肉瘤-180小鼠体液中抑癌因子的研究

本文报告从肉瘤-180小鼠腹水液中分离出抑癌因子,并将其与艾氏腹水瘤小鼠腹水中的抑癌因子进行比较。结果表明,二者具有相同的生物学特性和理化性质:1.它们均能杀伤不同的癌细胞株,但对正常细胞没有影响;2.经聚丙烯酰胺凝胶电泳,为泳动速率相同的均一多肽。     

抑癌蛋白PTEN的原核表达、抑癌活性研究和抗体制备

PTEN是具有蛋白质和酯类双重特异性磷酸酶活性的抑癌蛋白,在肿瘤治疗中具有广阔的应用前景。鉴于原核表达PTEN蛋白并用于抑癌实验的研究尚未见报道,因此尝试利用大肠杆菌表达有活性的PTEN蛋白,检测其抑癌效果。利用本室克隆的PTEN基因cDNA和原核表达载体pET44a( )分别构建带6×His和Nus标签的两种诱导型原核融合表达载体pETPTEN和pETNusPTEN,在不同的大肠杆菌表达宿主BL21(DE3)(简写为BL)和Rosettagami(DE3)pLysS(简写为RG)中诱导表达。SDSPAGE和Westernblot检测表明:在可溶性组分和包涵体中均含有目的蛋白,在BL中目的蛋白的表达量较高(18.7%)而在RG中可溶性蛋白的比例较高(6.6%)。经纯化和包涵体蛋白复性处理后,重组融合蛋白经Chariot转运入小鼠实体瘤及人前列腺癌DU145细胞。抑癌实验表明:与对照组相比,重组PTEN蛋白对小鼠实体瘤的生长抑制率为58.76%;对癌细胞DU145的生长抑制率可达46.16%;并可导致明显的G0G1期阻滞,其中在宿主RG中表达的重组蛋白抑癌效果明显高于BL宿主中表达的目的蛋白。证实在原核系统中表达的重组PTEN蛋白具有抑癌活性,同时制备了PTEN的高效价腹水多抗,为深入研究PTEN蛋白在癌症治疗中的应用打下了良好的基础。     

骆驼蓬籽蛋白提取物体外抑癌活性的研究

目的:探讨骆驼蓬籽蛋白提取物的体外抑癌活性.方法:采用MTT法检测硫酸铵沉淀的骆驼蓬籽蛋白提取物体外抗肿瘤活性,应用流式细胞术和Annexin V/PI双荧光染色法检测其对细胞凋亡的影响.结果:80%硫酸铵饱和度下的蛋白提取物对宫颈癌HeLa、食道癌Ecal09和肝癌BEL-7404细胞的IC50分别为72.11±3.88μg/mL、257.58±1.16μg/mL和174.07±1.32μg/mL.对HeLa细胞的抑制率与空白对照组相比有显著性差异(P＜0.01),呈现较好的量效关系,且有促进细胞凋亡的作用.结论:骆驼蓬籽蛋白中对HeLa细胞增殖具有较好的抑制活性,可能是通过诱导细胞凋亡来发挥其抗肿瘤作用.     

酶解骨胶原多肽的抗氧化特性研究

本文主要研究了酶解法制备的骨胶原多肽的总抗氧化能力、羟自由基清除作用和抑制超氧阴离子自由基的能力.通过与谷胱甘肽(GSH)的比较发现,溶液浓度在1130～150 mg/mL时,该骨胶原多肽的总抗氧化能力为GSH的71.92%.溶液浓度在2.5～20 mg/mL时,该多肽羟自由基清除作用为谷胱甘肽的1.36倍.溶液浓度为10～150 mg/mL时,多肽抑制超氧阴离子自由基的能力低于谷胱甘肽.     

基于液相色谱-质谱技术的早期胃癌血清多肽组学研究

目的:建立早期胃癌外周血清特征性多肽谱,分析其生物学特征,以探索一种特异且敏感的早期胃癌血清学诊断方法。方法:采集10例早期胃癌和10例正常对照血清,使用蛋白沉淀法去除高丰度血清蛋白质后,通过液相色谱-质谱联用技术重复三次进行多肽分离、离子化、质谱检测,将原始数据应用Label free方法中Max Quant算法对肽段进行相对定量,分析两组差异多肽及差异多肽匹配蛋白。结果:EGC组和N组重复三次所得色谱图总体比较一致,多肽重复检出率分别为87.54%和85.67%。其中EGC组可重复检测到的Unique peptide有65条,匹配对应31个蛋白;在EGC组和N组血清中显著差异的Unique peptide有22条,匹配对应11个蛋白。对血清显著差异多肽所匹配蛋白进行生物学分析,发现这些蛋白质多数位于细胞外,功能类别主要涉及分子水平的序列变换、信号肽、N-糖基化位点等,信号通路主要富集在凝血级联通路和补体级联通路。结论:早期胃癌血清特异性多肽谱图的建立可望成为胃癌早期诊断的血清学标志物,后续试验将进一步针对这些差异多肽进一步分析验证,筛选出早期胃癌标志性多肽,同时应用大样本进行验证,筛选出特异高效的早期胃癌血清标志物。     

不同单位保存的艾氏腹水癌细胞(EAC)的某些生物学特性比较研究

目的比较研究不同单位保种的EAC瘤株生物学特性的差异.方法选择研究了北京市肿瘤研究所、山东省医学科学院药物研究所及武汉大学保种中心保存EAC细胞的大小,细胞株体外培养时对血清的依赖性,接种小鼠皮下建立肿瘤动物模型肿块生长曲线,建立的动物模型对环磷酰胺CTX治疗的敏感性等指标.结果发现三个单位保存的EAC细胞形态及大小基本一致;北京EAC体外培养容易生长,7.5%NCS即可生长良好,山东及武汉EAC体外培养生长力较弱;皮下接种BALB/c小鼠后,北京EAC最易生长肿瘤,武汉EAC次之,山东EAC最差;建立的动物模型对CTX治疗的敏感性,依次为北京EAC,武汉EAC,山东EAC,用CTX治疗的抑瘤率相应为82.9%,71.9%,50%.结果可见三个不同单位保种的EAC瘤细胞生物学特性确已存在显著差异.     

PKD1基因与抑癌基因P53多态性与非综合征性唇腭裂的关联研究

目的:探讨多囊性肾病基因1(polystic kidney disease,PKD1)多态位点rs8049367与抑癌基因P53(anti-oncogene P53)多态位点rs4791774单核苷酸多态性与中国北方人群非综合征性唇腭裂(NSCL/P)的相关性。方法:运用聚合酶链式反应-连接酶检测反应(PCR-LDR)的检测方法,对602例NSCL/P患者和510例对照人群的PKD1基因的rs8049367位点和抑癌基因P53的rs4791774位点进行基因分型。利用SPSS12.0软件分析PKD1基因,抑癌基因P53多态性与NSCL/P的相关性。结果:rs8049367位点和rs4791774位点基因型及基因频率在两组的分布中差异无统计学意义(P0.05)。结论:PKD1基因的rs8049367位点和抑癌基因P53的rs4791774位点单核苷酸多态性可能与中国北方人群非综合征性唇腭裂的发生无相关性。     

ApoB-100-containing lipoproteins are major carriers of 3-iodothyronamine in circulation

3-Iodothyronamine (T(1)AM) is a biogenic amine derivative of thyroid hormone present in tissue and blood of vertebrates. Approximately 99% of the circulating thyroid hormones are bound to plasma proteins, including three major thyroid hormone-binding proteins, and the question arises as to whether circulating T(1)AM is also bound to serum factors. We report here that T(1)AM is largely bound to a single protein component of human serum. Using T(1)AM-affinity chromatography, we isolated this protein, and sequence analysis identified it as apolipoprotein B-100 (apoB-100), the protein component of several low density lipoprotein particles. Consistent with this finding, we demonstrate that >90% of specifically bound T(1)AM in human serum resides in the apoB-100-containing low density lipoprotein fraction. T(1)AM reversibly binds to apoB-100-containing lipoprotein particles with an equilibrium dissociation constant (K(D)) of 17 nm and a T(1)AM/apoB-100 stoichiometry of 1:1. Competition binding assays demonstrate that this binding site is highly selective for T(1)AM. Intracellular T(1)AM uptake is significantly enhanced by apoB-100-containing lipoprotein particles. Modest enhancements to apoB-100 cellular uptake and secretion by T(1)AM were observed; however, multidose T(1)AM treatment did not affect lipid or lipoprotein inventory in vivo. Thus, it appears that apoB-100 serves as a carrier of circulating T(1)AM and affords a novel mechanism by which T(1)AM gains entry to cells.     

江豚血液学的初步研究

本文对从长江中获得的江豚进行了血液学、血液化学特征的测定和血红蛋白、血浆蛋白电泳图谱的研究。发现江豚的嗜中性粒细胞所占百分比较高,淋巴细胞的百分比较低,具有嗜碱性粒细胞。江豚血红蛋白的电泳类型为Ⅰ-Ⅱ,即电泳相对迁移率在人类Hb S和Hb F之间,并有4条非血红素蛋白带(NHP)。用醋酸纤维薄膜作支持物进行电泳,血浆的总蛋白(TP)带可分成8条主要的区带,从阳极到阴极依次是:前白蛋白(ζ)、白蛋白(A)和6条后白蛋白带(PA);然而用聚丙烯酰胺凝胶作支持物共可分成16条区带,其中14条后白蛋白带。脂蛋白(Lp)在聚丙烯酰胺凝胶上可分成4条区带:α_1-脂蛋白、α_2-脂蛋白和β-脂蛋白,分别相当于聚丙烯酰胺凝胶上总蛋白带电泳图谱中的PA9、PA10和PA11,而前-β-脂蛋白和乳糜微粒在起点处。运铁蛋白(Tf)的电泳图为一条区带,相当于PA6。     

脂代谢的测定对老年2型糖尿病患者临床意义

目的探讨老年2型糖尿病与血脂、载脂蛋白、脂蛋白(a)之间的关系及其临床测定的意义。方法对97例老年2型糖尿病患者及65例老年对照组进行了血清总胆固醇(TC)、甘油三脂(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、载脂蛋白AI(ApoAI)、载脂蛋白B(ApoB)及脂蛋白(a)[Lp(a)]的测定。结果老年2型糖尿病患者血清TC、TG、LDL-C、ApoB及Lp(a)水平均显著高于老年对照组。而血清HDL-C和ApoAI水平老年2型糖尿病组显著低于老年对照组。结论老年2型糖尿病患者由于体内胰岛素相对不足及胰岛素抵抗,使血脂、载脂蛋白、脂蛋白浓度和组成成分发生变化及功能发生异常,从而促进动脉粥样硬化,并伴随着血管并发症的发生。因此,在对老年糖尿病并发症的预防和控制上,应在控制血糖的基础上减少脂肪的摄入,以降低高脂血症的发生,从而降低血管并发症的发生。     

Apolipoproteins C-I and C-III Inhibit Lipoprotein Lipase Activity by Displacement of the Enzyme from Lipid Droplets

Apolipoproteins (apo) C-I and C-III are known to inhibit lipoprotein lipase (LPL) activity, but the molecular mechanisms for this remain obscure. We present evidence that either apoC-I or apoC-III, when bound to triglyceride-rich lipoproteins, prevent binding of LPL to the lipid/water interface. This results in decreased lipolytic activity of the enzyme. Site-directed mutagenesis revealed that hydrophobic amino acid residues centrally located in the apoC-III molecule are critical for attachment to lipid emulsion particles and consequently inhibition of LPL activity. Triglyceride-rich lipoproteins stabilize LPL and protect the enzyme from inactivating factors such as angiopoietin-like protein 4 (angptl4). The addition of either apoC-I or apoC-III to triglyceride-rich particles severely diminished their protective effect on LPL and rendered the enzyme more susceptible to inactivation by angptl4. These observations were seen using chylomicrons as well as the synthetic lipid emulsion Intralipid. In the presence of the LPL activator protein apoC-II, more of apoC-I or apoC-III was needed for displacement of LPL from the lipid/water interface. In conclusion, we show that apoC-I and apoC-III inhibit lipolysis by displacing LPL from lipid emulsion particles. We also propose a role for these apolipoproteins in the irreversible inactivation of LPL by factors such as angptl4.     

Aggregated Electronegative Low Density Lipoprotein in Human Plasma Shows a High Tendency toward Phospholipolysis and Particle Fusion

Aggregation and fusion of lipoproteins trigger subendothelial retention of cholesterol, promoting atherosclerosis. The tendency of a lipoprotein to form fused particles is considered to be related to its atherogenic potential. We aimed to isolate and characterize aggregated and nonaggregated subfractions of LDL from human plasma, paying special attention to particle fusion mechanisms. Aggregated LDL was almost exclusively found in electronegative LDL (LDL(−)), a minor modified LDL subfraction, but not in native LDL (LDL(+)). The main difference between aggregated (agLDL(−)) and nonaggregated LDL(−) (nagLDL(−)) was a 6-fold increased phospholipase C-like activity in agLDL(−). agLDL(−) promoted the aggregation of LDL(+) and nagLDL(−). Lipoprotein fusion induced by α-chymotrypsin proteolysis was monitored by NMR and visualized by transmission electron microscopy. Particle fusion kinetics was much faster in agLDL(−) than in nagLDL(−) or LDL(+). NMR and chromatographic analysis revealed a rapid and massive phospholipid degradation in agLDL(−) but not in nagLDL(−) or LDL(+). Choline-containing phospholipids were extensively degraded, and ceramide, diacylglycerol, monoacylglycerol, and phosphorylcholine were the main products generated, suggesting the involvement of phospholipase C-like activity. The properties of agLDL(−) suggest that this subfraction plays a major role in atherogenesis by triggering lipoprotein fusion and cholesterol accumulation in the arterial wall.     

黄瓜香等中药调整腹水瘤小鼠肠道菌群减轻化疗副作用的研究

目的通过黄瓜香等中药调整肝癌腹水瘤小鼠肠道菌群以减轻化疗的副作用达到预防和治疗肿瘤的目的。方法以小鼠腹腔内注射肝癌H22腹水瘤细胞造成肝癌腹水瘤模型,分别于造模型前1周用黄瓜香等和造模型后5 d用顺铂加上黄瓜香等进行治疗,并于注射后做HE染色检测肿瘤种植情况、血中白细胞、血小板的数量及巨噬细胞吞噬能力等。结果中药预防组与对照组相比肿瘤种植明显降低,中药加化疗组与阴性对照组比血中红细胞、白细胞和血小板均有回升(P0.01),巨噬细胞吞噬率增加明显。结论黄瓜香等能调节机体免疫力,防治腹水瘤和减轻化疗的副作用。     

Structures of Discoidal High Density Lipoproteins: A COMBINED COMPUTATIONAL-EXPERIMENTAL APPROACH*

Conversion of discoidal phospholipid (PL)-rich high density lipoprotein (HDL) to spheroidal cholesteryl ester-rich HDL is a central step in reverse cholesterol transport. A detailed understanding of this process and the atheroprotective role of apolipoprotein A-I (apoA-I) requires knowledge of the structure and dynamics of these various particles. This study, combining computation with experimentation, illuminates structural features of apoA-I allowing it to incorporate varying amounts of PL. Molecular dynamics simulated annealing of PL-rich HDL models containing unesterified cholesterol results in double belt structures with the same general saddle-shaped conformation of both our previous molecular dynamics simulations at 310 K and the x-ray structure of lipid-free apoA-I. Conversion from a discoidal to a saddle-shaped particle involves loss of helicity and formation of loops in opposing antiparallel parts of the double belt. During surface expansion caused by the temperature-jump step, the curved palmitoyloleoylphosphatidylcholine bilayer surfaces approach planarity. Relaxation back into saddle-shaped structures after cool down and equilibration further supports the saddle-shaped particle model. Our kinetic analyses of reconstituted particles demonstrate that PL-rich particles exist in discrete sizes corresponding to local energetic minima. Agreement of experimental and computational determinations of particle size/shape and apoA-I helicity provide additional support for the saddle-shaped particle model. Truncation experiments combined with simulations suggest that the N-terminal proline-rich domain of apoA-I influences the stability of PL-rich HDL particles. We propose that apoA-I incorporates increasing PL in the form of minimal surface bilayers through the incremental unwinding of an initially twisted saddle-shaped apoA-I double belt structure.     

Glycine dimerization motif in the N-terminal transmembrane domain of the high density lipoprotein receptor SR-BI required for normal receptor oligomerization and lipid transport

Scavenger receptor class B, type I (SR-BI), a CD36 superfamily member, is an oligomeric high density lipoprotein (HDL) receptor that mediates negatively cooperative HDL binding and selective lipid uptake. We identified in the N-terminal transmembrane (N-TM) domain of SR-BI a conserved glycine dimerization motif, G(15)X(2)G(18)X(3)AX(2)G(25), of which the submotif G(18)X(3)AX(2)G(25) significantly contributes to homodimerization and lipid uptake activity. SR-BI variants were generated by mutations (single or multiple Gly → Leu substitutions) or by replacing the N-TM domain with those from other CD36 superfamily members containing (croquemort) or lacking (lysosomal integral membrane protein (LIMP) II) this glycine motif (chimeras). None of the SR-BI variants exhibited altered surface expression (based on antibody binding) or HDL binding. However, the G15L/G18L/G25L triple mutant exhibited reductions in cell surface homo-oligomerization (>10-fold) and the rate of selective lipid uptake (～ 2-fold). Gly(18) and Gly(25) were necessary for normal lipid uptake activity of SR-BI and the SR-BI/croquemort chimera. The lipid uptake activity of the glycine motif-deficient SR-BI/LIMP II chimera was low but could be increased by introducing glycines at positions 18 and 25. The rate of lipid uptake mediated by SR-BI/LIMP II chimeras was proportional to the extent of receptor oligomerization. Thus, the glycine dimerization motif G(18)X(3)AX(2)G(25) in the N-TM domain of SR-BI contributes substantially to the homo-oligomerization and lipid transport activity of SR-BI but does not influence the negative cooperativity of HDL binding. Oligomerization-independent binding cooperativity suggests that classic allostery is not involved and that the negative cooperativity is probably the consequence of a "lattice effect" (interligand steric interference accompanying binding to adjacent receptors).