Background and purpose:Small cell lung cancer(SCLC) is a highly aggressive malignancy with a 5-year survival rate of less than 10%.These features suggest the enrichment in cancer stem cells.Our study was aimed to establish a small cell lung cancer cell line from primary cultured from lung cancer tissue,and to identify and isolate cancer stem cells from the early generations of the established cell line.Methods:By using a serum-free medium,lung cancer primary cells were cultured from a small fresh lung cancer cell sample,and a small cell lung cancer cell line was established after passaging the cultured cells.Lung cancer stem cell markers were searched by using flow cytometry analysis to sort through the third or forth generation cells of the established cell line.Biological characteristics of lung cancer stem cells were studied by using the single cell clone formation test,plat colony formation test and cell sphere formation test.Results:A small cell lung cancer cell line was established by primarily culturing a fresh lung cancer sample.The cell line could easily passaged more than 25 generations.When the third or fourth generation cells of the established cell line were checked by flow cytometry,there was a small population of cells that were obviously with CD44 stronger positive(CD44++ cells,5.1%) than the main population cells,which were CD44 weak positive(CD44+ cells).CD44++ cells showed stronger colony formation ability than the CD44+ cells.Furthermore,only the CD44++ cells could form stem cells when a single cell was seeded in a well of 96 well plates.Most importantly,only the CD44++ cells could form cell spheres in ultra low attachment 96 well plates.These results indicated that the CD44++ cells enriched more cancer stem cells in the small cell lung cancer cell line.When CD44 and CD90 antibodies were co-stained,the cells of the established cell line could be separated into 4 populations,i.e.CD44+CD90-,CD44+ CD90+,CD44++ CD90+ and CD44++ CD90-cells.CD44+ CD90+ cells were the smallest population cells in the cell line,with a ratio of about 1.9%.When cultured in ultra low attachment 96 well plates,CD44++ CD90+ cells had the strongest cell sphere formation ability when compared with other population cells,indicating that CD90 might also be a small cell lung cancer stem cell marker.Conclusion:There were cancer stem-like cells in primary cultured small cell lung cancer cell lines,CD44 and CD90,which might mean that they could be lung cancer stem cell markers.

Objective:To mutate the codons which code the lysine at loci ? 99 ,? 82 in human hemoglobin gene to the codons which code the cystein.Methods:The objective gene to be mutated was cloned into pAlter Ex2 plasmis and then was mutated with the site directed mutagenesis mediated by oligonucleotide.Results:The sequencing of clones obtained by preliminary screening showed that the anticipated results were reached without any unexpected mutation.Conclusion:The results lay a foundation for the further development of research on expression and purification of recombinated mutant human hemoglobin gene.

Objective To make an attempt to express serialy the human ?,? globin in Escherichia Coli in order to carry out the research on blood substitute based on the genetic recombination of hemoglobin.Methods The routine molecular biology technology was used.Results The serial genetic fragment of ?,? globins containing the hemoglobin was amplified by PCR.The genetic sequencing showed that there was no unexpected mutation.And then,the serial genes were cloned into the pBV220 expression carrier.Undergoing thermal inducement,the expression product could reach about 20% of total bacterial protein.The expression product assumed the form of inclusion body,and it was vertified by Western Blotting.Conclusion This much can lay the solid foundations for further launching the research on expression and pruification of recombinant human hemoglobin genes.

Objective To obtain optimum scheme on reaction system for randomly amplified polymorphic DNA(RAPD) of Pseudomonas aeruginosa. Methods The optimum reaction systems of RAPD were obtained by single factor design and response surface design(RSD). Results The multiple optimum reaction systems caused by effect of interaction on multifactor were observed in single factor design, and the concentrations of key components of the optimum reaction system with good stability obtained by response surface design were 2.0 mmol/L of Mg 2+ , 0.5 ?mol/L of primer and 0.5 ng/?l of template. Conclusion To optimize the reaction system of RAPD, RSD is simpler, more scientific, and more reasonable than single factor design.

Yeast surface display is a new technology developed in recent years,which displays protein of interest on the surface of yeast cell.It can be used to display complex eukaryotic proteins requiring post-translational processing including glycosylation and efficient folding for functional activity. The basis of this technology, its development,methods of screening,its applications and perspective are mainly described in this paper.

Objective ToexpresschimericantigenofHCVwithmultipleimmunodominantepitopes inE .coliforimprovingthequalityofreagentsforHCVscreening .Methods Thegenefragmentencoding HCVchimericantigenwasobtainedbymolecularcloningmethodandclonedintopQE 30 plasmidforex pressioninE .coliM 15 .TheexpressedchimericantigenwaspurifiedbyNi NTAandcoatedonELISA platestoanalyzeitssensitivityandspecificity .Results Thechimericantigenof 5 30 0 0washighlyex pressed .ELISAassayofserumsamples (including 18HCVpositiveand 17negativesera)indicatedthatthe HCVchimericantigenhadhighsensitivityandspecificity .Conclusions ChimericantigenofHCVwith typicalimmunodominantepitopescanbeusedtodevelopgoodreagentsforHCVimmunoassay .

Fragments of ? and ? genes were cloned into expression plasmid pET-21b. Bacteria harbouring expression plasmid was induced with IPTG,and a band of expressed protein of 16 kD was found in PAGE. They expressed as soluble products. ? expression product reached about 5% of total bacterial protein,? expression product reached about 15% of total bacterial protein. The products were confirmed by Western-blotting.

Objective:To construct pIRES-I-Ad?? bicistronic eukaryotic expression vector. Methods: Total RNA was acquired by TRIZOL method, the genes of I-Ad ?? chains were amplified through RT-PCR, respectively. The target genes were connected to pGEM-T vector and sequenced. Then the target genes were subcloned into pIRES bicistronic eukaiyotic expression vector and NIH3T3 cell line was transfected. Transfectant was screened by G418 antibiotics. Total RNA of transfectant was obtained by TRIZOL method, mRNA of foreign gene was examined by RT-PCR. Flow cytometry( FCM) was used to detennine foreign gene expression in protein level and surface expression in NIH3T3 cell line. Results: Bicistronic eukaryotic expression vector pIRES-I-AdaB was established. Foreign gene in mRNA level in transfectants was examined. Verified Ⅰ-Ad ?? chain wa3 expressed in high level expressed on transfected NIH3T3 cell line surface by FCM. Conclusion:Bicistronic eukaryotic expression vector pIRES-I-Ad ?? was constructed successfully. It is useful for studying antigen presentation and interaction between epitopes and MHC- Ⅱ molecule of BALB/c mouse.

Objective To study the correlation between the genes of killer immunoglobulin (Ig)-like receptors (KIR) in natural killer cells and leukemia. MethodsPrimers of 16 KIR genes, including KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1 and 2 pseudogenes 2DP1 and 3DP1, were designed and synthesized. A polymerase chain reaction with sequence-specific primers (PCR-SSP) was developed to detect the mRNA expressions of the 16 genes. Totally 46 healthy individuals and 46 leukemic patients (15 cases of acute myelocytic leukemia, 13 cases of chronic myelocytic leukemia and 18 cases of acute lymphocytic leukemia) were detected and the frequencies of the KIR genes were analyzed. ResultsThe frequency of 2DL4, 3DL2, 3DL3 and 2DP1 in healthy and leukemic patients were 100%, and were detected in all patients and healthy individuals. The other KIR genes had similar frequencies except those of 2DL2 and 2DS2, which were 47.8% of the healthy individuals and 89.1% in leukemic patients. There was a significant difference between the 2 groups.ConclusionThere may be an association between pathogenesis of leukemia and 2DL2 and 2DS2 genes.

Adoptive cellular immunotherapy (ACI) achieves the elimination and control of tumor by mobilizing the body's immune function.It also has targeted efficacy and mild untoward effects.Cytokineinduced killer cells and tumor-infiltrating lymphocytes have been widely used in clinic and have obtained preliminary efficacy.With the development and clinical application of the specific gene transfer of T cell,it will further increase the efficacy of immunotherapy.At present,improving cell culture technology and cell function and using with other treatment are the key links to improve the efficacy of ACI.