Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA最新文献

Objective: To prepare and identify the monoclonal antibodies (mAb) against human zeta globin chain.

Methods: BALB/c mice were immunized with purified recombinant zeta globin chain, and the hybridomas were generated by fusion of mouse spleen cells and myeloma cells NS-1. After three fusions and successive cloning, 3 hybridoma cell lines secreting the mAb against zeta were obtained and the antibodies were purified from the ascites, followed by identification with indirect enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA in combination with rabbit anti-zeta serum.

Results and conclusion: Three hybridoma cell lines secreting anti-zeta mAb were established, designated as 1A12, 3H9 and 4D11, respectively. Both of 3H9 and 4D11 mAbs belonged to IgG1 isotype and mAb 1A12 to IgG2a. All the mAbs could bind specifically to recombinant zeta and natural zeta globin from hemolysate of --(SEA) gene carrier. In addition, 1A12 and 3H9 mAbs could recognize different epitopes on zeta globin, suggesting the possibility of developing a detection system for screening alpha-thalassemia with these mAbs.

Objective: To construct the standard recombinant plasmids for 7 common haplotypes of mannan-binding lectin (MBL) gene.

Methods: The DNA samples with known haplotypes and genotypes of MBL gene were used as the templates for amplifying the fragments of MBL gene haplotypes including the promoter region and exon 1 with sequence-specific primer-polymerase chain reaction (SSP-PCR) method. The amplified fragments were cloned into T vector and the bases located at codon 52 and codon 57 of exon 1 in MBL gene were mutated respectively by site-directed mutagenesis. All the 7 recombinant plasmids were identified by PCR and direct sequence analysis.

Results: From the DNA samples with known haplotypes and genotypes of MBL gene, the standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA and LYPB of MBL gene were constructed by SSP-PCR and molecular cloning technique. From the recombinant plasmids of HYPA and LYQA, the standard plasmids of haplotypes HYPD and LYQC of MBL gene were constructed by site-directed mutagenesis, respectively.

Conclusion: The constructed standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA, LYPB, HYPD and LYQC of MBL gene provide standard controls for detecting the SNPs, haplotypes and genotypes of MBL gene with such genotyping methods us SSP-PCR and real-time PCR.

Objective: To construct eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 (HIF-1alpha) and study their expressions in human microvascular endothelial cells (HMVECs).

Methods: Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Pro564 (ccc) in HIF-1alpha into gcc (Ala) in pcDNA3.1(+)-HIF-1alphato obtain single-site-mutated vector pcDNA3.1(+)-HIF-1alpha-564Ala, which was then subjected to a second site-directed mutagenesis to convert the codons for Asn803 into that of Ala (gct) to acquire double-site-mutated pcDNA3.1(+)-HIF-1alpha-564Ala-803Ala. After lipofectin-mediated transient transformation of HMVECs with the 3 recombinant plasmids including the two plasmids containing the mutations and the one without mutation, respectively, the expression levels of HIF-1alpha mRNA and protein were determined using RT-PCR, immunofluorescent staining and Western blotting.

Results: DNA sequence analysis demonstrated success of the two-step mutagenesis and the two plasmids of pcDNA3.1+-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha-564Ala- 803Ala were obtained, both of which could produce HIF-1alpha protein resistant to oxidation degradation in HMVECs as compared with the non-mutated one.

Conclusion: The recombinant plasmids pcDNA3.1(+)-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha- 564Ala-803Ala have been successfully constructed with efficient expressions in HMVECs.

Objective: To investigate the effects of local carbon ion irradiation on the length of survival and peripheral blood leukocyte and platelet counts of mice inoculated with pulmonary tumor cells.

Methods: Thirty tumor-bearing mice were randomly divided into control group (tumor-bearing but without carbon ion irradiation, n=10), 12 Gy group (n=10) and 24 Gy group (n=10). The right hind limbs of the mice, where the tumor cells were inoculated, were irradiated with carbon-ion beams at a single dose in 12 Gy and 24 Gy groups, and those of the control group received no irradiation. The peripheral blood leukocytes and platelets of the mice in all the 3 groups were counted immediately before and 7 and 14 days after irradiation, respectively, with the survival time of the mice recorded.

Results: There was no significant difference in the survival time of the mice between the 3 groups. The peripheral blood leukocyte counts in all groups increased after irradiation, no significant difference was noted between the two irradiation the groups and the control group. Irradiation at 24 Gy resulted in significant increase in peripheral blood platelet count on the 14th day (P=0.032, F=4.062), but no such increment was observed in the other 2 groups or on day 7 in the 24 Gy group.

Conclusion: Local carbon ion irradiation may not produce significant effects on the length of survival and hemogram of the tumor-bearing mice.

Objective: To explore the relationship between the differentiation of L6 myoblasts and oxidative stress.

Methods: MTT assay was used to determine the viability of L6 myoblasts, from which the total RNA was extracted for amplification of the myogenin gene fragment by RT-PCR. H(2)O(2)-induced morphological changes of the cells were observed.

Results: The myoblasts treated with low concentration of reactive oxygen (50 micromol/L H(2)O(2)) for 1 h exhibited accelerated cell growth (P<0.05), and treatment with 50 and 150 micromol/L H(2)O(2) induced the gene expression of myogenin, a molecular marker for differentiation of myoblasts. Morphological study revealed myotube formation and accelerated differentiation of the myoblasts induced by H(2)O(2).

Conclusion: The reactive oxygen may serve as the intracellular signal molecules to induce the growth and differentiation of the myoblasts.

Objective: To construct and characterize a mutant Enteroaggregative E. coli(EAggEC) O42 strain with in-frame deletion of high-pathogenicity island (HPI).

Methods: The kanamycin resistance gene (kan) was inserted between the sequences of irp8 and irp5 genes as the two homologous sequences for construction of the recombinant plasmid pCO85 by subcloning the recombined sequence into the suicide vector pCVD442. By homologous recombination and conjunction mobilization, EAO85 mutant with deletion of the core region of HPI about 24 kb spanning from irp8 to irp5 sequences was screened.

Results: Irp8 and irp5 genes of EAggEC O42 were exchanged by pCO85 by conjunction mobilization with the selection by sucrose. The EAO85 mutant was identified by their failure to yield PCR products with primers specific for the internal regions of HPI.

Conclusion: We have successfully constructed a mutant of EAggEC O42 strain with HPI in-frame deletion, which may facilitate the exploration of the role of HPI in EAggEC strain.

Objective: To investigate the role of all-trans retinoic acid (atRA) in inducing differentiation of neonatal rat striatal neural stem cells (NSCs).

Methods: Neonatal rat striatal NSCs were obtained by mechanical isolation and serum-free culture. The roles of atRA at different concentrations in inducing the differentiation of NSCs were observed by immunofluorescent cytochemical staining, and the expression of retinoic acid receptor (RAR) gene was determined by semi-quantitative RT-PCR.

Results: atRA could dose-dependently accelerate the differentiation of NSCs into neuron-like cells, and the physiological concentration of atRA was optimal for inducing NSC differentiation. atRA could induce the expression of RARbeta mRNA in a dose- and time-dependent manner.

Conclusion: atRA can accelerate differentiation of NSCs into neuron-likes cells and up-regulate the expression of RAR-beta mRNA in neonatal rat striatal NSCs.

Objective: To study the expression of cathepsins B and L in first-trimester gestational decidua and chorionic villi.

Methods: The decidua and chorionic villi in the first trimester of gestation were obtained from 30 women undergoing induced abortion, 25 with spontaneous abortion, 10 with normal endometrium in secretory phase, and 15 with bydatidiform mole in whom the expression of cathepsins B and L was determined by immunohistochemistry.

Results: Positive staining for cathepsins B and L were mainly detected in the trophoblasts and decidual cells. The positive expression rate of cathepsin B in the normal endometrium of secretory phase, decidua in the first trimester of gestation and spontaneous abortion were 10% (1/10), 83.3% (25/30), and 32.0% (8/25), respectively, and the rate of cathepsin L expression in the endometrium in secretory phase, decidua of induced abortion and spontaneous abortion were 0.0%, 63.3% (19/30), and 32.0% (8/25) respectively, showing significant difference in the expression rates of cathepsins B and L between the 3 groups (P<0.05). Strong positive expression of cathepsin B in chorionic villi of induced abortion, spontaneous abortion and hydatidiform mole were detected at the rates of 10.0% (3/30), 0.0%, and 66.7% (10/15), respectively, and cathepsin L at the rates of 26.7% (8/30), 4.0% (1/25), and 80.0% (12/15), respectively, with significant difference in strong cathepsins B and L expressions between the 3 groups (P<0.05).

Conclusion: The expression of cathepsins B and L differs in normal and abnormal deciduas and chorionic villi of early pregnancy, suggesting that the cathepsins may play important roles in the process of implantation.