Objective To optimize the preparation condition of Ginkgolides drop pill. Methods The technique of preparation drop pill were optimized by orthogonal test. Results The optimized technique to prepare Ginkgolides drop pills:PEG4000∶PEG6000=1∶1,the temperature for the aqueous mixture was 85 ℃,the temperature for cool aqueous was 5 ℃,active ingredients∶excipient was 1∶3. Conclusion good quality of Ginkgolides drop pill can produced.

BACKGROUND:Bone marrow mesenchymal stem cel s have immunomodulatory properties and have potential applications in immunosuppression. OBJECTIVE:To investigate the isolation and culture of bone marrow mesenchymal stem cel s and the immunoregulation of CD8+T lymphocytes. METHODS:Primary bone marrow mesenchymal stem cel s were isolated from Wistar rats by bone marrow adherence method. The primary cel s were purified by differential adherence and digestion, and col agen type I was added as extracel ular matrix to expand bone marrow mesenchymal stem cel s. Passage 2 bone marrow mesenchymal stem cel s at densities of 0, 1×103, 1×104 and 1×105/wel were co-cultured with CD8+T lymphocytes, fol owed by phytohemagglutinin stimulation for 68 hours. T lymphocyte aggregation and proliferation were detected by staining with staining with 5,6-carboxyfluorescein diacetate succinimidyl ester and MTT, respectively. RESULTS AND CONCLUSION:Different concentrations of bone marrow mesenchymal stem cel s had an inhibitory effect on T lymphocytes, but this effect was weakest for the cel s at the density of 1×103 per wel and strongest for the cel s at the density of 1×105 per wel . Obvious agglomeration was observed in the same media. The proliferation rates of CD8+T lymphocytes were (44.83±4.92)%, (31.94±6.28)%and (15.77±3.98)%, respectively, after co-culture with 1×103, 1×104, 1×105/wel bone marrow mesenchymal stem cel s. There were significant differences between groups (P<0.05). These results showed that primary rat bone marrow mesenchymal stem cel s could be obtained by direct adherence method of the whole bone marrow, and the cel s could be purified by differential adherence combined with digestion and control method. Bone marrow mesenchymal stem cel s could inhibit T lymphocyte proliferation in a concentration-dependent manner.

Child, Preschool ; Female ; Humans ; Sarcoma ; diagnosis ; surgery ; Stomach Neoplasms ; diagnosis ; surgery ; Stromal Cells ; pathology

OBJECTIVE: To analyze the balance function of injured lower limb by dynamic posturography. METHODS: Using the dynamic posturography instrument, the postural evoked responses of sixty-two normal people and two hundred and fifty-eight people with injured lower limb bones and joints were detected. The test was included sensory organization test (SOT) and adaption test (ADT). The results of two groups were compared by t test. RESULTS: Compared with the normal people, the impaired people had significant statistical differences in balance scores of SOT3-SOT6 and proportion score of dynamic proprioception (P < 0.05). There was no obvious decrease in ADT. CONCLUSION The balance function of injured lower limb significantly decreases.
Case-Control Studies ; Humans ; Lower Extremity/physiopathology* ; Postural Balance/physiology* ; Posture/physiology* ; Proprioception/physiology*

BACKGROUND: Bone marrow mesenchymal stem cell transplantation is superior to neural stem cell transplantation to repair spinal cord injury; however, the therapeutic effect is unstable and possibly related to microenvironment.OBJECTIVE: To study the differentiation of cultured in vitro bone marrow mesenchymal stem cells (BMSCs) into neurocytes by establishing a microenvironment and to observe differential expression of protein.DESIGN, TIME, AND SETTING: Observational contrast study was performed at the Laboratory of Neurobiology, Basic Medical College, Harbin Medical University from July 2005 to May 2007.MATERIALS: Adult Wistar rats and newborn fetal rats were used in this study.METHODS: Spinal cord was obtained from fetal rats to culture neurocytes. While, BMSCs were separated from bone marrow of adult rats, and they were then cultured in vitro, proliferated, and labeled with red fluorescin PKH26. BMSCs and neurocytes were individually cultured in the BMSCs group and the neurocyte group, respectively. In addition, BMSCs and neurocytes were co-cultured in vitro in double-layer culture dish in the co-culture group and the layered combination group, respectively.MAIN OUTCOME MEASURES: The obtained cells after 7-day culture were immunofluorescently detected by neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP). Surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) technique was used to analyze associated protein that was apparently changed during the differentiation from BMSCs into neurocytes.RESULTS: Seven days after co-culture, BMSCs were morphologically shared like neurocytes. Immunofluorescence indicated that NSE- and GFAP-positive ratios of BMSCs in the co-culture group were significantly higher than the layered combination group (P < 0.05); while, the ratios in the layered combination group were significantly higher than BMSCs alone group (P < 0.05). Five protein expressions were changed during the differentiation from BMSCs into neurocytes, for example, TIP39_RAT and CALC_RAT expressions increased in the layered combination group, which were 5.344 and 2.805 times as the primary expressions; INSL6_RAT, PNOC_RAT, and PCSKI_RAT expressions decreased, which were 0.380, 0.499, and 0.437 times as the primary expressions.CONCLUSION: By a microenvironment, both BMSCs and neurocytes in the co-culture and layered combination groups can differentiate into neuroblasts; while, contact differentiation ratio is higher than non-contract one. The differentiation is closely related to five proteins, including TIP39_RAT, CALC_RAT, INSL6_RAT, PNOC_RAT, and PCSK1_RAT.

Objective To improve the understanding,the incidence and imaging findings of tuberous sclerosis complex (TSC) combined with cardiac rhabdomyomas (CRs) in fetuses and infants.Methods The imaging findings of 9 infants with TSC combined with CRs and 4 fetuses with TSC combined with CRs from our hospital between June,2006 and November,2013 were retrospectively reviewed.Results The brain MRI of 9 with TSC combined with CRs showed bilateral subependy-mal nodules,subcortical white matter and cortical tubers.Subependymal nodules were isointense or hypointense on spin-echo T1WI and hypointense or hyperintense on spin-echo T2WI.Subcortical white matter and cortical tubers were hypointense or hyperintense on T1WI and hypointense or hyperintense on T2WI.There was varying contrast enhancement.Three of 9 infants presented single cardiac tumor and 6 of 9 infants presented multiply cardiac lesions.CRs on contrast cardiac MRI showed round solid masses in ventricular septums,ventricular outflow tract,ventricle or atrial free walls.The masses were isointense relative to the cardiac muscles on T1WI,T2WI and B-TFE sequence.There was varying contrast enhancement.Four fetuses with TSC on ultrafast MRI showed bilateral multiply subependymal nodules,the nodules were isointense or hyperintense on TFE T1WI and isointense or hypointense signals on SSTSE or B-FFE sequence,Four fetuses with CRs showed isointense to hyperintense solid masses in ventricular septums on ultrafast MRI,ventricle or atrial free walls on B-FFE sequence and SSTSE sequence images.Conclusions TSC in infant and fetus is a kind of neurocutaneous syndrome,usually combines with CRs.Fetal ultrafast and routine MRI is a useful method to make a definite diagnosis for cranial and cardiac lesions.The development of MRI might improve the timeliness and accuracy of the assessment for this disease.

BackgroundOur previous study determined that tetrandrine (Tet) has an inhibitory effect on the proliferation of human Tenon capsule fibroblasts ( TCFs ) in vitro,but its mechanism is poorly understood.ObjectiveThis study was to investigate the effect mechanism of Tet on human TCFs.MethodsHuman TCFs were isolated and cultured from scleral tissue of donor using explant technique.The cells were identified by vimentin antibody staining and morphology.The third generation of cells were seeded in the culture plate at the density of 1 × 105 cells/ml.Twenty-four hours after inoculation,the Tet of 1 × 10-5 mol/L was added in the well of culture plate,and the cells cultured only in 1640 medium served as the control group.The apoptosis of the cells was assessed by TUNEL,and the expressions of bax,bel-2,transforming growth factor-β2 (TGF-β2 ) in TCFs were detected using immunochemistry.Results The cultured cells showed the features of the fibroblasts in shape with the positive response for vimentin.A number of TUNEL positive cells were seen in Tet group and no TUNEL positive response was found in control group.The expression levels (A value) of bax,bcl-2 and TGF-~ protein in TCFs were 0.577 ± 0.009,0.430±0.012 and 0.341 ±0.017 in Tet group,and those in control group were 0.320±0.015,0.819±0.021 and 0.624±0.014 respectively,showing statistically significant differences between two groups( t =33.277,-35.356,-28.093,P＜0.01 ).Conclusions Tet suppresses the proliferation of human TCFs through up-regulating the expression of bax and down-regulating the expressions of bcl-2 and TGF-β2 in vitro.

Background Growth factor-induced proliferation and migration of retinal pigment epithelium (RPE) cells are the major pathological changes of proliferative vitreoretinopathy (PVR).Arsenic trioxide ( As2O3 ) is an active ingredient of Chinese traditional medicines,which has an inhibition on proliferation and migration of tumor cells.However,it is not clear whether As2O3 could inhibit growth factor-induced proliferation and migration of RPE cells. Objective This study was to explore the effects of As2O3 on epidermal growth factor (EGF)-induced proliferation and migration of ARPE-19 cells. Methods RPE cell line (ARPE-19 cells) were cultured.Different concentrations of As2O3(0,0.5,1.0,2.0,5.0,10.0,20.0 μmol/L) were added in the culture plate to treat ARPE-19 cells with or without 10 mg/L EGF in serum-free group for 24 and 48 hours,respectively.The MTT colorimetric assay was used to check the cell viability and evaluate the drug toxicity.The effects of As2O3 on EGF-induced proliferation of ARPE-19 cells were analyzed to get an effective and avirulent concentrations of As2O3.The effects of As2O3 on EGF-induced migration of ARPE-19 cells were observed by scratch-wound assay and the Boyden chamber assay.Results MTT assay showed that the A values were gradually declined with the increase of As2O3 concentrations after As2O3 treatment without EGF for 24 hours and 48 hours ( Fgroup =38.269,P =0.000 ; Ftime =0.874,P =0.358 ).Compared with the control group,no significant differences were seen in the A values of ARPE-19 cells in 0.5-5.0 μmol/L groups (all P＞0.05).Meantime,As2O3 reduced the A values of ARPE-19 cell with 10 mg/L EGF in dose- and time-dependent manner ( Fgroup =152.155,P =0.000 ; Ftime =51.649,P =0.000 ).There were not significant differences in 10 mg/L EGF-induced cell growth after 0.5,1.0,2.0 μmol/L As2O3 was added for 24 and 48 hours ( Fgroup =2.215,P =0.126 ;Ftime =2.230,P =0.155).However,when 5.0-20.0 μmol/L As2O3 added,the A values of 10 mg/L EGF-induced ARPE-19 cells lowed,showing a significant difference in comparison with the control groups ( all P＜0.05),with the cellular inhibiting rate 12％,32％,37％ in 24 hours and 39％,44％ and 53％ in 48 hours.Scratch-wound assay showed that EGF-induced horizontal migration of ARPE-19 cells was slow after 0.5-2.0 μmol/L As2O3 treated,and the same results also appeared in cell lognitudinal migration by Boyden chamber assay,with the inhibitory rates 22％,33％ and 46％ respectively. Conclusions As2O3 is avirulent on ARPE-19 cells within definite concentration range.At ≤ 2.0 μmol/L concentrations,As2O3 dose not affect EGF-induced proliferation of ARPE-19 cells,but it suppresses EGF-induced cell migration.At ≥ 5.0 μmol/L concentrations,As2O3 plays an inhibitory role to EGF-induced proliferation of ARPE-19 cells.

Animals ; Bone and Bones ; chemistry ; Female ; Ovariectomy ; adverse effects ; Ovary ; surgery ; Rats ; Rats, Sprague-Dawley ; Receptors, Estrogen ; analysis ; Transforming Growth Factors ; analysis

OBJECTIVEConditioned taste preference (CTP) is a taste learning reflex by which an animal learns to prefer a substance which tastes not well and has been studied with much interest in recent years. However, the neural substrates of CTP are less known. This study aimed to determine the possible neural path- ways of CTP and whether serum leptin level and the leptin receptor (OB-Rb) in the hind brain are involved following CTP formation.

METHODSWe established CTP of quinine in rats with a 2-bottle preference test. The serum leptin concentrations were detected, the expression of c-fos in the rat brain was tested to determine the nuclei in relation with establishment of CTR Finally, the OB-Rb mRNA expression was examined by RT-qPCR assay in parabrachial nucleus (PBN) and the nucleus of the solitary tract (NST) of the hind brain.

RESULTSCompared with control group, the level of serum leptin was higher in the CTP group (4.58 ± 0.52 vs 1.67 ± 0.25 µg/L, P < 0.01); increased c-fos positive cells were found in the anterior hypothalamus (AH, 221.75 ± 4.96 vs. 178.50 ± 6.63 cells/mm², P < 0.05), the basal lateral amygdala (BLA, 70.75 ± 6.17 vs 56.50 ± 3.62 cells/ mm², P < 0.05) and the nucleus of the solitary tract (NST, 41.25 ± 1.32 vs 32.50 ± 1.02 cells/mm², P < 0.05). But in ventromedial nucleus of the hypothalamus (VMH, 20.75 ± 2.73 vs 38.5 ± 1.54 per 1 mm², P < 005), PBN (21.50 ± 2.24 vs 36.25 ± 1.49 cells/mm², P < 0.05) and the central nucleus of the amygdala (CeA, 22.25 ± 1.53 vs 35.50 ± 2.11 cells/mm², P < 0.05), the number of c-fos positive cells was decreased in the CTP group. In addition, we found OB-Rb mRNA expression in PBN of CTP group rats was higher than that of control group (0.95 ± 0.055 vs 0.57 ± 0.034, P < 0.05), while there was no significant difference of OB-Rb mRNA expression in NST between the two groups.

CONCLUSIONNuclei AH, BLA, NST, VMH, PBN and CeA participate in the formation of CTP. Leptin and its receptor in PBN may be involved in the formation and maintenance of CTP.

Animals ; Conditioning (Psychology) ; Leptin ; blood ; Rats ; Receptors, Leptin ; physiology ; Rhombencephalon ; physiology ; Taste ; physiology