Methotrexate in type 1 diabetes mellitus:advances research / 军事医学

Type 1 diabetes mellitus(T1DM),also known as insulin dependent diabetes mellitus (IDDM), is classified as an autoimmune disease.Academia currently define it as one of the important autoimmune diseases(IAIDs).The most significant pathophysiological characteristics of T1DM are the pathological humoral and cellular autoimmune response which results in damage to pancreaticβ-cell and absolute reduction insulin.Thus, the mainstream traditional therapy for T1DM has been to chronically replenish insulin or substitutes.Nevertheless, this approach may generate a number of side effects, especially hypoglycemia reaction, hypoglycemic coma and insulin resistance.In recent years, the clinical use of methotrex-ate(MTX) as an immunity inhibitor or regulator aiming at pathogenesis of T1DM has received increasing attention and will become another significant case where an old drug is used for new purposes.The advances in research on MTX in T1DM are discussed in this paper.

CONSTRUCTION OF CD34 EXTRACELLULAR REGION ENCODING cDNA CLONE, AND EUKARYON EXPRESSION VECTOR AND ITS PRELIMINARY APPLICATION IN MONOCLONAL ANTIBODY PREPARATION BY GENE IMMUNIZATION / 解放军医学杂志

Objective To clone CD34 extracellular region encoding cDNA and to construct its eukaryon expression vector to explore the feasibility of its monoclonal antibody preparation by gene immunization. Methods Total RNA was extracted from KG-1a cell lines. CD34 extracellular region encoding genes were amplified by RT-PCR method and then confirmed by enzymatic lysis and DNA sequencing, and its eukaryon expression vector was constructed as pcDNA3.1-CD34. Twelve BABL/c mice aged 4-6 weeks were selected and randomly divided into 3 groups. Before immunization, 50?l 25% saccharin solution was injected into mouse musculus quadriceps femoris. Fifteen minutes later, blank control PBS, PBS diluted empty vector pcDNA3.1(50?g), or PBS diluted pcDNA3.1-CD34 was injected into the same site of the above three groups, respectively. Immunization was taken every two weeks for a total of three times. The antibody was detected regularly by FACS using tail blood from immunized mice. Results The results demonstrated that the length of CD34 cDNA was 886bp which was identical to the theoretical value and its sequence was confirmed by DNA sequencing which was identical to the registered sequence. The accuracy of CD34 expression vector recombination was confirmed by restriction enzymatic lysis. Hind III and EcoRI restriction enzymatic lysis sites were introduced into 5 and 3 terminals of amplified cDNA sequence, respectively. Terminate code TGA was also introduced into CD34 extracellular encoding cDNA. The expression vector possessing target genes was named as pcDNA3.1-CD34. FACS detection indicated that only 1/4(25%) immunized mice had a lower titer CD34 antibody in their tail vein serum 2-6 weeks after immunization, and the others did not show no antibody production. Conclusion The eukaryon expression vector pcDNA3.1-CD34, which express CD34 extracellular region, has been constructed. The feasibility of CD34 McAb preparation can primarily be confirmed by gene immunization.

Progress in Study on MHC Structure and Function / 中国实验血液学杂志

The MHC molecules were first discovered as major histocompatibility antigens. When their essential biological function as antigen-binding molecules and informers for T cells was unveiled, an explanation of why MHC also acts as major histocompatibility antigens was provided. When the antigen-binding cleft of MHC molecules was firstly clarified more than 10 years ago, the study of MHC structure and function has stepped into a prosperous new era, and many achievements have in this area have been achieved. This paper summarized the exciting progress in study on MHC structure and function in recent years.

Density and Affinity of IL-6 Receptors in Human Leukemic Cells / 中国肿瘤生物治疗杂志

Objective: To make a study of density and affinity of IL-6R in human leukemic cell lines, and discuss the affection of high affinity IL-6R to the targeted treatment of leukemia with IL-6-PE40 fusion protein. Methods: Radial binding assay with scatchard plot and FACS were used to analysis the density and affinity of IL-6R and protein expression of IL-6Rα and β subunits in totally 8 representative human leukemic cell lines. Results: Myelocytie, monocytic and erythrocytic leukemic cell lines U937, HL-60, KG1 and TF1 express high affinity IL-6R, whose average density per cell is 2 502,2 874, 2 319 and 9 329 respectively, however no 125I-IL-6 binding was detected on chronic myelocytic leukemic cell line K562 and lymphoblastic leukemic cell lines such as Raji, CEM and HUT28. These results correlate with those of FACS highly. Conclusion:These observations suggest that acute nonlymphoblastic leukemic cells may be more suitable for targeted treatment with IL-6-PFA0 fusion protein.

Cloning, expression and protein structure prediction of a novel SCF-TPO fusion gene / 中国病理生理杂志

AIM: To obtain the high expression of recombinant human stem cell factor - thrombopoitin (SCF-TPO) fusion gene and predict its structure property. METHODS: Tour primers were designed according to known sequence of TPO and SCF to amplify the functional amino acid domain of TPO and SCF by RT- PCR, respectively from fetus hepatocytes. The expression plasmid pET32a/SCF- TPO was constructed by VOE gene fusion technique and expressed in BL21(DE3)plysS. The fusion protein property, such as second structure, flexibility, and hydrophilicity were predicted by DS Gene and Protscale software. RESULTS: The expression vector, pET32a/SCF - TPO was constructed and the high expression of the SCF/TPO fusion protein was obtained, with the expression amount of up to 40% of the total cellular protein. DS Genel .5 and Protscale predict no new antigenicity in fusion protein, and the second structure and ioelectric point have no changes except four amino acids change in first structure. There are high flexibility and low hydrophilieity in the linker peptide. CONCLUSION: High expression of SCF- TPO fusion protein has been obtained and protein prediction shows that the fusion protein design is reasonable, which lay foundation for further study of biological fundation of SCF - TPO fusion protein.

The rapid prediction of GVHD by three-dimensional structure differences of HLA / 中国输血杂志

Objective Trying to predict the degree of GVHD after partly matched hematopoietic stem cell transplantation. Methods Analysis of the relationship between three-dimensional structure differences of donor-patient unmatched HLA and the GVHD levels after hematopoietic stem cell transplantation. Results GVHD levels were related to donor-patient unmatched HLA structure differences. The HLA structure differences forⅠ - Ⅱ degree GVHD were much smaller than that for Ⅲ - Ⅳ degree GVHD. Conclusion Prediction of GVHD by HLA structure differences is simple, rapid, specific and could help select proper conditioning regimens before transplantation and the proper immune suppressive agents after transplantation.

Analysis of biochemical and physical properties for a new recombinant B7-Pseudomonas exotoxin fusion protein B7-2-PE40KDEL / 中国免疫学杂志

Objective:Identification of some biochemical and physical properties for a new recombinant B7-2-PE40KDEL exotoxin fusion protein.Methods:12%SDS-PAGE separating and gel imaging analyzing,peptide mass fingerprinting,Western blot and MTT assasying were used respectively for identification of the protein.Results:Molecular weight of the recombinant B7-2-PE40KDEL was 72 628,5% of the difference to its theoretical value 69 561.The result of Western blot indicated that the purified recombinant B7-2-PE40KDEL could specifically bind with mAb anti-human B7-2 and the antibody against PEA,while the negative control did not.The recombinant B7-2-PE40KDEL digested with trypsin and then detected by MOLTI-TOF-MS.It was shown that the detected 15 peptides lied in the extracellular part of B7-2 and the truncated Pseudomonas extoxin PE40KDEL.Searching in the peptident data bank of Expasy website,we did not find any known proteins which was accordant with the above terms.The cytotoxic activity of the recombinant toxin with MTT method showed that the B7-2-PE40KDEL selectively killed Jurkat cell line which expressesed CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor.Conclusion:Recombinant B7-2-PE40KDEL exotoxin fusion protein we construct proves to be a new one with targeted killing bioactivity to B7:CD28 system.

DEVELOPMENT OF AN HIGH-EFFICIENT IMMUNOMAGNETIC ISOLATOR FOR THE PURIFICATION OF CD34~+ HEMATOPOIETIC CELLS / 解放军医学杂志

According to the principle of immunomagnetic separation, a novel high-efficient immunomagnetic isolator termed as CMSI 100 for the purification of human CD34 + hematopoietic cells was designed and successfully developed. To do this , neodymium iron boron having magnetic properties superior to any other permanent magnet materials was selected as the key parts of CMSI 100 isolator. The core of the magnet assembly was constructed by sandwiching mild steel bars between 4 pieces of magnet bars of neodymium iron boron. Each piece of magnet bars must be limited to the specifications of 3.5cm?2.0cm?0.3cm so that 2 700 gauss magnetic attractive force could be exerted at the magnet surface. Otherwise magnetic field above the surface of the magnet assembly is either very stronger or weaker, both of which are not beneficial to the enrichment of CD34 + hematopoietic cells. With CMSI 100 immunomagnetic isolator, more than 90% purity and over 95% viability of CD34 + hematopoietic cells could be obtained. These data indicate that CMSI 100 immunomagnetic isolator is as good as the internationally used Isolex TM 50 made by Baxter Company in USA.