The Biological Characteristics Of Human Umbilical Cord Blood Mononuclear Cells Cultured In Vitro

Backgrounds:The clinically therapeutic tools of vicious diseases, including various tumors, have been enlarged from operation, radiotherapy and chemotherapy to immuno therapy, among which cytotherapy is in a leading position. Human umbilical cord blood (UCB) is an alternative source to bone marrow in cytotherapy recently,as UCB contains abundant hematopoietic stem cells,weaker immunocyte antigenicity, and simple collection and conservation. It is a investigative hot spot about biological and immunological characteristics and mechanisms of antitumor effects of UCB. Objective:To study the biological and immunological characteristics of UCBmononuclear cells (MNC) stimulated by mitogen in vitro, and compare to the peripheral blood(PB) of healthy people, in order to provid objective index and rationale for clinical application of the infusion of UCBMNC. Materials and Methods:UCBMNC and PBMNC were separated by Ficoll and cultured in vitro without serum. One group was added phytohemagglutinin(PHA) and the other was not. Observe the cell clones and harvest the cells before cultivation and various time points after cultivation. We calculate the viable count after staining of trypan blue,detect the cell phenotype of UCBMNC by flowcytometry, and detect the lever of IFN-γ in culture supernatant by ELISA. The RNA of MNC were extracted by Trizol and reverse transcripted into cDNA. Then the cDNA were amplified by FRQ-PCR. The transcriptional lever of IFN-γ was relatively quantitated according to the ratio of the cycle threshold(Ct) of IFN-γ and the Ct of internal contrl gene β-actin. Results:1. MNC growth: PHA(+)groups: the volume, quantity and intensive degree of UCBMNC clones were increased endlessly,while that of PBMNC clones could not be compared with UCBMNC clones. PHA(-)groups: there were no obvious cell clones of UCBMNC and PBMNC.2. MNC cell population: PHA(+) groups: the maximum of UCBMNC cell population was (12.47 + 1. 30) × 10~6/ml,while that of PBMNC cell population was (5.70 ± 1.16) × 10~6/ml . PHA(-)groups: the increase of UCBMNC was slow,while the cell population of PBMNC tapered.3. Cell phenotype of UCBMNC: the T lymphocyte series were mobilized overall after the stimulation of PHA.4. Lever of IFN-γ in culture supernatant: IFN-γ in plasm of UCB was undetectable. IFN-γ in plasm of healthy people was (22. 945 ± 8. 013) ng/L. PHA (+) groups: the maximum of IFN-y in culture supernatant of UCBMNC was (5443.467± 26.385 ) ng/L,while that of PBMNC was about 1500 ng/L. The lever of IFN-y of UCBMNC was still higher than that of PBMNC when the factor of increase of cell population was eliminated.PHA(-) groups: the maximum of IFN- γ in culture supernatant of UCBMNCwas<150ng/L,while that of PBMNC was undetectable. 5. The transcriptional lever of IFN-y: PHA(+)groups: the transcription of UCBMNC increased significantly 8 hours after the stimulation of PHA,while that of PBMNC increased slowly. PHA(-)groups: the transcription of UCBMNC was not obvious, while that of PBMNC was decreased significantly 12 hours after cultivation. Conclusions:1. UCBMNC stimulated by one stimulant can be cultured well in vitro in short-time without serum.2. The reactiveness to PHA of UCBMNC surpass that of PBMNC. UCBMNC surpass PBMNC at amplification of cell population, ability of IFN-y secrete, and the transcriptional lever of IFN-y.3. The biological characteristics of UCBMNC of PHA (+) group: increase of cell number was gentle in the first 2 days, then the speed of increase accelerated after the 2nd day. The cell number reached the peak at the 3rd day. The T series cells of various phenotypes reached the peak at the 2nd day. The ability of IFN-y secrete by unit cell population was strongest at the 2nd day. The transcriptional lever of IFN-Y entered acceleration phase 8 hours after the stimulation of PHA.4. The best time of infusion is 12h ~ 24h after the UCBMNC,which are stimulated by PHA, are cultured in vitro without serum.5. The possible mechanism of DLI:a complicate immunocytes-cytokines network,which is made u...