Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363 bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved.

Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA from Zhikong scallop Chlamys farreri

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter for the TLR/IL-1R family. In this report, the first mollusk Myd88 ortholog (named as CfMyd88) was cloned from Zhikong scallop (Chlamys farreri). The full-length cDNA of CfMyd88 was of 1554 bp, including a 5 '-terminal untranslated region (UTR) of 427 bp, a polyA tail, and an open reading frame (ORF) of 1104 bp encoding a polypeptide of 367 amino acids containing the typical TLR and IL-1R-related (TIR) domain and death domain (DD). Homology analysis revealed that the predicted amino acid sequence of CfMyd88 was homologous to a variety of previously identified Myd88s with more than 30% identity. The temporal expressions of CfMyd88 mRNA in the mixed primary cultured haemocytes stimulated by lipopolysaccharide (LPS) and peptidoglycans (PGN) were measured by real-time RT-PCR system. The mRNA expression of CfMyd88 decreased after stimulation with both LPS and PGN, and the lowest level was about 1/3 times (at 6 h) and 1/10 times (at 9 h) to that in the control group, respectively. The expression then recovered and was upregulated to two-fold at 9 h after LPS stimulation or to the original level at 12 It after PGN stimulation. The results suggest that the MyD88-dependent signaling pathway exists in scallop and was involved in the defense system. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a putative lipopolysaccharide-induced TNF-alpha factor (LITAF) gene homologue from Zhikong scallop Chlamys farreri

LPS-induced TNF-alpha factor (LITAF) is a novel transcriptional factor that was first discovered in LPS-stimulated human macrophage cell line THP-1. LITAF can bind to TNF-a promoter to regulate its expression. The first scallop LITAF (named as CfLITAF) was cloned from Zhikong scallop Chlamys farreri by Expressed Sequence Tag (EST) and Polymerase Chain Reaction (PCR) techniques. The cDNA of CfLITAF was of 1240 bp and consisted of a 5' untranslated region (UTR) of 112 bp, a 3' UTR of 678 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 16.08 kDa and theoretical isoelectric point of 6.77. A typical conserved LITAF-domain was identified in CfLITAF by SMART analysis. Homology analysis of the deduced amino acid sequence of CfLITAF with other known sequences by using the BLAST program revealed that CfLITAF was homologous to the LITAF from human and rat (Identity = 46%), cattle, horse, mouse and chicken (Identity = 48%), western clawed frog (Identity = 42%), and zebrafish (Identity = 50%). The mRNA expression of CfLITAF in different tissues including haemocytes, muscle, mantle, heart, gill and gonad, and the temporal expression in haemocytes challenged by LPS or peptidoglycan (PGN) were measured by Real-time RT-PCR. CfLITAF mRNA transcripts could be detected in all tissues examined and be up-regulated in haemocytes after LPS challenge. No significant changes were observed after PGN stimulation. All these data indicated the existence of LITAF in scallop and also provided clue on the presence of TNF-alpha-like molecules in invertebrates. (C) 2007 Elsevier Ltd. All rights reserved.

A novel tumor necrosis factor ligand superfamily member (CsTL) from Ciona savignyi: Molecular identification and expression analysis

A novel invertebrate TNF ligand was identified and characterized in Ciona savignyi. The CsTL cDNA consisted of 995 nucleotides and encoded 281 amino acids. A conserved TNF family signature and several motifs of TNF ligand superfamily were identified in deduced amino acid sequence of CsTL. Phylogenetic analysis grouped CsTL, CiTNF (predicted TNF ligand superfamily homolog in Ciona intestinalis) and urchin TL1A with their own cluster apart from mammalian TNF alpha, LTA, TNFSF15 and fish TNFa proteins. Expression studies demonstrated that CsTL mRNA is present in all tested tissues from unchallenged ascidians and its expression was significantly upregulated in hemocytes following LIPS injection. The recombinant CsTL protein expressed using a baculovirus expression system showed potential cytotoxic activity in L929 cells. Present results indicated that TNF ligand superfamity molecules are present in marine invertebrates. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus

Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular structure and expression patterns of flounder (Paralichthys olivaceus) Myf-5, a myogenic regulatory factor

Myf-5, a member of the myogenic regulatory factors (MRF), has been shown to be expressed in muscle precursors in early stage zebrafish embryos. The MRFs, including MyoD, Myf-5, Myogenin and MR-F4, belong to the basic Helix-Loop-Helix transcription factors that contain a conserved basic Helix-Loop-Helix (bHLH) domain. To better understand the role of Myf-5 in the development of fish muscles, we have isolated the Myf-5 genomic sequence and cDNA from Flounder (Paralichthys olivaceus), and analyzed its structures and patterns of expression. Promoter analysis identified several putative transcription factor binding sites such as an E-box, NF-Y sites that might confer muscle-specific expression. Myf-5 transcripts were first detected in the paraxial mesoderm that gives rise to slow muscles. During somitogenesis, Myf-5 expression was found in developing somites. Myf-5 expression decreased gradually in somites in the anterior region, but remained strong in the newly formed somites. In the hatching stage, the expression was also detected in other muscle cells such as head muscle and fin muscle. In the growing fish, RT-PCR results showed that Myf-5 was expressed in the skeletal muscle and intestine. (c) 2006 Elsevier Inc. All rights reserved.

The second anti-lipopolysaccharide factor (EsALF-2) with antimicrobial activity from Eriocheir sinensis

The anti-lipopolysaccharide factor CALF) is a small basic protein that can bind and neutralize lipopolysaccharide (LPS), mediating degranulation and activation of an intracellular coagulation cascade. In the present study, cDNA of the second Eriocheir sinensis ALF (designated as EsALF-2) was cloned and the full-length cDNA of EsALF-2 was of 724 bp, consisting of an open reading frame (ORF) of 363 bp encoding a polypeptide of 120 amino acids. The deduced amino acid of EsALF-2 shared 82% similarity with EsALF-1 from E. sinensis and about 53-65% similarity with ALFs from other crustaceans. The potential tertiary structures of EsALF-1 and EsALF-2 contained two highly conserved-cysteine residues to define the LPS binding site, but the N-terminal of EsALF-1 formed a single additional alpha-helix compared to EsALF-2, implying that EsALF-1 and EsALF-2 might represent different biological functions in E. sinensis. The mRNA transcript of EsALF-2 was detected in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad, which suggested that EsALF-2 could be a multifunctional molecule for the host immune defense responses and thereby provided systemic protection against pathogens. The mRNA expression of EsALF-2 was up-regulated after Listonelln anguillarum and Pichia pastoris challenge and the recombinant protein of EsALF-2 showed antimicrobial activity against L. anguillarum and P. pastoris. indicating that EsALF-2 was involved in the immune defense responses in Chinese mitten crab against L. anguillarum and P. pastoris. These results together indicated that there were abundant and diverse ALFs in E. sinensis with various biological functions and these ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control of crab aquaculture. (C) 2010 Elsevier Ltd. All rights reserved.

Hypoxia-inducible factor 1 alpha cDNA cloning and its mRNA and protein tissue specific expression in domestic yak (Bos grunniens) from Qinghai-Tibetan plateau

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated a-subunit and a constitutively expressed beta-subunit. How animals living on Qinghai-Tibetan plateau adapt to the extreme hypoxia environment is known indistinctly. In this study, the Qinghai yak which has been living at 3000-5000 m attitude for at least two millions of years was selected as the model of high hypoxia-tolerant adaptation species. The HIF-1 alpha ORFs (open reading frames) encoding for two isoforms of HIF-1 alpha have been cloned from the brain of the domestic yak. Its expression of HIF-1 alpha was analyzed at both mRNA and protein levels in various tissues. Both its HIF-1 alpha mRNA and protein are tissue specific expression. Its HIF-1 alpha protein's high expression in the brain, lung, and kidney showed us that HIF-1 alpha protein may play an important role in the adaptation to hypoxia environment. (c) 2006 Elsevier Inc. All rights reserved.

Cloning of hypoxia-inducible factor 1 alpha cDNA from a high hypoxia tolerant mammal - plateau pika (Ochotona curzoniae)

Hypoxia-inducible factor I is a transcription factor composed of HIF-1alpha and HIF-1beta. It plays an important role in the signal transduction of cell response to hypoxia. Plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant and cold adaptation species livin only at 3000-5000m above sea level on the Qinghai-Tibet Plateau. In this study, HIF-1alpha cDNA of plateau pika was cloned and its expression in various tissues was studied. The results indicated that plateau pika HIF-1alpha cDNA was highly identical to those of the human (82%), bovine (89%), mouse (82%), and Norway rat (77%). The deduced amino acid sequence (822 bp) showed 90%, 92%, 86%, and 86% identities with those of the human, bovine, house mouse, and Norway rat, respectively. Northern blot analyses detected two isoforms named pLHIF-1alpha and pSHIF-1alpha. The HIF-1alpha mRNA was highly expressed in the brain and kidney, and much less in the heart, lung, liver, muscle, and spleen, which was quite different from the expression pattern of mouse mRNA. Meanwhile, a new variant of plateau pika HIF-1alpha mRNA was identified by RT-PCR and characterized. The deduced protein, composed of 536 amino acids, lacks a part of the oxygen-dependent degradation domain (ODD), both transactivation domains (TADs), and the nuclear localization signal motif (NLS). Our results suggest that HIF-1alpha may play an important role in the pika's adaptation to hypoxia, especially in brain and kidney, and pika HIF-1alpha function pattern may be different from that of mouse HIF-1alpha. Further-more, for the high ratio of HIF-1alpha homology among the animals, the HIF-1alpha gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa. (C) 2004 Elsevier Inc. All rights reserved.

Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation

It has been suggested that endothelial apoptosis is a primary lesion in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). We tested this hypothesis by examining the phenotypic signatures of endothelial microparticles (EMP) in TTP patients. In addition, the effect of TTP plasma on microvascular endothelial cells (MVEC) in culture was further delineated. EMP released by endothelial cells (EC) express markers of the parent EC; EMP released in activation carry predominantly CD54 and CD62E, while those in apoptosis CD31 and CD105. We investigated EMP release in vitro and in TTP patients. Following incubation of MVEC with TTP plasma, EMP and EC were analysed by flow cytometry for the expression of CD31, CD51, CD54, CD62E, CD105, CD106 and von Willebrand factor (VWF) antigen. EMP were also analysed in 12 TTP patients. In both EC and EMP, CD62E and CD54 expression were increased 3- to 10-fold and 8- to 10-fold respectively. However, CD31 and CD105 were reduced 40-60% in EC but increased twofold in EMP. VWF expression was found in 55 +/- 15% of CD62E(+) EMP. Markers of apoptosis were negative. In TTP patients, CD62E(+) and CD31(+)/CD42b(-) EMP were markedly elevated, and preceded and correlated well with a rise in platelet counts and a fall in lactate dehydrogenase. CD62E(+) EMP (60 +/- 20%) co-expressed VWF and CD62E. The ratio of CD31(+)/42b(-) to CD62E(+) EMP exhibited a pattern consistent with activation. In conclusion, our studies indicate endothelial activation in TTP. EMP that co-express VWF and CD62E could play a role in the pathogenesis of TTP.

降雨条件下碎石土滑坡稳定性研究

Debris Landslide is one of the types of landslides with the widest distribution, largest quantity, and the closest relationship with engineering construction. It is also one of the most important types of landslides that can cause disaster. This kind of landslide often occurs in the loose slopes which are made up of loose congeries formed by earth filling, residual soil, slope wash, dilapidation, landslide or full weathered material of hard rock. Rainfall is always the chief inducing factor of debris Landslide. Therefore, to research stability of debris Landslide during rainfall not only has important theoretical significance for understanding developing law and deformation and failure mechanism of debris landslide, but also has important practical significance for investigating, appraising, forecasting, preventing and controlling debris landslides. This thesis systematically summarized the relationships between rainfall and landslide, the method to survey water table in the landslides, the deformation and failure mechanism of debris landslide, and the progress in the stability analysis of landslides based on the analyses of data collected widely at home and abroad. The problems in the study of the stability of debris landslide during rainfall was reviewed and discussed. Due to the complicated geological conditions and the random rainfall conditions, the research on the landslides' stability must be based on engineering geological qualitative analysis. Through the collection of the data about the Panxi region and the Three Gorges Reservoir region, the author systematically summarized the engineering geological conditions, hydro-geological condition, distribution characteristics of stress field in the slope, physical and mechanical properties and hydro-mechanical properties of debris. In the viewpoint of dynamics of soil water and hydromechanics, physical process of rainfall to supply groundwater of debris landslides can be divided into two phases, i.e. non-saturated steady infiltrating phase and saturated unsteady supplying phase. The former can be described by mathematical model of surface water infiltration while the latter can be described by equivalent continuous medium model of groundwater seepage. With regard to specific hydrological geology system, we can obtain the dynamic variation law of water content, water table, landslide stability of rock and soil mass, along with quantity and duration of rainfall after the boundary condition on hydrological geology has been ascertained. This is a new way to study the response law of groundwater in the landslides during rainfall. After wet face of rock and soil mass connects with ground water table, the raising of water table will occur due to the supply of rainfall. Then interaction between ground water and rock and soil mass will occur, such as the action of physics, water, chemistry and mechanics, which caused the decrease of shearing strength of sliding zone. According to the action of groundwater on rock and soil mass, a concise mechanical model of debris landslide’s deformation was established during rainfall. The static equilibrium condition of landslide mass system was achieved according to the concise mechanical model, and then the typical deformation and failure process and failure mode of debris landslide during rainfall were discussed. In this thesis, the former limiting equilibrium slice method was modified and improved based on shearing strength theory of , a stability analysis program of debris landslide was established and developed taking account of the saturated-unsaturated seepage, by introducing the shearing strength theory of unsaturated soil mass made by (1978). The program has reasonable data storage and simple interface and is easy to operate, and can be perfectly used to carry out sensitivity analysis of influencing factors of landslides' stability, integrated with the program of Office Excel. The design of drainage engineering are always bases on empirical methods and is short of effective quantitative analysis and appraise, therefore, the conception of critical water table of debris landslide was put forward. For debris landslides with different kinds of slide face in the engineering practice, a program to search the critical water table of debris landslide was developed based on native groundwater table. And groundwater table in the slope should be declined below the critical water table in the drainage works, so the program can be directly used to guide drainage works in the debris landslide. Taking the slope deformation body in the back of former factory building of Muli Shawan hydroelectric power station as an example, a systematic and detailed research on debris landslides' stability during rainfall was researched systematically, the relationship among quantity of rainfall, water table and stability of slope was established, the debris landslides' stability in process of rainfall from dynamic viewpoint was analyzed and researched.

膨胀土化学改性实验研究

Expansive soil is a kind of typical unsaturated soil with characteristics of high swelling-shrinking deformation, cracks and over consolidation. It is very harmful to civil engineering, As a new processing method deal with expansive soil, Chemistry treatment has widespread applied in developed countries such as Europe and America, and also gained remarkable result. Based on the embankment filling soil improving testing projects in Meng-Xin freeway, this paper proposed a new processing method to expansive soil embankment wrapped with PAS-treated soil, experimental study of expansive soil chemical improved by PAS is been carried out. The water content change is the external factor which causes expansive soil to have swelling-shrinkage deformation. this reflected that the soil body swelling-shrinkage characteristic mainly depends on its mineral ingredient and the soil-water mutual function. This paper takes expansive soil as one kind of ordinary high plastic clay from angle of clay-water mutual function explained the expansive soil swelling-shrinkage deformation mechanism on microscopic. And take this swelling-shrinkage mechanism as the master line, Cooperates with the China Academy of Chemistry, we developed the new method PAS treatment, trough ionic exchange, joint, package and flocculation, the stronger static electricity function weakened the level through adsorption and the stronger static electricity function, PAS can weakened the negative charge repulsion between levels, causes the electric potential to reduce, diffusion layer thickness to be thinner, and improves the water affinity performance of expansive soil effectively. Moreover the space network architecture compromised with PAS and soil enhanced the joint strength between the clay particles , enable the soil body to have comparatively high strength and the distortion rate. pointed proposed the PAS modified principle. Combine with the construction of experimented road, this paper sums up and presents the construction craft and technology requirement of PAS treatment to expansive soil embankment. Through many experimental studied the basic physical property, the intensity characteristic and water stability changes of expansive soil and PAS-treated soil. The results of study indicate that adding lime into the expansive soil can reduce the content of clay gain obviously, reduce the plasticity notably, increase the strength greatly, control the property of swelling and shrinking effectively, and can meliorate the stability of sucking water clearly. Simultaneity PAS don’t change the cultivate capacity of the soil, the modified slope of the embankment can adopt plant fixed slope method as ecology protection. Finally the processing effect of use different treatment has analyzed through numerical simulation, summarized the PAS chemical wrapping treatment process in the actual project application, and appraised its processing effect and the project efficiency. The research indicated that PAS chemical treatment is one effective method to improve expansive soil. Compare with long-distance replacement, especially in the high plastic expansive soil massive distribution area, PAS treatment has the very greatly economical superiority to be promoted. The study in the paper not only afforded technique method to Meng-Xin expressway construction but also important for improvement of the expressway construction theory in swelling soil areas. Key words: PAS; expansive soil; swelling-shrinkage deformation mechanism; wrapping embankment; chemical modified treatment.

典型人类活动对边坡变形及稳定性的影响研究

China is a mountainous country in which geological hazards occurred frequently, especially in the east of China. Except the geology, topography and extreme climate, the large scale human activities have become a major factor to landslides. Typical human activities which induced landslides are fill, cut and underground mining. On the topic of the deformation mechanism and slope stability, taking three different man-made slopes as examples, deformation mechanism and slope stability were studied by several methods, such as field work, numerical modeling and monitor. The details are as following: (1) The numerical modeling approach advantages over other conventional methods such as limit methods, so the numerical modeling is the major tool in this thesis. So far, there is no uniform failure criterion for numerical simulation. The failure criterion were summarized and analyzed firstly, subsequently the appropriate criterion was determinated. (2) Taking 220kV Yanjin transformation substation fill slope as example, the deformable characteristic, unstable mode and laboratory tests were studied systematically. The results show: the slope deformation was probably caused by a combination effect of unfavorable topographic, geological and hydro geological conditions, and external loading due to filling. It was concluded that the creep deformation of the slope was triggered by external loading applied at the back of the slope. In order to define the calculating parameters, a set of consolidated drained (CD) tests, consolidated undrained (CU) tests, repeated direct shear tests and UCS tests were carried out. The stability of the slope before and after reinforcement was assessed using 3D numerical modeling and shear strength reduction technique. The numerical modeling results showed: the factor of safety (FOS) of the slope was 1.10 in the natural state, and reduced to 1.03 after fill, which was close to the critical state and it caused creeping slip or deformation under rainfall. The failure surface in the slope is in active shear failure, whereas tensile failure occurs at the slope crest. After the site was reinforced with piles, the FOS was 1.27. Therefore, the slope is stable after reinforcement measures were taken. (3) The cut slope stability is a complex problem. Taking the left cut slope of Xiangjiaba as example in this thesis, the deformation and slope stability were studied systematically by numerical modeling and monitor methods. The numerical results show: the displacement is gradually increasing along with the cutting, and the largest displacement is 27.5mm which located at the bench between the elevation 340 and 380. Some failure state units distribute near the undermining part and there is no linked failure state occurred from crest to bottom during cutting. After cutting, some failure units appeared at the ground surface between elevation 340 and 360. The increasing tense stress made the disturbed rock failed. The slope is stable after cutting by the monitor method, such as surface monitor, multipoint displacement meter, inclinometer and anchor cable tensometer. (4) The interaction between underground mining and slope stability is a common situation in mountainous. The slope deformation mechanism induced by underground mining may contributed significantly to slope destabilization. The Mabukan slope in xiangjiaba was analyzed to illustrate this. Failure mechanism and the slope stability were presented by numerical modeling and residual deformation monitor. The results show: the roof deformed to the free face and the floor uplift lightly to the free face. The subsidence basin is formed, but the subsidence and the horizontal movement is small, and there is no failure zone occurred. When the underground mining is going on, the roof deformation, subsidence and the horizontal movements begin increasing. The rock deformation near the free face is larger than the ground surface, and the interaction between these coal seams appeared. There are some tensile failures and shear failures occurred on the roof and floor, and a majority of failure is tensile failure. The roof deformation, subsidence and the horizontal movements increased obviously along with the underground mining. The failure characteristic is shear failure which means the tensile stress transformed to the compressive stress. So the underground mining will induced tensile stress first which lead to structure crack, subsequently the compressive stress appeared which result in slippage. The crest was subjected to horizontal tension which made the rock crack along with the joint. The long term residual deformation monitor demonstrates that the slope is stable after the underground mining stopped.

石油二次运移机理物理模拟研究

Oil and gas migration is very important for theoretical hydrocarbon geology study and exploration practice, but related research is weak. Physical simulation is a main method to study oil migration. Systematic experiments were done to quantitatively describe the migration patterns, path characters and oil saturation by adjusting the possible dynamic factors respectively. The following conclusions were drawn. 1. Darcy velocity and pore throat diameter were calculated according to seepage cross-sectional area and glass beads arrangement. With such normalized Darcy velocity and pore throat diameter, the date from one and two dimensional experiments can be reasonably drawn in two phase diagrams. It is found that the migration pattern can be identified using only one dimensionless number L which is defined as the ration of capillary number and Bond number. 2. Oil saturated in the pores between glass beads was used as calibration and oil saturation in the path was measured by magnetic resonance imaging. The results show that oil saturation in the center of migration path can reach 100%, is higher than oil saturation in the edge of migration path. 3. Percolation backbone during secondary oil migration was identified experimentally using Hele-Shaw cell. The backbone formed mainly because of the spatial variation of the cluster conductivity caused by oil saturation heterogeneity, main resistant force change, and path shrinkage and snap-off. Percolation backbone improves hydrocarbon migration efficiency and is a favorable factor for reservoir forming. 4. In the three dimensional filling models, the thickness of the secondary migration path is mall. It is only 2.5cm even for the piston pattern. Inclination of the model is the main influencing factor of the secondary path width.

生物－热催化过渡带沉积成岩演化及其成烃作用研究

The biothermocatalytic transitional zone gas is a new type of natural gas genetic theory, and also an clean, effective and high quality energy with shallow burial depth, wide distribution and few investment. Meanwhile, this puts biothermocatalytic transitional zone gas in important position to the energy resource and it is a challenging front study project. This paper introduces the concept, the present situation of study and developmental trend about biothermocatalytic transitional zone gas in detail. Then by using heat simulating of source rocks and catalysis mechanism analysis in the laboratory and studying structural evolution, sedimentation, diagenesis and the conditions of accumulation formation and so on, this paper also discusses catalytic mechanism and evolutionary model of the biothermocatalytic transitional zone gas formation, and establishes the methods of appraisal parameter and resources prediction about the biothermocatalytic transitional zone gas. At last, it shows that geochemical characteristics and differentiated mark of the biothermocatalytic transitional zone gas, and perfect natural gas genetic theory, and points out the conditions of accumulation formation, distribution characteristics and potential distribution region on the biothermocatalytic transitional zone gas m China. The paper mainly focuses on the formation mechanism and the resources potential about the biothermocatalytic transitional zone gas. Based on filed work, it is attached importance to a combination of macroscopic and microcosmic analysis, and the firsthand data are obtained to build up framework and model of the study by applying geologic theory. Based on sedimentary structure, it is expounded that structural actions have an effect on filling space and developmental cource of sediments and evolution of source rocks. Carried out sedimentary environment, sequence stratigraphy, sedimentary system and diagenesis and so on, it is concluded that diagenesis influences developmental evolution of source rocks, and basic geologic conditions of the biothermocatalytic transitional zone gas. Applying experiment simulating and catalytic simulating as well as chemical analysis, catalytic mechanism of clay minerals is discussed. Combined diagenecic dynamics with isotope fractionation dynamics, it is established that basis and method of resource appraisal about the biothermocatalytic transitional zone gas. All these results effectively assess and predict oil&gas resources about the biothermocatalytic transitional zone gas-bearing typical basin in China. I read more than 170 volumes on the biothermocatalytic transitional zone gas and complete the dissertation' summary with some 2.4 ten thousand words, draw up study contents in some detail and set up feasible experimental method and technologic course. 160 pieces of samples are obtained in oilfield such as Liaohe, Shengli, Dagang and Subei and so on, some 86 natural gas samples and more than 30 crude oil samples. Core profiles about 12 wells were observed and some 300 geologic photos were taken. Six papers were published in the center academic journal at home and abroad. Collected samples were analysised more than 1000 times, at last I complete this dissertation with more than 8 ten thousand words, and with 40 figures and 4 plates. According to these studies, it is concluded the following results and understandings. 1. The study indicates structural evolution and action of sedimentary basin influence and control the formation and accumulation the biothermocatalytic transitional zone gas. Then, the structural action can not only control accommodation space of sediments and the origin, migration and accumulation of hydrocarbon matters, but also can supply the origin of energy for hygrocarbon matters foramtion. 2. Sedimentary environments of the biothermocatalytic transitional zone gas are lake, river and swamp delta- alluvial fan sedimentary systems, having a warm, hot and humid climate. Fluctuation of lake level is from low to high., frequency, and piling rate of sedimentary center is high, which reflect a stable depression and rapidly filling sedimentary course, then resulting in source rocks with organic matter. 3. The paper perfects the natural gas genetic theory which is compound and continuous. It expounds the biothermocatalytic transitional zone gas is a special gas formation stage in continuous evolutionary sequence of organic matter, whose exogenic force is temperture and catalysis of clay minerals, at the same time, having decarbxylation, deamination and so on. 4. The methodology is established which is a combination of SEM, TEM and Engery spectrum analysis to identify microstructure of crystal morphology about clay minerals. Using differential thermal-chromatographic analysis, it can understand that hydrocarbon formation potential of different typies kerogens and catalytic method of all kinds of mineral matrix, and improve the surface acidity technology of clay minerals measured by the pyridine analytic method. 5. The experiments confirm catalysis of clay minerals to organic matter hygrocarbon formation. At low temperature (<300 ℃), there is mainly catalysis of montmorillonite, which can improve 2-3 times about produced gas of organic matters and the pyrolyzed temperature decreased 50 ℃; while at the high temperature, there is mainly catalysis of illite which can improve more than 2 times about produced gas of organic matters. 6. It is established the function relationship between organic matter (reactant) concentration and temperature, pressure, time, water and so on, that is C=f (D, t). Using Rali isotope fractionation effect to get methane isotope fractionation formula. According to the relationship between isotope fractionation of diagenesis and depth, and combined with sedimentary rate of the region, it is estimated that relict gas of the biothermocatalytic transitional zone gas in the representative basin. 7. It is revealed that hydrocarbon formation mechanism of the biothermocatalytic transitional zone gas is mainly from montmorillonite to mixed minerals during diagenesis. In interlayer, a lot of Al~(3+) substitute for Si~(4+), resulting in a imbalance between surface charge and interlayer charge of clay minerals and the occurrence of the Lewis and Bronsted acid sites, which promote to form the carbon cation. The cation can form alkene or small carbon cation. 8. It is addressed the comprehensive identification mark of the biothermo - catalytic transitional zone gas. In the temproal-spatial' distribution, its source rocks is mainly Palaeogene, secondly Cretaceous and Jurassic of Mesozoic, Triassic, having mudy rocks and coal-rich, their organic carbon being 0.2% and 0.4% respectively. The vitrinite reflection factor in source rocks Ro is 0.3-0.65%, a few up to 0.2%. The burial depth is 1000-3000m, being characterized by emerge of itself, reservoir of itself, shallow burial depth. In the transitional zone, from shallow to deep, contents of montmorillonites are progressively reduced while contents of illites increasing. Under SEM, it is observed that montmorillonites change into illite.s, firstly being mixed illite/ montmorillonite with burr-like, then itlite with silk-like. Carbon isotope of methane in the biothermocatatytic transitional zone gas , namely δ~(13)C_1-45‰- -60 ‰. 9. From the evolutionary sequence of time, distribution of the biothermocatalytic transitional zone gas is mainly oil&gas bearing basin in the Mesozoic-Neozoic Era. From the distribution region, it is mainly eastern stuctural active region and three large depressions in Bohaiwang basin. But most of them are located in evolutionary stage of the transitional zone, having the better relationship between produced, reservoir and seal layers, which is favorable about forming the biothermocatalytic transitional zone gas reservoir, and finding large gas (oil) field.