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<p><b>BACKGROUND</b>MMPs and TIMPs play important roles in tumor angiogenesis and invasion. Studies have shown that TIMP-2 has two roles in tumor invasion. However, its role in leukemic infiltration has not been well investigated. This study explored the roles of TIMP-2 in extramedullary infiltration of acute monocytic leukemic SHI-1 cells both in vitro and in vitro.</p><p><b>METHODS</b>A retroviral vector carrying the human TIMP-2 cDNA was constructed and transfected into the monocytic leukemic cell line SHI-1. The expression of TIMP-2 in the positive clones was determined. The proliferation of SHI-1 cells was examined by MTT assay. Trans-Matrigel invasion assays were used to investigate the infiltration ability in vitro. SHI-1 cells were intravenously injected into pre-treated nu/nu mice to investigate the infiltration ability feature in vitro.</p><p><b>RESULTS</b>The expression of TIMP-2 on the cell membrane was significantly elevated in SHI-1/TIMP-2 cells. Over-expression of TIMP-2 promoted the cells proliferation and the invasions in vitro. The SHI-1/TIMP-2 cells demonstrated higher infiltration ability when intravenously injected into nu/nu mice.</p><p><b>CONCLUSION</b>Over-expression of TIMP-2, especially on the cell membrane, may play important roles in promoting the proliferation and infiltration of SHI-1 leukemic cells.</p>
Subject(s)
Adult , Animals , Humans , Male , Mice , Cell Line , Cell Proliferation , Physiology , Leukemic Infiltration , Mesenchymal Stem Cells , Metabolism , Physiology , Mice, Inbred BALB C , Tissue Inhibitor of Metalloproteinase-2 , Genetics , MetabolismABSTRACT
ObjectiveTo explore the procedures and methods for genetic diagnosis in one nonsyndromic variants of congenital neutropenia (NSVCN) patient and its pathogenic mutation.Methods Genomic DNA was prepared from one NSVCN patient who had progressed to chronic myelomonocytic leukemia and ELA2,HAX1,WASp and GFI1 genes were amplified and sequenced.Results A novel compound heterogeneous mutation consisting of two frame-shift mutations (c.430-1insG and c.655- 9del5bp) was found in HAX1 gene.ConclusionA practically genetic diagnosis procedure for NSVCN has been established,and the novel HAX1 gene mutation may contribute to the etiology of NSVCN.
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Aim To pick out the siRNA which could most effectively inhibit the expression of TLR4 in microglial cells and to detect the cytotoxicity of the transfection complex.Methods Five siRNAs were chemicaly synthesized:four of them were used to inhibit TLR4 expression in microglial cells,the rest was fluorescence-labeled mismatch siRNA as a nagative control.They were all transfected into microglial cells,respectively.TLR4 mRNA was detected 24 h after transfection by RT-PCR and its protein expression wasobserved by Western blot 48 h later.The cytotoxicity of complex was detected using MTT.Results ① The transfection rate was high enough in microglial cells with siRNA(40 pmol)and LipofectamineTM 2000(1 μl).② The TLR4 siRNA pool reduced TLR4 mRNA by 85%(siRNA_(439)),73%(siRNA_(312)),67%(siRNA_(1495))and 33%(siRNA_(2062))respectively compared with mismatch siRNA-treated group 24 h after transfection in a microglial cell line.③ The TLR4 siRNA439 was the most effective siRNA(P<0.01).④ The cell survival rates were above 85% in the groups of Lipofectamine~(TM) 2000 1 μl compound less than 40 nmol·L~(-1) siRNA.Conclusions ① The TLR4 siRNA_(439) can inhibit TLR4 expression most effectively in microglial.② 40 nmol·L~(-1) siRNA and 1 μl Lipofectamine~(TM) 2000 have low cytotoxicity,which are suitable for transfection.
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Objective To investigate the prediction of anti-human leukocyte antigen antibodies (HLA) and anti-major histocompatibility complex class I-related chain A antibodies (MICA) to the development of acute rejection (AR) and kidney allograft function. Methods Forty-one kidney transplant patients were prospectively tested for anti-HLA and anti-MICA. Thirty-seven patients were screened using Luminex/single-antigen beads to determine the HLA and MICA-specific antibody levels at 0,30,90, 180,360,720 and 1080 days post-transplantation. The patients and donors of HLA and MICA allele typing were determined by PCR-SSOP, and donor specific antibody (DSA) and non-donor specific antibody (NDSA) were identified.Simultaneously,their serum creatinine (SCr) levels and clinical data were analyzed. Results Nine patients (21.95 % ,9/41 ) had pre-existing anti-HLA and(or) anti-MICA, including 6 cases of anti-MICA,2 cases of anti-HLA, and one case of anti-MICA and anti-HLA. Nine patients had pre-existing DSA and NDSA. In the 37 patients, 6 patients (16.2% ) developed de novo anti-HLA, and 3 (8.1%) developed de novo antiMICA. In patients positive for de novo anti-HLA, the titer of antibody was gradually increased during the follow-up of three years. Four patients out of 9 patients with pre-existing antibodies were suffered from AR (44.4%); In 6 patients positive for de novo anti-HLA,three cases (50.0%) were suffered from AR; In three patients positive for de novo anti-MICA,no AR occurred (P＜0.05). In two patients positive for DSA of HLAⅡ antibody detected at the third and seventh day after transplantation, the renal grafts were renovecd due to rejection. The Scr levels in patients positive for pre-existing MICA with AR were higher than in those positive for pre-existing MICA without AR at each scheduled time point during the follow-up period (P＜0.05). The Scr levels in patients negative for antibodies pre-transplantation and having AR were higher than in those having no AR at each scheduled time point during the follow-up period (P＜0. 01 ). The Scr levels in patients positive for de novo HLA and MICA and having AR one month following transplantation were higher than in those negative for antibodies and having no AR (P＜0.01 ). Conclusion Pre-existing and de novo anti-HLA were the irnportant factors for the development of AR, but the mismatch of HLA and MICA alleles in donors and patients was primary causes for generation of de novo antibodies.
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Objective To evaluate the changes in expression of NF-κB, IL-6 and TNF-α in spinal cord in a rat model of bone cancer. Methods Seventy-two female SD rats weighing 150-180 g were randomly divided into 3 groups (n = 24 each): control group (group C);sham operation group (group S) and bone cancer pain group (group BP). Bone cancer was induced by intra-tibial inoculation of 1 × 105 Walker 256 breast cancer cells. Paw withdrawal threshold to mechanical stimulation was measured with yon Frey filaments. The expression of NF-κB p65, IL-6 and TNF-α mRNA in the spinal cord was determined by RT-PCR and the expression of NF-κB p65 by immuno-histochemistry and NF-κB p65 positive cell count was determined. Results The paw withdrawal threshold was significantly lower and the expression of NF-κB p65, NF-κB p65 mRNA, IL-6 mRNA, TNF-α mRNA and NF-κB p65 positive cell count in the spinal cord were significantly higher in group BP than in group C and S ( P ＜0.05 or 0.01 ). Conclusion Intra-tibial inoculation of Walker 256 breast cancer cells activates NF-κB in the spinal cord, leading to the increased release of IL-6 and TNF-α and mechanical hyperalgesia.
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Objective To investigate the effect of KIR-HLA receptor-ligand model on the unrelated allo-hematopoietic stem cell transplantation (Allo-HSCT) of acute lymphoblastic leukemia (ALL). Methods The KIR genotype of 23 pairs of ALL patients and their HLA-matched unrelated donors obtained from the Database of China Marrow Donor Program. KIR genotype was determined using PCR-SSP. The expression of inhibitory KIR(iKIR) was determined by flow cytometry analysis on recipients after HSCT. Results Among all 23 donor/recipient pairs, 17 donors with KIR2DL2/L3 could find corresponding HLA-Cw1, 3, 7, 8, 12, 14 ligands in their recipients. Six donors with KIR2DL1 could match with HLA-Cw6, 15 in recipients. Sixteen donors with KIR3DL1 could recognize HLA-Bw4 and 12 donors with 3DL2 could find HLA-AI1 in their corresponding recipients, respectively. Ninteen patients were successfully transplanted, and the death rate of transplantation were 33.3% (2/6)and 40.0% (2/5) in KIR receptor-ligand matched model and the graft versus leukemia(HVG) KIR ligand-mismatching pattern. The frequency of acute graft versus host disease(GVHD) was 50.0% and death rate was 12.5% (1/8) in GVH KIR ligand-mismatching. The incidence rate of activated GVHD(aGVHD) was 20.0% in the HVG KIR ligand-mismatching. Five donor/recipient pairs of KIR gene typing were the KIR-haplotype A, 2 donor/recipient pairs with KIR2DS4 * 001/002 were died, 3 donor/recipient pairs with KIR2DS4 * 003-007 were obtained the disease free survival. The expression of CD158a/2DL1 was low when the patient had no aGVHD, but became much higher when aGVHD occurred. The percentage of NK cell of the patients was decreasing since transplantation, but still higher than normal after HSCT[ (23.4 ± 3.8 ) % vs (2.04 ± 0.58) %, P < 0.05 ]. Conclusion Analysis on KIR-HLA gene loci pattern may provide a useful parameter in predicting the clinical outcome of HLA-matched unrelated allogeneic hematopoietic stem cell transplantation for leukemia patients. Moreover, it may help to increase overall survival and disease free survival after HSCT by preventing the development of GVHD.
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Objective To explore the differences in the expression of inhibin(INH)receptors and activin (ACT)receptors in the follicular/luteinic phase in normal human ovaries and their relationship with female endocrine hormone levels.Methods Real time PCR and immunohistochemistry were used to determine the expression of inhibin receptors(INHR)genes,activin receptors(ACTR)genes.Serum estradiol(E2),follicle stimulating hormone(FSH),luteinizing hormone(LH),INHB,ACTA levels were determined by a solid quantitative sandwich enzyme immunoassay technique(Sandwich ELISA)in 21 women during follicular phase and another 21 women during luteinic phase,the correlations between each gene and each hormone were analyzed.Results(1)ACT type Ⅰ and Ⅱ receptors genes(ACTR Ⅰ A,ACTR Ⅰ B,ACTRⅡA,ACTR Ⅱ B)and INH receptor β-glycan genes were expressed higher in the follicular phase than in the luteinic phase:ACTR Ⅰ A(0.50±0.17 vs 0.36±0.18;P＜0.05),ACTR Ⅰ B(0.050±0.019 vs0.036±0.020;P＜0.05),ACTRⅡ A(0.10±0.04 vs 0.07±0.04;P＜0.05),ACTR Ⅱ B(0.28±0.10vs 0.19±0.11;P＜0.05),β-glycan(0.26±0.10 vs 0.17±0.09;P＜0.01).(2)The intensities of ACTR I A,ACTR Ⅱ A,β-glycan immunostaining in human normal ovaries in the follicular phase were significantly stronger compared to those in luteinic phase.In the follicular phase β-glycan expression was positively correlated with serum E2,FSH,LH,INHB levels.The correlation coefficient was 0.53(P＜0.05).0.74(P＜0.01),0.85(P＜0.01)and 0.76(P＜0.01)respectively.Conclusion In normal human ovary in the follicular phase INH and ACT bind their receptors and down-regulate or up-regulate FSH,thus influencing the follicular development.
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Objective To study the influence of human leucocyte antigen(HLA) and major his-tocompatibility complex class Ⅰ chain-related gene A (MICA) specific antibodies on renal allograft function and graft rejective reaction by monitoring their changes from preoperative to postoperative pe-riods. Methods Twenty-seven patients with renal aliografts were tested with the specificity of anti-HLA antibodies (anti-HLA class Ⅰ and anti-HLA class Ⅱ) and anti-MICA antibodies and their posi-tive value changes by flow PRATM beads. The HLA genotype was integrated to distinguish donor specific antibody(DSA) and non-donor specific antibody(NDSA). Their serum creatinine levels and clinical data were analyzed simultaneously. Results Of the 27 patients, 22 cases accepted renal transplantation from dead bodies and 5 eases accepted from live donors. Except 1 failed patient, the other 26 patients had good functional renal allografts. Twenty-four survival patients were followed up on month 1, 3, 6 and 12 after transplantation. Seven out of 27 patients had pre-exist antibody before transplantation. Among them, 2 patients had anti-HLA antibody; 3 patients had anti-MICA antibody; 2 patients had both anti-HLA and anti-MICA antibody. Three patients with no anti-HLA and anti-MICA antibodies before transplantation created antibodies after transplantation from 3 to 6 months. One patient created NDSA after transplantation and appeared chronic rejection. There were 3 patients who had anti-MICA antibodies before transplantation. The expression levels of antibodies had changed from high to low, but the specific anti-MICA antibody had not changed during the follow-up on month 1, 3, 6 and 12 after transplantation. The patient with pre-transplantation low level of anti-HLA class Ⅱ antibody appeared acute rejection with fever and his CMV was positive as well. The patient's SCr levels changed from 171 μmol/L to 236 μmol/L after I to 3 months post-transplantation. Twenty-four patients were divided into positive and negative groups according to the specific antibody. There was significant difference of SCr levels between the 2 groups 1 month and 1 year after transplantation(P= 0.03, 0.05). Conclusions It is important to detect the specificity and positive value of anti-HLA antibodies and anti-MICA antibody regularly during the post transplantation follow-up. This will make an effective therapy for decreasing the occurrenee and development of acute or chronic rejection and hy-pofunction on renal allograft.