OBJECTIVE:To establish the method for the content determination of total saponins in Naomaitong.METHODS:Colormetric method was applied using panoxadiol as control.The detection wavelength was set at 556 nm.RESULTS:The linear range of panoxadiol was 2.03～10.15 ?g?mL-1(r=0.999 6) with an average recovery of 100.69%(RSD=1.76%,n=6).CONCLUSION:The method is easy to operate with high precision,accuracy and good reproducibility for the quality control of Naomaitong.

ObjectiveTo determine the dose of butorphanol at which 50％ and 95％ effective dose (ED50 and ED95) of patients inhibition uterine contraction pain on analgesic artificial abortion.Methods Twenty-six patients undergoing analgesic artificial abortion were sequentially given different doses butorphanol so that the ED50 and ED95 could be determined by up-and-down sequential test.Anesthetic depth was observed when giving propofol with 200 mg/min speed,uterine contraction pain at awaking immediately,10,20 min after awaleing.Data was analyzed by Probit regression analysis for calculating ED50 and ED95..ResultsButorphanol could restrain uterine contraction pain on analgesic artificial abortion and the ED50 was 246 μg with the 95％ confidence interval of 201 μg to 281 μg,the ED95 was 324 μg with the 95％ confidence interval of 287 μg to 548 μg.ConclusionED50 and ED95 value can be expected as a parameter to optimize analgesic artificial abortion.

Objective:To discuss the screening results and clinical characteristics of children of Miao and Dong nationalities with mediterranean anemia in ethnic minority areas of Qiandongnan State of Guizhou Province,and to clarify the differences of the mediterranean anemia among different minorities.Methods:A total of 1 623 children of Miao and Dong nationalities with mediterranean anemia in minority areas of Qiandongnan State were selected by multistage stratified random sampling method;quantitative analysis of HbA2 and HbF was used to screen the selected children with mediterranean anemia initially;phenol chloroform extraction method was applied to extract the DNA from the children with mediterranean anemia;ASO/RDB-PCR reverse dot blot hybridization method was used to analyze the gene characteristics of the children with mediterranean anemia.Results:A total of 1 623 children of Miao and Dong nationalities were selected as the subjects.Among 938 children with Miao nationality,there were 18 children with positive α-mediterranean anemia and 36 children with positive β-mediterranean anemia,and the positive detection rate was 1.92%.Among 685 children with Tong nationality,there were 13 children with positive α-mediterranean anemia and 24 children with positive β-mediterranean anemia,and the positive detection rate was 3.50%.The detection rates of composite of α-and β-mediterranean anemia in the children of Miao nationality and Tong nationality were 1.49% and 4.61%.There was no significant difference in the detection rates of different kinds of mealiterranean anemia between two nationalities (P<0.05).The major gene mutations in α-mediterranean anemia were——SEA/-αα and-α3.7,and the major gene mutations in β-mediterranean anemia were CD17/N and CD14-15/N,while the major gene types of the composite of α-and β-mediterranean anemia were——SEA/β41-42 and——SEA/β17.There was no difference in the positive rates of major gene types of different kinds of mediter ranean anemia between two nationalities(P<0.05).Conclusion:There is no difference in the positive rate of children of Miao and Dong nationalities with mediterranean anemia in minority areas of Qiandongnan State.CD17/N,——SEA/-αα and ——SEA/β41-42 are the major gene types of α-,β-,and αβ-mediterranean anemia,respectively.

ObjectiveTo investigate the effect of combined spinal epidural analgesia (CSEA) on immune function by observing the changed level of T lymphocyte subsets in maternity sera in labor.MethodsFifty healthy primipara with single birth,vertex present and ASA I between July 2007 and Dec 2007 at the first Affiliated Hospital of Nangchang University,who were in spontaneous labor,were randomly divided into two subgroups when their rerviral dilations were in 2～3 cm.In interfering subgroup( n =25),the puncture point of CSEA was at L3-4 interspace,the fentanyl (20 μg) was used in lumbar anesthesia,the ropivaraine (0.1％) rombined with fentanyl (2 μg/ml) was used in epidural analgesia.Blood samples were taken from the mother vein at cervical dilation in 2 ～ 3 m (T1),fetal disengagement(T2),24 hrs after childbirth ( T3 ).Flow cytometry was used to measure T lymphocyte subsets,Radioimmunoassay (RIA) was used to measure cortisol.In addition,Data on labor progress,VAS score,and neonatal Apgar score were recorded for each patient.Results(1)The active phase in the first stage of labor after analgesia in the CSEA group [ ( 177.64 ± 67.98 ) min ] was significantly lower than that in control group [ (219.40 ± 67.37) min ].No significant difference was found for the active phase in the second stage and the third stage,and for the neonatal Apgar score between the CSEA group [ (32.92 ± 11.59 ) min,( 7.56 ± 2.47 )min,9.20 ± 0.82,respectively ] and the control group [ ( 31.44 ± 13.93 ) min,( 7.28 ± 2.25 ) min,8.84± 1.31,respectively ].(2)The level of cortisol in A group [ ( 548.11 ± 75.67) ng/ml ] was significantly lower than that in C group[ (789.32±96.07) ng/ml] at T2.(3) In two groups,the levels of the CD3+,CD4+,CD4+/CD8 + degraded in different degree at each point,more significantly decreased at T3,and these in C group[ (48.43 ± 6.46) ％,( 31.35 ± 8.93 ) ％,(0.96 ± 0.21 ) ％,respectively ] were significantly lower than those in A group [ (52.3 ± 5.62 ) ％,( 36.90 ± 7.91 ) ％,( 1.16 ± 0.25 ) ％,respectively ].ConclusionsCSEA could shorten the active delivery phase in the first stage of labor,and did not affect the neonatal Apgar score.It can alleviate the inhibitory effect of pain stress response on the immune function.

Objective To investigate whether ultrasound-mediated microbubble destruction can effectively deliver pEGFP-N1 plasmid into human gingival fibroblasts (HGFs).Methods The primary cultured HGFs were divided into 4 groups,that is,plasmid,microbubble+plasmid,ultrasound+plasmid,and ultrasound+microbubble+plasmid groups.pEGFP-N1 plasmid was used to transfect to HGFs as a gene marker with ultrasound-mediated microbubble destruction.The last group was further divided into subgroups to optimize the transfection conditions.After 48 h,phase-contrast fluorescent microscopy was employed to evaluate the expression of green fluorescent protein (GFP).The cell vitality was measured by the MTT assay.Results The transfection efficiency of the ultrasound+microbubble+plasmid group was higher than other experiment groups.Optimal gene expression was found when ultrasound was radiated at 1.5 W/cm2 for 60 s,microbubble was at a concentration of 10% and plasmid was at a concentration of 6.67 ?g/ml,and the transfection efficiency was highest under this condition.Conclusion Under specific conditions,ultrasound mediated microbubble destruction enhances the reporter gene transfection and expression in HGFs.

BACKGROUND: The investigation of culturing, passaging and establishing human limbal stem cells can strengthen the recognition of the stem cells and provide the enough cellular reserve for the basic and clinical research of limbal stem cell transplantation.OBJECTIVE: To explore a method of pessaging and establishing cell line of human limbal stem cells cultured in vitro.DESIGN: Randomized controlled observation.SETTING: Gannan Medical College.MATERIALS: The experiment was performed at Scientific Center of Gannan Medical College and the National Key Laboratory of Ophthalmology Hospital Affiliated to Sun Yat-Sen University from June 2003 to April 2004. Fresh human limbus corneae were isolated from two healthy donors. Procedures were performed according to the informed consent of the donors. Main reagents contained RPMI-1640 (Sigma R8755, containing L-glutamine) and 200 g/L fetal calf serum (FCS) (Gibco 16140-071). DMEM medium, chondroitin sulfatase and human epidermal growth factor (hEGF) were purchased from Sigma Co. USA; HEPES and DMSO were bought from Gibco, USA; 100% glycerinum was purchased from Yunjia Huangpu Pharmaceutical Product Limited Company, PRC; glutaraldehyde was bought from E.Merk, Germany; Alcohol, chlorhydric acid, acetone and methyl aldehyde were purchased from Beijing Chemical Agent Company, PRC; 0.25% parenzyme was bought from Shanghai Xinhua Pharmaceutical Factory, PRC.Above-mentioned reagents were analytical pure grade.METHODS: After digestion, human limbal tissues in limbal basilar part with an abundant pigment were cultured in the culture flask containing RPMI-1640 and 200 g/L FCS and in culture dish containing amniotic extracellular matrix (AECM) as the cultural supporter. Primary and passage cells were observed under light microscope and scanning electron microscope (SEM). The revival ratio of stem cell refrigeration of every generation was calculated by the trypanblau exclusion experiment.MAIN OUTCOME MEASURES: ① Observational results of limbal stem cells during the primary culture and serial subcultivation in vitro, and ② revival ratio of stem cell refrigeration.RESULTS: ①Findings of primary culture: Most limbal stem cells in the culture flask had the adherence and were arrayed uniformly sparsely to form monolayer and adhered to the bottom of culture flask under the inverted phase contrast microscope after 1-day culture. ② Findings of serial subcultivation: After human epidermal growth factor (hEGF) was added into the second passage, cells were scattered into the monolayer and adhered to grow quickly.Morphological variability of all the cells increased obviously when passage the 30th generation. The cellular volume was obviously increasing, and the round or irregular round cells gathered together. The 33rd generation human limbal stem cells still could vigorously differentiate, proliferate and grow in ACEM. ③ The revival ratio of stem cell refrigeration was 82.2%.CONCLUSION: The human limbal stem cell lines were preliminarily established by culturing and freezing the cells of 33 generations in vitro. The human limbal stem cell lines preferred to grow in the culture dish containing AECM as the cultural supporter.

AIM: To establish an HPLC method for determing content of puerarin isoflavones in effective parts of Naomaitong Granules.METHODS: Gradient elution with methanol-water-glacial acetic acid was used as the mobile phase.The flow rate was 1.0 mL/min,and the detection wavelength was set at 250 nm.RESULTS: The average recoveries of puerarin,daizein-8-capiosy(1→6) glucoside,3-methoxyl-puerarin,daidzin,daidzein were 100.89%(RSD = 2.1%),100.23%(RSD = 1.06%),101.04%(RSD = 1.92%),99.82%(RSD = 2.02%),102.06%(RSD = 1.34%) respectively.CONCLUSION: The method is simple,accurate and can be used for quality control of the effective parts in Naomaitong Granules.

OBJECTIVE:To establish HPLC fmgerprints of Naodesheng solid dispersion capsules.METHODS:HPLC method was adopted.The determination was performed on Hyspersil ODS2 column with mobile phase consisted of acetonitrile-0.05％ phosphoric acid (gradient elution) at the flow rate of 1.0 mL/min.The detection wavelengths were 210 nm (puerarin),345 nm (hydroxfsaffior yellow A).The column temperature was 30 ℃,and sample size was 10 μL.Using puerarin and hydroxysaffior yellow A as reference,HPLC fingerprints of 10 batches of sample were determined.Similarity evaluation,common peak identification and chemical components confirmation were performed by using Similarity Evaluation System of TCM Chromatographic Fingerprints (2012 edition).RESULTS:There were 29 and 23 common peaks in HPLC fingerprints of 10 batches of samples at 210 nm and 345 nm,respectively.The similarity was higher than 0.90.The medicinal material attribute of common peaks were Panax notoginseng,Ligusticum chuanxiong,Carthamus tinctorius,Pueraia lobata and Crataegus pinnatifida.Moreover,7 chemical components were identified at 210,345 nm,respectively.CONCLUSIONS:Established HPLC fingerprints can provide reference for identification and quality evaluation of Naodesheng solid dispersion capsules.

OBJECTIVETo investigate the possible role and significance of soluble programmed death-1 (sPD-1)/soluble programmed death ligand 1 (sPD-L1) in the pathogenesis of oral lichen planus (OLP).

METHODSThirty-six patients with OLP (20 cases of reticular OLP and 16 cases of erosive OLP) were enrolled in this study, and 18 healthy people served as controls. Lymphocyte subsets (CD3⁺, CD4⁺, CD8⁺, CD19⁺, CD16⁺ + 56⁺) were examined by flow cytometric analysis and humoral immunity indexes (IgG, IgA, IgM, C3, C4) tested by nephelometry immunoassay. The levels of sPD-1 and sPD-L1 proteins in serum of patients with OLP were determined by enzyme-linked immunosorbent assay. The correlations between the level of sPD-1, sPD-L1 proteins and the immune status and clinical characteristics of patients with OLP were analyzed by SPSS 19.0.

RESULTSCD3⁺, CD4⁺, CD8⁺, CD16⁺ + 56⁺ in patients with OLP were decreased compared with the normal value, while CD19⁺ in patients with OLP was increased compared with the normal value (P < 0.05). C3 and C4 in patients with OLP were decreased compared with the normal value, but IgM in patients with OLP was increased (P < 0.05). The levels of sPD-1 and sPD-L1 proteins in patients with OLP were significantly higher than that in control group [26.10(8.81, 40.00) ng/L vs 17.65(0.00, 26.10) ng/L, 29.53(21.47, 36.76) ng/L vs 22.79(1.19, 28.29) ng/L] (P < 0.05), but the expression of sPD-1 and sPD-L1 was not related with clinical characteristics of OLP. There were negative correlations between the levels of sPD-1 protein and CD4⁺ T cells or CD16⁺ + 56⁺ cells (r1 = -0.378, P1 = 0.007; r2 = -0.365, P2 = 0.009), while there was a positive correlation between the levels of sPD-1 and CD19⁺ B cells (r = 0.482, P = 0.000). There was a negative correlation between sPD-L1 expression level and CD4⁺ and a positive correlation between sPD-L1 expression level and IgG (r¹ = -0.286, P1 = 0.044; r² = 0.365, P₂= 0.029).

CONCLUSIONSIn patients with OLP, the cellular immune function is low with humoral immunity function disorder. PD-1/PD-L1 signaling pathway, which might be influenced by the involvement of sPD-1 and sPD-L1 proteins in a certain extent, may play an important role in the immune pathogenesis of OLP.

B-Lymphocytes ; B7-H1 Antigen ; blood ; Case-Control Studies ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Immunoglobulins ; blood ; Lichen Planus, Oral ; blood ; immunology ; Nephelometry and Turbidimetry ; methods ; Programmed Cell Death 1 Receptor ; blood ; Signal Transduction ; T-Lymphocytes

Objective To analyse the effect of mobilization and collection's time of peripheral blood stem cells(PBSC) from 8 cases of healthy donors. Methods The 10 donors were studied by self-control design.The number of aphereses was two times every donors. Healthy donors received rhG-CSF according to two different PBSC collection starting time: group 1:PBSC collection was starts 2 hours(2 h) after the fourth day or the fifth day of rhG-CSF. group 2:PBSC collection was starts 4 hours(4 h) after the fourth day or the fifth day of rhG-CSF.(The first dose of rhG-CSF was given on day 1, considering day 0 as the day before starting mobilization). In this study we have compared with two groups of apheresis product. Results The MNC count was significantly higher for donors 4 h collection (groups 2) then 2 h. ( groups 1)(P