BACKGROUND:Mesenchymal stem cells (MSCs) exist in human tissues.Presently,cell source is single;culture method has great differences;obtained results are not consistent.Thus,it cannot verfy that isolated and cultured cells are identical calls,which is difficult to compare.OBJECTIVE:To compare the biological features of MSCs derived form bone marrow (BM),perpheral blood (PB) and cord blood (CB) under in vitro culture conditions.DESIGN,TIME AND SETTING:The cytological in vitro controlled study was performed at the Department of Hematology,Navy General Hospital of Chinese PLA from June 2007 to December 2008.MATERIALS:A total of 10 donors of hemopoietic stem cell transplantation at the Department of Hematology,Navy General Hospital of Chinese PLA were selected.MB and PB cells were obtained from the same donor,and cell volumes were respectively 20 mL and 2 mL.CB cells (30 mL) were obtained from healthy primipara at the Department of Obstetrics,Navy General Hospital of Chinese PLA.METHODS:MSCs were obtained from BM,PB and CB by Percoll density gradient + adherence method,and then incubated in DMEM/F12 medium containing 10% fetal bovine serum.When 80%-90% confluency,cells were digested in trypsin-EDTA and made into 5×10~8/L cell suspension as P_0.Above-described operation was performed as P_1,and the rest may be deduced by analogy as P_2-P_5.MAIN OUTCOME MEASURES:The following parameters were measured:cell growth morphology;results of Wright-Giemsa staining;results of cytochemistry;cell proliferation amount;cell surface markers using flow cytometry.RESULTS:Time of adherence,time to 50% confluency and time to 80% confluency of BMSCs were earlier comarped with the PBMSCs and UCMSCs.Adherent cells from BM grew in whirpool-like type,while CB and PB did not at 5-7 days.Majority of aderent cells from BM were fibroblast-like cells,and small parts were endothelioid cells.Aderent cells from PB and CB at the fifth generation contained more endothelioid cells and mononuclear and macrophage-like cells besides fibroblast-like cells.PAS stain,Sudan black B stein,alkaline phosphatase (AKP) staining of adherent cells from BM,PB and CB were negative from P_1 to P_5.Compared with P0 cells,number of BMMSCs till P5 was significantly more in PBMSCs and UCMSCs (P ＜ 0.05).Positive rates of CD29,CD44,CD90,CD71,CD105,CD166 and HLA-ABC were 55.9% 92.8% at P0 to P5,but ≤6% following BMMSCs were incubated;19.7%-33.4% at P0 to P5,but ≤10% following PBMSCs were incubated;35.4%-93.2% at P_0 to P_5,but ≤20% following CBMSCs were incubated.Positive rates of CD34,CD45 and HLA-DR were low in BM-,PB-and CB-MSCs.Positive rates of CD14 and CD31 were low in BMMSCs;12.1%-28.3% in PBMSCs,and 8.1%-21.3% in CBMSCs.CONCLUSION:MSCs can be attained from BM,PB and CB.Quantities of MSCs form BM are the highest,with single component,followed by CBMSCs and PBMSCs,with multiple components.

Objective To clone and express the encoding sequence of glyceraldehydes-3-phosphate dehydrogenase(GAPDH)from periodic Brugia molayi(Bm).Methods Total RNA was extraeted from periodic Brugic malayi.The BmGAPDH gene was amplified by RT-PCR.The PCR product was cloned and then subeloned into pcDNA3.1(+)vector.The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification,and were transformed into COS-7 cell subsequently.The expressed protein was identified by SDS-PAGE.Results BmGAPDH mRNA was highiy expressed in transfected COS-7 cell.The deduced amino acid sequence was identical with that of BmGAPDH.The recombinant pnotein wag about Nr 43 000.Conclusion The recombinant plasmid peDNA3.1(+)-BmGAPDH has been constructed and the protein has been expressed correctly.

To explore the feasibility of chemical and biological method in evaluation of the in vitro dissolution rate of Liuwei Wuling tablet (LWT), this experiment investigated the inhibitory effect of LWT dissolving solutions on LX-2 hepatic stellate cells in 0.1% SDS dissolution medium in different dissolving periods. From these results, the cumulative dissolution rate of LWT was obtained based on the cell inhibitory rate. The dissolution rates of deoxyschizandrin, phillyrin, and Specnuezhenide were determined by HPLC method. A novel approach of self-defined weighting coefficient had been created to establish the integrated dissolution rate model. Then f2 similar factor method was used to evaluate the relevance of these two methods. The results showed that f2 values for deoxyschizandrin, phillyrin, Specnuezhenide, and the integrated dissolution were 61, 43, 61 and 75 respectively, indicating that the dissolution of multi-component integration could fully reflect the biological potency of the whole recipe. The dissolution evaluation method for multicomponent integration based on biological activity is expected to be one of the effective means for in vitro dissolution test of LWT.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Kinetics ; Quality Control ; Solubility ; Tablets ; chemistry

OBJECTIVETo observe the clinical effects of Qingchang Huashi Recipe (QHR) for treating active ulcerative colitis (UC) patients of inner-accumulation of damp-heat syndrome (IADHS), and to evaluate its safety.

METHODSUsing a central random system, 60 patients with mild-to-moderately initial onset or relapsed active UC of IADHS were assigned to the test group (30 cases) and the control group (30 cases). Patients in the test group took QHR (Rhizoma Coptidis 6 g, Radix Scutellariae 10 g, Radix Pulsatillae 10 g, Radix Aucklandiae 10 g, parched Radix Angelicae sinensis 10 g, Radix Paeoniae alba 20 g, Cortex Cinnamomi 3 g, Radix Glycyrrhizae 6 g, and so on), 1 dose each time, decocted twice, mixed to 300 mL, taken in two portions. The components were modified according to the condition of illness. Enema of Guanchang Recipe (GCR) was combined (Cortex Phellodendri 30 g, Radix Sophorae flavescentis 10 g, Radix Sanguisorbae 30 g, Rhizoma bletillae 9 g, Radix notoginseng 3 g, Xilei powder 1.5 g), decocted twice, mixed and concentrated to 120 mL, applied before sleep every evening, with an interval of 12 days after 12 successive days). Those in the control group took Mesalazine Enteric-coated Tablet (MECT, 0.25 g/tablet), 1 g each time, 4 times daily. The therapeutic course for all was 8 weeks. The symptom integral, the colonoscopic results, the pathological efficacy, and the remission rate were compared between the two groups. The medication safety was monitored.

RESULTSBy the end of the treatment the improvement of symptoms was superior in the test group to that of the control group (P<0.05). The colonoscopic and pathological results were improved in the two groups, but with no statistical difference (P>0.05). There was no statistical difference in the mucosal healing rate (50.0% vs 43.3%) and the remission rate (36.7% vs 30.0%) between the two groups. Only 1 patient of the control group had moderate increase of ALT during the whole test.

CONCLUSIONSQHR was effective and safe in treating active UC patients of IADHS. Besides, its effect on improving the symptoms was better than that of MECT.

Adult ; Colitis, Ulcerative ; diagnosis ; drug therapy ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Male ; Mesalamine ; therapeutic use ; Middle Aged ; Phytotherapy ; methods ; Treatment Outcome

Adult ; Aged ; Coal Mining ; Health Promotion ; methods ; Humans ; Male ; Middle Aged ; Occupational Health ; statistics & numerical data ; Young Adult

The aim of this study is to develop monoclonal antibody against human hepatocyte growth factor activator inhibitor 1 (HAI-1) for future study of HAI-1. The cDNA fragments of human hepatocyte growth factor activator inhibitor 1 (HAI-1) were subcloned to construct GST-HAI-1 fusion protein expression vectors. The vectors were transformed into E. coli and fusion protein expression was induced by IPTG. The GST-HAI-1 fusion proteins were separated on preparative SDS-PAGE and recovered by electroelution, and used to immunize BALB/c mice. Hybridomas producing monoclonal antibodies against human HAI-1 were prepared by cell fusion technique and characterized by ELISA, Western Blot and immunohistochemical staining. One hybridoma cell line, ZMC6, was obtained, which produces specific antibody against the expressed GST-HAI-1 fusion protein. The monoclonal antibody recognizes both the membrane-type and secretory-type HAI-1 proteins of colorectal tissue. The successful development of anti-HAI-1 antibody provides a powerful tool for further investigation on HAI-1's function.
Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Glutathione Transferase ; genetics ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Proteinase Inhibitory Proteins, Secretory ; analysis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; immunology

This paper analyses the defects of bubble oxygen inhalators currently used, and investigates into their solutions for improvement.
Oxygen Inhalation Therapy ; instrumentation ; methods ; Oxygenators ; standards

ABSTRACT:China has a very high incidence of gastric cancer.Chinese medicine has advantages in the treating of gastric canc-er.Professor Zhou Zhong-yin,awarded National Chinese Medical Science Master,has put forward the theory of cancerous tox-in pathogenesis theory based on his long-term clinical experience.Better effect of the treatment of gastric cancer has been con-firmed by the application of cancerous toxin pathogenesis theory on gastric cancer patients.Cancerous toxin remaining in the stomach is the key pathogenesis of gastric cancer.Cancerous toxin always intermingled with phlegm turbidity,blood stasis, damp turbidity and toxic heat is very common in the patients with gastric cancer.So the principle and treatment of gastric canc-er is the combination of anti-cancer while removing toxic material and other therapeutic principle,such as regulating qi and har-monizing stomach,clearing heat and promoting diuresis,promoting blood circulation to remove blood stasis,reducing phlegm and resolving masses and reinforcing deficiency.

OBJECTIVETo investigate the influencing factors of polyethylenimine (PEI) in gene transfer in vitro.

METHODSCytotoxic effects of PEI on in vitro cultured NIH 3T3 cells were quantified by MTT assay. The interaction between PEI and DNA at different charge ratios was analyzed by agarose gel electrophoresis retardation assay. The expression of gene transfer was monitored in Cos-7 cells using pEGFP and pSV beta plasmids as the reporter gene systems. Influences of chloroquine, albumin, serum, salt ion strength, and Mg(2+) ion and other factors on PEI/DNA transfer efficiency were evaluated.

RESULTThe survival rate of NIH3T3 cells at 6 mg/L of PEI was 64.2% and at 7 mg/L of PEI was 54.4%. Gel electrophoresis retardation assays showed that PEI completely retarded DNA migration at 3.0 PEI nitrogen per DNA phosphate. Chloroquine enhanced the transfection efficiency of PEI. Albumin and serum in the culture medium decreased the transfection efficiency. HBS(HEPES buffered solution) or 150 mmol/L NaCl as the dilution solution of PEI/DNA was superior over 278 mmol/L glucose solution in the transfection efficiency. Mg(2+) in the dilution solution decreased the transfer efficiency of PEI/DNA.

CONCLUSIONPEI is efficient gene transfer agent of eukaryotes in vitro, and can be possibly used in vivo.

Animals ; COS Cells ; Cell Survival ; Chloroquine ; pharmacology ; Culture Media ; Gene Transfer Techniques ; Magnesium ; pharmacology ; Mice ; NIH 3T3 Cells ; Osmolar Concentration ; Polyethyleneimine ; pharmacology

OBJECTIVETo examine the potential function of NMDA receptor NR2A subunit C-terminus in assembling and surface expression of the receptor in HEK293 cells.

METHODSFive vectors GFP- NR2ADeltaC1- DeltaC5 were constructed for expressing N-terminally GFP-tagged NR2A with C-terminal deletion at different regions by using conventional techniques of molecular cloning. The deleted region for NR2ADeltaC1-Delta C5 was 897L-1017S, 1024D-1142P, 1149D-1347G, 1354S-1464V, and 897L-1464V. These plasmids were transfected alone or co-transfected with NR1-1a into HEK293 cells. The surface NMDA receptors were immuno-stained using rabbit antibody against GFP and Cy3 conjugated secondary antibody in living cells.

RESULTThe vectors GFP-NR2ADeltaC1-DeltaC5 were generated and all of them expressed GFP fluorescence in the transfected cells. Surface NMDA receptors were detected by immuno-labeling with anti-GFP in the cells co-transfected by NR1-1a and any one of GFP-NR2ADeltaC1-DeltaC5. However, no surface expression of NR2A proteins was found in the transfected cells with any one of these plasmids alone.

CONCLUSIONWithin the region downstream from the 897L of NR2A subunit, neither a particular domain directly interacted with ER retention domain in NR1-1a C1 cassette, nor that determining ER retention of NR2A subunit itself has been found, indicating that more complicated mechanisms might exist in which the subunit assembling and targeting to plasma membrane of NMDA receptors undergo.

Cell Line ; Gene Deletion ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; metabolism ; Mutation ; Receptors, N-Methyl-D-Aspartate ; analysis ; genetics