衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

单链特异核酸酶S_1的提取

Vogt从粗的Aspergillus oryzae α-淀粉酶制剂提纯了单链特异的核酸酶S_1(简称S_1酶),并详细描述了它的性质。目前已广泛利用这种酶于植物病毒和类病毒(RNA)的互补DNA(cDNA)的分子杂交中。本文报道从商品高峰淀粉酶粉制剂提取核酸酶S_1及其在cDNA杂交中的应用。     

抗人大肠癌重组单链抗体的研制

应用重组噬菌体抗体系统制备了重组单链抗体。首先从抗人结肠癌ND-1单抗杂交瘤细胞中提取mRNA,利用反转录多聚酶链反应(RT-PCR)扩增出单抗重链可变区(V_H)及轻链可变区(V_L)片段,再通过连接DNA合成单链抗体可变区片段(ScFv),然后经双酶切后克隆到pCANTAB5E载体中,在E.coliTGI细胞中表达出噬菌体融合蛋白,用抗原阳性噬菌体感染E.coliHB2151细胞,产生单链抗体,该单链抗体既保持了原单抗的特异性,应用上又优于原单抗。     

分子克隆中使用的其他酶(续)

核酸酶BaJ31: 此酶有以下两种活性:(1)高度专一的单链脱氧核糖核苷酸内切酶与外切酶活性,催化从双链DNA3′,5′两端切除寡核苷酸片段或单核苷酸的反应(DNA两条链的降解速度几乎是相同的)。(2)与核酸酶S_1相似的单链专一内切酶活性。     

用于转化玉米的载体的构建及其鉴定

将在植物中能表达的GUS基因插入到质粒pBS中,得到两种重组质粒pBSG_1和pBSG_2,经限制性内切酶分析表明,它们分别是GUS基因以不同方向插入到pBS中的结果,Southern杂交也证明GUS基因巳插入到pBS中。用类似的方法构建了含植物中能表达的NpTII报告基因及S_1片段的质粒pBIS,。所构建的三个质粒均含有与玉米核DNA有同源性的S_1类质粒片段,可用于转化玉米原生质体。     

大白鼠DNA拓扑异构酶Ⅰ生物学性质的研究

对大白鼠组织作DNA拓扑弄构酶Ⅰ(拓扑酶Ⅰ)活力测定,见酶活力出现在胚胎早期,在胚胎发育过程及出生后不同年龄期,酶活力基本稳定;几种成年大鼠组织的酶活力彼此无显著差异;肝细胞再生及癌变,酶活力亦无显著变化。     

分裂中期的CHO细胞能对其染色体DNA进行切除修补吗?

以中国仓鼠细胞CHO-K_1为材料,用放射自显术检查紫外线照射后细胞内DNA的切除修补,没有得到足以证明分裂中期细胞对其染色体DNA进行切除修补的可靠证据。此结果与1977年Ikushima所报告的不同。对于这种修补能力的缺乏,作者认为应考虑到的原因至少有:(1)分裂中期染色体上的DNA处在高度紧密的状态,难于进行切除修补;(2)分裂中期细胞中担任DNA修补合成的多聚酶β的活性可能下降;(3)分裂中期细胞的核膜解体。     

小鼠IL-27真核表达载体的构建及其在RAW264.7 细胞中的表达

目的:构建小鼠白介素27(Interleukin 27,IL-27)单链融合基因的真核表达载体并检验其在RAW264.7细胞中的表达情况。方法:提取小鼠脾细胞总RNA,通过RT-PCR扩增出小鼠EBI3和p28 c DNA。采用重叠延伸PCR(splicing by overlap extension PCR,SOE PCR)通过编码疏水性多肽接头(Gly4Ser)3的DNA序列连接小鼠EBI3和p28基因片段,构建小鼠IL-27单链融合基因(mouse single chain IL-27,msc IL-27),并将其克隆至pc DNA3.1(+)载体。通过酶切和测序鉴定阳性重组载体,将重组质粒pc DNA3.1-IL-27通过脂质体转染法转染小鼠巨噬细胞株RAW264.7,通过RT-PCR方法检测目的基因的表达。结果:测序分析表明,小鼠IL-27单链融合基因中EBI3、linker和p28的连接顺序、方向及碱基序列与预期相符。在转染后的RAW264.7细胞中检测到了小鼠IL-27 m RNA的表达。结论:成功构建了小鼠IL-27单链融合基因及其真核表达载体,并在RAW264.7细胞中实现表达,为进一步探讨IL-27的生物学功能奠定了基础。     

羟磷灰石离心法及其在细胞DNA辐射损伤和修复研究中的应用

Ahnstrm等在1973年首先用羟磷灰石(以下简称HA)层析法检测DNA单链断裂。该法较经典的硷性蔗糖梯度离心法操作简便,灵敏度高。我们根据本室条件,按照Kanter等的方法稍加改进,用国产HA建立了HA离心分离检测DNA单链断裂及其重接的技术,并用该法观察了小鼠白血病L_(7712)细胞DNA单链断裂及其重接。材料与方法(一)细胞取郑升等建立的小鼠腹水型淋巴性白血病细胞(L_(7712)),由615纯种小鼠     

末端终止法测定非特异性降解的DNA片段的顺序

用末端终止法测定DNA顺序的方法之一是用限制性内切酶将被测DNA降解,再与噬菌体M_(13)DNA重组、克隆。提取单链DNA做为模板,进行顺序测定。寻找一些非特异性的降解工具取代内切酶,有一定的经济价值,而且还可以扩大方法的应用范围。尤其在多酶切点的载体M_(13)mp系列出现后,更为DNA重组提供了方便。本文用酶法及超声波法切剪,得到了适合在M_(13)mp_8载体中以平齐末端重组的片段。一、材料与方法 1.材料小牛胸腺DNA及质粒PJDB     

PROLIFERATIVE BEHAVIOR OF DIFFERENTIATING CELLS IN THE DEVELOPING RAT PAROTID GLAND

Parotid glands of litters of rats at age intervals from 20 days in utero to 100 days were assayed for DNA content and examined by light- and electron-microscopy. The age differences in total DNA and DNA concentration indicated that there was a rapid rate of proliferation of parenchymal cells until 25 postnatal days, after which the rate declined rapidly, and that there was a rapid increase in cell size between 18 and 25 days. These findings were substantiated by histologic observations, such as the presence of numerous mitotic figures until 25 days of age, and the rapid maturation of the acinar cells between 18 and 25 days. These data suggest that the acinar cells of the rat parotid gland comprise an expanding cell population. Light- and electron-microscopic observations consistently indicated that cells with mitotic figures were about as well differentiated as other parenchymal cells at all stages of gland development, including mature acinar cells observed at ages 23 and 25 days. These observations support the view that the division of cells in advanced stages of differentiation may be important in the growth of certain organs and tissues.     

A QUANTITATIVE STEREOLOGICAL DESCRIPTION OF THE ULTRASTRUCTURE OF NORMAL RAT LIVER PARENCHYMAL CELLS

The principles of stereology have been applied to a morphometric analysis of parenchymal cells from the peripheral, midzonal, and central regions of normal rat liver lobules. The fractional volumes of cytoplasm occupied by mitochondria, peroxisomes, lysosomes, lipid, and glycogen have been determined. The surface densities of smooth- and rough-surfaced endoplasmic reticulum and of mitochondrial envelope and cristae have also been measured. The average number and dimensions of mitochondria and peroxisomes have been evaluated. By the use of an independent measurement of the average cytoplasmic volume, these data have been expressed as the actual volumes, areas, and numbers per cell in the different parts of the hepatic lobule. Similarly, the volumes of the envelope, cristae, and matrix compartments and the area of cristae membranes have been calculated for the average-sized mitochondrion in each lobular zone. Structural homogeneity is found in over 80% of normal rat liver parenchymal cells, with most of the significant differences being confined to those cells immediately surrounding the central veins.     

THE ENZYMATIC PREPARATION OF ISOLATED INTACT PARENCHYMAL CELLS FROM RAT LIVER

Suspensions of isolated parenchymal cells were prepared from rat liver by incubation with collagenase and hyaluronidase followed by mechanical treatment. Utilization of 0.15% collagenase together with 0.15% hyaluronidase yielded adequate numbers of cells for experimental purposes. As shown by light and electron microscopy, approximately 75% of the isolated cells retain their structural integrity. The cell suspensions are capable of maintaining endogenous respiration in the presence of 1% albumin for periods of time up to 8 hr. These cell preparations consist almost entirely of parenchymal cells and offer a unique tissue preparation for the study of hepatic metabolism.     

THE LOCALIZATION OF ALBUMIN AND FIBRINOGEN IN HUMAN LIVER CELLS

Human liver sections were stained with anti-human serum albumin and/or anti-human fibrin monomer fluorescent conjugates. Approximately 10 per cent of the hepatic cells stained specifically for human serum albumin,1 per cent for fibrinogen, and 0.1 per cent for both. Approximately 18 per cent of the Kupffer cells stained specifically for human serum albumin and 33 per cent for fibrinogen. Staining of both cell types was mainly cytoplasmic, although albumin was found in the nuclei of some parenchymal cells, depending on the method of fixation. Cytoplasmic granules staining specifically for fibrinogen could be seen just inside the cell membrane facing the bile caniculi in many more parenchymal cells than the 1 per cent showing diffuse cytoplasmic staining. The technical difficulties involved in preparing fluorescent conjugates against these antigens and in the fixation of these antigens for immunofluorescent staining are discussed.     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

MICROSPECTROPHOTOMETRY OF CELL NUCLEI STAINED WITH THE FEULGEN REACTION : IV. FORMATION OF TETRAPLOID NUCLEI IN RAT LIVER CELLS DURING POSTNATAL GROWTH

1. DNA contents of the individual parenchymal nuclei of rat livers during postnatal growth were estimated by microspectrophotometric apparatus, and different ploidy classes of nuclei were classified by their DNA contents. With the same material the total number of parenchymal nuclei in the liver was counted microscopically. 2. If the DNA content of nuclei encountered most frequently in several tissues represents the diploid class, the ploidy classes of the rat liver cell nuclei correspond to di-, tri-, tetra-, and octoploid, with the di- and tetraploid ones predominating considerably. 3. In suckling rats (below 25 gm. of body weight) the liver parenchyma is composed almost exclusively of cells with diploid nuclei, whereas in young rats (above 80 gm.), of tetraploid nuclei. In the growth stage between 25 and 80 gm., there is a remarkable replacement of the diploid nuclei by the tetraploid ones. However, in the liver of adult rats weighing more than 150 gm., any increase or decrease in the frequency of diploid and tetraploid nuclei is hardly observable. In such rats, the nuclear population of the liver parenchyma seems to reach a cell-ecological equilibrium which is considered to be a stable one. 4. It is shown that such nuclear populations and the total number of nuclei in a liver are controlled by the growth state, and not by the age. 5. The decrease in the total number of diploid nuclei and the increase in tetraploid nuclei in the growing livers of rats weighing from 40 up to 130 gm. can both be explained by the hypothesis that the tetraploid nuclei originate from the interphase diploid nuclei without involving mitosis. This hypothesis implies that mitosis is confined to the reproduction of diploid cells alone. 6. It is suggested that, in general, the synthesis of DNA does not necessarily result in the formation of visible mitotic chromosomes. 7. Mitotic time and generation time of diploid nuclei and the percentage of the tetraploidization from diploid nuclei are calculated and discussed.     

THE FINE STRUCTURE, POTASSIUM CONTENT, AND RESPIRATORY ACTIVITY OF ISOLATED RAT LIVER PARENCHYMAL CELLS PREPARED BY IMPROVED ENZYMATIC TECHNIQUES

Further modifications of the enzymatic technique for the preparation of isolated, intact, parenchymal cells from rat liver as previously described by this laboratory are presented together with a detailed account of several critical factors involved during the procedure. In addition, the fine structure of the cells as revealed by electron microscopy and the characteristics of their respiratory activity in different media and with several added substrates are described. It is shown that cells obtained by adding calcium during the preparative procedure retain approximately 34% more potassium than cells prepared solely in a calcium-free medium. The former cells also demonstrate a higher respiratory activity, which is not due to uncoupling of respiration. Electron microscopy reveals that the cells have an intact plasma membrane and well-preserved intracellular organelles. Glycogen particles are observed in all cells and are particularly abundant when either 20 mM pyruvate is added during the preparation or Eagle's Minimum Essential Medium is employed.     

Immunohistochemical and biochemical studies on DNA ligase from a rat liver parenchymal cell line

We have recently obtained a monospecific antibody against calf thymus DNA ligase composed of a single Mr = 130,000 polypeptide. Immunohistochemical studies using the antibody and immunoperoxidase detection methods indicated that DNA ligase in a rat liver parenchymal cell line (BB) is localized essentially in nucleus. The specific activity of DNA ligase from growing BB cells was more than 10-fold higher than that from rat hepatocytes. The molecular forms of DNA ligase in these cell-free extracts were also analyzed.     

SHR和SD鼠脑梗塞后外侧皮质细胞的代谢变化

运用酶组织化学方法结合计算机图像分析,对自发性高血压大鼠（ＳＨＲ）和正常血压的ＳＤ大鼠在大脑中动脉迅速阻断之后皮质细胞的代谢变化进行了比较观察。实验结果显示在缺血１５ｍｉｎ,皮质细胞的细胞色素Ｃ氧化酶（ＣＣＯ）和乳酸脱氢酶（ＬＤＨ）的活性即已发生变化,而且随缺血时间的延长而更加明显,ＳＨＲ酶活性的变化更为显著,提示ＳＨＲ皮质细胞对缺血缺氧更为敏感。     

THE REPLICATION TIME AND PATTERN OF CARCINOGEN-INDUCED HEPATOMA CELLS

The replication time and pattern have been investigated in hepatoma cells induced by feeding 3'Me-DAB to male rats for 5 months. With the use of tritiated thymidine as a DNA label along with autoradiography, mitotic nuclear labeling has been studied 0.5 to 72 hours after the administration of the label. The following time intervals have been estimated: replication time, 31 hours; DNA synthesis, 17 hours; G₂ plus Mitosis, 2 hours; G₁, 12 hours. Only about 8 per cent of the tumor cell (interphase) population is "flash" labeled, following a single dose of 50 µC of H³TDR. This group of cells has been followed through three cycles of division. The repeated rhythmic passage of tumor cells through cell division is similar to that previously reported for normal liver cells in the growing rat. However, tumor cells have longer replication and DNA synthesis times. In addition, the several time intervals studied vary more in the tumor cell population than they do in the growing normal cell population.