Hematopoietic stem cells (HSCs), defined as cells capable of both self-renewal and differentiation into all blood lineages, sustain hematopoiesis throughout life of individuals. It has been considered that HSCs can infinitely regenerate cells with the same potential because telomere length is maintained upon their self-renewal division. However, it has reported that telomere shortening occurs in human HSCs with aging or after bone marrow transplantation. It is well known that serial bone marrow transplantation results poor reconstitution in mouse. These observations rather suggest that the decrease in stem cell activity is linked to telomere shortening. We therefore examined a relationship between level of repopulating activity and telomere length in HSCs isolated from TERT knockout mice which had null mutation in a catalytic subunit of telomerase and wild-type mice by using competitive repopulation assay and Flow-FISH on bone marrow cells after transplantation. Data clearly show a shortening of telomere length in bone marrow cells reconstituted with single HSCs as compared with those reconstituted with bone marrow competitor cells. There was no remarkable difference in reduction ratio of telomere length between wild-type and TERT KO mice. There however appeared no change in telomere length after transplantation with 100 HSCs. Long-term reconstitution was unsuccessful when 1 or 10 HSCs from third generation of the KO mice were transplantation. These results together indicate that the reconstitution capacity proportionally decreases with reduction in telomere length and that telomerase activity in HSCs is insufficient to prevent their telomere shortening though cell division.
Necrosis or apoptosis is characterized by typical morphological alterations. The morphological characteristic is possible to analyze by the change of cell population in forward scatter (FS) versus side scatter (SS) by flow cytometry. In this study the HL60 cells were treated with 20mM hydrochloric acid (HCl) and were induced to necrosis. The cell population on histogram was divided into 5 regions by FS versus SS. The each region seems to represent cell condition. The cells in each region were analyzed by fluorescence intensities of fluorescein diacetate (FDA), propidium iodide(PI), CD45 and Rhodamin123. In parallel with these experiments, we chased also morphological changes of treated HL60 cells under the light microscope. These results suggest this method make possible to evaluate exactly the process of cell death by HCl.
Malignant fibrous histiocytoma (MFH) can occur rarely in the intracranial meninges. Here, we report a rare case of MFH with multiple recurrences and distant metastases to the lung, thymus, parotid gland and subcutaneous regions, with special reference to cytogenetic analyses. Comparative genomic hybridization analysis revealed loss of 13q and gains of chromosomes 19 and 20 in all of three specimens obtained from the primary, recurrent and subcutaneous metastatic tumors. Additional cytogenetic changes involved loss of chromosome X, and losses of X and 15q at the sites of recurrence and metastasis, respectively. DNA aneuploidy was detected only in the metastasis. The MIB-1 indices at primary, recurrent and subcutaneous sites were 10.9%,17.3% and 19.2%, respectively. Our results suggest that MFH acquire chromosomal imbalance and proliferative potential during the process of recurrence or metastasis.
A cross-talk among a variety of cytokines in local lesions is suspected to play a significant role in the development and healing of the lesions including pleural effusion. A new flow cytometric technique, cytometric beads array (CBA), is a tool to simultaneously measure not only absolute abundance of various cytokines but also their relative abundance in a small amount of liquid sample. Therefore CBA is expected to be a very useful method to provide information for understanding a feedback mechanism of cytokines regulating synthesis and / or secretion of each other in local lesions. The present article reviews this technique and our study using a commercially available CBA kit and pleural effusion obtained from the patients in National Sanyo Hospital.
We assessed the PCR-primer ratio for synthesis of single stranded DNA in the in situ RT-PCR indirect. We tested 4 different primer ratio (sense versus antisense), 50:1, 50:0.2, 50:0.1 or 50:0.05 for detecting rat insulin mRNA. To detect the single stranded DNA synthesized in the RT-PCR, we used the specific probe in three different concentrations, 4, 2 or 1 μg/ml. The intensity of the detection was dependent on the concentration, strong positive by 4.0 μg/ml probe, intermediate positive by 2.0 μg/ml and weak positive by 1.0 μg/ml. The detection level of the control products, which were produced using primers whose ratio were 50:50 or 50:0, were weak positive by 2.0 μg/ml probe and negative by 1.0μg/ml. The results indicated that the primer ratio of 50:1 to 0.05 gave the sufficient intensity in the hybridization. Single stranded DNA in the specimen synthesized prior RT-PCR makes the hybridization supersensitive.