HMG＿（14）单克隆抗体杂交瘤株的建立

以北京鸭红细胞提取的ＨＭＧｓ（ＨＭＧ＿１，ＨＭＧ＿（２ａ），ＨＭＧ＿（２ｂ），ＨＭＧ＿（１４），ＨＭＧ＿（１７））免疫Ｂａｌｂ／ｃ小鼠，取其脏压细胞与Ｓｐ２／０（小鼠骨髓瘤细胞）融合，经选择培养，阳性筛选，克隆化以及冻存复苏，获得了三株稳定分泌抗ＨＭＧ＿（１４）的杂交瘤细胞株（１Ｃ＿（１２），１Ｏ＿３和２Ｆ＿２），经ＷｅｓｔｅｒｎＢｌｏｔ鉴定，与其它几种ＨＭＧ无反应，抗体为ＩｇＧ＿１亚型。腹水ＨＭＧ＿（１４）单克隆抗体经ＳｅｐｈａｄａｘＣＬ－４Ｂ亲和柱得到了纯化。     

小鼠集落刺激因子（CSFs）的体外诱导研究

用体外淋巴细胞培养系统，对一些非淋巴因子诱导小鼠脾细胞产生集落刺激因子（ＣＳＦｓ）进行研究的结果发现，静止的小鼠脾细胞对原激酶（ＵＫ）不产生应答，只有当ＣｏｎＡ刺激后才能产生ＣＳＦｓ；表皮生长因子（ＥＧＦ）与ＣｏｎＡ协同作用能明显诱导ＣＳＦｓ的产生．人绒毛膜促性腺激素（ＨｃＧ）可直接作用于静止的脾细胞，使其产生ＣＳＦＳ．剂量依赖试验表明当ＵＫ为１６００ＩＵ／ｍｌ，ＥＧＦ为２０ｎｇ／ｍｌ，ＨＣＧ为４００ＩＵ／ｍｌ时所诱导的ＣＳＦｓ活性达到最高值．此外，ＣＳＦｓ的诱导还具有时间依赖关系．ＵＫ作用６０ｈ，ＥＧＦ和ＨＣＧ作用４８ｈ诱导产生的ＣＳＦｓ活性达到最高值．ＨＣＧ抗体中试验进一步说明ＨＣＧ可直接作用于脾细胞．联苯胺染色结果表明ＵＫ．ＥＧＦ和ＨＣＧ诱导的条件培养液中均不含红系集落刺激因子．     

基因重组牛碱性成纤维细胞生长因子的纯化和鉴定

牛碱性成纤维细胞生长因子（ｂＦＧＲ）ｃＤＮＡ在Ｅ．ｃｏｌｉ中表达，高压均质破碎菌体后，上清液以Ｈｅｐａｒｉｎ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ亲和层析、Ｃ１８反相ＨＰＬＣ分高纯化得到一分子量为２．２５×１０￣４的蛋白质，其等电点为９．３４，并能与抗牛ｂＦＧＦＭｃＡｂ反应；其氨基酸组成与ｂＦＧＦ文献值一致；活性分析结果表明此蛋白质不仅能促进ＢＡＬＢ／ｃ３Ｔ３细胞增值，ＥＤ＿（５０）＝０．８５ｎｇ／ｍｌ，而且能促进鸡胚尿囊膜微血管增生，所得蛋白为具有生物活性的重组碱性成纤维细胞生长因子。     

GHRP—scu—PA—32K突变体的构建,表达及纯化产物的部分性 …

在ｓｃｕ－ＰＡ３２Ｋ的ｃＤＮＡ分子基础上经定点突变,在Ｎ端紧接Ｌｅｕ＾１以前引入编码ＧＨＲＰ四肽的寡核苷酸序列（ＧＧＴＣＡＴＡＧＧＣＣＴ）,构建了ＧＨＲＰ－ｓｃｕ－ＰＡ－３２Ｋ的突变体ｃＤＮＡ。将它克隆到表达载体ｐＣＭ－β－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ＾－细胞。筛选到的稳定表达株在无 清培养基的表达量为５８０ＩＲ／（１０＾６细胞．２４ｈ）。经锌离子螯合亲和柱纯化的产物,ＳＤＳ－ＰＡＧＥ显示为一蛋     

HMC14单克隆抗体杂交瘤株的建立

以北京鸭红细胞提取的ＨＭＧｓ免疫Ｂａｌｂ／ｃ小鼠，取其脏压细胞与Ｓｐ２／０（小鼠骨髓瘤细胞）融合，经选择培养，阳性筛选，克隆化以及冻存复苏，获得了三株稳定分泌抗ＨＭＧ１４的杂交瘤细胞株，经ＷｗｓｔｅｒｎＢｌｏｔ鉴定，与其它几种ＨＭＧ无反应，抗体为ＩｇＧ１亚型。腹水ＨＭＧ１４单隆抗体经ＳｅｐｈａｄｘＣＬ－４Ｂ亲和柱得到了纯化。     

肝素—琼脂糖凝胶4B的制备及在肝生长因子提取纯化中的…

以琼脂糖凝胶为载体，肝为配基，乙腈活化扣经环氧氯丙烷偶联，制得肝素－琼脂糖凝胶，经间接法测定，偶联率为２．４７５ｍｇ肝素／ｇ湿重琼脂糖凝胶。与Ｐｈａｒｍａｃｉａ公司的Ｈｅｐａｒｉｎ－Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ比较，两种凝胶对肝细胞生长因子的亲和能力、层析行为及重复使用性能等方面非常相似，且自制凝胶的成本仅为进口的１／２０，解决了ＨＧＦ纯化过程中的关键步骤，使大量提取生产ＨＧＦ成为可能。     

肝素－琼脂糖凝胶4B的制备及在肝生长因子提取纯化中的应用

以琼脂糖凝胶为载体，肝素为配基，乙睛活化后经环氧氯丙烷偶联，制得肝素——琼脂糖凝胶．经间接法测定，偶联率为２．４７５ｍｇ肝素／ｇ湿重琼脂糖凝胶，与Ｐｈａｒｍａｃｉａ公司的Ｈｅｐａｒｉｎ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ比较，两种凝胶对肝细胞生长因子（ＨＧＦ）的亲和能力、层析行为及重复使用性能等方面非常相似，且自制凝胶的成本仅为进口的１／２０，解决了ＨＧＦ纯化过程中的关键步骤，使大量提取生产ＨＧＦ成为可能．     

无血清培养细胞HICSF的自分泌生长因子的检测

为了探讨无血清培养细胞ＨＩＣＳＦ的自分泌调控机制，采用ＲＴ－ＰＣＲ技术检测生长因子ｂＦＧＦ，ＴＧＦβ１和细胞因子ＩＬ－６，ＩＬ－８，Ｇ－ＣＳＦ以及细胞因子受体ＩＬ－６Ｒ和ＩＬ－８Ｒ在ＨＩＣＳＦ细胞及其同基因来源的血清培养细胞ＨＩＣ中的表达状况，同时用＾３Ｈ－ＴｄＲ掺入法观察重组ＩＬ－６和ＴＧＦβ１对这２种细胞生长的影响，以观察癌细胞在有血清和无血清培养条件下部分生长因子和细胞因子的表达及反应性差异     

DISCOVERY OF MICROFOSSILS FROM THE 1848－MA－OLD CHANGZHOUGOU FORMATION，JIXIAN SECTION，NORTH CHINA

ＤＩＳＣＯＶＥＲＹＯＦＭＩＣＲＯＦＯＳＳＩＬＳＦＲＯＭＴＨＥ１８４８－ＭＡ－ＯＬＤＣＨＡＮＧＺＨＯＵＧＯＵＦＯＲＭＡＴＩＯＮ，ＪＩＸＩＡＮＳＥＣＴＩＯＮ，ＮＯＲＴＨＣＨＩＮＡＺｈａｎｇＺｈｏｎｇｙｉｎｇ（ＤｅｐａｒｔｍｅｎｔｏｆＥａｒｔｈＳｃｉｅｎｃ...     

血清促肝细胞生长素的检测与意义

目的；建立人血清中促肝细胞生长素（ＨＧＦ）的免疫学检测方法，厂家直病患血清中ＨＧＦ水平的变化及其临床意义。方法：自人胎肝匀浆中提取、纯化ＨＧＦ，免疫家兔制备抗血清，建立双抗体夹心法的ＨＧＦ测定技术。结果：Ｘ经的ＨＧＦ于ＳＤＳ＿ＰＡＧＥ呈条带，分子量１７５００。正常人血清中ＨＧＦ水平为２８－１４０μｇ／Ｌ。各型肝病患和ＳＬＥ患血清中ＨＧＦ都有不同程度的升高。结论：动态观察肝病患血清中ＨＧＦ     

ATP-stimulated interaction between epidermal growth factor receptor and supercoiled DNA

The receptor for epidermal growth factor (EGF) has been identified as a transmembrane glycoprotein that has tyrosine-specific kinase activity. The kinase activity of the receptor is enhanced in the presence of EGF (or related peptides), and the phosphorylation of a number of substrates, as well as autophosphorylation of the receptor, has been reported. Analogous findings have been described for the insulin receptor and the receptor for platelet-derived growth factor (PDGF). Thus, a number of hormone receptors and several viral transforming proteins appear to share the highly unusual property of tyrosine-specific kinase activity. Nevertheless, the specific relationship between tyrosine kinase activity and the control of cell growth and replication is unknown. It is known that after the initial binding of EGF to the plasma membrane, the hormone together with its receptor is rapidly internalized in endocytic vesicles and the hormone is eventually degraded in lysosomes. It is possible that the function of EGF is simply to stimulate internalization of its receptor, and that as a result of its altered location the receptor is able to phosphorylate a cytoplasmic component or even interact directly with a nuclear component. We now report that the purified receptor for EGF is able to interact with and nick supercoiled double-stranded DNA in an ATP-stimulated manner.     

超氧化物歧化酶模型化合物对离体肝癌细胞的影响

探讨了外源抗氧化剂超氧化物歧化酶模型化合物MSOD(Cu2C21N14H27C13)对人肝癌细胞增殖的影响．采用离体培养的人肝癌细胞SSMC-7721细胞株,接种入96孔板,24h后加入MSOD模型化合物,采用MTT(methyl thiazolyl tetrazolium)实验,计算肝癌细胞生长抑制率;结果表明,不同浓度组的SOD模型化合物对肝癌细胞的增殖均有抑制作用,而且这种抑制作用呈时间和剂量效应．     

Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts

Fibroblastic cultures derived from normal human tissues undergo a finite number of population doublings when serially subcultivated in vitro (see refs 1, 2 for reviews). Epidermal growth factor (EGF) serves as a mitogen for early doubling level cultures of the human fetal lung-derived cell strain, WI-38, under serum-free conditions. The ability of cells from late doubling level cultures to respond mitogenically to EGF is lost, however, despite undiminished binding of EGF throughout the replicative lifespan. The ultimate effects of EGF, that is DNA synthesis and mitosis (see ref. 4 for review), occur after a sequence of events initiated by binding of ligand to specific cellular receptors. The receptor for EGF has been characterized as a 145,000-165,000 (145 K-165 K) molecular weight doublet, and, like the receptors for platelet-derived growth factor and insulin, and the transforming proteins of certain of the RNA tumour viruses, is a tyrosine-specific protein kinase with autophosphorylating activity. Moreover, several of the cellular target molecules of tyrosine phosphorylation have been found to be substrates for two or more of these kinases. The hypothesis that tyrosine phosphorylation underlies a common mechanism of growth control prompted us to ask whether the loss of responsiveness to EGF by late doubling level WI-38 cells is accompanied by altered expression of the EGF receptor, and specifically whether changes occur in the ability of receptors from populations of cells of various in vitro ages to catalyse tyrosine autophosphorylation. We show here that autophosphorylating activity is absent from the EGF receptor of cells which have lost their mitogenic responsiveness to EGF.     

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.     

小鼠睾丸特异表达新基因(mtLR1)在大肠杆菌中的表达

根据已经克隆的mtLR1基因序列,采用RT-PCR的方法获得mtLR1基因。将所得的PCR产物插入原核表达载体pMAL-P-2x中,得重组质粒(pMAL-P-2x/mtLR1)并转化大肠杆菌(E．coli)DH5α．经IPTG诱导表达,SDS-PAGE电泳分析显示,重组蛋白得到了正确表达,表达的融合蛋白占菌体蛋白总量的52％,分子质量约为73 kDa。mtLR1蛋白的高效表达,为研究其生物学功能和制备单克隆抗体奠定了基础。     

Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor

Epidermal growth factor (EGF) and transforming growth factor type I (TGF) are polypeptides of 53 and 50 amino acid residues, respectively. Both bind to EGF receptor, a 1,200-residue transmembranous glycoprotein, leading to phosphorylation of the receptor, enhancement of its tyrosine-specific kinase activity and ultimately to stimulation of cell growth. We report here that a 140-residue polypeptide encoded by one of the early genes of vaccinia virus (VV) is related closely to EGF and TGF. The presence of putative signal and transmembranous sequences further suggests that the viral protein might be an integral membrane protein, but that, as in the case of EGF itself, the membrane-associated form may be the precursor of a soluble growth factor. Production of EGF-like growth factors by virally infected cells could account for the proliferative diseases associated with members of the poxvirus family such as Shope fibroma virus, Yaba tumour virus, and molluscum contagiosum virus (MCV).     

Desensitisation of cultured glial cells to epidermal growth factor by receptor down-regulation.

Epidermal growth factor (EGF), which can be purified from the mouse submaxillary gland or from pregnant human urine, is a potent multiplication-stimulating factor for several types of cultured cells, including human fibroblasts and glial cells. The molecule binds with high affinity and saturation kinetics to a cell-surface receptor, is subsequently internalised and finally degraded. The binding event is accompanied by a reduction in the number of EGF receptors. This phenomenon--'receptor down-regulation'--has been demonstrated with several hormones and may be a general principle for the modulation of binding groups on the outer cell surface. Further, it has been proposed that receptor loss acts to regulate the cellular response to the binding ligand. The present study provides direct experimental support for this hypothesis. It demonstrates that down-regulation of EGF receptors on glial cells causes desensitisation of the mitogenic response of these cells to subsequent stimulation with EGF.