[Establishment of Flow Cytometric Immunobead Array Assay for Quantitation of Platelet-Specific Antibodies and Its Application].

Objective: To establish a flow cytometric immunobead array assay (FCIA) to quantify platelet antibodies and to explore its application in the diagnosis and treatment of ITP.

Methods: The guantitative standard curve was established by binding the human IgG of known concentration on antibody-coated microbeads; at the same time, the platelet-specific antigen and antibody complex was captured and levels of platelet antibodies were detected using the microbeads coated by 5 kinds of antibodies against platelets suca as GPIX (SZ1), GPⅠb (SZ2), GpⅢa (SZ21), GPⅡb (SZ22) and p-selection (SZ51). The fluorescence signal detected by flow cytometry were transformed into the conentration of platelet antibodies in samples through the quantitative standard curve, thereby establishing the method for quantititive detection of platelet-specific antibodies in plasm samples (FCIA), moreover the property, efficiency and clinical application of establishod FCIA method were evaluated.

[Effect of Recombinant Protein in the Spacer Domain on ADAMTS13 Activity].

Objective: To obtain the recombinant protein of spacer domain in von Willebrand factor cleaving protease (ADAMTS13), and further study its biological function in ADAMTS13.

Methods: The prokaryotic expression vector was constructed by using the template of plasmid with full-length ADAMTS13, and then transfected into E coli., following the induction of IPTG with the low temperature (30 ℃).

[Establishment of Double Antibody Sandwich ELISA for the Determination of Human Soluble VE-Cadherin in Human Plasma and Its Application].

Objective: To establish double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human soluble VE-cadherin in human plasma and to investigate its value in clinical use.

Methods: The monoclonal antibodies against human VE-cadherin were prepared from BALB/c mice immunized with prokaryotic expression recombinant proteins, and the best combination of double antibodies was selected by checkerboard titration method. Double antibody sandwich ELISA for the determination of human VE-cadherin was established by using HRP-labeled McAb as a detection antibody and a capture antibody.

[Preparation and application of monoclonal antibody against human vascular endothelial cadherin 5].

Objective: To prepare and identify monoclonal antibody (mAb) against human vascular endothelial cadherin 5 (VECDH5).

Methods: BALB/c mice were immunized with recombinant VECDH5 protein for preparing mAb using hybridoma technique. The positive clones were confirmed and selected by indirect ELISA for titer determination.

[Clinical and Laboratorial Characteristics of Primary Acute Myeloid leukemia with Philadelphia Chromosome and Inversion 16].

Objective: To summarize the clinical characteristics as well as diagnosis and treatment in 1 case of acute myeloid leukemia(AML) with coexpression of Ph and inv(16).

Methods: A series of clinical tests, the cellular morphological, immunological, cytogenetic and molecular biological examinations of leukemia cells were performed.

Results: The clinical characteristics of this patient were very common.

[Effect of decitabine on immune regulation in patients with acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation].

Based on the representative articles in recent years, the different mechanisms of decitabine on immune regulation in patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT) are summarized. Decitabine improves the expression of WT1 gene to stimulate specific cytotoxic T cells which can enhance graft versus leukemia effect (GVL) and improve the expression of FOXP3 gene to stimulate regulatory T cells so as to inhibit the acute graft versus host disease (GVHD). Through the above-mentimed mechanisms, decitabine can improve both therapeutic effect and quality of life in the patients with AML after allogeneic HSCT.

[Eukaryotic expression of von Willebrand factor A1A2A3 triplet and its biological activity].

The aim of this study was to construct the eukaryotic expression vectors harboring human vWF-A1A2A3 gene and to investigate its expression in CHO cells and biologic function so as to provide a basis for further exploring the biologic activity of vWF-A1A2A3. The primers were designed according to published sequences; the human vWF-A1A2A3 was amplified by PCR from vWF cDNA; the fragment of interest was inserted into eukaryotic expression vector pSectag2b by using restriction enzyme and ligase after vWF-A1A2A3 was confirmed by sequencing. The recombinant expression plasmid was transfected into CHO cells and the stable expression product (rvWF-A1A2A3) was detected by using Western blot.

[Immunophenotypic and cytogenetic features in 51 cases of chronic lymphocytic leukemia].

The study was aimed to investigate the immunophenotypic and cytogenetic features of chronic lymphocytic leukemia (CLL) in order to provide an evidence for diagnosis and therapy. Immunophenotypic analysis was performed by using a panel of monoclonal antibodies and three-color immunofluorescence staining methods of flow cytometry in 51 patients with CLL, and the cytogenetic features were analyzed by R-banding technique. The results indicated that among 51 CLL cases, the positive rate of CD19 and CD23 was 96.

[Inhibitory effect of WT1 gene isoform transfection on proliferation of leukemia cell line NB4].

Background & Objective: The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor, and is highly expressed in leukemia cells and negatively correlated with the prognosis of leukemia. WT1 mRNA undergoes alternative splicing at 2 sites resulting in 4 isoforms. The mRNA splice isoforms are constantly expressed in all tissues expressing WT1 with fixed proportions.

[A2 domain of human von Willebrand factor expressed in E. coli and its biological activity].

Von Willebrand factor (vWF) is the unique substrate for the metalloprotease, ADAMTS-13, and plays a pivotal role in the pathology of von Willebrand disease (vWD) and thrombotic thrombocytopenic purpure (TTP). To study the pathogenesis of TTP and to establish a method to diagnose TTP, the DNA fragment of vWF-A2 domain was amplified and inserted into expression vector with 6 x His tag (pQE-30), the recombinant expression vector was transformed into E. coli (strain M15) and induced by IPTG.

[Anti-pancreatic cancer immune response induced by K-ras mutated peptide].

Background & Objective: Since most tumor antigens are uncertain, the immunotherapy against tumors still lingers in clinical trials. Because k-ras proto-oncogene specifically expresses in pancreatic cancer cells with constant mutation site, it may be an ideal target for immunotherapy against pancreatic cancer. This study was to evaluate the feasibility of inducing immunotherapy on pancreatic cancer by K-ras mutated peptides, give experimental evidence to clinical individual therapy on pancreatic cancer.