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OBJECTIVE：To analysis the correlation between chrom aticity value and quality index of Atractylodis chinensis decoction piece powder stir-fired with bran ，and to determine its processing time. METHODS ：The processed samples of 16 batches of A. chinensis decoction piece stir-fired with bran （S0-S15，S0 is the raw product of A. chinensis ）were prepared ，and chromaticity values of all samples were determined ，such as lightness value （L*），yellow blue value （b*），red green value （a*）. UPLC fingerprint of sample were analyzed ，and the contents of extract and volatile oil were also determined. Pearson correlation was used to analyze the correlation between the chromaticity value and quality index （relative peak area of each chromatographic peak in UPLC fingerprint ，water-soluble extract content ，alcohol-soluble extract content and volatile oil content ）. Multivariate statistical analysis （principal component analysis ，cluster analysis ，partial least squares discriminant analysis ）was carried out with chromaticity value and quality index ，and the processing time of A. chinensis decoction piece stir-fired with bran was determined by grey correlation method. RESULTS ：In the process of bran frying ，with the extension of processing time ，L* and b* of decoction pieces powder decreased ，and a* increased first and then decreased ；relative areas of peak 1 and peak 2 increased first and then decreased，while relative areas of peak 3（5-hydroxymethyl furfural ）increased，and the areas of the other peaks decreased. The content of the extract did not change significantly with time ，and the content of the volatile oil decreased. The results of correlation analysis showed that the relative peak area of peak 2-27，alcohol-soluble extract content and volatile oil content had a certain correlation with the chromaticity value ，while the relative peak area of peak 1 and water-soluble extract content had no linear correlation with the chromaticity value. Results of multivariate statistical analysis showed that the samples were divided into mild （S0-S5），excessive （S12-S15），moderate （S6-processing time of 18-33 min）. The results of grey correlation method showed that the processing time of A. chinensis decoction piece stir-fired w ith bran should be controlled in the range of 18-24 min，and the optimal processing time was 18 min. CONCLUSIONS ：There is a correlation between chromaticity value of A. chinensis decoction piece powder stir-fired with bran and the relative peak area of 27 chromatographic peaks ，and content of extract and volatile oil. It is suggested that the processing time should be 18 min.
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Objective To establish a novel PCR method that can differentiate the two biotype of Mycoplasma urealytium rapidly based on the disparities of parC gene sequences for clinical routine examination.Methods Design two pairs of specific primers and probes for the targeted gene according to the differences of the parC gene sequences from 14 standard serotypes.The specificity of this amplification was verified by detecting two Mycoplasma urealyticum standard strains including serovar1 and serovar4,50 clinical isolated strains ( 12 are Uu strains and 38 are Up strains) and 7 common bacterias from vagina.Collected 70 swab specimens from patients of Sexually Transmit Department with urogenital inflammation symptom and 71 swab specimens from the gynecology health examination population.All those specimens were detected by culture and our method respectively.The sensitivity of this method was evaluated by comparing with the culture.Differences of the infection rate and distribution of biotypes between different populations were analyzed using statistical software.Results Standard strains of Mycoplasma urealyticum and clinical isolates can be typed into two species by the PCR method without nonspecific amplification.The sensitivity of PCR method is much higher than that of culture ( P＜0.05).The infection rates of Uu,Up and the mixing were 8.57％,61.40％,24.30％ respectively for the patients with vaginitis.However,it was 8.80％,67.60％,8.45％ respectively for the gynecology health examination population.There is a significant difference of the total infection rate between the two population(P＜0.05).It showed no significant difference with the distribution of the two types of simple infection in the two groups ( P＞0.05).But the rate of mixing infection is much higher in the patients with vaginitis ( P＜0.05 ).Conclusion The pathology of Mycoplasma urealyticum may be related to the mixed infection of different biotypes.
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Objective To detect systematic oxidative stress in preeclampsia.Methods (1)Morphological features of placenta hypoxia were observed by histological method ; (2) Level of granulocyte intracellular reactive oxygen species was monitored by dyeing full blood with 2' ,7'-dichlorodihydrofluorescein diacetate (H2DCFDA) ; (3) Level of H_2O_2 in sera was detected by special kits.Results Compared to normal pregnancy,placentas from preeclampsia showed distinct features of hypoxic stress injury,such as more syncytial knots formation,fibrosis emerged,vein in-jury and loss its normal configuration; Fluorescence values of ROS probe in neutrophils from different women were 45.61±12.20(n =49),51.02 ± 13.60(n =56,P <0.01)and 85.10 ± 16.30(n =47,P <0.01); Concentra-tions of H_2O_2were (24.57±5.17)μmol/L(n =49),(26.61±3.25)μmol/L(n =56,P 0.01) and (39.84±9.67)μmol/L(n=47,P<0.01) respectively.Conclusion With the help of histological method,flow cytometry and special kits,systematic oxidative stress can be detected through checking placentic tissues,netrophils and sera of preeclampsia.
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Objective To construct drug-resistant variant recombinant human cytomegalovirus (rHCMV)and identify drug susceptibility by phenotypic and genotypic assays.Methods The UL97 fragments containing Pine I recongnition site and resistant mutation were introduced by site-directed mutagenesis using gene splicing by overlap extension polymerase chain reaction(PCR),and blended with human cytomegalo-virus(HCMV)standard strain ADl 69 genomic DNA proportionally,then the DNA mixture were transfected into MRC-5 fibroblasts by the vector of liposomes.HCMV-PP65 antigen was detected by indirect-immunofluorescent assay to verify rHCMV infection of MRC-5 fibroblasts.When the eytopathic effect(CPE)of homologous recombinant virus reached 100%,the virus was harvested.The purified target virus was screened by plague with different concentrations of ganciclovir(GCV)and the recombinant virus was identified by plague reduction test and DNA sequencing of drug-resistant genes(UL97 and UL54).Results The UL97 fragments containing intended mutations for transfection were constructed successfully.After cotransfected with AD169DNA mixture for 7 days,rHCMV formed cytopathology was obviously visible,which was verified as rHCMV infected focus by HCMV-PP65 antigen test.The UL97 genotypic analysis of recombinant virus obtained by cloning was as expected.No mutation was found by UL54 gene sequencing.The GCV susceptibility of rHCMV positive clone was 15 μmol/L (50% inhibiting concentration),which was 12-fold of standard AD169 strain and was drug-resistant phenotype.Conclusions The rHCMV containing intended mutations is constructed successfully by cotransfeetion into MRC-5 cells and the recombinant virus strain is obtained by GCV screening and plaque purifying.The establishment of this method provides technique platform for identifications of new drug-resistant mutations of HCMV during anti-viral therapy.
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Objective To investigate the effects of protein kinase C (PKC) on proliferation, extracelluar matrix synthesis and transcriptional factor SOX9 (SRY-related high mobility group-box gene 9) expression of rat growth plate chondrocytes in vitro. Methods Rat costochondral growth plate chondrocytes (RGC) were isolated and cultured. The 1st serum free cultured passage RGCs were treated with 1, 10 and 100 nmol/L phorbol 12,13-dibutyrate (PDBu), PKC agonist, cell morphology were observed with inverted microscopy, cell proliferation, COLLAGEN and GAG synthesis were detected by isotope incorporation COLLAGEN TYPEⅡ and AGGRECAN mRNA transcription and SOX9 expression were revealed by RT-PCR and Western blot.Results 100 nmol/L PDBu treatment made the cell morphology of serum free cultured RGC closed to serum group and inhibited proliferation but promoted COLLAGEN and AGGRECAN synthesis, 3H-TdR、3H-proline and 35S-sulfate incorporation of 100nmol/L PDBu group were as 83%, 52.6% and 146.5% as those of serum free control(P
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OBJECTIVE To establish a fingerprinting method by randomly amplified polymorphic DNA.Epidemiological study was carried out on Pseudomonas aeruginosa in Ruijin Hospital. METHODS To obtain optimum scheme on reaction system for randomly amplified polymorphic DNA(RAPD) of P.aeruginosa. RESULTS P.aeruginosa strains isolated from the same ward shared the same RAPD fingerprint type,except for pulmonary ward.Different ward was with different fingerprint type. CONCLUSIONS Prevalent strain was not found in the whole hospital,but within ward exists hospital-acquired infection phenomenon.
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Aim To investigate effects of genistein (5, 7, 4′-trihydroxyisoflavone) on rat costochondral growth plate chondrocyte (RGC) collagen and Sox9 expression. Methods Primary cultured RGC, effects of genistein on collagen synthesis, col2a1 mRNA transcription and Sox9 protein expression of 1st passage RGC were detected by isotope incorporation, RT-PCR and western blotting. Results Genistein inhibited collagen synthesis of 1st passage RGC (P
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To investigate the damage on macrophage of the commercial peritoneal dialysis solution(CDS). Methods Macrophages were seperated from peritoneal fluid remained overnight of seven CAPD patients and TNF-a level of supernatant was determined and compared with those macrophages from uremic patients not yet recieving peritoneal dialysis. Results TNF-a levels of different glucose concentration decreased obviously in experimental group compared with control group, especially lower in 2.5% and 4.25% group. Conclusion In vivo experiment confirms that CDS possesses a long time inhibition on macrophage and this inhibition varies with different glucose concentrations.
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AIM: To confirm that CD4~+CD25~+ regulatory T cells don't have an instinctive defection in IL-2 secretion, and to have an insight into the maturation state of CD4~+CD25~+ T cells in cord blood. METHODS: CD4~+CD25~+ and CD4~+CD25~- T cells were purified from cord blood of term infants (CB) and adult peripheral blood (PB) by autoMACS, and stimulated with PDB plus ionomycin. After 45 hours of culture, cells were detected for expression of CD69 and CD25 by flow cytometry, and the supernatants were measured for 7 kinds of cytokines by Luminex. RESULTS: CD4~+CD25~+ T cells from both CB and PB proliferated comparably with CD4~+CD25~- T cells when stimulated with PDB plus ionomycin. After 45 hours of culture, however, the CD4~+CD25~+ T cells underwent a tendency of cell death. Expression of CD25 was further upregulated when CD25~+ cells were activated. Under stimulation of PDB plus ionomycin, both CD4~+CD25~+ and CD4~+CD25~- T cells in PB secreted high levels of IFN-?, IL-2 and TNF-?, with CD25~+ cells secreted much higher level of IL-5, IL-4 and IL-10 than those in CD25~- cells; CD4~+CD25~+ and CD4~+CD25~- T cells in CB also secreted high level of IL-2 and TNF-? but much lower level of IFN-? than those in PB, and no secretion of IL-5, IL-4 and IL-10 was observed. CONCLUSION: CD4~+CD25~+ regulatory T cells don't have an instinctive defection in IL-2 secretion, otherwise there may be a different TCR signaling pattern in CD4~+CD25~+ T cells from traditional T cells. The CD4~+CD25~+ T cells in cord blood have not fully matured in function.
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AIM: To study the effects of ultraviolet (UV) on mitochondrial functions and apoptosis in HaCaT cells.METHODS: After irradiation by UV at low dose(UVA 2 J/cm~2,UVB 10 mJ/cm~2) and high dose(UVA 6 J/cm~2,UVB(30 mJ/cm~2),) HaCaT cells were cultured for 15 hours.Flow cytometry was used to measure mitochondrial membrane potential,mitochondrial mass and apoptotic rate.Annexin V-FITC/PI staining of apoptotic cells was analyzed by laser confocal microscopy.RESULTS: After UV irradiation,cell proportion with low mitochondrial membrane potential increased with irradiation doses.The proportion of control group,low dose group and high dose group were 7.94%?1.02%,25.87%?4.55% and 39.27%?5.32%,respectively.Cells proportion with low mitochondrial mass increased with irradiation doses.The proportion of control group,low dose group and high dose group were 15.19%?1.58%,40.36%?4.41% and 68.79%?5.46%,respectively.The hypodiploid peaks of DNA content analysis represented the apoptotic rate of HaCaT cells.The apoptotic rate of control group,low dose group and high dose group were 1.82%?0.51%,30.16%?5.47% and 58.49%?5.98%,respectively.To analyze the cells apoptosis by staining with annexin V-FITC and PI,the results were consistent with those of DNA content analysis.Cells in control group showed almost no positive staining cells.Single annexin V-FITC positive cells in low dose group and double positive cells in high dose group were predominant,respectively.CONCLUSION: UV irradiation induces HaCaT cell mitochondrial depolarization,as well as mitochondrial mass loss.These changes are related to cell apoptosis.
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AIM: To establish the methods for rat costochondral growth plate chondrocyte (RGC) separation and culture and investigate their biological features, thereby provide experimental bases for studying the regulation of chondrocyte proliferation and differentiation. METHODS: RGC were obtained by microdissection and digestion, and cultured in monolayer. Morphological changes of the serial passage of RGC and the cell growth kinectics were observed. The cellular GAG and collagen type Ⅱ expression were detected by histochemistry and ICC. RESULTS: There were more than 98% viable cells in the obtained RGC. The morphology of primary cultured RGC was round or polygon. In this experiment, the sixth passage RGC was still maintained and showed polygonal morphology. The index of duplicatings/day increased in the preceding fourth passage RGC and decreased afterwards. There were more than 95% cells expressed collagen type Ⅱ and alcine blue stained positively in the primary RGC, as the passage number increased, the ratio of collagen type Ⅱ expression and alcine blue positive stained RGC dropped abruptly. CONCLUSION: The separation and culture methods adopted in this study can obtain high pure and viable RGC. The preceding three passage RGC maintains their in vivo phenotype, they are idle experimental materials for studying the regulation of proliferation and differentiation of chondrocyte and tissue engineering.
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Objective To observe the effects of methylene blue photochemical(MB-P) inactivation of human cytomegalovirus in blood products.Methods Plasma and red blood cell suspensions containing 10% HCMV and 5?mol/L MB were illuminated by fluorescent light of 38000 Lux for 1 hour, HCMV was used as model viruses for validation of virus inactivation.Results The 50% tissue culture infective dose (TCID_ 50 ) contained in plasma and red blood cell suspensions decreased by 4~6 times.Conclusion MB-P treatment is effective in inactivating the infectivity of HCMV in plasma and red blood cell suspension.
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AIM: To study the effect of VEGF on extracellular H2O2 production in HUVECs and the role of H2O2 in the VEGF-induced proliferation. METHODS: HUVECs was stimulated with 500 ?g/L VEGF. Products of extracellular H2O2 was detected by H2DCFDA staining. MTT method was used to value the influences of 3?106 U/L catalase and 5-20 mmol/L H2O2 to VEGF function. RESULTS: After treatment for 15 min with VEGF, HUVECs appeared fluorescence, and continued to become stronger, peaked at 45 min then decreased. HUVECs, which was treated simultaneity with VEGF and 3?106 U/L catalase, only appeared very faint fluorescence. The proliferation of HUVECs by VEGF was restrained when treated with 3?106 U/L catalase. The extrinsic H2O2 at concentration of 5-10 mmol/L promoted the proliferation of HUVECs but inhibited the proliferation effect of VEGF on HUVECs (P
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0.05). [3H]-proline incorporation in testing groups was 20% higher than that in control. CONCLUSION: The present study suggests EGF is able to enhance RGCs proliferation and collagen synthesis. Dedifferentiation caused by serial passage decreases proliferative effect of EGF on RGCs, but has no effect on collagen synthesis enhancement.
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Objective To investigate the effects of laser irradiation on intracellular ROS(reactive oxidant species),intracellular calcium concentration(_i,and cell membrane integrity in the process of live cell imaging with confocal laser scanning microscopy. Methods The effects of a given laser irradiation on ROS,intracellular calcium concentration(_i and cell viability were revealed respectively by stained ECV-304 with H_2DCFDA,Fluo-4AM and calcein-AM/PI,and visualized and analyzed using ultra view LCI(live cell image)confocal microscopy. Results The irradiation of 488nm laser induced fluorescent intensity of DCF to increase abruptly and attain the climax in about 80 seconds,afterwards the fluorescent intensity fell and returned to the baseline.In the 70 minutes of the irradiation,the fluorescent intensity of intracellular Fluo-4 kept a slightly ascending tendency.The fluorescent intensity of calcein decreased 15minutes after the irradiation,and serval cells were PI positively stained.Conclusion 488nm laser irradiation induces intracellular reactive oxidant species(ROS) and calcium concentration to increase,but there is no significant influence on cell membrane integrity.