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.

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.

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.

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 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.

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

This study established superparamagnetic iron oxide (SPIO)-labeled nerve growth factor-beta (NGF-beta) gene-modified spinal cord-derived neural stem cells (NSCs). The E14 rat embryonic spinal cord-derived NSCs were isolated and cultured. The cells of the third passage were transfected with plasmid pcDNA3-hNGFbeta by using FuGENE HD transfection reagent. The expression of NGF-beta was measured by immunocytochemistry and Western blotting. The positive clones were selected, allowed to proliferate and then labeled with SPIO, which was mediated by FuGENE HD transfection reagent. Prussian blue staining and transmission electron microscopy (TEM) were used to identify the SPIO particles in the cells. The distinctive markers for stem cells (nestin), neuron (beta-III-tubulin), oligodendrocyte (CNPase) and astrocyte (GFAP) were employed to evaluate the differentiation ability of the labeled cells. The immunocytochemistry and western blotting showed that NGF-beta was expressed in spinal cord-derived NSCs. Prussian blue staining indicated that numerous blue-stained particles appeared in the cytoplasma of the labeled cells. TEM showed that SPIO particles were found in vacuolar structures of different sizes and the cytoplasma. The immunocytochemistry demonstrated that the labeled cells were nestin-positive. After differentiation, the cells expressed beta-III-tubulin, CNPase and GFAP. It was concluded that the SPIO-labeled NGF-beta gene-modified spinal cord-derived NSC were successfully established, which are multipotent and capable of self-renewal.
Cells, Cultured ; Dextrans/*diagnostic use ; Embryo, Mammalian ; Magnetic Resonance Imaging ; Magnetics ; Magnetite Nanoparticles/*diagnostic use ; Nerve Growth Factor/*genetics ; Nerve Growth Factor/pharmacology ; Neural Stem Cells/*cytology ; Spinal Cord/*cytology ; Transfection

Objective To investigate the clinical features,karyotype,and the prenatal diagnosis for his sibling of a Chinese patient with rare ring chromosome 20 syndrome induced intractable epilepsy.Methods The clinical data of the patient diagnosed in Peking University People's Hospital were collected.The clinical manifestations,chromosome karyotype were summarized.Results The proband,a boy,started to show intermittent tonic seizures or atypical absence seizures and psychomotor retardation from the age of 11 months.Several anti-epilepsy drugs and globulin had been tried without effect.Common karyotype analysis and epilepsy-related genes analysis revealed no abnormality.However,abnormal karyotype 46,XY,r(20)(p13q13.3) in his peripheral blood lymphocytes was found by high resolution chromosome karyotype analysis with 550 G-banding,and the diagnosis of ring chromosome 20 syndrome,type Ⅱ was confirmed.The mother of the patient underwent amniocentesis at the midterm of the second pregnancy.The cultured amniocytes karyotypes were normal.The second child(a boy) of the family was 1 year old without epilepsy and the psychomotor development was normal.Conclusions Ring chromosome 20 syndrome is a rare human chromosome abnormality.The syndrome is associated with epileptic seizures,behavior disorders and mental retardation.Since karyotype testing is not a routine investigation for the patient with epilepsy,the diagnosis of ring chromosome 20 syndrome is usually delayed or misdiagnosed.The karyotype analysis should be considered for the etiological study of the patients with intractable epilepsy with unknown origin.

Aim To study the effects of EVn-50 on human gastric carcinoma SGC-7901 cells in vitro and invivo. Methods Human gastric carcinoma SGC-7901 cells were cultured in vitro. The inhibitory rate of cells was determined by cell counting and the cell growth curve was made. Plate clone formation assay was carried out to detect the phenotypes of colony formation. Trail of human gastric carcinoma xenografts in nude mouse model was used to draw the transplant tumor growth curve and test inhibition rate of EVn-50 on human gastric carcinoma. The histopathological changes were observed by lightmicroscopy and electronmicroscopy. Results In vitro,EVn-50 at 1,10,100 mg?L-1inhibited the growth and proliferation of SGC-7901 cells in a dose-dependent manner and a time-dependent manner; The colony-forming rate was reduced drastically compared with control group(P