BACKGROUND:Currently,there is not a standard method for in vitro culture and large scale amplification of umbilical cord blood mesenchymal stem cells(UCB-MSCs).OBJECTIVE:To investigate the isolation,purification and culture of UCB-MSCs in vitro,and to detect its surface marker variation.METHODS:The monocytes were harvested from UCB using 1.077 g/cm3 lymphocytes separating solution and density gradient centrifugation,followed by incubation in an incubator containing 5%CO2 at 37℃.The cell morphological changes were observed at different time points and the expression of surface marker was detected using flow cytometry.RESULTS AND CONCLUSION:The monocytes isolated from the UCB grew initially into numerous hematopoietic cell clones,most of which were granulocyte/macrophage colony-forming units and burst forming unit-erithroid,increasing by(37.1±2.3)and (10.4±1.7),respectively.Switzerland staining showed most of them were granulocyte clones(80,1±85.2)%,next was erythroid clones(14.2±1.8)%.At 7 days after culture,some shuttle fibroblast-like cells and fiat osteogenic-like cell spread the whole plastic well.At 14 days after culture,flow cytometry showed CD38+ cells accounted for 1.64%,and CD34+/CD38+ cells accounted for 1,71%,and CD34+/CD38- were 0.55%.PI+ and Annexin-V+ cells accounted for 0.05% and 0.18% respectively.At 21 days after culture,CD38+,CD34+/CD38+ and CD34+/CD38- cells were 74.32%,1.61%,and 0.24%.The results reveled that UCB-MSCs can be isolated and cultured in vitro.

OBJECTIVE: To investigate the effects of heterogenous suberect spatholobus stem (Spatholobus suberectu, Mucuna birdwoodiana, Millettia oosperma and Millettia dielsiana) on peripheral blood cell counts of mice with bone marrow suppression induced by (60)Co gamma ray irradiation. METHODS: Bone marrow suppression was induced by sublethal dose of (60)Co gamma ray in mice. White blood cell (WBC), red blood cell (RBC) and platelet (PLT) counts in peripheral blood of the mice were detected one day, 3, 7, 14 and 21 days after intragastric administration of different doses of the four kinds of suberect spatholobus stem, respectively. RESULTS: A slowdown of the decrease of WBC, RBC and PLT counts in peripheral blood of the mice with bone marrow suppression was observed after intragastric administration. The WBC, RBC and PLT counts in the Mucuna birdwoodiana-treated and Spatholobus suberectu-treated groups were significantly higher than those in the untreated group (P<0.05). CONCLUSION: All the four kinds of heterogenous suberect spatholobus stem can accelerate the recovery of WBC, RBC and PLT counts in peripheral blood of mice with bone marrow suppression, while the effects of Spatholobus suberectu and Mucuna birdwoodiana are relatively good.

BACKGROUND: Hematogenesis of a body mainly depends on the proliferation of hematopoietic progenitor cell (HPC). Hematopoietic functional impairment will occur when hematopoietic cells are injured by radioactive ray or chemical drug. The proliferation of HPC is the key link of promoting hematogenesis.OBJECTIVE: To study the effects of nine monomers extracted from Spatholobus suberectus Dunn (SSD) on proliferation of HPC in marrow-depressed mice. DESIGN: Randomized controlled trial.SETTING: Department of Pharmacy, General Hospital of Chinese PLA.MATERIALS: This experiment was conducted in the Department of Clinical Pharmacology, General Hospital of Chinese PLA from November 2002 to February 2003. Totally 348 healthy Kunming mice, weighing 22-25g, clean grade, of irrespective gender, were selected in this study (certification: SCXK-2001-001). The animal experiment was approved by the local ethics committee. SSD was provided by Dispensary of Traditional Chinese Medicine, General Hospital of Chinese PLA; monomers (gallocatechin, formononetin, catechin, pyromucic acid, syringic acid, Demethylvestitol, 1,3,5-benzenetriol, ononin, and epicatechin) were extracted from SSD acetoacetate; TGL-16 centrifuger was made in Shanghai 6th Medical Equipment Factory; CO2 incubator was made in SANYO Company, Japan; MK inverted microscope was provided by OLYMPUS Company, Japan.METHODS: Experimental grouping: Mice were randomly divided into 29 groups, including normal group; control group; gallocatechin high-, medium-, low-dose groups; formononetin high-, medium-, low-dose groups; catechin high-, medium-, low-dose groups; pyromucic acid high-, medium-, low-dose groups; syringic acid high-, medium-, low-dose groups; Demethylvestitol high-, medium-, low-dose groups; 1,3,5-benzenetriol high-, medium-, low-dose groups; ononin high-, medium-, low-dose groups; epicatechin high-, medium-, low-dose groups with 12 mice in each group. Experimental intervention: All the mice except the mice in normal group had been given total body sublethal dose of irradiation by 60Co γ-ray (215.3 rontgen/min, 4 Gy dose rate, irradiation time of 107.5 seconds). Normal saline was injected intraperitoneally into 8 mice in normal group and control group at the third day after inadiation. Stored solution 2,0.4,0.08g/L of each monomer was intraperitoneally injected into the mice in each monomer high-, medium-, low-dose groups, respectively, at the third day after irradiation. Experimental evaluation: Thirty minutes after administration, blood of 8 mice in normal, control group and 12 mice in other groups was collected and normal, control and each dose monomer-containing serums were obtained after centrifugation for 15 minutes, filtering through 0.45μm filter membrane. Then 4 mice in normal and control group were killed to study the effects of nine monomers on proliferation of HPC in marrow-depressed mice by counting erythrocyte colony-forming unit (CFU-E), burst-forming uniterythroid (BFU-E), granulocyte-macrophage colony-forming unit (CFU-GM), and megakaryocyte colony-forming unit (CFU-Meg).MAIN OUTCOME MEASURES: CFU-GM, CFU-E, BFU-E, and CFU-Meg in each group. RESULTS: Totally 348 mice were included in the final analysis. CFU-E: The quantity of CFU-E in high-dose of catechin, gallocatechin, syningic acid, and epicatechin groups was significantly higher than that in the control group (P<0.05-0.01) while the quantity of CFU-E in medium-and low-dose of catechin and medium-dose of gallocatechin was also significantly higher than that in the control group (P<0.05). CFU-GM: Except pyromucic acid and ononin groups, the amount of CFU-GM in other groups was significantly higher than that in the control group (P<0.01). BFU-E: Compared with control group, the amount of BFU-E remarkably increased under the effect of each dose of catechin, gallocatechin, syringic acid and high-, medium-dose of epicatechin (P<0.05). CFU-Meg: The amount of CFU-Meg in high-, low-dose syringic acid groups, low-dose gallocatechin groups and each dose group of catechin and epicatechin was significantly higher than that in the control group (P<0.05). Amount of all colonies in the control group was significantly lower than that in the normal group (P<0.01). CONCLUSION: Nine monomers extracted from SSD can promote the proliferation of HPC in bone marrow depressed mice. In particular, the activity of catechin to stimulate proliferation is the strongest.

Objective To observe the effects of fructose,fibroblast growth supplement(FGS) and ethylamine sulfonic acid on the total number,the survival rate and the survival number of Human Retinal Pigment Epithelial(hRPE) Cell.Methods Microencapsulated hRPE cells were plated and cultured in four kinds of mediums,which contained fructose,fibroblast growth supplement(FGS),ethylamine sulfonic acid or no extra ingredient respectively.The total cell number,survival rate and viable cell number of the microencapsulated hRPE cell on day 0th,1st,3rd,7th were calculated.Results After 7days of culture,the lowest cell survival rate of microencapsulated hRPE cells in the four groups was(75.00±3.00)%,but there were no significantly differences(Ps＞0.05) among the groups.The total number of cells in the fibroblast growth factor group([8.00±0.46]×104) and ethylamine sulfonic acid group([7.20±0.36]×104) were significantly higher than the blank group(([6.10±0.56]×104),Ps＜0.05),while no statistical difference was observed in the comparison between the fructose group([6.00±0.46]×104) and blank control(P＞0.05).Conclusion The FGS and ethylamine sulfonic acid can promote the proliferation of the microencapsulated hRPE cells.

BACKGROUND:Several studies have demonstrated that stem cells can differentiate into vascular endothelial cells, and then further differentiate and form blood capillary. Based on this principle, autologous bone marrow mesenchyrnal stem cell (BMSC) transplantation promotes angiogenesis to treat ischemia in lower limb.OBJECTIVE: To evaluate the angiogenesis in rabbit ischemic limbs following autologous BMSC transplantation using high-frequency two-dimensional ultrasound detection in conjunction with Doppler color-flow imaging examination. DESIGN, TIME AND SEI-FING: A randomized, controlled, animal experiment was performed in the Third People's Hospital of Wuxi between March 2007 and April 2008.MATERIALS: Twenty-four New Zealand rabbits were randomized to a control group and a cell transplantation group, with 12 rabbits in each.METHODS: Ischemia in lower limbs was induced in all rabbits. One week following ischemia induction, the cell transplantation group was injected with 0.5 mL cell suspension, comprising 2×106 BrdU-labeled autologous BMSCs cultured in vitro, through multiple sites in the region of gastrocnemius muscle. Simultaneously, the control group received the same amount of physical saline in the same region.MAIN OUTCOME MEASURES: The initial segment of rabbit femoral artery and superficial femoral artery was subjected to high-frequency two-dimensional ultrasound and Doppler color-flow imaging examinations to measure femoral artery vascular internal diameter, blood flow peak velocity, and blood flow acceleration time. Ischemic muscular tissue was taken for immunohistochemical staining to detect transplanted cell distribution and for pathological examination of angiogenesis. RESULTS: Two weeks following autologous BMSC transplantation, high-frequency ultrasound results revealed that femoral artery internal diameter and blood flow peak velocity were greater, but blood flow acceleration time was shorter, in the cell transplantation group than in the control group (P<0.01). Immunohistochemical staining results demonstrated the presence of BrdU-positive cells. Pathological sections displayed that vascular density was significantly higher in the cell transplantation group than ih the control group. CONCLUSION: Autologous BMSC transplantation is a promising, simple, and effective method of treating ischemia in lower limbs owing to its promotion of angiogenesis. Meanwhile, high-frequency ultrasound detection of femoral artery is an effective, practical method to evaluate the clinical outcomes of autologous BMSC transplantation.

Aim To study effects of nine compounds extracted from Spatholobus suberectus Dunn (SSD) on proliferation of hematopoietic progenitor cell (HPC) in marrow-depressed mice. Methods Serum pharmacology experiment was used to observe the influence of nine compounds on growth of CFU-E、BFU-E、CFU-GM、CFU-Meg in marrow-depressed mice. Results Compared with the control, all compounds except pyromucic acid and ononin could significantly stimulate the growth of CFU-GM (P

OBJECTIVE:To establish the GC fingerpriont for Lingyang ganmao tablets.METHODS:The determination was performed on DB-5 ms capillary column.Flame ionization detector was adopted with temperature of 250 ℃ (temperature programming).The temperature of injector was 240 ℃.Carrier gas was nitrogen with flow rate of 1.0 mL/min;the sample size was 1 μL by split sampling with split ratio of 20 ∶ 1.Using peppermint ketone as reference,Similarity Evaluation Software for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 edition) was used for the similarity analysis of 10 batches of Lingyang ganmao tablets.RESULTS:There were 14 common peaks in the batches of Lingyang ganmao tablets,similarity degrees were higher than 0.98.It was proved that the GC profiles and control fingerprint of 10 batches of samples had good consistency.CONCLUSIONS:The established fingerprint can provide reference for the identification and quality evaluation of Lingyang ganmao tablets.

Objective To obtain alpaca single domain antibody targeting Her2.Methods An alpaca was immunized with human recombination Her2 protein mixed with Freund's adjuvant.Total RNA was extracted from the alpaca's blood and was used to synthesize first strand cDNA.Single domain antibody variable region (VHH) gene of the alpaca was amplified by PCR and cloned into pMES4 vector for library construction.After screening, E.coli BL21 (DE3) was transformed with selected clones and was induced with IPTG for the expression of recombinant proteins.The nanobody was purified by nickel ion affinity chromatography column.The affinity of the nanobodies to Her2 was tested.Results After the second round of screening, two antibody clones were selected, H3 and H5.The affinity of H5 was 8.106×10-10mol/L.Histochemistry results showed that H5 could recognize Her2 antigen in breast tumor tissue.Conclusion An Her2 specific nanobody derived from alpaca is obtained through phage display library screening, which can recognize human Her2 antibody in human breast tumor tissue.

To investigate the effects of cembrane-type diterpenes extracted from Sinularia flexibilis on the proliferation of PC12 cells and their protective effects on PC12 cells exposed to glutamate.

Objective To explore the effect of dendritic cells (DC) primed by total RNA extracted from human colon cancer Lovo cell on specific cytotoxicity of cytokine-induced killer cells (CIK) in vitro.Methods Cord blood mononuclear cells extracted by Ficoll density gradient centrifuge were induced into CIK and DC cells separately, and their Immunophenotype was detected by Flow cytometer. Trizol harvested total RNA from colon cancer cell Lovo and the RNAs were loaded to DCs obtained from cord blood as tumor anti gens. Effectors were grouped accordingly as CIK cells co-cultured with DCs transfected with Lovo RNA, CIK cells co- cultured with unloaded DCs and CIK cells. Targets was Lovo cells. In vitro cytotoxicity of CHK cells was extured with DCs loaded with Lovo RNA(76.49%±4.21%), DC + CIK group was lower(53.84% ± 2.15%),and CIK cells group possessed the lowest cytotoxicity(32.20% ± 3.07%), showing statistic significance( P ＜0.05). Conclusion Extraction of total RNA from tumor cells is simple and easy for clinical implementation.Total RNAs acted as antigen to pulse DCs can strengthen the specific cytotoxicity of CIK cells, which will have good prospects for clinical application.