis B virus (HBV) s (HBsAg) and e (HBeAg) in 2.2.15 cells, and to explore the potential therapeutic effects of DNAzymes on replication of HBV genome. METHODS: DNAzymes DrzBS and DrzBC specific to HBV (aywsubtype) s gene ORF A^157UG and e gene ORF A^1816UG, were designed and synthesized. Inhibitory effects of DrzBS or DrzBC on the expressions of HBV s and e genes as well as HBV DNA levels in culture supernatants were observed in 2.2.15 cells. RESULTS: After being treated with DrzBS or DrzBC, the expression of HBV s or e genes in 2.2.15 cells was depressed dramatically. The maximum inhibition rate was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The concentration for effective inhibition of both DrzBS and DrzBC was within 0.1-2.5 μmol/L, showing a dosedependence. The efficiency of inhibiting HBsAg, HBeAg in 2.2.15 cells by DrzBS or DrzBC was higher than that of the same target genes by antisense oligonucleotides (ASON). The concentration for effective inhibition of DNAzymes was at least 10-fold lower compared with ASON controls. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed. CONCLUSION: DrzBS and DrzBC can block the expression of HBV s- and e-genes in 2.2.15 cells and provide a specific and effective anti-HBV gene therapeutic means.

INDUCTION OF C-MYC GENE AMPLIFICATION BY HYDROXYUREA AND ITS INHIBITION BY HOMOHARRINGTONINE

Induction of c-myc gene amplification in L1210 cells by hydroxyurea and its inhibition by homohar-ringtonine were investigated using the DNA-DNA molecular hybridization technique. When the cells were treated with hydroxyurea 1.0 mM for 16 hours, and incubated a further 16 hours in a drug-free medium, the c-myc gene amplified 23.5-fold. If homohar-ringtonine 50 μM was used at the same time as hydroxyurea, gene amplification did not occur. Cycloheximide, an inhibitor of protein biosynthesis, produced a similar effect. Our results indicated that a (or some) protein factor(s) might be involved in gene amplification. Detailed analysis showed that the synthesis of this protein factor(s) started 4 hours before the initiation of the S phase but did not continue in the S phase. It was also found that this protein factor(s) was very labile and began to degrade 2 hours after its appearance.

Performance of Antoregldatory Senescence-inhibition Gene in Rice

The performance of autoregulatory senescence-inhibition gene PSAG12--IPT in rice has been investigated in the study. 422 transgenic plants from 134 independent resistant calli were obtained from 4 rice varieties through Agrobacterium-mediated transformation. Among them, 233were positive PSAG12-IPT transgenic plants identified by GUS histochemical assay and PCR analysis.Southern analysis showed the transgene was randomly integrated into rice genome, of which 42.29 % was single copy. Investigations on photosynthesis function and agronomic characters of R1 generation showed that chlorophy Ⅱ content and photosynthesis rate of flag leaves in transgenic plants, were 41.23 96 and 60.24 % higher than the control wild-type rice, respectively. The growth duration and plant height of the transgenic plants were similar to the control. Variations of other characters were dependent on the varieties. For the variety Millin with significant aging phenomenon in China, its total grains per hill, its seed setting rate and 1000-grain weight were increased by 40.44%, 8.05% and 8.32% respectively. The results indicated that after leaf senescence of varieties liable to age was delayed, the seed setting rate and the filling degree of seeds were improved, which finally resulted in significantly increased seed yield and biomass per hill. The new variety Wuyujing 2 without serious aging problem, was also increased in the panicles per hill, the total grains per hill, the seed yield per hill and biomass in different degrees.

Down-regulation of Notch-1 gene expression inhibits growth of TJ-905 glioblastoma

Objective To study the inhibitory effect of siRNA on glioblastoma (GBM) Notch-1 gene expression in addition to the growth of TJ-905 glioblastoma. Methods Three small interference RNAs (siRNAs) targeting Notch1 gene,named siRNA1,siRNA2,siRNA3,synthesized chemically in vitro with gene bank BLAST. TJ-905 cells were transfected twice with the siRNA by using Oligofectamine

Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5-1.2 × 107IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.

Gene transfer of huCTLA4-Ig to inhibit the acute rejection of liver allograft in rats

To observe the effect of gene transfer of huCTLA4-Ig to inhibit the acute rejection of liver allograft in rats.Methods With AdEasy vector system,the recombinant adenovirus containing huCTLA4-Ig gene was constructed.Using ex vivo gene transfer technique,exogenous gene was introduced to the liver graft during cold preservation and expressed locally in the graft.The effect of inhibition of acute rejection and inducing liver graft tolerance was observed.Results No recipients in group A (without any treatment,n=5) or group B (treated with Ad-GFP,n=4) died within 3 weeks after transplantation and severe acute rejection (massive periportal infiltration,endothelilitis,damage to biliary epithelium and severe tissue destruction) was confirmed pathologically in the graft.In contrast,all recipients in group C (treated with Ad-huCTLA4-Ig,n=5) achieved long-term liver allograft survival (>150 days).Histological examination of Ad-huCTLA4-Ig transduced allografts demonstrated a mild to moderate periportal inflammation and mild injury to liver graft on day 8 posttransplant.A mild mononuclear infiltration was observed;however,there was complete preservation of the bild ducts and no evidence of vascular injury on day 150 posttransplant.The mean IL-2 concentration in serum was (362.09±45.84) ng/L at day 1 pretransplant.In control animals (groups A and B),serum IL-2 concentration was elevated to a high level within 7 days posttransplant,which was about 1.5 to 2.5 times as much as that before transplant.In contrast,in huCTLA4-Ig-treated animals (groups C),IL-2 concentration in serum was maintained at a relative low level,which was near or less than that before transplant (P<0.01).Conclusion Using ex vivo gene transfer technique,huCTLA4-Ig gene can be introduced to the liver graft during cold preservation.The modified graft can express and excrete immunoregulatory protein locally,which can suppress acute alloimmune response and is responsible for prolongation of graft survival without using routine immunosuppressive drugs.These findings provide some experimental evidence that gene delivery of sequences encoding immunoregulatory proteins can be applied to clinical liver transplantation for inhibiting the acute alloimmune response and achieving graft tolerance.7 refs,2 tabs.

共载吲哚菁绿和Nrf2-siRNA的多功能纳米粒子对抗口腔鳞状细胞癌的体外作用研究

目的:构建共载吲哚菁绿(indo cyanine green,ICG)和核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)-短干扰RNA(small interfering RNA,siRNA)的多功能纳米载药粒子,并探究其联合光热疗法与抗氧化抑制作用增效的光动力疗法协同对抗口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)的体外作用与机制。方法:利用超声乳化法和静电力吸附作用制备基于聚氨基酯/聚乳酸-羟基乙酸共聚物且共载ICG和Nrf2-siRNA的纳米粒子PPI-siRNA。表征PPI-siRNA纳米粒子的形貌、粒径大小和分布、表面电位及其载药情况;评价PPI-siRNA纳米粒子对ICG和Nrf2-siRNA的胞内递送以及Nrf2-siRNA逃逸溶酶体吞噬的性能;通过检测舌鳞状细胞癌细胞SCC-25随激光照射的升温效应、热休克蛋白60表达和活性氧生成水平考察PPI-siRNA纳米粒子的光热和光动力性能;考察细胞内Nrf2及其调控的下游基因谷氨酸-半胱氨酸连接酶修饰亚基和谷氨酸-半胱氨酸连接酶的表达水平,从而探究Nrf2-siRNA助力光动力效应的作用机制;运用噻唑蓝比色法考察PPI-siRNA联合激光照射对SCC-25的细胞毒性作用。结果:PPI-siRNA纳米粒子呈形貌规则的球状结构,粒径约为180 nm,并成功共载ICG与Nrf2-siRNA;PPI-siRNA纳米粒子可以高效递送ICG和Nrf2-siRNA入胞,且可以确保Nrf2-siRNA逃逸溶酶体吞噬,从而发挥基因沉默作用;PPI-siRNA纳米粒子具备良好的光热和光动力性能,Nrf2-siRNA可以通过下调抗氧化蛋白和基因的表达显著提升光学疗法的抗肿瘤效率。结论:PPI-siRNA纳米粒子可以联合光热疗法与基因沉默作用增效的光动力疗法并有效抑制SCC-25的体外生长,在OSCC的临床治疗方面具有广阔的应用前景。

Effects of Exogenous p16^(ink4a) Gene on Biological Behaviors of Human Lung Cancer Cells

The effects of exogenous p16^ink4a gene on biological behaviors of human lung cancer cell line with homozygous deletion of p16^ink4a gene were investigated. Exogenous p16^ink4a gene was transfected by lipofectin into human lung cell line A549, in which p16^ink4a gene was homozygously deleted. The expression of p16^ink4a mRNA and protein was detected by RT-PCR and immunocytochemistry, respectively. The changes in the behaviors of the transfected cell lines in vitro and in vivo were observed. In the transfected cell line A549, the exogenous p16^ink4a gene could be stably expressed. The growth of A549 cells transfected with p16^ink4a gene was obviously slowed down. Flow cytometry revealed that transfection of the exogenous p16^ink4a gene resulted in A549 cell lines arrest in G1 phase of cell cycle. The tumorigenicity of these transfected cells in nude mice could be inhibited, and the tumor growth of nude mice was significantly suppressed. It was concluded that exogenous p16^ink4a gene may be stably expressed in human lung cancer cell line A549. The expression of the introduced p16^ink4a could block lung cancer cells to entry into S phase of cell cycle and inhibit tumor malignant growth both in vitro and in vivo.

Growth-inhibitory effects of MOB2 on human hepatic carcinoma cell line SMMC-7721

AIM:To investigate the growth-inhibiting and apoptosis-inducing effects of the gene MOB2 on human hepatic carcinoma cell line SMMC-7721.METHODS:The full-length cDNA of the MOB2 gene was amplified from human umbilical vein endothelial cells.The correct full-length MOB2 cDNA was subcloned into the eukaryotic expression vector pEGFP-C1.After lipofection of the MOB2 gene into cancer cells,the levels of MOB2 protein in the cancer cells were detected by immunoblotting.To transfect the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells,the cells were cultured in Dulbecco's Modified Eagle'sMedium with 10% fetal calf serum and glutamine,and then mixed with liposomes,Lipofectamine 2000 and the plasmid vector pEGFP-CI-MOB2.RESULTS:We observed the growth and proliferation of SMMC-7721 cells containing pEGFP-CI-MOB2 and analyzed their apoptosis and growth cycle phases by flow cytometry.We successfully transfected the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells and screened for a single clone cell containing MOB2.After transfection,MOB2 enhanced growth suppression,induced apoptosis,increased the ratio of G0/G1,significantly inhibited the advance of cell cycle phase,and arrested cells in G0/G1 phase.CONCLUSION:MOB2 overexpression induces apoptosis and inhibits the growth of human hepatic cancer cells,which may be useful in gene therapy for hepatic carcinoma.

Lentivirus mediated shRNA interference targeting MAT2B induces growth-inhibition and apoptosis in hepatocelluar carcinoma

AIM: To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2β gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. METHODS: We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-xL and bcl-xS were detected with western blot. RESULTS: We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved indown-regulating bcl-xL and up-regulating bcl-xS. CONCLUSION: LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.

Transactivation of the TIEG1 confers growth inhibition of transforming growth factor-β-susceptible hepatocellular carcinoma cells

AIM:To investigate the role of transforming growth factor(TGF)-β-inducible early gene 1(TIEG1) in TGF-β-induced growth inhibition in hepatocellular carcinoma(HCC) cells.METHODS:Human hepatocyte and HCC cell lines with varied susceptibilities to TGF-β1 were tested by methylthiazoletetrazolium(MTT) assay.The expression changes of Smad2,Smad3,Smad4,Smad7,TIEG1 and TIEG2 gene following treatment with TGF-β1 in a TGF-β-sensitive hepatocyte cell line(MIHA),a TGF-β-sensitive hepatoma cell line(Hep3B) and two TGF-β-insensitive hepatoma cell lines(HepG2 and Bel7404) were examined.SiRNA targeting TIEG1 was transfected into Hep3B cells and the sensitivity of cells to TGF-β1 was examined.Overexpression of TIEG1 was induced by lentiviral-mediated transduction in TGF-β1-resistant hepatoma cell lines(Bel7404 and HepG2).MTT assay and 4',6-Diamidino-2-phenylindole staining were used to identify cell viability and apoptosis,respectively.The expression level of stathmin was measured by reverse transcriptase polymerase chain reaction and Western-blotting analysis,and stathmin promoter activity by TIEG1 was monitored by a luciferase reporter gene system.RESULTS:TIEG1 was significantly upregulated by TGF-β1 in the TGF-β1-sensitive HCC cell line,Hep3B,but not in the resistant cell lines.The suppression of TIEG1 by siRNAs decreased the sensitivity of Hep3B cells to TGF-β1,whereas the overexpression of TIEG1 mediated growth inhibition and apoptosis in TGF-β1-resistant HCC cell lines,which resembled those of TGF-β1-sensitive HCC cells treated with TGF-β1.Our data further suggested that stathmin was a direct target of TIEG1,as stathmin was signif icantly downregulated by TIEG1 overexpression,and stathmin promoter activity was inhibited by TIEG1 in a dose-dependent manner.CONCLUSION:Our data suggest that transactivation of TIEG1 conferred growth inhibition of TGF-β-susceptible human HCC cells.

Arginine kinase in Toxocara canis:Exon-intron organization,functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors

Objective: To determine exon/intron organization of the Toxocara canis(T. canis) AK(TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Methods: Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame(1 200 bp) of TCAK(wild type) was cloned into the BamH 1/SalI site of pM AL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pM AL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25℃ for the forward reaction(phosphagen synthesis). Results: Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K_m value of the mutant(Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K_m value of the mutant(Serine to Glycine) increased to 0.19 mM. The Km value(0.19 mM) of the double mutant(Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type(0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica(A. indica), an aminoglycoside antibiotic(aminosidine), a citrus flavonoid glycoside(rutin) and a commercially available catechin mixture against TCAK. Green and black tea(1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK. Conclusions: Arginine kinase in T. canis has a seven-exon/six-intron gene structure. However, further studies are needed to identify a specific compound within the extract causing the inhibitory effect and also to determine the molecular mechanisms behind inhibition of arginine kinase in T. canis.

Study on Formation and Inhibition Mechanism of Biofilm of Pig Actinobacillus pleuropneumoniae

Pig Actinobacillus pleuropneumoniae(App) could induce chronic respiratory tract infection in pigs, which causes major economic losses on pig industry. Bacterial biofilm(BBF) is bacterial community adsorbed on the surface of biomaterials or body cavity, to protect bacteria escape, and recurrent outbreaks of related infectious diseases and chronic infections resulting therefrom are called bacterial biofilm diseases. App BF belongs to polymers with spatial structure in vitro, and its formation is regulated by multiple genes. Among them, gene deletion of the key component TolC of multidrug efflux pumps and type I secretion systems causes that App BF adhesion weakens; gene deletion of catalytic core ClpP of Clp proteolytic complex induces the inhibition of BF formation; outer membrane lipoprotein VacJ of App promotes BF formation; gene deletion of active enzyme LuxS enhances the formation of App BF and decreases bacterial adhesion ability; gene deletion of Adh obviously declines bacterial accumulation, BF formation and adhesion to host cells. In this paper, BF formation or inhibition mechanism in App is elaborated from molecular level, which could provide reference basis for exploring the prevention of its biofilm diseases.

Changes of tumorigenicity of the B16 melanoma cells transfected with MIP-1α gene

Macrophage inflammatory protein-l, a recentlycharacterized chemokine, consists of two chains (αand β). MIP-lα has been shown to exert strongchemotactic effect on neutrophils, monocytes and Tlymphocytes. In the present study, the B16 melanomacells were transfected with recombinant adenoviruscontaining MIP-lα gene. The biological characteri-zation of the MIP-1α gene transfected B16 melanomacells was investigated. The level of MIP-1α in thesupernatant of gene-transfected melanoma cells was368±24 ng/ml/10~6/24hr.. By using Boyden chambersystem, this supernatant showed strong chemotacticactivity for NK cells, CD4^+ T cells, CD8^+ T cells orthe freshly isolated peritoneal macrophages in vitro.Though the in vitro growth of the gene-transfected B16 melanoma cclls was not aftered, the in vivogrowth of the tumor cells subcutaneously inoculatedwas significantly inhibited. The infiltration ofinflammatory cells into the tumor mass formed bygene-transfected B16 cells was much more obviousthan that by

急性非致残性缺血性脑血管事件高危人群CYP2C19基因多态性与氯吡格雷抵抗的关系

目的探讨急性非致残性缺血性脑血管事件高危人群CYP2C19基因多态性与氯吡格雷抵抗的关系。方法选取2020年1月至2021年3月就诊于九江市第一人民医院的160例急性非致残性缺血性脑血管事件高危人群作为研究对象,采用京因药物基因床旁检测系统来实现CYP2C19基因床边快速检测,依据CYP2C19基因位点分为快代谢组64例、中间代谢组76例、慢代谢组20例。比较不同代谢基因组患者的血小板ADP抑制率及氯吡格雷抵抗情况的差异。结果不同代谢基因组中氯吡格雷抵抗率情况比较,差异无统计学意义(P>0.05);不同代谢基因组血小板ADP抑制率比较,差异无统计学意义(P>0.05)。结论对于服用氯吡格雷的急性非致残性缺血性脑血管事件高危人群的患者,CYP2C19基因多态性与氯吡格雷抵抗尚不能确定有确切关联。

Inhibiting effect of antisense hTRT on telomerase activity of human liver cancer cell line SMMC-7721

Objective: To induce changes in biological character of human liver cancer cell line SMMC-7721 by blocking the expression of telomerase genes hTRT and to explore its value in cancer gene therapy. Methods: The vehicle for eukaryotic expression of antisense hTRT was constructed and then transfected into SMMC-7721 cells. The effects of antisense hTRT gene on telomerase activity, cancer cell growth and malignant phenotypes were analyzed. Results: The obtained transfectants that could express antisense hTRT gene stably showed marked decrease in telomerase activity; the shortening of telomere was obvious; cells presented contact growth inhibition; in nude mice transplantation, the rate of tumor induction dramatically decreased. Conclusion: Antisense hTRT gene expression can significantly inhibit telomerase activity of cancer cells and decrease malignant phenotypes in vitro and in vivo. Therefore, as a telomerase inhibitor, antisense hTRT gene may be a new pathway for cancer therapy.

Reversion of malignancy in human gastric cancer MKN-45 cells through the transfection of transforming growth factor-β type Ⅱ receptor gene

Human gastric cancer MKN-45 cells which are resistant to TGF-β growth inhibition and possess TGF-β type Ⅰ and type Ⅲ receptors, but not type Ⅱ receptors, have been used as a model system to reconstitute these caflcer cells with TGF-β RII cDNA. The results of these experiments indicated that the reexpression of TGF-g RII gene in MKN-45 cells can restore their sensitivity to TGFβ growth inhibition, decrease their growth rate, reduce their cloning efficiency in soft agar and tumorigenicity in nude mice in stable transfectants, in comparison with their control MKN-45 cells. Among different RII transfectants,their difference in the changes of these parameters, as a result of the regain of autocrine negative growth control by TGF-β, is roughly proportional to their level of expression of transfected RII mRNA. From these data, it is concluded that the inactivation of TGF-β RII gene is related to the escape of growth control by TGF-β in MKN-45 cells. The importance of the study of the interplay of TGF-β and its receptor system in the negative growth control of gastric cancer, and possibly also of other cancers, is discussed.

油菜抗除草剂新种质PN19的交互抗性鉴定及其抗性基因的表达研究

鉴定抗除草剂油菜种质对不同种类除草剂的交互抗性,获得可安全交替使用的其他类型除草剂,可延长抗性品种使用时间。本研究以油菜抗SU类除草剂新种质PN19及转抗性基因的拟南芥和烟草为材料,在3-5叶期喷施不同浓度梯度的五大类ALS抑制类除草剂(SU、IMI、TP、PB和SCT类),通过抗性鉴定以确定PN19对各类除草剂的交互抗性。结果表明:SU和SCT类交互抗性最强,IMI和TP类其次,PB类最低。转基因植株除草剂抗性评价及烟草ALS酶体外活性分析表明,五类除草剂交互抗性功能源于靶标ALS酶对各除草剂敏感性下降。且五类除草剂处理后PN19抗性基因较野生型表达上调。本研究为田间ALS类除草剂混合使用进行杂草防控提供了可能性。