Study On Effect Of Angelica Polysaccharides On Resistant Ability Of Cryopreservation Damage And Recoverable Ability From Cryopreservation Damage Of Hemapotoietic Cells

There are thousands of years that Angelica is an important Chinese traditional medicine to nourish the blood and promote blood flow. Modern empirical studies have showed that the chemical composition of angelica is very complex, and Angelica polysaccharides (APS) is one of the main chemical composition in it. The past researches discovered that APS could obviously promote the proliferation and differentiation of BFU-E, CFU-E, CFU-GM and CFU-MK in health and anaemic mice. APS also could promote the proliferation and differentiation of BFU-E, CFU-E, CFU-GM and CFU-Mix in the human being. APS with hemopoietic growth factors(HGFs)could promote expansion of human umbilical cord blood (UCB) the number of mononuclear cells (MNC),CFU-GM,CFU-E in vitro , furthermore could promote formation and adherence of stromal cell . But now there was no report about effect of APS on resistant ability of cryopresevation damage and recoverable ability from cryopersevation damage of hematopoietic cells.Hematopoietic stem cell transplantation(HSCT) is a method that transplant separated hematopoietic stem cells (HSC) from bone marrow, peripheral blood and umbilical cord blood to patients ,and could reconstruct hematopoietic and immunologic function. HSCT is an effective method to treat malignant blood diseases and many kinds of solid tumor. The advantage of cord blood as a source of hematopoietic stem cells for transplantation have become clear: firstly, the proliferative capacity of HSC in cord blood is superior to that of cells in bone marrow. Secondly, the use of cord blood reduces the risk of graft vs host disease. Considering a limited number of HSC in a single unit ,there is a potential obstacle that the cord blood is applied to adult. To widely carry out HSCT and establish umbilical cord blood banks, we must long-term cryopreservate hematopoietic cells. It is an important link of the technique of HSCT to cryopreservate and thaw hematopoietic cells effectively.During the procedure of cryopreservation and thaw, the viability of hematopoietic was damaged partly. There was many studies on cryopreservation of UCB hematopoietic cells to improve the recovery viability , such as different cryoprotectants﹑ colling rates﹑ thawing methods, and so on. The present study discovered that some of HGFs could enhance the proliferative potentiality.But they were very expensive . Investigation discovered that total saponins of panax ginsng (TSPG) could induce the proliferation of thawed hematopoietic stem cells and raise the post-freezing recoverable ability of hematopoietic stem cell. Many polysacchride in plants,especial the polysacchride of Chinese traditional medicine could promote hematopoiesis markedly, It could raise the proliferative potentiality of cryopreservated HSC or not , worthwhile to study deeply.In this research the experimental object is cord blood of healthy ,uncomplicated and term delivery parturient. There are two parts in this research: 1. Study on effect APS on resistant ability of cryopreservation damage on hematopoietic cells. 2. Study on effect of APS on recoverable ability from cryopreservation damage of UCB hematopoietic cells, and explore the effect of APS with hematopoietic growth factors (HGFs) on expansion of UCB hematopoietic cells. The objective of this research is to provid experimental evidence for cryopreservation of application and development of APS.1. Study on effect of APS on resistant ability of cryopreservation damage on UCB hematopoietic cellsObjective: Study on effect of APS on resistant ability of cryopreservation damage on UCB hematopoietic cells.Methods: 1. UCB mononuclear cells (MNC) was separated by three methods of density gradient centrifugation of Ficoll, precipitation method with gelatinum and precipitation method with hydroxyethyl starch(HES) . Before and after separated, the number of MNC was counted , the yield of MNC was calculated . then we compared the efficiency of three separating methods. 2. MNC was cryopreservated for 1, 3 or 6 monthes with 5% final concentration of dimethyl sulfoxide (DMSO) . Techniques of cell counting , typan blue exclusion , flow cytometry and hematopoietic cell culture in vitro were used. Through compared the rate of typan blue exclusion, recovery rate of MNC, CFU-Mix and CD34+ cells, the effect of cryopreservation for different-term on the viability of UCB MNC was known. 3. The MNC were randomly divided into 5 groups: APS 50μg/ml group , APS 100μg/ml group, APS 200μg/ml group, APS 400μg/ml group and control group. MNC of every group were cultured 24h with various concentrations of APS or tales doses culture fluid , the number of MNC was counted before and after culture and calculated the proliferative folds of cord blood MNC. 4. After cultured 24h , MNC of every group were placed in liquid nitrogen with 5% DMSO for 1 month. After thawed, the techniques were used of cell counting , experiment of typan blue exclusin, colong-forming assay and flow cytometry, Through compared the rate of typan blue exclusion, recovery rate of MNC, CFU-Mix and CD34+ cells, the effect of APS on resistant ability of cryopreservation damage on UCB hematopoietic cells was known. Results: 1.The separating effeciency of gelatinum method and HES method was much higher than Ficoll method(P<0.05), HES and gelatinum method have on significant difference (P>0.05). 2. Recovery rate of MNC, CFU-Mix, CD34+ cells and viability of MNC after different-term freezing, there was no significant difference ( P > 0.05 ) . 3. The proliferative folds of APS 100μg/ml group and APS 200μg/ml group was markedly higher than that of control group (P<0.05). 4.The recovery rate of MNC and CFU-Mix of APS100μg/ml group and APS 200μg/ml group was significantly more than that of control group (P<0.05); The rate of typan blue exclusion of APS 50μg/ml,APS 100μg/ml group and APS 200μg/ml group markedly higher than that of control group (P<0.05); The recovery rate of CD34+ cells in cord blood MNC had no significant difference between APS groups and control group(P>0.05).Conclusion: 1. The separating efficiency of precipitation method with gelatinum and HES was higher than Ficoll method. 2. A fraction of MNC lost their proliferatory ability after thawed , but the damage was not deteriorated with freezing time. 3. APS could promote the proliferation ability of cord blood MNC. 4. APS could enhance the resistant ability of cryopreservation damage of UCB hematopoietic cells 2. Effect of APS on recoverable ability from cryopreservation damage of UCB hematopoietic cellsObjective: To study the effect of APS on recoverable ability from cryopreservation damage of UCB hematopoietic cells and APS with hematopoietic growth factors (HGFs) on expansion of UCB hematopoietic cells after thawing.Methods: 1. The MNC after thawed were randomly divided into 5 groups: APS 50μg/ml group ,APS 100μg/ml group,APS 200μg/ml group,APS 400μg/ml group and control group. Thawed MNC were cultured 24h with various concentrations of APS, the effect of APS on the recoverable ability from cryopreservation damage were detected by cell counting assay, typan blue exclusion assay , colorimetric MTT assay for cell proliferation , colong-forming assay and flow cytometry. 2.The MNC after thawed randomly divided into 5 groups: APS 50μg/ml + HGFs group, APS 100μg/ml+HGFs group, APS200μg/ml+HGFs group, APS400μg/ml+HGFs group and control group. The effects of different concentration of APS and combination of HGFs( including SCF, IL-3, GM-CSF, G-CSF and EPO) on the ability of proliferation and differentiation thawed MNC were detected by counting the number of MNC ,colong-forming assay and flow cytometry ,after 14-day liquid cultured. Results: 1.Compared with control group, the number of MNC ,the rate of trypan blue exclusion , the proliferation of MNC and the colony production of CFU-Mix of APS groups. APS 100μg/ml group and 200μg/ml group was significantly enhanced(P<0.05). The percentage of CD34+ cells in cord blood of APS 100μg/ml group was markedly higher than that of control group (P<0.05). 2. Compared with control group, APS(100μg/ml and 200μg/ml) combined with HGFs could raise significantly the expansion fold and the colony production of CFU-Mix of thawed UCB hematopoietic cells(P<0.05).Conclusion: 1. APS could raise the post-freezing recoverable ability of hematopoietic cells. 2.APS combined with HGFs could promote expansion of hematopoietic cells after thawed.