去细胞带瓣牛颈静脉管道支架的制备及形态结构观察

目的研究去细胞带瓣牛颈静脉组织工程支架的制备,并观察其形态结构。方法取新鲜牛颈静脉48条,随机分为对照组、实验组,每组24条。对照组为新鲜牛颈静脉;实验组为去细胞牛颈静脉,用40 g/L脱氧胆酸钠+50 m l/L Triton-X-100为主要成分的试剂进行处理,去除内皮细胞及成纤维细胞。HE染色、苦味酸-天狼猩红染色、弹力纤维染色、电子扫描显微镜检查、基因组DNA检测,观察其形态结构,评价去细胞效果。结果实验组瓣膜及血管壁中内皮细胞、成纤维细胞去除完全,无细胞核碎片;瓣膜及血管壁胶原纤维和弹力纤维呈波浪状排列、整齐,结构完整;瓣膜、血管壁基因组DNA含量,分别较对照组下降97.58%、97.25%。结论脱氧胆酸钠+Triton-X-100是一种有效的去除牛颈静脉细胞,制备组织工程带瓣管道支架的方法。     

乙醇、丁二醇改性对牛颈静脉的抗钙化作用

目的探讨两种醇类改性对牛颈静脉带瓣管道的抗钙化作用。方法将戊二醛固定的牛颈静脉带瓣管道分别用80%乙醇(A组)和100%2,3-丁二醇(B组)进行化学改性,分别将两组管壁片及瓣膜片植入12只刚离乳的SD大鼠背部两侧皮下,90 d后处死大鼠,取出植入的组织片进行组织钙质量分数、总蛋白水平测定与光镜、电镜检查。结果A组的牛颈静脉管壁及瓣膜组织钙质量分数分别为(173±61)μg/m g和(2.13±0.85)μg/m g,B组钙质量分数分别为(181±29)μg/m g和(1.73±1.20)μg/m g,两组比较,均无显著性差异(P>0.05);组织学观察显示两组改性管壁及瓣膜组织炎性细胞浸润较少,胶原纤维结构保持良好。A组管壁及瓣膜片总蛋白水平分别为(337±293)m g/L和(142±82)m g/L,B组管壁及瓣膜片总蛋白水平分别为(269±230)m g/L和(139±111)m g/L,两组比较,均无显著性差异(P>0.05)。结论两种醇类均可作为牛颈静脉带瓣管道的改性剂;生物材料钙化与蛋白吸附相关;丁二醇及乙醇改性减少了生物材料的蛋白吸附,从而增强其抗钙化性能。     

猪主动脉瓣去细胞基质的制备

目的完全去除猪主动脉瓣细胞,取得良好的去细胞支架材料。方法用5 g/L胰酶对标本中的某些基质和细胞加以消化后,再应用不同去垢剂（十二烷基硫酸钠、脱氧胆脂酸钠、Triton X-100）加以洗脱,结合溶液渗透压改变等去除猪主动脉瓣细胞;标本进行大体、光镜、电镜观察和热皱缩温度的检测。结果胆脂酸钠无法完全脱除猪主动脉瓣细胞,十二烷基硫酸钠和曲拉通均可完全去除猪主动脉瓣细胞,Triton X-100组的主动脉瓣胶原纤维和弹性纤维得以较好保持。结论Triton X-100可以成功脱除猪主动脉瓣细胞,为组织工程瓣膜的研究提供材料。     

制备犬胸主动脉脱细胞血管支架的新方法

目的 研究制备犬胸主动脉脱细胞血管支架的新方法,制备出理想的犬胸主动脉脱细胞基质,从而为构建组织工程血管提供支架材料.方法 在无菌条件下从成年比格犬体内取出胸主动脉血管段（28根）,随机分成4组：A组血管段设为正常对照组;B组血管段置于-70℃的冰箱和4℃冰箱内反复冻融2次;C组血管段在0.1％的SDS溶液中持续震荡1d;D组血管段经历反复冻融后置于1％ Triton X-100的PBS溶液和1μmol/L PMSF的混合液中常温下震荡2d,于0.01％的SDS溶液中持续震荡1d,最后加入核酸酶Dnase 0.2 mg/L和Rnase0.02 mg/L消化24h.四组血管段经历不同的过程后,全部从大体形态、光镜、电子显微镜观察、力学性能测试、免疫组化等角度进行检测并做统计学分析.结果 0.1％ SDS法虽能完全脱除细胞,但制备的脱细胞基质弹力纤维排列紊乱,部分发生断裂,且不能保持良好的形状和张力强度,管腔有不同程度的塌陷;反复冻融＋1％ Triton X-100＋PMSF＋0.01％ SDS法不仅能完全脱除细胞,保持基质纤维的正常排列结构,而且制备的脱细胞基质能保持良好的形状和力学性能,管腔无塌陷.结论 联合应用反复冻融、Triton X-100、PMSF和SDS的方法能够将犬胸主动脉的细胞成分除去,是一种制备脱细胞血管支架的有效方法.     

基质细胞衍生因子1α促进大鼠骨髓源性内皮祖细胞血管样结构形成

目的观察基质细胞衍生因子1α对体外培养的大鼠骨髓源性内皮祖细胞血管样结构形成的影响。方法微孔法获取大鼠骨髓内皮祖细胞,采用免疫荧光鉴定内皮祖细胞特异性标记物VEGFR-2/CD133。1、10和100μg/L基质细胞衍生因子1α以及100μg/L基质细胞衍生因子1α+CXCR4拮抗剂AMD3100共处理内皮祖细胞,采用细胞培养、MTT等方法检测内皮祖细胞血管样结构形成和细胞增殖能力。结果体外培养的大鼠骨髓源性内皮祖细胞出现典型铺路石形状及血管样结构,与此同时内皮祖细胞分化成熟,表达内皮细胞特异性标记物vWF。MTT法检测发现,10和100μg/L基质细胞衍生因子1α处理组的OD值(分别为0.813±0.056和1.029±0.078)与对照组(0.591±0.054)比明显升高(P<0.01),而100μg/L基质细胞衍生因子1α+AMD3100组OD值(0.607±0.077)与对照组比差异无显著性,与100μg/L基质细胞衍生因子1α组比较明显降低(P<0.01)。100μg/L基质细胞衍生因子1α处理组血管样结构长度是对照组的近6倍(10.890±0.360比1.930±0.279,P<0.01),10...     

基于体外巨噬细胞极化作用进行异种血管组织脱细胞评价的实验研究

目的 通过体外巨噬细胞极化诱导的实验方法,客观评价异种血管组织进行除垢剂脱细胞处理后的纯度和免疫原性。方法 对新鲜牛主动脉采用3-[(3-胆酰胺基丙基)二甲氨基]-1-丙磺酸内盐[3-(3-Cholamidopropyl)dimethylammonium)-1-propanesulfonate,CHAPS]和十二烷基硫酸钠（sodium dodecyl sulfate,SDS）的双除垢剂脱细胞方法进行脱细胞处理,其中SDS脱细胞时间设置为24 h和36 h两组,定量检测脱细胞处理后组织材料DNA浓度。进一步将脱细胞血管基质材料消化为溶液状态,在体外对SD大鼠来源的巨噬细胞进行刺激诱导。采用定量聚合酶链反应（quantitative polymerase chain reaction,qPCR）分析巨噬细胞表面标记物诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）、CD80、CD206和CD163,以及分泌的细胞因子白细胞介素（interleukin,IL）-1β、IL-10和肿瘤坏死因子-α(tumor necrosis factor-α,...     

组织工程化肺动脉带瓣管道纤维支架制作方法初步探讨

目的:观察采用酶加去污剂的方法脱去猪肺动脉带瓣管道的细胞和基质的效果,并通过物理和化学等方法对其进行测定,评价其作为组织工程支架材料的可行性。方法:采用0.25%胰蛋白酶和1%的Triton依次对新鲜猪肺动脉带瓣管道进行脱细胞脱基质处理。通过厚度、含水量、抗拉强度及热皱缩温度测定其物理性质与新鲜猪肺动脉对比;通过测定其蛋白多糖含量间接反应基质脱除的大小;通过新西兰幼鼠皮下埋植试验观察异种移植后炎症反应的大小。结果:采用0.25%胰蛋白酶和去污剂1%Triton能够有效脱除肺动脉壁和瓣膜中的细胞和基质成分,产生多孔隙性、低免疫原性的支架材料,初步具备良好组织工程支架材料的基本特点。     

光氧化处理牛颈静脉带瓣管道重建肺动脉与右心室连接

目的观察光氧化处理的牛颈静脉带瓣管道用于重建右心室—肺动脉连接后,在体内循环系统中与血流接触条件下的抗钙化性能及其血流动力学性能。方法以经光氧化反应处理的牛颈静脉带瓣管道为研究对象,以单纯戊二醛交联固定的牛颈静脉带瓣管道作为对照,建立犬重建右心室—肺动脉连接的动物模型;实验动物饲养观察10个月后,通过超声心动图和心导管检查等方法评价两种不同方法固定处理的牛颈静脉带瓣管道重建犬右心室—肺动脉连接后的血流动力学性能,并了解牛颈静脉管道内的瓣膜在犬的右心系统内的功能。屠宰实验动物后取出标本,通过原子吸收光谱法测定组织钙含量,von Kossa钙盐染色观察组织钙化情况。结果心脏超声显示两组管道均通畅,瓣叶活动良好。光氧化处理组结扎肺动脉前、后直接测压及术后9~10个月通过心导管测压,所测得的跨瓣压差维持在较低水平,跨瓣压差无明显升高;戊二醛组结扎肺动脉前、后及术后9~10个月测得的跨瓣压差与光氧化组相似。光氧化组管道血管壁钙含量为7.60±8.02 mg/g,戊二醛组管道血管壁钙含量为22.05±10.78 mg/g,两组有显著性差异(P<0.05);光氧化组和戊二醛组管道瓣膜钙含量分别为0.74±0.23 mg/g和0.88±0.69 mg/g,两组相比无统计学差异(P>0.05)。结论光氧化反应处理固定的牛颈静脉带瓣管道可用于重建右心室—肺动脉连接,血流动力性能良好,与自体肺动脉相当,远期血流动力学性能尚待进一步观察研究。光氧化反应处理固定的牛颈静脉带瓣管道在狗的动物模型中,抗钙化性能优于戊二醛固定的牛颈静脉管道。     

京尼平抑制饱和脂肪酸诱导的HepG2细胞凋亡和内质网应激

目的 探讨京尼平减轻棕榈酸对HepG2细胞毒性的作用及机制.方法 将HepG2细胞分为4组,分别用牛血清白蛋白、棕榈酸(1 mmol/L)、京尼平(20 μmol/L)或京尼平(20 μmol/L)预处理30 min后棕榈酸(1 mmol/L)孵育24 h,检测细胞活力及乳酸脱氧酶(LDH)释放;孵育16 h后经流式细...     

小口径全生物化异种移植血管制备方法的实验研究

目的 探索和评价酶-去污剂法联合肝素结合处理犬颈动脉,以构建小口径全生物化移植血管.方法 将犬颈动脉分为两组,均采用酶-去污剂法脱细胞处理,其中一组再行肝素结合处理;对两组样本进行组织学和扫描电镜观察及机械性能测试以评价脱细胞效果;甲苯胺蓝染色观察肝素结合情况;凝血时间实验观察肝素发挥作用的持续时间.结果犬颈动脉经脱细胞后,细胞成分去除完全,细胞外基质保持完好,机械性能无明显改变;肝素结合于管壁全层;经肝素处理后的血管标本凝血时间明显延长.结论 酶-去污剂法联合肝素结合处理可以作为制备小口径异种移植血管的新方法 .     

Histological/biological characterization of decellularized bovine jugular vein

Several deficiencies in currently available right ventricular valved conduits make them problematic for use in infants and children. A solution would be to develop a tissue-engineered valved conduit containing autologous cells. A method was devised to produce a decellularized bovine matrix scaffold for developing a tissue-engineered right ventricular valved conduit. Fresh bovine jugular veins were treated with sodium deoxycholate and Triton X-100. The major structural proteins of the fresh and decellularized jugular venous valves and vessel walls were detected by histological methods. Thickness, water absorption rate, water maintenance rate, disruption strength, and extensibility were determined. Circumferential and radial specimens of valves and vessel walls were subjected to tensile testing. Histological analysis showed that no cell fragments were retained within the decellularized matrix scaffold and the major structural proteins had been retained intact. There were no significant differences in thickness, rates of absorption and maintenance of water, disruption strength, and extensibility between the decellularized and fresh veins. It was concluded that this treatment can successfully remove cellular components while maintaining the major structural components and the histological and biological properties of bovine jugular veins.     

Separation and properties of DNA polymerase from murine leukemia L1210 cells.

The possible existence of several species of DNA-dependent DNA polymerases in mammalian cells in addition to those 2 polymerases which are the smaller enzyme from nucleus and larger one from cytoplasm each having distinct characteristics, have been reported recently. In order to examine the heterogeneity of DNA polymerases in murine leukemia L1210 cells and to characterize their general properties, we have attempted to separate the DNA polymerase activities from L1210 cells. By diethylaminoethyl (DEAE)-cellulose chromatography (0.2 M-1M KCl) of the whole cell extract from L1210 solubilized by 1% Triton X-100 and 0.5 mM ethylenediaminetetraacetate (EDTA), 4 fractions with DNA-dependent DNA polymerase activities were obtained and designated as DD-1, DD-2, DD-3, and DD-4 for eluents with each corresponding concentration of 0.2, 0.3, 0.5, and 0.7 M KCl, respectively. They were distinguishable in properties such as template preference, divalent cation requirement, DNase sensitivity, isoelectric point (pI) and the behavior on the phosphocellulose chromatography. DD-1 preferred native DNA as template exhibiting similar characteristic as nuclear polymerase with low molecular weight and insensitivity to SH-inhibitors. DD-2, DD-3, and DD-4 utilized activated DNA most efficiently, while activity of DD-3 increased even in the presence of DNase 1 under the condition where the others were completely inhibited. Distribution of DNA polymerase activities in the cells is discussed briefly.     

Prevention of porcine aortic wall calcification by acellularization: necessity for a non-glutaraldehyde-based fixation treatment

BACKGROUND AND AIM OF THE STUDY: Acellularization prevents cell-mediated calcification of the aortic wall, but the inflammatory response towards the unfixed tissue is problematic. Two additional fixation methods, applied after tissue acellularization, were studied. METHODS: Porcine aortic wall samples were randomized into four groups: (1) Standard fixation with glutaraldehyde (GA); (2) acellularization by a combined method of enzymes (DNase, RNase) and a detergent (Triton X-100); (3) acellularization followed by standard GA fixation; (4) acellularization followed by photo-oxidation. Samples were implanted into the wall of both jugular veins of six juvenile sheep. Tissue was explanted after three months and evaluated by X-radiography, light and electron microscopy, and calcium content (cc) measurement (atomic absorption spectrometry). Auto-fluorescence of elastic fibers was used to identify the relationship between calcific deposits and elastin. RESULTS: GA-fixed aortic wall samples showed clear mineralization (cc 41.6 +/- 17.8 microg/mg), occurring predominantly at the level of cell remnants, as confirmed by electron- and fluorescence microscopy, locating calcific deposits in between elastic fibers. Acellularized aortic wall fragments were calcified significantly less, but an important (non-infectious) inflammatory response caused elastolysis and subsequent calcification of the elastic fibers (cc 5.6 +/- 2.8 microg/mg). Acellularized and GA-fixed fragments revealed important, inhomogeneously spread calcific deposits (cc 24.7 +/- 10.0 pg/mg). Photo-oxidized samples remained free from calcification (cc 0.82 +/- 1.6 microg/mg). CONCLUSION: Acellularization is a promising tool in the prevention of porcine aortic wall calcification, but additional tissue fixation is necessary to prevent structural degeneration. GA fixation after acellularization causes important inhomogeneous tissue mineralization. Photo-oxidation combines optimal tissue fixation with superior anticalcification characteristics.     

A highly phosphorylated subpopulation of insulin-like growth factor II/mannose 6-phosphate receptors is concentrated in a clathrin-enriched plasma membrane fraction.

Insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) receptors immunoprecipitated from purified plasma membranes of 32P-labeled rat adipocytes are markedly heterogenous in their phosphorylation state. Approximately 80% of the plasma membrane receptors are solubilized in 1% (vol/vol) Triton X-100 and are phosphorylated on serine residues at a stoichiometry of approximately 0.1-0.2 mol of phosphate per mol of receptor. In contrast, 15-20% of the receptors are Triton X-100-insoluble and are phosphorylated on serine and threonine residues at approximately 4 or 5 mol of phosphate per mol of receptor. This Triton X-100-insoluble membrane subfraction contains only 5% of the total plasma membrane protein and yet contains all of the clathrin heavy chain associated with plasma membrane, as detected by immunoblotting with a monoclonal antibody. Based on the relative yields of protein in the detergent-insoluble material, IGF-II/Man-6-P receptors are concentrated approximately equal to 3-fold in this clathrin-enriched subfraction. Insulin treatment of intact cells increased the total IGF-II/Man-6-P receptors in the Triton X-100-soluble fraction of the plasma membrane, whereas no change in receptor number in the detergent-insoluble fraction was seen. However, insulin markedly decreased the phosphorylation stoichiometry of the Triton X-100-insoluble receptors. Taken together, these results indicate that insulin decreases the phosphorylation state of a highly phosphorylated subpopulation of IGF-II/Man-6-P receptors on the plasma membrane. In addition, insulin action may prevent the concentration of these receptors in a clathrin-enriched membrane subfraction.     

Ribonuclease-sensitive DNA-synthesizing complex in human sperm heads and seminal fluid.

An endogenous DNA-synthesizing complex sensitive to ribonuclease has been found in purified preparations of swollen human sperm heads. Incorporation of [3H]dTTP into acid-precipitable material occurred in the presence of actinomycin D and required addition of dGTP, dCTP, dATP, plus Mg++. Polymerization was sensitive to pretreatment of the complex with pancreatic RNase A or Triton X-100. Exogenous activity was elicited by the synthetic template (dT)12--18-(rA)n but not by (dT)12--18-(dA)n or (dT)10. The complex sedimented from a 10,000 X g supernatant by centrifugation at 165,000 X g for 60 min and banded in sucrose at a density of 1.21--1.25 g/cm3. Endogenous RNase-sensitive DNA polymerase activity from cell-free seminal fluid was also detected in a fraction in sucrose at a density of 1.22 g/cm3. This activity was labile to freezing and stimulated by 0.04% Triton X-100, and thus differed from that of sperm heads.     

Bovine renal mitochondrial vitamin D3 hydroxylases: regulation of in vitro activities by inhibitors and antioxidants

The regulation of bovine renal 1 alpha- and 24-hydroxylase activities was examined in primary bovine proximal tubule cell cultures. Maximal 1 alpha- and 24-hydroxylase activities in primary bovine proximal tubule cultures ranged from 1.5-1.8 and 2.0-2.7 pmol/min X 10(6) cells, respectively. The apparent Km was 795 nM for 1 alpha-hydroxylase activity and 1130 nM for 24-hydroxylase activity. 1 alpha- and 24-hydroxylase activities decreased in primary culture after cell plating. Activities decreased both as a function of cell number and as a function of the culture dish. 1 alpha-Hydroxylase activity decayed with a t1/2 of 37 h, while 24-hydroxylase activity decayed with a t1/2 of 45 h. Decreasing cell densities, at which cells were plated, increased the t1/2 for the decay of both activities [t1/2 = 21 h at 5,000 cells/cm vs. t1/2 = 37 h at 25,000 cells/cm for 1 alpha-hydroxylase (P greater than 0.001); t1/2 = 33 h at 5,000 cells/cm vs. t1/2 = 45 h at 25,000 cells/cm for 24-hydroxylase, (P greater than 0.0001)]. Direct addition of 0.25 mM metyrapone inhibited 1 alpha-hydroxylase activity by 33% and 24-hydroxylase activity by 51%. Long term incubation of cell cultures with 0.25 mM metyrapone resulted in a slowing in the loss of both hydroxylase activities, but did not stop the decay. 1 alpha-Hydroxylase activity in 4-day metyrapone-treated cultures was 35% higher than in 4-day untreated cultures. 24-Hydroxylase activity was increased 42% in treated cultures vs. that in untreated cell cultures. Both 1 alpha- and 24-hydroxylase activities were inhibited by direct addition of antioxidants. 1 alpha-Hydroxylase activity was directly inhibited 74% by the addition of 0.1 mM butylated hydroxyanisole (BHA), 69% by the addition of 0.1 mM butylated hydroxytoluene (BHT), and 56% by the addition of 0.05 mM benzyl sulfoxide (BS). 24-Hydroxylase activity was also directly inhibited 72% by 0.1 mM BHA, 55% by 0.1 mM BHT, and 73% 0.05 mM BS. There was no significant difference between the inhibition of either hydroxylase by each antioxidant. Antioxidant mixtures increased the inhibition of hydroxylase activities above that with single antioxidant. The addition of 0.1 mM BHA and 0.05 mM BS to cultures resulted in 100% inhibition of 24-hydroxylase activity and 95% inhibition of 1 alpha-hydroxylase activity. The results were very similar when 0.01 mM BS and 0.1 mM BHT were added to cultures, i.e. 100% and 91% inhibition of 24- and 1 alpha-hydroxylase activities.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction between thyrotropin (TSH) binding inhibitor immunoglobulins (TBII) and soluble TSH receptors in fat cells

To clarify whether TSH binding inhibitor immunoglobulins (TBII) are antibodies to the membrane site associated with the TSH receptor itself or its neighboring sites, the interactions of TSH and TBII with soluble TSH receptor were investigated with a TSH radioreceptor assay using labeled highly purified bovine TSH (bTSH) and Triton extracts from guinea pig crude fat cell membranes (800-10,000 x g fraction). Treatment of the crude fat cell membranes with 0.5% (vol/vol) Triton X-100 resulted in solubilization of membrane proteins with recoveries of 25-30%, whereas increasing the concentration of Triton X-100 in the assay medium caused a decrease of [125I]bTSH binding to the solubilized membranes. Thus, the solubilized membranes were found to retain the capacity to bind [125I]bTSH below the Triton X-100 concentration of 0.4% in the assay medium. Incubation of the solubilized membranes with [125I]bTSH for 24 h at 4 C led to a steady state of specific binding, while incubation at 37 C resulted in more rapid but less specific binding, with a shorter duration of the steady state. Maximum binding occurred within the physiological pH range. Both TSH and Graves' immunoglobulins (Igs) specifically inhibited [125I]bTSH binding to the solubilized membranes, as demonstrated by polyethylene glycol separation of the [125I]bTSH-solubilized membrane complex from unbound [125I]-bTSH. Scatchard analysis of [125I]bTSh displacement curves for both TSH and Graves' Igs indicated a single class of binding site for each, with an affinity constant of 1.8 x 10(9) M-1. In addition, Igs capable of inhibiting [125I]bTSH binding to the solubilized membranes were detected predominantly in the serum of patients with Graves' disease. These results strongly suggest that TBII are antibodies directed to the TSh receptor itself without strict organ and species specificity.     

Immunohistochemical and biochemical characterization of antisera raised to human tumor nucleoli

Summary Antinucleolar antisera were raised in rabbits, goats and sheep to nucleoli isolated from three human tumor cell lines. The antisera were shown to cross-react by immunofluorescence with human tumor cell lines originating from different organs and with frozen sections from a wide variety of human malignant and non-malignant tissues. Tumor versus normal tissue discrimination by several antisera was significantly improved by treatment of frozen tissues with a buffered glutaraldelyde/Triton X-100 solution prior to immunofluorescent staining. The molecular specificity of these antisera was determined by immunostaining electrotransfer nitrocellulose strips following SDS-PAGE of nucleolar preparations and nuclear extracts. Although different immunostaining patterns were obtained for individual antinucleolar antisera, nucleolar proteins of molecular weight 120, 100, 94, 68, 54, 38, 33, and 32 kDa were the most often recognized by antisera raised in the three different species. G187 antiserum strongly reacted with 100, 94, and 38 kDa proteins from freshly obtained leukemic specimens. The Immunoreactivity of the 100, 94, and 38 kDa proteins was unaffected by glutaraldehyde/Triton X-100 treatment when immunostained with antisera that demonstrated the greatest tumor specificity on sections treated with glutaraldehyde/Triton X-100. These three nucleolar proteins may be more highly associated with nucleoli of malignant cells than with nucleoli of normal cells.Abbreviations used G-T 0.05% glutaraldehyde, 0.5% Triton X-100 in 0.2 M NaPO₄, pH 7.4 - SDS-PAGE sodium dodecylsulfate/polyacrylamide gel electrophoresis - PBS phosphate-buffered saline: 0.01 M phosphate, 0.15 M NaCl, pH 7.4     

同型半胱氨酸对人THP-1巨噬细胞CD36表达的影响

目的:探讨同型半胱氨酸(Hcy)对人THP-1巨噬细胞CD36表达的影响.方法:在应用佛波酯(PMA)诱导分化成功的人THP-1巨噬细胞中加入0、0.01、0.05、0.1、0.2 mmol/L的Hcy孵育24 h(为阴性对照组、0.01 mmol/L Hcy组、0.05 mmol/L Hcy组、0.1 mmol/L Hcy组和0.2 mmo]/L Hcy组);并与0.1 mmol/L Hcy分别培养0、3、6、12、24,48 h,同时以上述各个时点为参照设对照,用流式细胞仪检测THP-1巨噬细胞CD36分子的表达.结果:THP-1巨噬细胞与不同浓度(0、0.01、0.05、0.1、0.2 mmol/L)Hcy孵育24 h后,0.2 mmol/L Hcy组与阴性对照组比较,THP-1巨噬细胞CD36表达增加最明显(P＜0.05),差异有统计学意义.THP-1巨噬细胞与0.1 mmol/L Hcy培养0、3、6、12、24、48 h,各时间点CD36表达均高于其相应的对照(P均＜0.05),差异均有统计学意义;48 h时与0 h时比较,THP-1巨噬细胞CD36表达增加最明显(P＜0.05),差异有统计学意义.其他组问比较差异无统计学意义.结论.THP-1巨噬细胞与不同浓度Hcy(0、0.01、0.05、0.1、0.2 mmol/L)孵育24 h后,Hcy可使THP-1巨噬细胞CD36的表达增加,且呈剂量依赖关系;与0.1 mmol/L Hcy培养不同时间(0、3、6、12、24、48 h),也可使THP-1巨噬细胞CD36的表达增加,并呈时间依赖关系.高浓度Hcy可以上调巨噬细胞CD36分子的表达.