Isolation and identification of leukemia stem cells from acute myeloid leukemia patients / 白血病·淋巴瘤

Objective To isolate and identify leukemia stem cells from acute myeloid leukemia patients for further research. Methods By Ficoll density gradient centrifugation, mononuclear cells were firstly separated from bone marrow of patients. According to specific surface markers, CD+34 CD+123 of leukemic stem cells were sorted by flow cytometer. Their ability of self-renewal and differentiation were evaluated by colony formation and cobblestone forming ability. At the same time, the purity and cell morphology of CD+34 CD+123 cells was analysed. Results Comparared with total mononuclear cells, the proportion of the CD+34 CD+123 cells after sorting was 10.7 %, and these cells showed the ability of colony forming and cobblestone forming, and the purity proportion of CD+34 CD+123 cells was 62.1%. Conclusion The leukemia stem cells were isolated successfully and could be used in further study.

Transplantation of atrial natriuretic peptide-expressing fibroblasts reduces blood pressure and increases urine volume in spontaneously hypertensive rats / 生物工程学报

To investigate the potential of gene therapy for the treatment of chronic diseases such as hypertension, chronic heart failure, and chronic renal failure, we established the neonatal rat fibroblast line engineered to secrete the mutant human atrial natriuretic peptide (mhANP), and then transplanted the cell line into young spontaneously hypertensive rats (SHR) subcutaneously. We found that a single transplantation of the cell line caused an obvious rise in the concentration of mhANP in serum 7 d after transplantation ((135 +/- 8) vs (106 +/- 7) pg/mL, P < 0.01). The animals' blood pressure in test group was always remarkably lower than that of empty vector group within 42 d after transplantation, even though the blood pressure in all groups was constantly increasing in the process of ontogeny ((175 +/- 10) mm Hg vs (189 +/- 12) mm Hg, P < 0.05). A maximal blood pressure reduction of 33 mm Hg ((157 +/- 9) mm Hg vs (124 +/- 112) mm Hg, P < 0.01) was observed 14 d post cell transplantation. There was a marked increase in urine volume in test group from second week after treatment beginning ((5.9 +/- 0.7) mL/6 h vs (4.3 +/- 0.8) mL/6 h, P < 0.01) and the effect lasted 14 d ((6.1 +/- 1.1) mL/6 h vs (4.0 +/- 0.8) mL/6 h, P < 0.01), however the statistical difference in concentration of K+ and Na+ in serum and urine was not observed. The results suggested that subcutaneous implantation of fibroblasts-expressing mhANP significantly reduced blood pressure in young SHR during the period of ontogeny and efficiently improved their renal function and the somatic gene transfer of mhANP may have potential value in the treatment of human chronic diseases such as hypertension, chronic heart failure, and chronic renal failure.

High-level expression of foreign genes via multiple joined operons and a new concept regarding the restricted constant of total amount of plasmid DNA per Escherichia coli cell / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell.</p><p><b>METHODS</b>Two series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed. The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments. The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments. The expression level of desired protein was assayed by SDS-PAGE and laser density scanning. Plasmid copy number was determined by incorporation with (3)H-thymidine ((3)H-TdR).</p><p><b>RESULTS</b>No influence of the tandem-joined operons on host growth and plasmid stability was observed. Upon induction, the desired protein accumulations in the CW11 series were 44.9% +/- 3.9%, 51.3% +/- 4.1%, 54.8% +/- 3.3% and 58.2% +/- 3.4% of total cell protein. In the CW12 series, the yields were 32.2% +/- 5.0%, 42.8% +/- 4.1% and 46.9% +/- 4.0% of total cell protein. As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P < 0.01). Further calculation showed that the total amount of plasmid DNA per cell was not significantly different in each series (P > 0.05) and restricted to some extent.</p><p><b>CONCLUSIONS</b>Increasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein. Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E. coli strains under identical incubation condition.</p>