Human hepatoma cell line QGY-7703 was treated with the medium containing 10, 20, 40 and 60 micromol/L carotenoids extracts from Potamogoton crispus L. (CEPC) for 48 h, 96 h and 144 h, respectively. The inhibition rates to hepatoma cells were determined by MTT assay. The results showed the average inhibition rates of the three treatments varied from 0.14%-23.07%, 39.59%-70.61% and 71.65%-87.01%, respectively. After hepatoma cells were treated with the medium containing 10, 20 and 40 micromol/L of CEPC for 24 h, 48 h and 72 h, respectively, many typical morphology features of apoptotic cells were observed by Laser Scanning Confocal Microscope (LSCM), such as the distinct decrease in number and volume of cells, the shrinkage and deformation of cells, the shape of cell nucleuses appearing like crescent even piece, the decrease in area of yellow DNA in cell nuclei. The percentage of hepatoma cells in every phase of cell cycle was analyzed by Flow Cytometer (FCM) after being treated with 10 micromol/L and 20 micromol/L CEPC for 48 h. Compared with the control group, the cells in G0/G1 phase increased 23.8% (P<0.01) and 35.6% (P<0.01) respectively, in S phase decreased relevantly, while the cells in G2/M phase had no significant change. The Ca2+ concentration (fluorescence intensity) in cytoplasm of hepatoma cells was determined by LSCM after being treated with 20 micromol/L CEPC for 48 h. The Ca2+ concentration increased significantly (P<0.01) and was 1.5 times that of control group. These results demonstrated that the CEPC inhibited the proliferation of hepatoma cells QGY-7703 significantly in a dose and time-dependent manner in vitro. The hepatoma cells treated with CEPC were evidently arrested at G0/G1 phase in the cell cycle. The concentration of Ca2+ in test group cells cytoplasm increased significantly, it might be the important cause that CEPC induced the apoptosis of hepatoma cells QGY-7703. This paper provided scientific basis for further studying and developing the function and value of carotenoids in Potamogoton crispus L.
Using maize anthers of 2 cultivars as experimental materials, the effects of mannitol, colchcine and proline concentration in pretreatment solution and pretreating temperature on the survival percentage of isolated microspore were investigated. The survival percentage of isolated microspores were able to be respectively and obviously improved by pretreating anthers with mannitol from low to high concentration (0.35 mol/L, 24h --> 0.40 mol/L, 24h --> 0.45 mol/L, 24h), or with colchicine 200 mg/L or with proline 200 mg/L and pretreating at 15 degrees C for 48h. The probable mechanism of pretreatments was discussed.
Endophytic SR-15, SR-25 and SL-37 strains screened from rice hybrid D you 527 in Sichuan were analyzed. Through penetration and microscopic test, the strains were found be mainly located in the cell gap, vacuole and cytoplasm. PUC18 transferring and ERIC-PCR showed that the recombination strain SR-15 could grow in the plant stably. The strain was identified as Bacillus halmapalus based on its chemical components of cell wall, physiological and biochemical characters. It was also shown that the strain was not injurious to rice plant, instead, it promoted rice plant growth by penetration. The Cry1Ac gene was transferred into the stain and verified by Southern analysis. Cry1Ac-transferred SR-15 was toxic to the Chilo suppressalis, brought about death ratio as high as 84.2%.
Bulked segregant analysis method was used to identify random amplified polymorphic DNA (RAPD) markers linked to the fertility restoring gene for the cytoplasmic male sterility (CMS) capsicum annum L. Totally 336 random primers were screened on the DNA samples of restorer and sterile bulks. Primer S418 produced a special band in restorer line. It was about 3000 bp, including two fragments 1515 bp and 1162 bp. Fluorescence in situ hybridization(FISH) indicated the fragment of 1515 bp only existed in restorer line.It was designed to S418(1515). Analysis of the sequence indicated S418(1515) was unknown before. The homology of blastn was less than 40%, however the homology of tBlastx indicated this sequence was high homologous with the part sequences of rice which were distributed on 2,4,7,10 chromosomes. It suggested this sequence might have the similar function with them. This result offered a good foundation to research the molecular mechanism of fertility restoration for CMS capsicum. Based on the sequence, special primers were designed to transform the RAPD marker to PCR marker. The result indicated that these primers could be used to screen the restorer lines from a large quantitive of candidate lines.
Mlo gene in wheat is critical to determine wheat broad spectrum disease resistance against the pathogenic powdery mildew fungus. According to wheat TaMlo3 cDNA sequence, the two DNA fragments encoding the intracellular loop 2 and C-terminus of wheat Mlo protein were cloned respectively into the vector pET-30a, and then the fusion polypeptides were expressed in E. coli BL21. The recombinant proteins were purified by Ni2+ -NTA agarose column, and then were used to immunize the rabbits respectively. Two polyclonal antisera were obtained and they could be specifically bound on the extrahaustorial membrane and extrahaustorial matrix of the powdery mildew fungus by immuno-gold labeling.
Using inter-subspecific hybrid rice of IIyou 2070 and IIyou 419 , their restorer lines 2070 and Zhong419, the various forms and levels of root-derived cytokinins(CTKs) in root exudates, and changes of various forms of cytokinins in rice flag leaves and grains were analyzed by HPLC and ELISA after initial heading stage. The results indicated that zeatin had 62.8% to 89.1% of total cytokinins and was the dominant form of cytokinins in root exudates during early grain filling, whereas, the contents of diHZ and diHZR greatly increased during milk ripening. This change was consistent with the dynamic changes of ZRs (Z+ZR)and diHZRs (diHZ+diHZR)in flag leaves of IIyou 2070 and 2070. Different from main cytokinin composition in root exudates, IPAs took high proportion of total CTKs in leaf and grain. We suggested that there were changes of various forms and activities of root-derived cytokinins in root exudates, and this change was related to catabolic enzymes of cytokinins and the different expression of enzyme genes. Meanwhile, the relationship between cytokinin changes of vigorous and weak spikelets and grain filling was disscused.
The alteration of mitochondrion in Pisum sativum leaf cells infected with B935,isolates of BBWV 2, were investigated by electron microscopy, immunogold-labeling technique and stereologic quantity. The results showed that mitochondria were hyperplastic and aggregated nearby the membranous proliferation in cytoplasm. Many mitochondria were enlarged and distorted. There are some square paracrystalline inclusions within abnormal mitochondria. The difference between infected and healthy leaf cells at mitochondrion was observed to reveal significances in volume density (V(V)), surface density (S(V)), numerical density (Nv)(P < 0.01), but it was varied in form factor (PE), circumference index (CI), ratio of surface to volume (R(SV)). Some immunogold particles were localized to the crystal substance between the mitochondria. A high density of immunogold particles were localized to the crystalline aggregates and tubules composed of virus particles. This indicated that hyperplasia and aggregation of mitochondrion related to the assembly of virion in infection cells. BBWV 2 particles were assembled at the position of adherent mitochondria. The paracrystalline inclusions within mitochondria could represent the accumulation of a certain protein.
除mtDNA突变以外,LHON的突变基因的杂合性、临床表现的不完全外显性和明显的性别偏向等问题使其发病机制尚不明确,本研究拟了解LHON与氧化应激的关系,探索其进一步可能的发病机制和治疗.抽取有mtDNA*LHON G11778A突变的LHON患者7人、正常携带者10人及正常健康非母系家庭成员13人的外周血,用PHA(植物血凝素)作为细胞氧化应激的促发剂,运用化学发光法测定全血中氧自由基的变化.家系人群(包括病人和正常携带者)中全血氧自由基的变化在即时组要明显高于对照人群(P＜0.05),而家系人群二组间、即时组及10min组间的变化差异不大(P＞0.05).在外界氧化应激刺激下,LHON家系人群组织或细胞中自由基/抗氧化物间的平衡状态更容易被打破,提示氧化应激在其发病机制中起重要作用.
To investigate the expression patterns of histone acetyltransferase (GCN5) and histone deacetylase 1 (HDAC1) in preimplantation mouse embryos and the effects of in-vitro cultures on their expressions, immunocytochemistry was used to detect the expressions of GCN5 and HDAC1 in mouse embryos at the stages of two-cell, four-cell, eight-cell, morula and blastocyst in both in-vivo and invitro groups. In in-vivo group, the obvious expressions of GCN5 were observed in the cytoplasm of all kinds of mouse embryos but blastocysts. Meanwhile, HDAC1 was highly expressed in the nuclei of four and eight-cell embryos and morula but mainly seen in the cytoplasm of two-cell embryos. In blastocysts, the HDAC1 fluorescence was limited to the nuclei of trophoblast cells. In in-vitro group, there were no obvious GCN5 expressions in all kinds of embryos and the expression of HDAC1 was significantly reduced although its expression pattern was similar to that of in-vivo group. Those results showed that in -vitro culture environments could inhibit GCN5 expression and decrease the expression of HDAC1 in mouse preimplantation embryos, which might affect the correct embryonic gene expression.
The expression of estrogen receptor(ER) was examined in the oogenesis of termites (Reticulitermes aculabialis) with immunocytochemical localization method. The results showed ER existed in oocytes at the process of differentiation and the process of growth. ER immunopositive substance was localized in the nucleus of oocytes at the differentiation stage and (the cytoplasm of oocytes at the growth stage. On the other hand,previous studies have demonstrated the procerebrum of termites could secrete FSH (Follicle Stimulating Hormone)and LH (Luteinizing Hormone) which stimulated estrogen secreting. These results provide a new morphological proof that "brain-gonad" participates in the regulation of the oogenesis of termites, as have been shown in mammal.
Our previous studies suggested that GPI-80 expression might be associated with myeloid differentiation. Dimethyl sulfoxide (DMSO) and all-trans-retinoic acid (RA) are two main inducers that can induce HL-60 cell to differentiate down the way of neutrophilic lineage. GPI-80 is a novel member of a human glycosylphosphatidylinositol (GPI)-anchored family that has been proposed as a potential regulator of beta2-integrin-dependent leukocyte adhesion, and is expressed mainly in human neutrophils. RT-PCR, flow cytometry and western-blot assays were used to detect the GPI-80 expression on HL-60 cells induced by DMSO or RA and to analyze the relationship between GPI-80 expression and CD11b or transferrin receptor (CD71) expression. The results showed GPI-80 was slightly expressed on the level of mRNA on RA-induced HL-60 cells by RT-PCR, while using flow cytometry and western-blot assays, We could not detect any GPI-80 expression on these cells, Furthermore, RA suppressed the induction of GPI-80 expression. However, DMSO could clearly derive the GPI-80 expression and this expression correlated well with the increasing dose and duration of DMSO stimulation. GPI-80 positive cells, which were included in CD11b-strongly positive and transferrin receptor-completely negative populations, were sufficiently differentiated cells suggesting that GPI-80 expression is tightly associated with the neutrophilic maturation and can be used as a mature neutrophilic marker. RA inhibits the GPI-80 expression on DMSO induced HL-60 cells suggests that different signal conduction has been activated by DMSO and RA when they induce the HL-60 cells to the neutrophilic differentiation.
A recombinant phage vaccine expressing EGFR on it's capsid was constructed and used to study the anti-tumor effect. The T7 phage display system was applied to display seven xenogenic (human, chicken) epidermal growth factor receptor extracellular domain fragments. The EGFR fragment was expressed as fused protein with 10B capsid on the surface of T7 phage. The T7-EGFR phage vaccines were injected into C57BL/6J mice, and then Lewis lung cancer cells were inoculated after 4 weeks immunization. The tumor tissue was excised and weighed after 10 days to evaluate the anti-tumor effect of each experimental group. The EGFR expression of the phage vaccine was verified by western-blot analysis. The A431 cells with high expressed EGFR was used to detect the anti-EGFR antibody by flow cytometry analysis. The results showed that the A431 cell can react with the serum obtained from the mice after three-week immunization. The experimental results confirmed that special EGFR antibody could be induced by the T7-EGFR phage vaccine. The T7-EGFR phage vaccine can elicit endogenous special EGFR antibody in mice and is capable of suppressing the tumor proliferation and retarding the growth of Lewis lung cancer. This research can be used to develop an anti-tumor vaccine for the target-therapy of EGFR(+) tumor.
The anther of Magnolia biloba is tetrasporangiate with glandular tapetum, which consists of one or two layers of cells. Cytokinesis during meiosis of its microspore mother cell is modified simultaneous type, and the microspore tetrads are isobilateral. Mature pollen grains are two-celled. Tetrad cells and microspores are irregularly shaped during the microsporogenesis. There were two ovules on the ventral surface of unicarpellate ovary wall. Ovules were anatropous, bitegmnous and crassinucellar. Archesporial cell was one cell and differentiated from cell in the second layer beneath epidermis. The development of the embryo sac conformed to the polygonum type. The embryological characteristics of Magnolia biloba are very similar to those of other species in Magnoliaceae. The megasporogenesis and microsporogenesis and the development of their female and male gametophyte are partially abnormal. Abnormal phenomena in the process of reproduction of Magnolia biloba causing this species to be endangered was discussed.
To investigate microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396, D17S579 and D17S855, and their effect on the expression of nm23H1 and BRCA1 of gastric cancer, which would provide experimental basis for clinical treatment and prognosis analysis of gastric cancer. DNA was extracted from paraffin-embedded materials. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to analyze MSI and LOH. Expression of nm23H, and BRCA1 was detected by Envision immuno-histochemistry and Leica-Qwin computer imaging techniques. In the forty cases of gastric cancer, the frequency of MSI, LOH and nm23H1 protein were 20.00%, 17.50% and 55.00% respectively at locus D17S396, while at locus D17S579, the frequency of MSI, LOH and BRCA1 protein were 22.50%, 15.00% and 37.50% respectively; at locus D17S855, the frequency of MSI, LOH and BRCA1 of thirty-seven cases were 18.92%, 18.92%, 37.84% respectively. In tumor node metastasis (TNM) staging, at locus D17S396, D17S579 and D17S855, MSI in stages I + II appeared more frequently than that in stages III + IV, while LOH appeared the contrary tendency. In the group of metastasis of gastric cancer, MSI had a less frequency (5.00%) than that with no metastasis (35.00%, P < 0.05) at locus D17S396, but LOH appeared more frequently (30.00%) than that with no metastasis (5.00%, P < 0.05). At locus D17S579, MSI had an increasing tendency with the degree of tumor differentiation (50.00% in high differentiation cases, 20.00% in middle differentiation cases, and 0% in low differentiation cases, P < 0.05). The frequency of nm23H1 and BRCA1 protein in stages TNM I + II was higher than that in stages TNM III + IV; and that in higher differentiation cases was higher than in poor differentiation cases. The frequency of nm23H1 protein in the group of metastasis (30.00%) was less than that with no metastasis significantly (80.00%, P<0.01). The frequency of nm23H1 protein in the group positive to MSI (87.50%) was higher than that in the group negative to MSI (46.88%, P < 0.05). However, nm23H1 protein in group positive to LOH (14.29%) was lower than that in the group negative to LOH (63.64%, P < 0.05). The frequency of BRCA1 protein in the group positive to MSI (66.67%) was more than that in the group negative to MSI (29.03%, P < 0.05). The results of experiments indicate that MSI and LOH may separately control the development of sporadic colon cancer with different pathways. MSI may be an early period molecule marker for sporadic colon cancer, enhanced expression of nm23H1 protein can effectively inhibit colon cancer metastasis and improve prognosis of sporadic colon cancer patients. By comparison, LOH mostly arises in the late period of sporadic colon cancer and endows a high aggressive and poor prognostic phenotype. nm23H1 protein could effectively restrain gastric cancer metastasis and development; and BRCA1 protein could restain tumor from becoming lower differentiation.
In order to improve the development rate of preimplantation nuclear transfer embryos (NT embryos) after transplanting nuclei derived from transgenic goat fetal cells, the donor fetal fibroblasts starved for 5 days in DMEM containing 0.5% FCS were divided into three groups and treated with different methods respectively before using as donor cell. Group 1 was frozen at -80 degrees C or in liquid nitrogen for several days or months. Group 2 was at first treated as the same as group 1, then cultured for 2-5 days in DMEM containing 10% FCS and starved for another 5 days subsequently. Group 3 was cultured for 2-5 days in DMEM containing 10% FCS and starved for another 5 days subsequently. The rate of G0/G1 phase cells from group 2 was 95.68% and significantly different from group 1's 88.66%. The rate of survival cells from group 2 was 99.9% and significantly different from group 1's 80.00% (P < 0.05).The morula- blastocyst stage NT embryos development rate of group 2 was 66.09% and significantly different from group 1's 22.00% and group 3's 50.51% (P < 0.05). All NT embryos of above three groups were transferred into synchronous oestrus recipients and the pregnant status of recipients was checked by B-mode ultrasound diagnosis after 35 days. The recipient pregnancy rate of group 2 was 45.83%, much higher than that of group 1(20.00%) and group 3 (29.58%). The result of this experiment showed that donor cells treated with freezing and two times starvation could significantly improve the rate of G0/G1 phase cells, the rate of survival cells, the NT embryos development rate and the recipient pregnancy rate.
The Chromosomes of Macrobrachium nipponense had been prepared using the materials of testis or embryo with the chromosome low-osmosis method or cell low-osmosis method. After 5.5 h, the bivalents at meiotic diplotene were appeared. The comparison of the two methods for two different materials showed that the morpha of the chromosome of testis using the low-osmosis method was better than that of using the cell low-osmosis method. Contrarily, the low-osmosis was better than the cell low-osmosis for the morpha of the chromosome of embryo. The bivalents at meiotic diplotene were rod-shaped, and centromere position could be identified easily at that time. The diploid chromosomes numbers were 104. The chromosome formula was N=11M+26SM+4ST+11T. The technique of the chromosome preparation of Macrobrachium nipponense was preliminarily discussed as well.
The expressions of VEGF-C mRNA, VEGFR-3 and CD31 were studied in order to investigate the correlation between them and neoangiogenesis, hyperplasia of micro-lymphatics and tumor metastases. 34 cases of prostate cancer tissue and 12 cases of adjacent nontumorous tissue specimens were tested. They were marked by VEGFR-3 and CD31 with immunohistochemistic staining and analyzed with image, the micro-lymphatics count (MLC) and microvessel density (MVD) were counted using Weidner's highest vessel density count method; the expression of VEGF-C mRNA was inspected in situ hybridization. The expression of VEGF-C mRNA was 44.12% positive in 34 cases of prostate cancer, the MLC (8.26 +/- 2.73)mm2 and MVD (74.82 +/- 11.76)mm2 in prostate cancer were significantly higher than those in adjacent nontumorous tissue (MLC, 4.82 +/- 3.48/mm2; MVD, 32.86 +/- 5.41/mm2). In addition, there was a correlation between the expression of VEGF-C mRNA and micro-lymphatics metastases and there was a positive correlation between the expression of VEGFR-3 and CD31. The expressions of VEGF-C mRNA , MLC, MVD in stage III and IV and those who have lymph metastasis were higher than those in stage I and II and those who have no lymph metastasis; the expressions of VEGFR-3 and CD31 in VEGF-C mRNA positive groups were significantly higher than those in negative groups. The difference of histopathologic grading in prostate cancer had no statistical significance. VEGF-C could accelerate the hyperplasia of micro-lymphatics and neoangiogenesis induced by tumor and play an important role in tumor lymph metastases. There was a close correlation between the expressions of VEGFR-3, CD31 and tumor metastases. The increase of MLC and MVD on prostate cancer indicated the hyperplasia of new micro-lymphatics and neoangiogenesis in the tumor tissue, which could also be a signal to determine the tumor metastases in clinic.
Genetic diversity of 28 cultivars of yam (Dioscorea opposita Thunb) was assessed by means of Inter-simple sequence repeat (ISSR) markers. The results showed that seven proper primers, with rich polymorphism, could be selected from a total of forty four ISSR ones; distinct differences appeared among 28 cultivars amplified bands, and the rate of polymorphic bands was 83.01%; Shannon's Information index was 0.3191; a Jaccard's genetic similarity matrix and a dendrogram for these cultivars were formed, in which they could be divided into four groups: Group 1 was composed of D. opposita. cv. Ribenbai, D. opposita. cv. Huashanyao and D. opposita. cv. Ribenyuan; Group2 contained D. opposita. cv. Xiaoye; Group 3 contained D. opposita. cv. No.1 Songye; other 23 cultivars were put into Group4. PCA(Principal component analysis) was employed to evaluate the resolving power of the markers to differentiate among them. This laid the foundation of the identification of yam cultivars and the efficient use of its germplasm resources.
MFUT-II, a DNA fragment encoding murine alpha1,2-fucosyltransferase,was cloned by RT-PCR. The open-reading frame was ligated into mammalian expression vector pcDNA 3.1 (pcDNA3.1-MFUT-II) and was transiently transfected into COS-7 cells using a Cellphect Transfection kit. Moreover, the expression of the gene in murine tissues was analyzed with Northern hybridization showing a 3.5-Kb mRNA transcript product in several tissues. The existence of MFUT-II was detected by Southern blot. MFUT-II was found to be a new member of the murine alpha1,2-FT gene family. Its open reading frame encoded 347 amino acids with a predicted molecular mass of 39.21 kDa and exhibited sequence homology with murine H & Sec1 gene. MFUT-II also exhibited sequence homology with Human Se gene (79.0%), Rat FTB gene (89.0%) and Rabbit FT-III gene (77.0%), respectively. COS-7 cells transfected with pcDNA3.1-MFUT- II showed alpha 1,2-fucosyltransferase activity. Southern analysis revealed that MFUT-II was present in the mouse genome as a single-copy gene. In conclusion, MFUT-II was a murine Se gene.
The present study has determined the histological changes, VEGF (Vascular en- dothelial growth factor) mRNA alternative expressions and VEGF mRNA levels in chondrogenic- callus, osteogenic-callus and total callus by histology, RT-PCR and Northern blot during rat frac- ture repair. The results showed that the cellular events of intramembranous ossification, chodrogen- esis and endochondral ossification appeared in the callus simultaneously and sequentially. The VEGF mRNAs were expressed in the osteogenic-callus and the chondrogenic-callus, and the strongest expressions were VEGF_(120) and VEGF_(164) mRNA. The VEGF mRNA levels in the total cal- lus increased with time after fracture. Take together, these findings suggest that osteogenesis-de- rived VEGF may play a potential role to modulate the differentiation process of osteogenic and chondrogenic cells during fracture repair.
