HIGH EFFICIENT TRANSFER AND EXPRESSION OF HUMAN CLOTTING FACTOR Ⅸ cDNA IN CULTURED HUMAN PRIMARY SKIN FIBROBLASTS FROM HEMOPHILIA B PATIENT BY RETROVIRAL VECTORS

To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human iibrosarcoma cell line, HT1080 cells, was used to assay the factor Ⅸ-virus titers of these four virus-producing PA317 cells, which ranged from 2×10~4 to 5×10~5 cfu/ml. The factor Ⅸ proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor Ⅸ at the rate of 584 rig/10~6 cells/24h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor Ⅸ than LTR promoter. The highest expressed retroviral vector XL-Ⅸ was used to infect a line of f     

ESTABLISHMENT OF AN EPITHELIAL CELL LINE ORIGINATED FROM LEG SKIN OF A PATIENT WITH PALATINE CARCINOMA

An epithelial cell line was established from the leg skin of a patient with palatine carcinoma. On the basis of the studies of biological characteristics, such as rapid cell growth, neoplastic morphology, a large group B marker chromosome, the ability of the transplanted cells to produce tumor nodes in Wistar newborn rat, the cell possessing the structure of low differentiated squamous carcinoma and skin-like tissue, etc., we conclude that the PeaSE-1 cell line is an abnormal epithelium cell line.     

EXPERIMENTAL STUDY OF INVASION AND METASTASIS OF CANCER CELLS AND ESTABLISHMENT OF THE MORPHOLOGICAL PATTERN IN THE PROCESS OF INVASION AND METASTATIC MODELS OF CANCER CELLS

For the study of invasion of tumor cells in vitro and in vivo methods were used to observe theinvasion process and morphological characteristics of tumor cells. The different animal tumor cells andcell lines from human tumors were used. The process of cancer cells in vitro invasion can be dividedinto 5 stages. We suggest that filopodia protruding from the tumor cell margin seem to play an im-portant role in the initiation of cell invasion. For experimental study of cancer metastasis we established three spontaneous metastatic models, in-cluding spontaneous lymphatic and hematogenous metastasis of mouse uterine cervix carcinoma No 14(U_(14))and No 27(U_(27)), and third spontaneous lung metastasis in mouse forestomach carcinoma (FC). Two tumorcell lines are established: One is MFC cell line which is derived from FC tumor; second cell line isMUCC which is derived from the U27 tumor. These metastasis models and their cell lines are veryuseful for studying the mechanism of metastasis, tumor heterogenicity an     

ESTABLISHMENT AND CHARACTERIZATION OF HUMAN MALIGNANT PLEURAL MESOTHELIOMA CELL LINE SMC-1

A cell line (SMC-1) has been established from the resected tissue of a patient with malignant pleural mesothelioma which has been pathologically confirmed to be the mixed type. The cell line is characterized by the pleomorphy and multilaycred growth of the cells with the population-doubling time of approximately 32h, the highest mitotic index of 46—50‰ and the modal chromosome number of 69—70. Transplantation of the cells into the anireals can produce tumors bearing histological resemblance to the original material. The cultured cells, xenografted tumor cells and the cells from the host specimen show similar histochemical expressions. These data meet the requirements of a human malignant pleural mesothelioma cell line. The establishment of this cell line will serve as a useful tool in clinical research of diagnosis and treatment of this devastating disease.     

In vivo INDUCTION OF MALIGNANT TRANSFORMATION OF NUDE MOUSE B LYMPHOCYTES BY A HUMAN MULTIPLE MYELOMA

The hone marrow puncture fluid of human multiple myeloma was inoculated subcutaneously in BALB/C/nu nude mice. After passage of the xenograft from nude mice in vitro, a murine malignant lymphosarcoma cell line was unexpectedly established, which was named NLB-SK. The murine NLBSK cell line has been cultivated in vitro over 72 passages for 10 months. Repeated cryopreservation showed that the murine NLB-SK cell line revived satisfactorily. Based on cell biological characteristics the malignant transformation migh the attributed to horizontal oncogenesis between human malignant tumor cells and nude mouse normal somatic cells.     

A MOUSE MACROPHAGE-LIKE CELL LINE (MMC-1) AND ITS BIOLOGICAL CHARACTERISTICS——BIOLOGICAL CHARACTERIZATION

The biological characteristics of mouse macrophage-like cell line (MMC-1) are described.This cell line has been cultured in vitro for more than 20 months. MMC-1 cells are adherent to typical morphology of macrophages, but bigger in size thanthose of the uncultivated. They are actively phagocytic with abundant Fc receptors. The kary-otypes are mostly pantaploidy. This cell line provides a useful tool for the biological andimmunological study of macrophage.     

TRANSFORMATION OF CULTURED HUMAN FETAL GASTRIC FIBROBLASTS WITH PHYSICO-CHEMICAL AGENTS

Treatment of early passage human fetal gastric fibrohlasts with ultraviolet (UV) light and the chemical carcinogen ethyl nitrosourea (ENU) in succession resulted in an immortally growing cell line, named GTS 8502. The cells of this line display typical transformation characteristics, such as irregularly shaped nuclei, heteroploidization of karyotype and frequent appearance of heteromorphic chromosomes, the enhanced volume ratio of nucleus to cytoplasm, multinucleoli, appearance of microvilli on the surface of the cells and agglutination reaction to lectin concanavalin A. The transformants have high growing and mitotic indices and the ability of focus-formation on monolayers and anchorage independent growth in soft agar medium. Moreover, these cells induced turnouts in nude mice or in immunosuppressed new-born rats through heterotransplantation. The results of various methods including electromicroscopy and histochemical analyses indicate that GTS 8502 cells are of fibroblast origin.Our results thus indi     

MOLECULAR CLONING AND NUCLEOTIDE SEQUENCE ANALYSIS OF A TRANSFORMING GENE FROM HUMAN GASTROCARCINOMA CELL LINE

Mouse and rat fibroblasts were transfected with total DNA from human gastrocarcinoma cell line BGC-823. It was shown by hybridization assay that the genome of one of the rat secondary foci contains transforming genes from the human gastrocarcinoma cell line, which are homologous to the protooncogene c-Ha-ras in the normal cells. The genomic library of the rat secondary foci was constructed, using λ phage EMBL3 as the vector. The transforming gene Ha-ras of the human gastrocarcinoma cell was thus cloned by screening the library with the probes of human Alu repeat sequence and c-Ha-ras. The nucleotide sequences of the first and second exons were analysed by M13-dideoxy method. The result shows that the nucleotide sequence of the transforming gene is the same as that of the normal protooncogene except one nucleotide difference in the first exon.     

ULTRASTRUCTURAL STUDY OF THE HATSCHEK''''S PIT EPITHELIAL CELLS OF AMPHIOXUS

The authors have observed, using an electron microscope, that the number of thesecretory granules and the development of various organelles in the apical epithelial cellof the Hatschek's pit showed a positive correlation with the gonadal development andmaturity. At the same time, only the epithelial cells respond to GnRH-A(gonadotropin-releasing hormone analogue). Both cytological and endocrinological experiments demon-strate that the cell may be the primitive gonadotropic cell of amphioxus.     

EFFECTS OF MONOCLONAL ANTIBODY ANTIEGF RECEPTOR ON HUMAN NASOPHARYNGEAL CARCINOMA CELL AND OTHER TUMOR CELLS

Three anti-EGF receptor MoAbs were used in these studies. Administration of MoAbs 3 and 176 inhibited tumor formation in nude mice by CNE-2, a poorly differentiated nasopharyngeal carcinoma cell line and A431, an epidermoid carcinoma cell line. When the same MoAbs were used in treatment against HeLa, a cervical carcinoma, tumor growth was not affected. The number of EGF receptors and apparent dissociation constants for ~(125)I-EGF on CNE-2 and A431 was 1.3×10~(?)/cell (Kd 7.7×10~(-8)mol/L) and 1.4×10~6/cell(Kd 2.4×10~(-9)mol/L), respectively. Both MoAbs 3 and 176, capable of competing with EGF for receptor binding, showed significant tumor growth inhibition. MoAb 101 was incapable of blocking the binding of EGF to its receptor, and not as effective as MoAbs 3 and 176 in tumor growth inhibition. Our observation is that the MoAb anti-EGF receptor is cytostatic rather than cytocidal, in vitro against CNE-2 and A431.     

RE-DEVELOPMENT OF SYNAPTIC CONNECTIONS AFTER IMPLANTED SINGLE 5-HT CONTAINING NEURON IN ISOLATED LEECH GANGLIA

Single mature Retzius cells which contain 5-HT have been implanted in isolated ganglia of Whitmania pigra. The properties of the implanted Retzius cell and its connections with the original identified nerve cells of the ganglia have been measured by intracellular microelectrodes. At 3 days in culture an implanted Retzius cell maintains its normal electrical membrane properties. It also forms the non-rectifying electronic coupling with the original Retzius cell and rectifying junctions with the L motor neuron of the host ganglion. In addition it receives chemical inputs of the impulse from T and N sensory neurons of ganglion. Impulses in the implanted Retzius cell evoke synaptic potentials in the L motor neuron, In this paper the appearance of novel synapses, which are formed by implanting an extra mature neuron as a pre- or post-synaptic element, is described. It might represent functional development of central nervous system at an earlier stage.     

STRUCTURAL EFFECT IN FORKED CONJUGATIVE SYSTEMS——BIFURCATION-TYPE OF FORKED POLYENIC NITRILES,CARBOXYLIC ACIDS AND ESTERS

Five homologous series of bifurcate systems of aliphatic and aromatic polyenic cyano andcarboxylic compounds have been prepared and studied. The electronic absorption spectra forthe series and the NMR chemical shifts for the methyl-, methylene- and beta-protons havebeen found to conform very well to the rule of homologous linearity. The mass spectra forthe α-cyano polyenic ester series show strong peaks for the fragments of M-COOEt but noneof M-CN, indicating that the CN group seems to be in stronger conjugation with the poly-enic chain than the COOEt group does. In all the forked series studided, a red shift in electronic spectra is brought about upon theintroduction of an electron-attractive branching group, just like the case of introducing anelectron-repelling substituent. This has been taken as an indication of the predominance of themolecular integrality over the group characteristics. By means of the method of similar triangles between a homologous line for a linearseries and that for the corres     

THE ANTISENSE C-HA-RAS OLIGODEOXY-NUCLEOTIDES INHIBIT THE PROLIFERATION OF THE GASTROCARCINOMA-DNA-TRANSFORMED RAT 3-3 CELLS AND REDUCE THE P21 EXPRESSION OF THE CELLS

The Rat 3-3 is a secondary transformant of the rat fibroblast cell line (Rat-1) transfectedwith total DNA of a gastrocarcinoma cell line BGC-823. The cells over-express the c-Ha-rasoncogene which contains point mutation at the 12th codon. In order to determine how theactivated c-Ha-ras oncogene expression governs the cell's transformation, two pendadecadeoxy-nucleotides AS-1 and AS-2 were synthesized. AS-1 was complementary to the single strandof the first three codons and the upstream sequence close to the ribosome binding site of c-Ha-ras mRNA. AS-2 was complementary to the 3' end of the first intron and the 5' end ofthe second exon of c-Ha-ras unripe RNA enclosed in the nucleus. The oligonucleotides couldblock either the translation of c-Ha-ras mRNA or the splicing of c-Ha-ras unripe RNA, thusinhibiting the expression of the activated c-Ha-ras oncogene and the proliferation of thetransformed cells Rat 3-3. The inhibitory effect increased with a growing concentration ofthe antisense oligodeoxynucleoti     

A PRELIMINARY STUDY ON THE MECHANISM OF NEONATAL ALLOTRANSPLANTATION TOLERANCE——PARTICIPATION OF SUPPRESSOR CELLS IN THE MEDIATION OF NEONATAL ALLOTRANSPLANTATION TOLERANCE

Using a method from Billingham, an animal model of the neonatal allotransplantation tolerance of SWI to C57BL inbred mice was established and the mechanism of neonatal tolerance established by us was preliminarily studied.Our experiment indicates that the clonal deletion theory is not the unique explanation for the neonatal tolerance and a peripheral active inhibitory mechanism including suppressor cells may participate in meditating and maintaining the neonatal tolerance.The suppressor cells in the neonatal tolerance are radioresistant, but rather sensitive to mitomyein C treatment. These results suggest that suppressor cell population may be nonhomogenous. Further studies about this problem are needed.     

A MOUSE MACROPHAGE-LIKE CELL LINE(MMC-1) AND ITS BIOLOGICAL CHARACTERISTICS——SCANNING ELECTRON MICROSCOPIC OBSERVATION

The surface characteristies and EA-rosette with opsonized chicken erythrocytes by mousemacrophage-like cell line (MAC-1) were studied by scanning clectron microscopy. The ultra-structures of the cell surface were characteristies of macrophage.     

Construction of HIV-1 Virus-like Particle Vaccine

The virus-like particle（VLPs） vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have been used, one can express the HIV-1 main structure proteins, Gagpol and Env, and the other contains an antibiotic gene. The two kinds of plasmids have been cotransfected into 293 cells. A stable cell line that can express Gagpol and Env proteins efficiently and lastingly has been screened. It has been confirmed that Gagpol and Env proteins in the cell culture supernatant can be self-assembled into virus-like particles. The authors have detected the secretion of VLPs in the cell medium, defined the peak of the secretion, and followed and monitored the stability of expression.     

MECHANISM OF INTERFERON ACTION——STRUCTURAL REQUIREMENT OF LIGAND AND CELLULAR SPECIFICITY

We had reported the existence of 2'-5'P_3A_3 receptor on the peritoneal macrophages from Wistar rat. In this paper data are given to demonstrate that the ligand, for binding with 2'-5'P_3A_3 receptor and enhancing the phagocytosis of macrophages, must have a three-phosphate radical on its 5'-terminus and at least three adenylate residues in 2'-5'oligoadenylate. (~3H)-2'-5'P_3A_3 can also bind on macrophages from other animals; and the binding of (~3H)-2'-5'P_3A_3 on Hepatoma cell line 7402 is lower than that on macrophages; whereas it is very low (binding ratio less than 1%) on erythrocytes (from rabbit, rat and mouse) or human embryo lung fibroblast cells. The binding conditions of the ligand with 2'-5'P_3A_3 receptor have also been investigated.     

STUDIES ON TiCI4/Mg (OEt)_2/EB SUPPORTED CATALYSTS FOR PROPYLENE POLYMERIZATION

The supported catalysts for propylene polymerization were prepared by milling Mg (OEt)_2 with EB (ethylbenzoate) and treating with TiCl_4 solution. When TiCl_4/(Mg(OEt)_2/EB) (mol.) ratio was increased, decrease in contents of-OEt and Ti of the catalysts was observed, while the content of EB increased. It is proved by analyses of IR, X-ray and XPS that during co-milling Mg(OEt)_2 with EB no reactions have taken place. But after treatment with TiCl_4 solution, Mg(OEt)_2 converts into MgCl_2 and EB coordinates on the resulting MgCl_2 carrier, a surface complex forms.The activity of catalysts,isotacticity and vicosimetric molecular weight of polypropylene increase with the decrease of the content of ethoxyl group. The kinetic curves of propene polymerization obtained with present catalysts system display decay curves. It is found from the triad tacticity calculated from the expanded spectra of methyl carbon region that, ethoxyl group in catalyst has an effect on the configuration of polymer chain.     

STUDIES ON 4-OXALYSINE-RESISTANT MUTANT CELL LINE AND ITS REGENERATED PLANT TISSUE FROM NICOTIANA TABACUM L.

A 4-oxalysine-resistant cell line from tobacco callus was selected and established through successive treatments with 1-4-oxalysine(OL, 1g/1), 2% EMS and then OL(4g/1). The mutant callus was not cross resistant to Eth and 5MT and accumulated twice amount of lysine as that of the parent. Mutant pleats were regenerated from the mutant cells. The mutant plants flowered and set seeds within the culture flasks. A secondary mutant cell line was induced from the mutant plant. The eL-resistance, lysine content and peroxidase isoenzyme of both the mutant cell and secondary cell line were examined and confirmed that all these traits, which were different from those of the parent line, were transmitted through the mutant cell line to its secondary callus. Experiment on sexual transmission is under way.     

STRUCTURE DETERMINATION OF THE SILVER CARP INSULIN CRYSTAL

After rotation search of an orthorhombic crystal form of Silver Carp insulin, based on the known porcine 2-Zn insulin structure, the Silver Carp insulin molecules were positioned in the unit cell by applying the translation function and R-factor search methods. The packing of the molecules in the cell is reasonably good. A preliminary refinement of this model at 3.5 resolution has been carried out through the use of restrained least-squares method. The segments of polypeptide chain, which were not involved in the refinement, could be visible in the electron density map calculated using the refined atomic parameters.