OBJECTIVE: To establish a method for the content determination of domiphen by potassium chromate indicator method.METHODS: The contents of domiphen were determined based on the theory that bromide ion in domiphon could react with AgNO3 to produce silver bromide precipitation.The method was compared with the sodium tetraphenylborate method issued in China Pharmacopeica(2005 edition).RESULTS: The RSD of contents was 0.18%,and the average recovery was 100.2% in the potassium chromate indicator method.There was no significant difference between the results of two determination methods by t-test.CONCLUSION: The potassium chromate indicator method is simple,fast and accurate,which can be used for the content determination of domiphen.

Objective To establish and optimize AFLP reaction system used in providing necessary technique basis for genetic diversity analysis in Asarum sieboldii.Methods Leaves of A.sieboldii were used as experimental materials to analyze various essential elements of the whole process,such as quality of extracted DNA,time of restriction digest and ligation,concentration of Mg2+,primers and Taq DNA polymerase during PCR amplification etc.,so that the most optimal AFLP reaction system could be built up. Results The optimal AFLP reaction system of A.sieboldii has been constructed: in the genomic DNA extraction,mercaptoethanol being utilized and samples being incubated about 30 min at 65 ℃;the genomic DNA being digested by Trul Ⅰ and Pst Ⅰ for 3 h respectively;in PCR amplification,the final concentration of Mg2+ being 1.5 mmol/L and the volume of Taq DNA polymerase being 0.2 ?L.Conclusion The present reaction system for A.sieboldii is able to gain the favorable results of AFLP analysis and can be used in the genetic diversity research of the species.

A quality control (QC) strategy for quantitative and qualitative analysis of “common peaks” in chemical fingerprint was proposed to analyze Yuanhu Zhitong tablet (YZT), using high performance liquid chromatography with diode array detector and tandem mass spectrometry (HPLC-DAD-MS/MS). The chromatographic separation was achieved on an Agilent Eclipse plus C18 column with a gradient elution using a mixture of 0.4‰ ammonium acetate aqueous (pH 6.0 adjusted with glacial acetic acid) and acetonitrile. In chemical fingerprint, 40 peaks were assigned as the “common peaks”. For quantification of “common peaks”, the detection wavelength was set at 254 nm, 270 nm, 280 nm and 345 nm, respectively. The method was validated and good results were obtained to simultaneously determine 10 analytes (protopine, jatrorrhizine, coptisine, palmatine, berberine, xanthotoxin, bergapten, tetrahydropalmatine, imperatorin and isoimperatorin). For qualification of “common peaks”, 33 compounds including 10 quantitative analytes were identified or tentatively characterized using LC-MS/MS. These results demonstrated that the present approach may be a powerful and useful tool to tackle the complex quality issue of YZT.

Objective To evaluate the sensitivity and specificity of prenatal ultrasound for detecting fetal cleft lip and palate,and the diagnosis rate of associated congenital structural and chromosomal abnormalities.Methods Thirty one thousand two hundred and forty five singleton pregnant women accepted prenatal examination and delivered in Guangzhou Women & Children' s Medical Center from Jan.2006 to Dec.2010 were recruited in this study.All pregnant women underwent prenatal ultrasound screening during second trimester,and whose fetuses were suspected to be cleft lip and palate were suggested to accept karyotype analysis.All babies delivered received oral examination to diagnose cleft lip and palate.Results Cleft lip and palate was diagnosed in 48 cases (1.5‰,48/31 245).Among which,there were 16 cases (33.3％,16/48) of cleft lip,21 cases (43.8％,21/48) of cleft lip with cleft palate and 11 cases (22.9％,11/48) of cleft palate.Prenatal ultrasound screening suggested 18 cases of cleft lip and 14 cases were comfirmed after birth with the accuracy rate of 77.8％,3 cases were diagnosed to be cleft lip with cleft palate and one cases was misdiagnosed.Prenatal ultrasound screening suggested 18 cases of cleft lip with cleft palate in accordance with the diagnosis after birth.Thirteen cases were normal in prenatal ultrasound screening,but two were diagnosed as cleft lip and 11 were diagnosed as cleft palate after birth.The sensitivity of prenatal ultrasound screening for cleft lip and cleft lip with cleft palate was 86.5％(32/37),and the sensitivity for cleft lip and palate was 66.7％ (32/48),the false positive rate was 2.1％ (1/48).Ten cases (27.8％,10/36) of cleft lip with cleft palate were found to be complicated with other abnormalities.Nine of the 18 cases prenatally diagnosed cleft lip with cleft palate accepted karyotype analysis and 7 were abnormal.Twenty-three of 36 cases with fetal cleft lip and palate in prenatal ultrasound screening were induced.Conclusions Ultrasound screening has a high sensitivity for detection of cleft lip with or without cleft palate,but difficult to detect cleft palate.The risk of combining with chromosomal defects in cleft lip fetus is very low,but might increase once associated with cleft palate.

Objective To explore the diagnosis and management of functional pancreatic endocrine tumor.Methods Clinical data of 19 cases of functional pancreatic endocrine tumor were retrospectively analyzed.Results 15 cases of insulinoma,2 cases of gastrinoma and 2 cases of glucagonoma were qualitatively diagnosed.The positive rate of preoperative diagosis for type B ultrasonic inspection,CT,MRI,EUS,selective portovenous sampling and intraoperative type B ultrasonic inspection was 15.8％ (3/19),67.5％ (10/16),71.4％ (5/7),87.5％ (7/8),100％(2/2) and 85.7％(6/7) respectively.Of the total 19 cases,7 cases underwent open surgery,11 cases unde rwent laparoscopic surgery,and one case didn't undergo any surgery as liver metastasis had occurred when glucagonoma was diagnosed.The operation methods included tumor enucleation (n=13),distal pancreatic resection (n=3),distal pancreatic resection plus splenectomy (n=1),and pancreatic head resection with duodenum preserved (n=1).Conclusions The measurement of serum insulin,gastrin and glucagon is the main basis for qualitative diagonosis of pancreatic endocrine tumor.Two stage spinal CT thin scanning is the main method for tumor location.Intraoperative type B ultrasonic inspection is the supplement to preoperative location.Tumor enucleation is the main choice of treatment.

Objective:To establish experimental teaching model for slippery pulse by mini-pig.Methods:The model of slippery pulse was established by driping low molecule dextran in vein.The normal and slippery pulse were extracted from axil artery of mini-pig by two experienced traditional Chinese physician through double blind method.Meanwile,the correlative parameters of pulse graph of axil artery such as MSAB,MSBC,HFF,HE/HB and TW were extracted through optimizational extraction method by using NX-8 multifunctional sphygmograph.Results:The pulse rate of slippery pulse of mini-pig was slightly fast than that of normal pulse.The rhythm of slippery pulse was regularity,the nger sensation was powerful,and the pulse syate was smooth.Compared with the normal pluse,the pulse graph of slippery pluse displayed a steep ascend ramus,high and narrow B wave,tiny D wave,lower E valley and obvious F wave.HB,MSAB and MSBC increased(P

AIM:To establish a real-time PCR technique for detection and quantification of TCR ? chain expression and to investigate TCR ? chain expression level in patients with chronic myeloid leukemia(CML).METHODS:Real-time PCR with SYBR GreenⅠ technology was used for detecting TCR ? chain expression level in peripheral blood mononuclear cells from 30 patients with CML and 30 normal individuals.?2-microglobulin gene(?2M) was used as an endogenous reference.Relative changes in TCR ? chain expression level were used by the 2-Ct method between patients with CML and normal individuals.RESULTS:The SYBR GreenⅠ real-time technique for quantitative detection of TCR ? chain expression levels was established successfully.The expression level of TCR ? chain in 18 patients with CML was reduced.However,the TCR ? chain expressed increased in 12 patients with CML.CONCLUSION:The TCR ? chain expression level is divided into down expression(60%) and over expression(40%) groups,and the down expression of TCR ? chain might related to cellular immunodeficiency in most of CML patients.

Objective:To explore the effect of the synthetic immunomodulator CH 2b with a thiazolidin-4-one ring on the pathogenesis of rheumatoid arthritis (RA) mice induced by glucose-6-phosphate isomerase(GPI) mixed peptides.Methods:hGPI325-339 ,hGPI469-483 peptide fragments were mixed with complete freund′s adjuvant fully ,DBA/1 mice were givien subcutaneous injection of the emulsifiers,pertussis toxin to strengthen immunity.And then RA mice were intervened with α-GalCer and CH2b,the changes of body weight ,ankle joint symptoms scores were observed .The joint tissues stained with hematoxylin and eosin ( HE) was used to evaluate the inflammatory cells.Fluorescence-activated cell sorting ( FACS) was used to detect the frequency changes of iNKT cells .Cytometric bead array(CBA) was used to analyze the levels of serum cytokines TNF-α,IL-6,IL-4,IFN-γ.Results: Compared with the model group,α-GalCer,CH2b could reduce the inflammation of the model mice ,significantly improve the body weight growth and the joint swelling(P<0.05).The inflammatory cells infiltration were decreased.More importantly,CH2b improved the frequency of iNKT cells in peak,the difference was statistically significant (P<0.05).The levels of TNF-α,IL-6,IFN-γin serum were significantly decreased(P<0.05) by CH2b.α-GalCer,CH2b could decrease the levels of TNF-α,IL-6,IFN-γ,and the two groups had no statistically significant difference(P>0.05).Conclusion:Immunomodulator CH2b by activating iNKT cells affect the secretion of inflammatory cytokines ,and it relieved the GPI induced arthritis .

Objective To investigate the effects of a novel synthetic immunostimulator CH2b containing thiazolidin-4-one on the function of invariant nature killer T (iNKT) cells isolated from patients with active rheumatoid arthritis (RA).Methods Peripheral blood mononuclear cells (PBMCs) isolated from patients with active RA were in vitro cultured with α-Galcer and IL-2.The iNKT cells were separated by using magnetic activated cell sorting (MACS) method.The effects of CH2b on the proliferation of iNKT cells were analyzed by using MTT assay.MILLIPLEX MAP Human Cytokine/Chemokine kit was used to measure the levels of IFN-γ and IL-4 in the supernatants of iNKT cell culture.The expressions of IFN-γand IL-4 at mRNA level in iNKT cells were analyzed by RT-PCR.Western blot assay was used to detect the levels of T-bet and GATA-3 in iNKT cells.Results CH2b significantly enhanced the proliferation of IL-2 activated iNKT cells isolated from the patients with active RA.CH2b promoted the secretion of IL-4,resulting in a decrease in the ratio of IFN-γ/IL-4.Moreover,CH2b promoted the expressions of GATA-3 and IL-4 at mRNA level in iNKT cells.Conclusion The novel immunostimulator,CH2b,might enhance the immunoregulatory effects of iNKT cells by promoting the GATA-3 pathway-mediated secretion of Th2-1ike cytokines and inducing the differentiation of Th0 to Th2 cells.

[ ABSTRACT] AIM:To establish an animal model of rheumatoid arthritis ( RA) in DBA/1 mice induced by im-munodominant mixed peptides derived from glucose-6-phosphate isomerase (GPI).METHODS: The DBA/1 mice were immunized with emulsified mixed peptide fragments of hGPI 325-339+hGPI469-483 or single peptide hGPI325-339 in com-plete Freund′s adjuvant by subcutaneous injection to induce the model of RA .Body weight , ankle joint symptom scores , the pathological change of the ankle joint , the levels of CD4 +T cells in the spleen and peripheral blood , the proportion of iNKT cells in the peripheral blood , and the levels of TNF-αand IL-6 in serum were detected to evaluate and analyze the model.RESULTS:The hind paw of the model mice appeared red swelling on the 8th day, and then aggravated gradually to the limbs.The red swelling reached peak on the 14th day, and then relieved gradually .Inflammation response dominated by lymphocytes and monocytes was observed in the ankle joint .The inflammatory effect of mixed peptides was more obvious than that of the single one (P<0.05).Compared with control group and the mice treated with single peptide , the weight gain was slow, the amount of CD4 +T cells in the peripheral blood and spleen were increased , the proportion of peripheral iNKT cells in the inflammatory peak was decreased (P<0.05), and the serum level of TNF-αwas increased significantly ( P<0.05) in the mice treated with mixed peptide fragments .CONCLUSION: The immunological characteristics of RA model induced by mixed GPI peptides in DBA/1 mice is closer to that in RA patients , especially in the immunopathology of iNKT cells.Therefore, this model can be used as a new tool for studying the mechanism and immunological intervention of RA.