Effect of ERK1/2 phosphorylation on the aggregation of the rat platelets in vitro

AIM: To study the influence of PD098059 on the rat platelet aggregation rate and the phosphorylation of ERK1/2 induced by the different agonists, and to observe the effects of phosphorylation of ERK1/2 on the platelet aggregation. METHODS: The maximal aggregation rate (MAR) was measured by nephelometry. The inhibitory rate of PD098059 and the appearing time of MAR were also observed. ERK1/2 phosphorylation was detected by Western blot. RESULTS: The phosphorylation of ERK1/2 was detected during aggregation induced by thrombin and ADP. PD098059 inhibited the MAR and phosphorylation of ERK1/2. Effects of PD098059 were different on the aggregation induced by thrombin and ADP. CONCLUSIONS: The phosphorylation of ERK1/2 is one of the cellular signal transduction mechanisms of platelets aggregation. Phosphorylation of ERK1/2 plays different roles during the platelet aggregation induced by thrombin and ADP.     

Primary study on biocompatibility of chitosan-Mg membrane in vitro

AIM: To prepare the chitosan-Mg membranes (CM) and explore the biocompatibility of membranes with PC12 cells and feasibility of its usage as bionmaterials in tissue engineering.METHODS: The appearance of the chitosan-Mg membranes was observed by scanning electromicroscope (SEM) and the element of membranes was analyzed by X-ray energy spectrometer. The expansion coefficient of CM was also detected. PC12 cells were co-cultured with CM in vitro. The morphological changes of PC12 cells on membranes were observed under SEM and the cell vitality was detected by MTT assay.RESULTS: The surface of chitosan membranes (CS) was smoother than that of CM, but the CM were full of tiny pores. The content of Mg element was related with the dose of MgSO4 added into chitosan solution in a dose dependent manner. Morphology observation showed that PC12 cells grew well with CM compared with CS. The cells were rich of microvilli and long processes on 7th day, and synapses like structure was formed in many PC12 cells. In addition, the cell viability in experiment group was higher than that in control group (P<0.05) on 7th day detected by MTT method.CONCLUSION: Chitosan can combine with Mg2+. The complexation rate is related with the content of Mg2+ but not in direct ratio. Chitosan/Mg membranes show good biocompatibility with PC12 cells.     

Peripheral blood mononuclear cells activated by graded zirconia-hydroxyapatite composite in vitro

AIM: To evaluate the immunogenicity of a novel orthopedics materials (graded zirconia-hydroxyapatite composite) in vitro by using peripheral blood mononuclear cells (PBMCs) from healthy young people, and simple zirconia-hydroxyapatite composited material was used as control materials. METHODS: Proliferation of PBMCs cultured in different liquid after 5 days was measured by MTT methods. ELISA was used to detect TNF-α and IL-6 concentration in the supernatant of PBMCs cultured in the extracts after 24 hours. Flow cytometery was used to measure CD69 and CD25 in activated PBMCs cultured in the extracts of the two kinds of materials after 24 hours. RESULTS: The proliferation rate of the simple composite group was significantly lower than that in negative group (P<0.05), but there was no difference between the graded composited group and negative group. After 24 hours culture with LPS, the concentrations of TNF-α and IL-6 in the simple composited group were significantly higher than that in graded composited group, respectively (P<0.05). Cultured with PHA for 24 hours, the ratio of CD69 and CD25 positive PBMCs in the simple composited group was all significantly higher than that in graded composited material group (P<0.01). CONCLUSION: The number of PBMCs activated by the graded composited material is less than the simple composited material and the immunogenicity of the graded composited material is lower than the simple composited material.     

The inhibitory effect of quercetin on in vitro activation of T lymphocytes

AIM:To investigate the inhibitory effect of quercetin on in vitro activation of T lymphocytes by polyclonal activators with CD69 expression as an activation marker.METHODS:After being separated from lymphoid nodes of a C57BL/6 mouse, the lymphocytes were exposed to polyclonal activators (PDB or Con A) with or without quercetin. Then they were harvested at 2 h, 6 h and 24 h, respectively. The expressional rates of CD69 on T lymphocytes were assessed by two-color immunofluorescent staining and flow cytometry, and the inhibitory rates of quercetin at different time points were estimated.RESULTS:Quercetin had no effect on the expressional rate of CD69 on T lymphocytes under resting states. After the stimulation with PDB or Con A, the expressional rates of CD69 on T lymphocytes in the present of quercetin (10 μmol/L) showed significant decrease compared with those of control groups at different time points (P＜0.01). The inhibitory rate of quercetin on CD69 expression stimulated by PDB dropped sharply from 2 h to 24 h, whereas the inhibitory rate of quercetin on Con A action were relatively stable.CONCLUSION:Quercetin has inhibitory effects on the activation of T lymphocytes by Con A or PDB, suggesting that the action site of quercetin may be on PKCθ or its downstream. Furthermore,these inhibitory effect seems to be reversible.     

Study on polarized Th1/Th2 cell in vitro from adult human peripheral blood

AIM: To investigate the details of CD4⁺ T cell polarized to Th1/Th2 in vitro. METHODS: After isolated the PBMCs and blood-plasma from adult human peripheral blood by Ficoll-Hypaque centrifugation, the PBMC culture procedure with or without the self-blood -plasma was applied to polarize T cells in vitro, these cells were polarized by PHA(20 mg/L),non-PHA respectively. The polarized rates of Th cell after 24 h,48 h,72 h were estimated respectively by flow cytometry following two-color immunofluorescent staining. RESULTS: CD4⁺T cell would polarize to Th1/Th2 two subsets after self-cytokines and PHA activation in vitro. The polarized rates of T cell after cultured for 24 h,48 h and 72 h were (13.28%±1.59%)/(12.70%±1.65%),(17.19%±1.03%)/(17.50%±1.30%),(19.49%±2.87%)/(18.58%±1.49%) respectively, but the polarized rates of T cell were very low if without self-blood-plasma. The difference between them was significant. The ratios of Th1/Th2 cells were about 1. CONCLUSION: CD4⁺T cell from adult human peripheral blood would polarize to Th1/Th2 two subsets in the presence of self-blood-plasma and PHA(20 mg/L) in vitro, and the cell number of Th1 and Th2 would be in balance.     

石榴试管嫁接技术研究

【目的】尝试建立高效的石榴试管嫁接技术体系,尝试解决根癌农杆菌介导的石榴遗传转化中再生的转化不定芽难以生根顺利发育成完整植株的问题。【方法】以‘突尼斯软籽’石榴试管实生苗为接穗‘,豫大籽’石榴试管实生苗为砧木,设置不同的砧穗组合进行嫁接;在添加不同浓度6-BA的培养基中进行嫁接,对嫁接成活的试管苗进行驯化移栽,统计嫁接成活率和移栽成活率。【结果】进行试管嫁接时适宜采用的接穗类型为带有4片叶片的茎尖,砧木类型为不带子叶的根和下胚轴连接体,嫁接成活率可达到73.33%;将四叶一心的‘突尼斯软籽’石榴嫁接至不带叶片的‘豫大籽’石榴,嫁接苗在添加1.5 mg·L~(-1)6-BA和0.1 mg·L~(-1)NAA的MS培养基上的生长效果最好,移栽成活率最高可达75.86%。【结论】研究所建立的石榴试管嫁接体系,为解决石榴遗传转化过程中不定芽生根困难提供了一条新的途径,并为通过基因工程手段进行石榴种质改良奠定基础。     

铁线蕨孢子离体繁殖技术研究

采用孢子离体培养技术对铁线蕨进行了快繁技术研究。结果表明:把经过消毒灭菌的孢子囊从孢子叶上切下,进行破碎取出孢子后接种到1/2MS培养基上,孢子萌发较快,萌发后的原叶体接种到MS培养基上能得到较大的扩繁速率,叶原体诱导孢子体在试管内诱导难度较大,可采用0.1%KH2PO4诱导剂在试管外诱导,更适合叶原体向孢子体的转化。     

八仙花茎尖离体培养技术研究

以八仙花带芽枝条为试材,采用茎尖离体培养技术对八仙花进行快繁研究。结果表明:把经过消毒灭菌的茎尖切割后转入芽诱导培养基MS+6-BA 1.0mg/L+NAA 0.1mg/L+GA31.0mg/L,萌发率达到85%;继代培养基采用MS+6-BA 0.5mg/L+NAA 0.04mg/L+腺嘌呤5.0mg/L,会具有较高的生长系数;生根培养基采用1/2MS+IBA 0.8mg/L+活性炭3g/L,能够完全生根成苗。     

风信子离体快繁技术研究

利用离体快繁技术,以风信子的鳞茎为外植体,添加不同浓度6-BA和NAA进行不定芽的诱导、不定芽的继代和生根培养.结果表明:鳞片的初代最佳诱导培养基为:MS+6-BA 1.5 mg/L+NAA 0.3 mg/L,诱导率达到80％;最佳继代培养基为:MS+6-BA 1.0 mg/L+NAA 0.2 mg/L;生根诱导最佳培养基:1/2MS+NAA0.3 mg/L,生根率可达90％.     

Inhibitory effects of propolis on platelet adhesion in flowing blood in vitro

AIM:To investigate effects of propolis-ethanol extract on platelet activity in flowing blood. METHODS:Intima-injury model was made by in vitro perfusion. Coverslips was coated by using fibrinogen and human Ⅲ type collagen to detect platelet adhesive rate when blood went through fibrinogen and collagen surface in 1 000/s shear rate. Three experimental groups were set up: negative control group (24% ethanol), experimental group (0.1 g/L, 24% propolis-ethanol extract) and positive control group (0.1 g/L, ferulic acid). RESULTS:Intima-injury model was made. On fibrinogen and collagen, platelet adhesion on area of experimental group was lower than that in negative control group (P<0.01). There was no significant difference between experimental group and positive control group (P>0.05). Propolis-ethanol extract decreased more obviously the platelet adhesion area on fibrinogen surface than that in collagen surface (P<0.01). CONCLUSION:Propolis-ethanol extract inhibits platelet activation and reduces its adhesion on fibrinogen and collagen surface, especially in fibrinogen surface.     

三角梅组培苗试管内外生根研究

以紫花三角梅和紫红重瓣三角梅组培苗为试材,研究其在试管内、外的生根情况.结果表明:试管内生根以1/4 MS+IBA 1.5 mg/L+NAA 0.05 mg/L处理的生根情况最好,生根率为92.53％,平均生根数量11条,平均根长4.75 cm;试管外的生根率为43％.     

Identification by isoenzymes of five cultivars of Citrus medica grafted on four rootstocks

Summary

Four enzymatic systems: esterase, acid phosphatase (both rapid and slow types) and peroxidase were studied on five cultivars of Citrus medica grafted on four rootstocks. The results showed that the analysis of esterase banding patterns permitted a clear distinction of the cultivars because esterase patterns were reproducible and were not affected by the rootstocks. In some instances differences in peroxidase patterns and staining intensities could be observed in certain combinations of scion/rootstock of the same cultivar.     

Effect of isoflavone genistein on activation and proliferation of mouse T cells in vitro

AIM: To study the effect of genistein on activation and proliferation of T cells, and explore the molecular mechanism of genistein. METHODS: Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the express of CD69 and CD25 by activated T cells in vitro in response to Concanavalin (ConA )and Phorbol 12, 13-dibutyrate(PDB) or T cell proliferation stained by CFSE in response to PDB / Ionomycin or ConA. RESULTS: Genistein inhibited the expression of CD69 and CD25 in activated T cells in response to Con A in a concentration-dependent manner and in response to PDB in a high concentration. Genistein inhibited proliferation of T cells in both groups in a concentration-dependent manner. CONCLUSION: Genistein inhibited activation and proliferation of T cells in vitro in response to polyclonal stimulus, and it may hold potential as a new immunosuppressant.     

不同砧木与甜瓜嫁接亲和性分析

为了研究不同砧木与甜瓜嫁接亲和性,分别用厚皮和薄皮甜瓜做接穗,用南瓜、葫芦和野生西瓜作砧木,通过嫁接成活率、植株长势、单株果实平均质量等8个指标研究嫁接亲和性。结果表明,厚皮和薄皮甜瓜用南瓜作砧木,平均成活率分别为87%和70%;用葫芦作砧木,平均成活率分别为76%和80%;用野生西瓜作砧木,平均成活率均为10%;定植后,南瓜砧木嫁接苗生长最快,果实平均质量比对照高30%以上,薄皮甜瓜处理单株结果数量低于对照,葫芦砧木嫁接苗生长比对照慢,在伸蔓期,80%植株黄化死亡。野生西瓜作砧木,定植20 d后,80%植株黄化死亡。综上所述,南瓜类型砧木与甜瓜嫁接亲和性最好,其次为葫芦,野生西瓜亲和性最差。     

Effect of fat-specific protein 27 on activation of rat hepatic stellate cells in vitro

AIM：To investigate the effects of fat-specific protein 27 (Fsp27) on the proliferation and activation of hepatic stellate cells (HSCs) in vitro. METHODS：HSCs were isolated from the liver of SD rats. The mRNA and protein expression of Fsp27 in primary HSCs and activated HSCs was detected by real-time fluorescence quantitative PCR, immunofluorescence staining and Western blotting. After 72 h of transfection with Fsp27-carrying lentivirus (pLV-Fsp27), the proliferation of HSCs was tested by CCK-8 assay, the protein expression of α-smooth muscle actin (α-SMA) in HSCs was detected by Western blotting, and the mRNA expression of fibrosis-related proteins, including matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinase 1 (TIMP-1) and transforming growth factor beta 1 (TGF-β1), was determined by real-time fluorescence quantitative PCR. RESULTS：Rat HSCs were successfully isolated and cultured. The difference of Fsp27 expression between primary HSCs and activated HSCs was significant (P<0.01). The proliferation and activation of HSCs was inhibited 72 h after pLV-Fsp27 transfection (P<0.05). Fsp27 enhanced the mRNA expression of MMP-2 and down-regulate the mRNA expression of TIMP-1 and TGF-β1 in activated HSCs (P<0.05). CONCLUSION：Fsp27 inhibits the proliferation and activation of HSCs and regulates the expression of fibrosis-related proteins. Fsp27 may play an important role in maintenance of the quiescent phenotype of HSCs.     

苗药红花龙胆体外抑菌试验

以采自六盘水的红花龙胆(Gentiana rhodantha)为试材,采用滤纸片扩散法检测红花龙胆全草、地上部分、组培苗根超声粗提物对大肠杆菌、枯草芽孢杆菌及金黄色葡萄球菌的抑菌活性,并检测氧化剂、温度对抑菌活性的影响。结果表明:对以上3种菌的抑菌效果,组培苗根全草地上部,全草与组培苗根的抑菌效果为枯草芽孢杆菌大肠杆菌金黄色葡萄球菌,地上部的抑菌效果为大肠杆菌枯草芽孢杆菌金黄色葡萄球菌;提取液浓度为0.125 g·m L-1时,对大肠杆菌、枯草芽孢杆菌及金黄色葡萄球菌的抑菌圈直径,组培苗根为(11.33±0.44)、(11.67±0.38)、(9.67±0.53)mm,全草为(9.42±0.45)、(9.05±0.72)、(8.36±0.44)mm,地上部为(8.34±0.42)、(6.00±0.00)、(9.33±0.44)mm;氧化剂对红花龙胆抑菌活性影响较小,红花龙胆提取物具有热稳定性。红花龙胆组培苗根可以开发成天然抑菌剂。     

彩色马铃薯新品系试管薯诱导因素研究

以彩色马铃薯新品系‘HS-15’脱毒试管苗为材料,在MS培养基下,添加2种不同浓度蔗糖、5种不同浓度6-BA诱导,通过2种不同光照处理,进行彩色马铃薯试管薯诱导研究。结果表明：在16h/d光照培养15d后转入8%蔗糖＋6-BA2mg/L的培养基全黑暗培养,对‘HS-15’试管薯诱导效果最好。     

薄皮甜瓜不同砧木嫁接效应比较

采用圣砧1号和新士佐2种南瓜砧木嫁接当地薄皮甜瓜品种花皮脆瓜,试验结果表明,2种砧木的嫁接苗亲和性表现均佳,嫁接成活率在95%以上,抗枯萎病效果极佳;圣砧1号嫁接苗生长速度快于新土佐嫁接苗,且第1雌花开放早1d左右,盛花期早2d左右;圣砧1号嫁接组合产量表现略逊于新土佐嫁接组合,2种砧木对果实口感无不良影响。     

Effects of herbs of activation blood on atherosclerotic plaque morphology in ApoE gene-deficient mice

AIM: To observe the effects of six common traditional Chinese herbs of activating blood, paeoniae rubra radix, salviae miltiorrhizae radix, ligustici, rhizome, notoginseng radix, pruni persicae semen and wine staemed radix et rhizome, on atherosclerotic plaque structure and stabilization in ApoE gene-deficient mice. METHODS: Four sections of the aortic root were choosen and stained with hematoxylin and masson. All sections were measured with Image-Pro Plus Version 4.5.1 (IPP) software. RESULTS: Compared with the model group, plaque area corrected by cross-sectional vessel wall area reduced significantly in salviae miltirrhizae radix treatment group, lipid core area reduced in paeoniae rubra radix group, pruni persicae semen and wine steamed radix et rhizome treatment group, minimum thickness of fibrous cap became thicker significantly in salviae miltiorrhizae radix, ligustici, rhizome, pruni persicae semen and wine steamed radix et rhizome treatment group. CONCLUSION: These Chinese herbs may stabilize the atherosclerotic plaques in ApoE gene-deficient mice by interfering their structure, but their effects do not parallel with their activating blood efficacy in traditional Chinese medicine.     

Effect of camptothecin on the activation,proliferation and cell cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of camptothecin (CPT) on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (ConA) in vitro. METHODS: A model of T cell activation and proliferation was established by stimulated the cells with Con A. T cells were treated with different concentrations of CPT. The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation index was determined by carboxyl fluorescin diacetate succinmidyl ester by flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining. RESULTS: After stimulation with Con A for 6 h, the activation rate of CD69+ T cell in Con A group was (58.88±0.55)%. The percentages of CD69 positive cells were (55.48±0.98)%, (54.67±1.05)%, (50.40±0.82)%, (42.47±1.32)%, correspond to the treatments with different concentrations of CPT (10 nmol/L, 20 nmol/L, 50 nmol/L, 100 nmol/L), respectively. After 48 h treatment with Con A, the proliferation index in different concentrations of CPT treatment (10 nmol/L, 20 nmol/L, 50 nmol/L and 100 nmol/L) exerted a definite inhibitory effect on the proliferation (P<0.01). Moreover, the cell-cycle distribution analysis showed that apoptosis peak was observed in different concentrations of CPT treatment after 48 h cultured with Con A. CONCLUSION: CPT significantly inhibits the early stages of the Con A-induced T cell activation and proliferation, and detents the T lymphocytes in G₀/G₁ phase.