癌变原理研究——Ⅵ.大鼠肝及肝癌中氨甲酰磷酸合成酶Ⅰ的免疫化学研究

在研究癌变过程CPS_1~*活性降低的机制中,我们由大鼠肝分离提纯了CPS_1。方法是用去污剂十六烷基三甲基溴化铵从大鼠肝线粒体分离CPS_1,经过硫酸铵分步沉淀以及QAE-Sephadex A-50柱层析,得到聚丙烯酰胺凝胶电泳均一的CPS_1制剂,可与OCT 完全分开。由纯化的CPS_1制备的单价抗血清可以完全抑制大鼠肝及肝癌的CPS_1活性。免疫化学定量滴定证明肝癌、癌前肝及新生鼠肝CPS_1活性的变化,系由于相应酶蛋白量的改变,在其免疫化学性质上未发现有质的差异。     

癌变原理研究——Ⅵ.大鼠肝及肝癌中氨甲酰磷酸合成酶Ⅰ的免疫化学研究

在研究癌变过程CPS_1活性降低的机制中,我们由大鼠肝分离提纯了CPS_1。方法是用去污剂十六烷基三甲基溴化铵从大鼠肝线粒体分离CPS_1,经过硫酸铵分步沉淀以及QAE-Sephadex A-50柱层析,得到聚丙烯酰胺凝胶电泳均一的CPS_1制剂,可与OCT完全分开。由纯化的CPS_1制备的单价抗血清可以完全抑制大鼠肝及肝癌的CPS_1活性。免疫化学定量滴定证明肝癌、癌前肝及新生鼠肝CPS_1活性的变化,系由于相应酶蛋白量的改变,在其免疫化学性质上未发现有质的差异。     

原发性肝癌的多药耐药

肝癌多药耐药的产生是多基因、多因素、多途径、多步骤综合作用的复杂过程,因此研究引起肝癌多药耐药的相关因素、作用机制及逆转MDR,提高肝癌化疗效果成为目前肝癌研究的热点,但不同的肝癌耐药细胞株有不同的多药耐药基因表型,如何针对不同的基因表型来逆转肝癌的多药耐药,是临床治疗肝癌需要面对的一个问题.     

大鼠肝癌模型建立的研究进展

肝癌动物模型是研究肝癌的重要依据,其在揭示肿瘤发病机制、评估治疗方法中的角色不可或缺。近年来,研究人员借助不同类型的动物模型不断获得有关肝癌及其治疗预后的丰富数据。本文通过总结常用的诱发性和移植性大鼠肝癌的建模方式及应用,以呈现近年来在构建大鼠肝癌模型实践中的进展。     

磷酸钙柱层析在分离动物组织核糖核酸中的应用及其对核糖核酸稳定性的研究

(1)本实验探索了磷酸钙柱层析在分离动物组织RNA提取液的洗脱条件。(2)磷酸钙柱层析应用于大鼠肝RNA总溶液的分离获得重复性较好的柱层析图谱。(3)对大鼠肝磷酸钙柱层析各峰的RNA性质进行初步分析,但对B峰的性质还不了解。(4)磷酸钙柱层析本身似对RNA分子的降解无甚影响。(5)比较了正常肝、再生肝及3′-甲基奶油黄诱发肝癌RNA的磷酸钙柱层析图谱。(6)观察了大鼠腎、胰、脑等组织RNA的磷酸钙柱层析图谱,与肝脏者有明显的差别。(7)观察了感染脑心肌炎病毒小鼠脑RNA的柱层析图谱,并试验了所洗脱的RNA的部分的致病活性。     

miRNAs在肝癌干细胞中的作用

MicroRNAs (miRNAs)是一类参与转录后调控的小分子RNA,它在调控细胞的增殖、分化及肿瘤的形成等多种生理及病理过程中发挥着重要的作用.肝癌干细胞是肝癌组织中具有自我更新能力和分化潜能的一个微群体,它能够启始肝癌的发生,并且与肝癌的抗药性及复发等密切相关.已经有研究表明miRNAs对肝癌干细胞的发生发展起着重要的调控作用,包括致癌和抑癌的作用,因此总结miRNAs在肝癌干细胞中作用,有助于更好理解肝癌干细胞的特性及肝癌肿瘤生物学.近年来,除了传统的分子生物学手段,组学和系统生物学研究策略的运用也为miRNAs在肝癌中的研究提供了新的思路.鉴于此,深入研究miRNAs在肝癌干细胞中的分子机制,将为靶向肝癌干细胞的临床治疗提供新的途径.     

KMT6在肝癌细胞转移中的作用及对肝癌预后的影响

目的:探讨组蛋白赖氨酸甲基转移酶6(histone lysine methyltransferase 6,KMT6)对人肝癌细胞转移的调控作用及对肝癌患者预后的影响。方法:采用RNA干涉技术合成靶向KMT6的小干扰RNA(KMT6 siRNA)片段,瞬时转染至人肝癌细胞系Huh-7。随后,分别采用细胞黏附实验、细胞侵袭实验以及划痕迁移实验检测肝癌细胞的黏附、侵袭和迁移等肿瘤细胞转移能力。运用Oncomine数据库和cBioportal肝癌数据库分析KMT6的表达情况及对肝癌预后的影响。结果:靶向KMT6的小干扰RNA(KMT6 siRNA)片段瞬时转染至人肝癌细胞系Huh-7后,KMT6蛋白的表达显著下降,且黏附率、侵袭率和划痕修复率均显著降低(P<0.05)。Oncomine肝癌数据库分析表明肝癌组织KMT6的表达增加,cBioportal肝癌数据库分析表明KMT6基因改变与肝癌患者预后不良密切相关。结论:KMT6分子可促进肝癌细胞黏附、侵袭和迁移,其基因改变与肝癌患者预后不良显著相关。KMT6分子有望成为肝癌治疗的新靶点。     

miR-155对肝癌细胞增殖的影响

目的:在肝癌细胞系中过表达miR-155,研究其对肝癌细胞增殖的影响。方法:将pc DNA3.0-miR-155表达载体瞬时转染Huh7.5.1及Hcclm3肝癌细胞系,通过实时定量PCR技术对miR-155在转录水平的表达进行检测,采用CCK8法及克隆形成实验检测miR-155过表达后对Huh7.5.1及Hcclm3肝癌细胞系增殖的影响。结果:转染细胞后72 h,经实时定量PCR检测,Huh7.5.1及Hcclm3肝癌细胞中成熟miR-155的表达分别上调约431及16倍(P0.01),说明其能有效高表达;CCK8法及克隆形成实验结果显示,miR-155能够明显促进肝癌细胞增殖(P0.01)。结论:pc DNA3.0-miR-155转染Huh7.5.1及Hcclm3肝癌细胞系后能高效表达成熟miR-155,同时证明过表达miR-155能使肝癌细胞的增殖受到非常明显的促进。     

肝癌基因调控网络研究进展

肝癌(Hepatocellular carcinoma,HCC)是我国常见的恶性肿瘤之一。肝癌基因调控网络(HCC regulatory network,HCC GRN)是研究肝癌分子机制的重要途径之一,其节点包括肝癌相关的分子,如mi RNA、TF等,网络的边由节点间相互作用关系构成。基于不同类型的数据构建的肝癌基因调控网络其类型及特征各有不同。综合近年来肝癌基因调控网络研究发现,由TF与mi RNA构建的肝癌转录调控网络更能揭露肝癌关键基因,反映关键基因在调控网络中的扰动情况。整合基因变异信息与调控网络成为研究肝癌基因调控网络的趋势,但相应的研究几乎是空白的。本文从HCC GRN的数据来源、分类及特征,及各类型调控网络的近年研究情况等方面进行综述,并结合相关研究工作对肝癌基因调控网络研究现状进行分析与讨论,对前景进行展望,为这一领域研究工作提供参考。     

腹腔镜肝癌切除术与开放式肝癌切除术的疗效比较研究

目的:探讨腹腔镜肝癌切除手术治疗原发性肝癌的可行性及安全性。方法:选取2008年6月至2011年1月在我院行腹腔镜肝癌切除术的30例患者作为研究对象,另外选择同期在我院行开放式肝癌切除术的30例患者作为对照。结果:30例均在腹腔镜下成功地完成手术,其中22例行腹腔镜局部切除术,8例行肝左外叶切除术。手术时间103-142 min,出血量60-480mL,术后均未发生严重并发症,术后平均住院8.6 d。术后随访18~36个月,局部复发或种植性转移率与对照组无显著差异。结论:腹腔镜肝癌切除术是安全可行治疗原发性肝癌的手术方式。     

THE FORMATION OF PEPTIDO-AMINES FROM CONSTITUENT AMINO ACIDS AND HISTAMINE IN HYPOTHALAMIC TISSUE

—The formation of peptido-amines from acetylaspartic acid and histamine incubated with amino acids Glu, Gly, Ser, Ala and GABA is described. The incubation was performed using a 16000 g× 30 min supernatant extract of hypothalamic tissue which had been cleared of small molecular weight compounds by passage through a G-25 Sephadex column.     

New and rapid fully automated method for determination of tazobactam and piperacillin in fatty tissue and serum by column-switching liquid chromatography

A sensitive and rapid HPLC assay for determining tazobactam and piperacillin in fatty tissue and serum is described. While the common methods need liquid-liquid extraction before the injection in a automated column switching HPLC, the new method works by direct injection of the filtered tissue extract or diluted serum in a automated column switching HPLC without any other pre-treatment. This was performed by the use of a NH2-precolumn and enrichment/transfer at different pH-level. During the analyses, the NH2-precolumn was automatically regenerated with acetonitrile-water. The chromatogram peaks for piperacillin and tazobactam were identified by the retention time and quantified by peak area. The calibration curve was linear between 1 and 16 microg/ml. The quantification limit of tazobactam was about 1 microg/ml in fatty tissue extracts and in diluted serum (calculated for pure serum 2 microg/ml), respectively. For piperacillin it was less. The described procedure allows sample clean-up and determination of the antibiotic within 35 min. The chromatograms with this easy sample treatment had the same quantity of matrix peaks and in contrast to liquid-liquid extraction no loss of piperacillin. Because of the automatically rinsing of the NH2-precolumn during the chromatographic separation, more than 50 different biological samples could be measured with one NH2-precolumn without loss of performance.     

Treatment of spasmodic torticollis by dorsal column stimulation.

A new method is described to treat spasmodic torticollis with the implantation of a dorsal column stimulator at the C1--2 level or with transcutaneous stimulation. 22 patients were evaluated. 3 had sufficient relief to be treated with transcutaneous stimulation only. An additional 6 patients had surgically implanted dorsal column stimulators. It was empirically determined that a frequency of 800--1,100 Hz gave the best relief from torticollis. 1 patient had an excellent result; 3 have had good results; 1 had a fair result, and 1 had a poor result. An additional patient with dystonia musculorum deformans was considerably improved by the use of dorsal column stimulation.     

Simple and rapid chromatographic purification of type V collagen from a pepsin digest of porcine intestinal connective tissue,an unmanageable starting material for conventional column chromatography

A chromatographic method is described for purification of type V collagen, a minor constituent in extracellular matrix, from a pepsin digest of porcine intestinal connective tissue. The starting material was a viscous and turbid solution even after centrifugation. Direct application of the sample to a commercially available DEAE-cellulose column resulted in clogging. On the other hand, type V collagen, [alpha1(V)](2)alpha2(V) form, was successfully captured by a filter paper-based DEAE-cellulose column chromatography and purified by a subsequent commercially available cation-exchange medium without clogging. This is a vast improvement over previously described salt fractionation methods.     

Analysis of free nucleotide pools of mouse liver tissue by high-pressure liquid chromatography (HPLC)

In this paper, analysis of free nucleotides from mouseliver tissue during different day times has been described. Perchloric acid extract of mouse liver tissue was neutralized with tri-N-octylamine in trichlorotriflouroethane and after removal of ClO4(-), subjected to preliminary purification on a Cu(2+)-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure used in the study gave a good resolution of free nucleotides on a single column.     

Routine amino acid capillary column chromatography of microquantities in the picomole range--procedures and modifications of a commercial analyzer

A commercial micro amino acid analyzer using capillary columns (internal diameter 0.7 mm) had to be modified greatly in order to get satisfactory separation and quantitative analysis on the microscale of free amino acids in biological fluids, tissue extracts, and hydrolysates. Furthermore, the usual analytical conditions were changed (i.e., lowering of the buffer pH) and a procedure and apparatus for sample deproteinization is described which allows simple handling of volumes in the range from 20 to 100 μl. The modifications described guarantee better and more reproducible analyses than reported so far. A design of a new “analytical unit” for capillary column chromatography is proposed.     

Radiometric determination of cytidine 5′-triphosphate in biological materials

A simple and sensitive radioenzymic method is described for the direct determination of cytidine 5′-triphosphate (CTP) in the range of 1 to 9 nmol in perchloric acid extracts of tissue. The method involves a specific conversion of CTP and [1,2-¹⁴C]phosphorylethanolamine to labeled CDP-ethanolamine using purified CTP:phosphorylethanolamine cytidylyltransferase (EC 2.7.7.14). The [¹⁴C]CDP-ethanolamine formed is isolated on a Dowex 1-formate column taking advantage of the selectivity of borate complexing. A spectrophotometric method for determining CTP in standard solutions is also described.     

Isolation and purification of cyanogen bromide-derived peptides of type II collagen directly from tissue (Swarm chondrosarcoma)

A preparative procedure is described for isolating type II collagen-fragments directly from tissue. Swarm chondrosarcoma from rat, a cartilagenous tissue rich in type II collagen, was digested by cyanogen bromide in 70% formic acid. The resulting crude extract was desalted (G 25 column chromatography) and lyophylized. The yield of peptide mixture was about 1 250 mg obtained from 100 g tissue. The method of purification commonly used for type II collagen prior to cyanogen bromide-cleavage yielded 20 mg peptides from 100 g tissue. Separation of the cyanogen bromide-derived fragments was performed by gel filtration. The column was run at 43 degrees C (denaturing-temperature of collagens) to avoid fibril formation, and a volatile buffer was used (ammonium formate buffer, pH 7.5) so that the effluent fractions could be easily lyophylized. Two-dimensional gel electrophoresis of the main peaks of the column profile demonstrated that this purification step resulted in a good separation of the fragment mixture, although additional steps may be necessary for complete separation of the peptides. The most striking advantages of the method for direct digestion of tissue outlined here are the increase in yield (about 60-fold) and the reduction of purification steps (avoiding type II collagen purification).     

Computer treatment of gas-liquid chromatographic data, with special reference to fatty acid methyl esters

A computer program is described which analyzes output punched directly onto paper tape from a gas-liquid chromatograph. Although this program was written specifically for samples of fatty acid methyl esters derived from adipose tissue triglycerides which are eluted within 1 hr, modification of the dimension statements in the program would enable it to deal with samples which require a longer time to come off the column. The salient features of the rationale of the program are discussed in detail, particularly the procedures for base line correction and for estimating the contributions from components which are not perfectly separated in the column. Examples are given of the program in practice, of comparing the results it gives with those obtained by manual triangulation of the areas on a recorder chart, and of indicating the range of column load over which we have found that it operates satisfactorily. A sample computer print-out from the program is presented and interpreted.     

论实轴型中柱皱纹珊瑚的分类

具实轴型中柱的皱纹珊瑚的分类位置至今尚未取得统一。这类珊瑚的轴部由纤状组织构成,它不同于复中柱或皱形复中柱,它的形成与主隔壁,对隔壁或一级隔壁内端没有关系,是皱纹珊瑚中一类独立发育的轴部构造。具实轴型中柱的珊瑚可以独立建立新目,称Ｃｙａｔｈａｘｏｎｉｉｄａ ｏｒｄ．ｎｏｖ．。按照组成实轴的纤状组织类型和实轴形态,可以划分为３科,即Ｃｙａｔｈａｘ－ｏｎｉｉｄａｅ ｅｄｗａｒｄｓ ｅｔ Ｈａｉｍｅ,１