Effects of c-fos antisense oligoneuleotide and p21 genetic transfection on the intimal proliferation of venous autografts in rabbits

AIM: To investigate the effects of c-fos antisense oligoneuleotide and p21 genetic transfection on the intimal proliferation of venous autografts. METHODS: The external jugule veins were autografted into common carotid arteries in the same side in 20 New Zealand rabbits, which were divided evenly into experimental and control group randomly. The transplanted veins of experimental group were immersed in the adenovirus-mediated p21 gene solution for 15 minutes just before anastomosis and coated with c-fos antisense oligoneucleotide glue gel just after anastomosis, while the control was only treated with empty vector. The transplanted vascular sample were taken at 2 weeks after operation. The intimal thickness (IT), degree of restenosis (DR), expression of proliferating cell nuclear antigen (PCNA), quantity of VSMC were determined by immunohischemistry. RESULTS: The IT, DR and expression of PCNA, VSMC were decreased, compared to control group. CONCLUSION: Transfection of c-fos antisense oligoneuleotide and p21 gene inhibits the intimal proliferation of venous antografs.     

Determination of genetic stability of grafted marula trees using AFLP markers

Genetic stability of grafted marula trees within seven lines, some of which exhibiting interline phenotypic variations, was evaluated using the AFLP technique. The study was conducted in two-fold using samples from the DR, LP, MR, OS, PH, SW and TR lines. The genetic analysis using leaf materials indicated varying levels of genetic variations between genotypes within the OS, MR, LP and PH lines. The genotypes of the DR, TR and SW lines showed high intraspecific genetic similarity and formed tight clusters on the UPGMA-based dendrogram. The genetic analysis using a subset of the initial sample set constituting both leaf and bark materials which represented the scion and the rootstock components of grafted trees showed high genetic similarities between the graft partners of some genotypes within the OS and PH lines. These high levels of genetic similarity between scion and rootstock partners of a grafted tree suggested that the rootstock graft developed shoots and grew into a tree following graft failure. The observed results imply that grafting is not always successful even within compatible species, however, it can be reliably monitored using molecular markers.     

植被和坡度对边坡稳定性的影响分析

边坡工程的稳定性问题是岩土工程的主要研究内容之一。随着我国经济、科技水平的不断发展和进步,人们对边坡稳定性的研究是从早期通过长期的观测统计出数据,慢慢发展到现在采用模拟试验来完成研究。边坡稳定性分析和评价是边坡工程研究的核心,在研究中人们发现:不同坡度对于边坡的稳定性有着不同程度的影响。本试验就从不同坡度条件下,有无植被2种坡度在降雨条件下进行研究分析。     

Ultrasound promotes the delivery of an antisense oligo-peptide nucleic acid targeting against PDGF-B mRNA to inhibit the vascular restenosis

AIM:To elucidate the effect of antisense oligo-peptide nucleic acid (PNA) against PDGF-B mRNA on in vivo proliferation of VSMCs and vascular restenosis. METHODS:A rabbit vascular restenotic model was constructed and a synthesized PNA was transfected into the vascular cells using a therapeutic ultrasound for the gene drug delivery. The proliferation of intimal VSMCs was observed by monitoring the expression of proliferating cell nuclear antigen (PCNA) and platelet-derived growth factor B chain mRNA (PDGF-B mRNA) with the LSAB and in situ hybridization, respectively. The intimal thickness and area were measured. RESULTS:PNA delivered by therapeutic ultrasound significantly inhibited the expression of PCNA and PDGF-B mRNA by intimal VSMCs 1 week after denudation with the inhibitory rates of 84.19% and 64.95%, respectively and reduced the intimal thickness and area. CONCLUSION:The PNA against PDGF-B mRNA delivered by therapeatic ultrasound inhibits the proliferation of rabbit VSMCs and the formation of intima.     

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.     

Enhancement effect of adenovirus mediated antisense c-myc gene and caffeine on chemotherapy of osteosarcoma cells to cisplatin

AIM: The cancer biology has showed that overexpression of oncogenes is responsible for the progression of human malignancies,antisense technology can block a certain gene expression.Caffeine has enhancement effect on chemotherapy of osteosarcoma cells to cisplatin,we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin.METHODS: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 750 base pairs in a reverse direction into adenovirus vector.Ad-Asc-myc and caffeine was used respectively or together to co-operate with cisplatin to treat the osteosarcoma MG-63 cells in vitro,and Western blotting,MTT,flow cytometry (FCM),electron microscope were used to evaluate expression of c-Myc protein,tumor cell proliferation in vitro,apoptosis and cell cycle analysis.RESULTS: Ad-Asc-myc was obtained with the titer of 2×10¹² pfu/L.Ad-Asc-myc down-regulated the expression of c-Myc protein,Ad-Asc-myc or caffeine enhanced the effects of 2.0,5.0 mg/L cisplatin on MG-63 cells.Moreover,Ad-Asc-myc combined with caffeine significantly enhanced this effects,not only on cisplatin-induced apoptosis,but also on tumor cells proliferation in vitro.The expression of bcl-2 was downregulated,bax were upregulated,while there was no change in the expression of E2F-1.FCM analysis showed that cisplatin treatment induced a block in S phase,and caffeine reversed this block and speeded up the progression of cells out of the S phase.Ad-Asc-myc induced obvious G₂/M phase arrest in transfected cells.CONCLUSION: Ad-Asc-myc combined with caffeine may enhance apoptosis-induced and chemotherapy effects of osteosarcoma MG-63 cells to cisplatin.     

Three kinds of antisense oligodeoxynucleotide enhance the sensitivity of leukemia cell K562 to cisplatin

AIM: To investigate whether antisense oligodeoxynucleotides of hTERT、bcl-2 and c-myc could enhance the sensitivity of leukemia cell K562 to cisplatin. METHODS: The inhibiting effects of cisplatin and cisplatin plus antisense oligodeoxynucleotide on K562 cells were determined by MTT. RESULTS: The inhibiting rate of 20 μmol/L cisplatin to K562 cell is 17.17%±1.36% and it becomes 25.41%±1.77%, 26.18%±1.43% and 28.29%±1.05%, respectively, as combinated with antisense oligodeoxynucleotide of hTERT, bcl-2 or c-myc. CONCLUSION: These results indicated that antisense oligodeoxynucleotides of hTERT, bcl-2 and c-myc enhanced efficacy of cisplatin in K562 leukemic cells.     

The effect of antisense bcr-abl oligonucleotides on the growth of K562 cells

AIM:To study the effect of bcr- abl gene antisense phosphorothioate oligonucleotides(Aspo) on K562 cell line and explore its significance in chrenic myelogeneous leukemia (CML) gene therapy.METHODS:Cells were exposed to oligomeis, observed by inverted microscope.Cells inhibitory rate were determined by 0.4 trypan blue exclusion . CFU-K562 were cultured in 0.8 % methylcellulose . P210 was measured by flow cytomety RESULTS: K562 cells were treated with Aspo, they still grew in clone state and show antisense sequence specific and dose dependent. When the concentration of Aspo was more than Spznol/L, the growth of cells was inhibited and P210 was down regulated or completely suppressed, and the greatest growth inhibition was at 120h . There was signifi-cant inhibition of cell proliferation in a rang‘cells number from 1×10⁴/mL to 5×10⁴/mL after treatment with 10unol/L Aspo. b2a2 Aspo was also effect on K562 cells which expressing b3a2 mRNA.CONCLUSION: bcr-abl Aspo has a specific growth inhibition effect on K562 cells, and worths further study in CML gene therapy.     

Effect of antisense locked nucleic acid blocking translation initiation region of c-myc exon 2 on apoptosis of hepatocellular carcinoma cells

AIM: To observe the effect of antisense locked nucleic acid (anti-LNA) blocking the translation initiation region of c-myc exon 2 on the viability and apoptosis of hepatocellular carcinoma cells.METHODS: The anti-LNA that was complementary to the translation initiation region of c-myc exon 2 was designed, synthesized, and introduced into the HepG2 cells by cationic liposome-mediated transfection. The mRNA and protein levels of c-Myc in the cells were determined by RT-PCR and Western blot. The change of cell apoptosis was analyzed by flow cytometry, and the toxicity of anti-LNA to the cells was detected by MTT assay.RESULTS: Five days after transfection, the mRNA level of c-Myc in anti-LNA group was 0.335±0.016, and the protein level was 0.448±0.037, significantly lower than those in control group (both P<0.05). The ratio of apoptotic cells in anti-LNA group was 32%±6%, which was higher than that in control group (P<0.05).CONCLUSION: Antisense locked nucleic acid targeting at the translation initiation region of c-myc exon 2 shows strong inhibitory effects on the apoptosis of hepatocellular carcinoma cells.     

A summary of the study on the homeobox gene Pem

Pem gene is one of the homeobox gene. Unlike members of Hox gene family, Pem gene is unique located at the proximal end of the X chromosome and its expression has been detected in several immortalized and transformed cells, placenta, embryos and reproductive tissues. Numeral studies showed that its expression is controlled by hormone such as androgen. This review discussed the possible role of Pem in regulating genes involved in the differentiation and development of extraembryonic tissue, spermatogenesis and sperm maturation.     

The antisense phosphorothioate oligodeoxynucleotides of bcl- 2 oncogene enhances the apoptotic effect of As2O3 on NB4 leukemic cells

AIM: To study and explore whether the antisense phosphorothioate oligodeoxynucleotides (ASODN) of bcl-2 oncogene would increase the apoptotic effect of As₂O₃ on NB4 leukemic cells. METHODS: The biological and morphological changes in NB4 cells from microculture with As₂O₃, bcl-2 ASODN or both were observed. The changes in DNA content and Bcl-2 protein of NB4 cells from microculture with As₂O₃, bcl-2 ASODN or both were determined by tissue chemistry and flowcytometry. RESULTS: There was much more apoptotic effect of As₂O₃ on NB4 cells while it combined with bcl- 2 ASODN (ASODN 10.0 μmol/L+ As₂O₃ 0.25 μmol/L) than alone(ASODN 10.0 μmol/L or As₂O₃ 0.25μmol/L),and so did inhibitory effect of Bcl-2 protein expression by flow cytometry. CONCLUSION:The bcl-2 ASODN can enhance the apoptotic effect of As₂O₃ on NB4 leukemic cells.     

甘肃中部梨资源遗传变异和亲缘关系的SSR分析

用6对SSR特异性引物对甘肃中部46个梨品种或类型的遗传变异和亲缘关系进行了分析,结果表明,SSR位点的期望杂合度为0.446～0.738,平均为0.639,显示了良好的品种鉴定能力。采用UPGMA聚类分析法构建的系统树将供试梨品种或类型分为7个大组:木梨组、白梨组、秋子梨组、褐梨组、西洋梨组和2个可能以秋子梨为基本种而形成的复杂的种间杂种组。唯一的砂梨品种黄花梨包含在白梨组中。新疆梨的5个品种没有独立成组,而是分别包含在木梨、白梨及秋子梨组中。一些原来根据形态学无法归类的品种和类型分别和木梨、白梨和西洋梨聚合在一起,显示出和这些梨系统的亲缘关系;木梨虽然采集地相对集中,但也显示出丰富的遗传多样性。     

Effect of historic landscape change on the genetic structure of the bush-cricket Metrioptera roeseli

This study investigates the impact of past and present landscape structure on the current genetic structure of the bush-cricket Metrioptera roeseli (Orthoptera, Tettigoniidae) in a rural landscape in Germany. Assuming that land-use types, such as grassland, arable land and forest, as well as linear structures, mainly roads, differentially affect the connectivity of the bush-cricket's habitat and therefore migration and gene flow, we correlated landscape parameters between sampling locations as derived from GIS-maps with genetic similarities between individual bush-crickets as estimated by RAPD-PCR. Fifty bush-crickets were sampled with distances between sampling locations varying between 15 m and 2 km. Corresponding landscape configurations were recorded in 8 years between 1945 and 1998. Landscape configuration 50 years ago appeared to have influenced the present genetic structure of the bush-cricket (R ² = 0.18). Crossing roads and land use other than grassland along the transect between sampling locations tended to decrease genetic similarity, whereas grassland and parallel roads tended to increase genetic similarity between bush-crickets. Following shifts in land use during 1953–1973 the correlation between landscape and present genetic structure decreased gradually. Our study suggests that it needs time for the landscape to build a visible effect on the genetic structure of the bush-cricket population, and that this effect cannot be detected if the landscape changes faster than the genetic structure responds to it.     

High multiplication frequency and genetic stability for commercialization of the three varieties of micropropagated tea plants (Camellia spp.)

RAPD and microsatellite markers were used to determine the genetic fidelity of micropropagated plants from the three varieties of tea plant derived from explants of field grown mother bush as well as in vitro germinated seedlings. Rate of shoot multiplication was better from nodal explants than from shoot tips. A maximum of 32–33 shoots was observed in cotyledonary node in 1/2 MS medium with BAP (6 mg/l), GA₃ (1 mg/l) and IBA (0.5 mg/l). 90% of the in vitro derived microshoots were micrografted into rootstocks. The micropropagated plantlets showed both cytological and genetical stability. SSR primers showed complete stability among the regenerants. The results convinced that plants derived from axillary as well as adventitious mode of propagation can be genetically true to type. This cost effective technique would help in fast clonal propagation at a commercial scale.     

阔叶猕猴桃遗传转化技术参数的优化

为了提高阔叶猕猴桃的遗传转化效率,以阔叶猕猴桃叶柄为试材,通过根癌农杆菌介导法进行了遗传转化技术参数的研究。结果表明,叶柄预培养3～4d、用农杆菌悬浮液(D600nm值0.5)感染10min、共培养48h、共培养时在培养基中加入200μmol/L乙酰丁香酮处理可以获得较高的gus基因表达率。在供试的1200个叶柄中获得了49个抗性芽,转化频率为4.1%。对转基因抗性材料进行PCR检测和gus基因组织化学染色,证实了外源基因已整合到阔叶猕猴桃的基因组中,并得到稳定表达。     

The influence of exposure to light on the phenolic content of ‘Fuji’ apple

In this study, the influence of changes in fruit lighting on individual and total phenols in ‘Fuji’ apple, as well as color development was studied. Content levels of eight quercetin glycosides, five anthocyanins, two catechins and a hydroxycinnamic acid in the skin of apples were analyzed, using high-performance liquid chromatography, tandem mass spectrometry (HPLC-MS). Total phenol, chlorophyll and carotenoid contents were determined by spectrometry. The purpose of this study was to compare content levels of those compounds in apple skin of fruit grown in different parts of the tree canopy, under and outside of the hail net. Lighting of fruit was measured during the last month before harvest. The lowest values were measured in the inner fruit and higher ones in the outer parts of the canopy, while the highest values were measured in fruit growing at the top of the tree. The hail net had no influence on the decrease of lighting in comparison to the control. Light conditions in the tree canopy influenced lower content levels of quercetin glycosides and most anthocyanins in the fruit skin in the inner part of the tree canopy, whereas fruit from the canopy top contained the highest levels of quercetin glycosides and cyanidin glycosides. Catechin, epicatechin and chlorogenic acid content levels in apple skin were independent of fruit position in the canopy or hail net usage, except for chlorogenic acid, where the content level was higher in cases when the orchard was covered with a hail net. Fruit from the top and outer parts of the canopy had a darker and redder coloration than inner fruit, while no influence of canopy position on chlorophyll and carotenoids was detected. Since quercetin glycosides and cyanidin glycosides are influential in red skin color development, better coloration of fruit from the outer and top canopy was observed. More intensive lighting stimulated a higher content level of flavonoids and, consequently, better coloration, which is an important factor in fruit quality.     

苹果属植物变叶海棠遗传多样性形成机理研究进展

过去20余年国内外学者对苹果属植物的起源、分类、区系地理、种内和种间遗传变异等开展了广泛的研究。然而,有关苹果属植物遗传多样性形成机理迄今未见任何直接报道。作者从变叶海棠的遗传多样性及其起源,物种的居群分化及其近缘种的形成几方面重点介绍过去15a西南大学在研究变叶海棠的遗传多样性和居群分化等方面所取得的进展。我们的研究工作为进一步探索苹果属植物遗传多样性形成机理奠定了基础,并为变叶海棠珍贵基因资源的保护和利用提供了理论依据。     

Effects of different chemotherapeutic agents on reversing the acquired resistance to TRAIL gene in DLD1 colon cancer cells

AIM:To evaluate effects of different chemotherapeutic agents on reversing the acquired resistance to TRAIL gene and clarify the involved mechanisms in DLD1-TRAIL/R colon cancer cells. METHODS:Human colon cancer cell line DLD1-TRAIL/R cells that were resistant to TRAIL-expressing adenovector (Ad/gTRAIL) were treated with Ad/gTRAIL combined with different chemotherapeutic agents. Then, the cell viability was measured by MTT method, and apoptotic signaling conditions, including activation of caspase-3 and caspase-8, expression of Bax and Bcl-XL, were measured by Western blotting analysis. RESULTS:In vitro data showed that several chemotherapeutic agents, including 5-fluorouracil (5-FU) and mitomycin c (MMC), overcome the acquired resistance to TRAIL gene in DLD1-TRAIL/R colon cancer cells. The combination of Ad/gTRAIL and 5-FU effectively suppressed tumor growth in vivo in subcutaneous tumors established from DLD1-TRAIL/R cells. Further data showed that treatment with the combination of Ad/gTRAIL and 5-FU or MMC led to enhance the activation of caspase-3. Moreover, MMC but not 5-FU induced overexpression of Bax gene that was sufficient to overcome the resistance to TRAIL gene in DLD1-TRAIL/R cells. CONCLUSION:Chemotherapeutic agents, such as 5-FU and MMC, overcome the acquired resistance to TRAIL gene in DLD1-TRAIL/R cells. The candidate mechanisms for MMC but not 5-FU to overcome this resistance might involve the induction of over-expressed Bax protein in DLD1-TRAIL/R cells.     

西瓜PRSV-W和ZYMV-CH抗性的遗传与连锁分析

番木瓜环斑病毒西瓜株系（PRSV—W）和小西葫芦黄化花叶病毒中国株系（ZYMV—CH）是危害我国西瓜的主要病害。以抗病毒病西瓜野生种质P．I．595203与感病普通西瓜自交系98R为亲本，采用单粒遗传方式得到109个F3代株系。分别对亲本、F1及F3代109个株系群体进行了苗期抗PRSV—W和ZYMV—CH接种鉴定，通过F3代群体的抗感分离情况来推测F2代单株的基因型，并分别对PRSV—W与ZYMV—CH抗性遗传及其这2个抗性基因的连锁关系进行分析。研究结果发现P．I．595203对PRSV—W的抗性是由单隐性基因控制，同时可能存在微效修饰基因，将此基因暂定名为prs—W；同时进一步证实了西瓜对ZYMV—CH的抗性由单隐性基因zym-CH控制，zym-CH和prs-W2个抗性基因在遗传上存在一定连锁关系，其遗传距离是27cM。