A semimicroquality evaluation method on Panax notoginseng and its application in analysis of continuous cropping obstacles research samples / 中国中药杂志

Panax notoginseng is a commonly used traditional Chinese medicine with blood activating effect while has continuous cropping obstacle problem in planting process. In present study, a semimicroextraction method with water-saturated n-butanol on 0.1 g notoginseng sample was established with good repeatability (RSD<2.5%) and 9.6%-20.6% higher extraction efficiency of seven saponins than the conventional method. A total of 16 characteristic peaks were identified by LC-MS-IT-TOF, including eight 20(S)-protopanaxatriol (PPT) type saponins and eight 20(S)-protopanaxadiol (PPD) type saponins. The established method was utilized to evaluate the quality of notoginseng samples cultivated by manual intervened methods to overcome continuous cropping obstacles.As a result, HPLC fingerprint similarity, content of Fa and ratio of notoginsenoside K and notoginsenoside Fa (N-K/Fa) were found out to be as valuatable markers of the quality of samples in continuous cropping obstacle research, of which N-K/Fa could also be applied to the analysis of notoginseng samples with different growth years.Notoginseng samples with continuous cropping obstacle had HPLC fingerprint similarity lower than 0.87, in consistent with normal sample, and had significant lower content of notoginsenoside Fa and significant higher N-K/Fa (2.35-4.74) than normal group (0.45-1.33). All samples in the first group with manual intervention showed high similarity with normal group (>0.87), similar content of common peaks and N-K/Fa (0.42-2.06). The content of notoginsenoside K in the second group with manual intervention was higher than normal group. All samples except two displayed similarity higher than 0.87 and possessed content of 16 saponins close to normal group. The result showed that notoginseng samples with continuous cropping obstacle had lower quality than normal sample. And manual intervened methods could improve their quality in different levels.The method established in this study was simple, fast and accurate, and the markers may provide new guides for quality control in continuous cropping obstacle research of notoginseng.

Determination of Th9 cells and IL-9 in children with Mycoplasma pneumoniae infection / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the clinical significance of T helper type 9 (Th9) cells and interleukin-9 (IL-9) in children suffering from Mycoplasma pneumoniae (MP) infection.</p><p><b>METHODS</b>A total of 86 children who were diagnosed with MP infection between January 2013 and June 2014 were classified into upper respiratory infection (URI) group (n=29), mild MP pneumonia (MPP) group (n=32) and severe MPP group (n=25). Twenty-eight healthy children were used as the control group. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and the percentage of Th9 cells in peripheral blood was measured by flow cytometry. Serum IL-9 level was determined using ELISA.</p><p><b>RESULTS</b>The URI, mild MPP, and severe MPP groups had significantly higher percentages of Th9 cells and IL-9 levels than the control group (P<0.05); the mild MPP and severe MPP groups had significantly higher percentages of Th9 cells and IL-9 levels than the URI group (P<0.05), and the two indices were significantly higher in the severe MPP group than in the mild MPP group (P<0.01).</p><p><b>CONCLUSIONS</b>Children with MP infection have an elevated percentage of Th9 cells and IL-9 expression, both of which are positively correlated with the severity of the disease. It can be predicted that Th9 cells and IL-9 can be used as evaluation indicators for the progression and outcome of children with MP infection.</p>

Effects of safflower injection on HEL leukemia cell proliferation and apoptosis and relevant molecular mechanisms / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effect of safflower injection on the proliferation and apoptosis of human leukemia cell line HEL and the relevant molecular mechanisms.</p><p><b>METHODS</b>HEL cells were treated with different concentrations of safflower injection. HEL cells without safflower injection treatment were used as the control group. MTT method was used to detect the inhibitory rate of the HEL cells at 24, 48 and 72 hours after various concentrations of safflower injection treatment (10, 20, 30, 40 and 50 mg/mL). The cell cycle and apoptosis were detected using flow cytometry and the HOXB3-mRNA expression was measured by RT-PCR at 48 hours after safflower injection treatment (10, 20 and 30 mg/mL).</p><p><b>RESULTS</b>Compared with the control group, various concentrations of safflower injection inhibited HEL cell proliferation in a dose-dependent manner (P<0.05). At 48 hours after various concentrations of safflower injection treatment, the number of treated cells in the G2/M phase increased, but that in the S phase decreased, and the apoptosis rate was significantly higher than that in the control group, with a dose-dependent manner (P<0.05). The expression of HOXB3-mRNA in safflower injection-treated cells decreased in a dose-dependent manner compared with the control group (P<0.05).</p><p><b>CONCLUSIONS</b>Safflower injection can inhibit proliferation and induce apoptosis of HEL cells in vitro, and its underlying mechanisms may involve down-regulation of the HOXB3-mRNA expression.</p>

Effect of Apollon siRNA combined with tetramethylpyrazine on proliferation and apoptosis of leukemia K562 cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the effect of small interfering RNA (siRNA) silencing Apollon gene combined with tetramethylpyrazine (TMP) on the proliferation and apoptosis of human chronic myeloid leukemia cell line K562.</p><p><b>METHODS</b>K562 cells were divided into blank control, negative control, and RNA interference (RNAi) group. For the RNAi group, the pGPHI-GFP-Neo-Apollon eukaryotic expression vector based on the best Apollon siRNA fragments screened out in previous experiments was constructed; the blank control group received no treatment, and the negative control group was transfected with negative plasmid vector. The mRNA and protein expression of Apollon was measured by RT-PCR and cell immunofluorescence, respectively. Additionally, TMP (320 μg/mL) was applied to set TMP, TMP+negative control, and TMP+RNAi groups. The cell viability and apoptosis rate were determined by MTT assay and flow cytometry, respectively.</p><p><b>RESULTS</b>The constructed vector was stably expressed in K562 cells. The RNAi group had significantly lower mRNA and protein expression of Apollon than the blank control group and negative control (P<0.05). The RNAi group had significantly increased proliferation inhibition rate and apoptosis rate, as compared with the blank contorl group (P<0.05). The TMP+RNAi group had significantly increased proliferation inhibition rate and apoptosis rate, as compared with the RNAi, and TMP groups (P<0.05).</p><p><b>CONCLUSIONS</b>Apollon siRNA can significantly inhibit the proliferation and promote the apoptosis of K562 cells, and the addition of TMP can further increase the proliferation inhibition rate and apoptosis rate, suggesting that siRNA technology combined with drugs has a significant potential value in the treatment of leukemia.</p>

Effect of astragalus injection on U937 leukemia cells proliferation and apoptosis and relevant molecular mechanisms / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effect of astragalus injection on U937 leukemia cells proliferation and apoptosis and relevant molecular mechanisms.</p><p><b>METHODS</b>Leukemia cell line U937 cells were treated with different concentrations of astragalus (62.5, 125, 250, 500, 1 000 μg/mL). The U937 cells without astragalus treatment were used as the control group. The ability of cell proliferation was measured by MTT method. Flow cytometry was used to explore cell apoptosis. The cell morphology changes were observed under a fluorescent microscope by dyeing Hoechst33258. mRNA expression of c-myc and p27 in U937 cells which was exposed in 1 000 μg/mL astragalus after 0, 12, 24 and 48 hours was detected by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Various concentrations of astragalus injection inhibited U937 cell proliferation effectively compared with the control group (P<0.05). They also induced U937 cells apoptosis and the apoptosis rate reached to (63 ± 4)% in the 1 000 μg/mL astragalus treatment group. mRNA expression level of c-myc was gradually declined and p27 mRNA expression was gradually increased with astragalus treatment time (P<0.01).</p><p><b>CONCLUSIONS</b>Astragalus injection may inhibit proliferation and induce apoptosis of leukemia cell line U937 in vitro. This contributes to down-regulation of c-myc expression and up-regulation of p27 expression.</p>

Effects of Apollon antisense oligonucleotide on proliferation and apoptosis of K562 cells / 中华实用儿科临床杂志

Objective To investigate the effects of Apollon antisense oligonucleotide (ASODN) on proliferation,apoptosis and drug resistance of human leukemia(K562) cells.Methods Specific phosphorothioate ASODN and missense oligonucleotide (MSODN) of Apollon mRNA were synthesized and transfected into K562 cells following cationic liposome.The proliferation inhibition of K562 cells was assessed by MTT.The apoptosis rate was detected by Annexin V-FITC.The sensitivity of K562 cells to etoposide and vincristine was detected by MTT.Results Apollon antisense oligonucleotide inhibition of K562 cells with the concentration and time increased.ASODN at a final concentration of 600 nmol/L could significantly inhibit the K562 cell proliferation.The apoptosis rate was apparently increased (P ＜ 0.01).Conclusions Apollon ASODN may decrease Apollon gene expression,suppress K562 cells proliferation effectively,and induce significant apoptosis of K562 cells.Apollon ASODN is able to reverse the drug resistance via inhibition of Apollon expression and inducement of apoptosis.

Expression of homeobox gene HOXA9 in childhood acute leukemia, and its clinical significance / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the expression of homeobox gene HOXA9 in the bone marrow mononuclear cells of children with acute leukemia (AL) and its clinical significance.</p><p><b>METHODS</b>Forty-six children with AL were divided into acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) groups. Fifteen children with idiopathic thrombocytopenic purpura were selected as a control group. The mRNA expression of HOXA9 was measured by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>HOXA9 expression was detected in 63% of the 52 bone marrow samples from 46 AL children. The positive HOXA9 expression rate in the AML group was significantly higher than in the ALL and control groups (86% vs 35% and 13%; P<0.05). The mRNA expression of HOXA9 in the AML group was significantly higher than in the ALL and control groups (P<0.05). Among the children with AML, those with M5 AML had the highest HOXA9 mRNA level, followed by children with M4 AML and children with M1 and/or M2 AML, but HOXA9 expression was not detected in children with M3 AML. The high-risk subgroup of AML children had relatively high levels of HOXA9 expression. In the children with AML, the initial treatment subgroup had significantly higher positive HOXA9 expression rate and HOXA9 mRNA levels than in the remission subgroup and control group (P<0.05), but there were no significant differences between the latter two groups (P>0.05). The non-remission subgroup had significantly higher HOXA9 expression than the remission subgroup and control group (P<0.05).</p><p><b>CONCLUSIONS</b>High expression of HOXA9 is associated with the occurrence of AL, and its expression level is significantly higher in children with AML than in those with ALL. There is a positive correlation between the expression level of HOXA9 and the risk of childhood leukemia, and high expression of HOXA9 suggests poor prognosis. Therefore, HOXA9 can be used as one of the indices in the diagnosis, treatment and prognosis prediction of childhood AL.</p>

Effect of HOXA10 gene silenced by shRNA on proliferation and apoptosis of U937cell line / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the effects of lentivirus-mediated RNA interference targeting HOXA10 gene on the proliferation, apoptosis and morphology of leukemic cell line U937.</p><p><b>METHODS</b>Four different shRNA plasmids were designed and built to interfere with HOXA10 gene. The four interference plasmids were transfected into 293T cells with the HOXA10 over expression plasmid and then the RNAi efficiency of the four interference plasmids was determined by Western blot. The best one was chosen to transfect 293T cells with lentiviral helping plasmids to produce packaged lentivirus (lenti-shHOXA10). U937 cells were divided into interference group (lenti-shHOXA10), negative control group and untreated group. After infection with the packaged lentivirus, infection efficiency of lentivirus for U937 was detected by flow cytometry, and the expression of HOXA10 gene mRNA and protein was detected by real-time PCR and Western blot. Cell survival was determined by MTT assay. Apoptosis rate was detected by flow cytometry.</p><p><b>RESULTS</b>Lentiviral-shRNA vector of HOXA10 gene was successfully constructed. Compared with the negative control and untreated groups, mRNA level of HOXA10 decreased by (92.3±1.3)%, protein levels decreased by 91.1%, and the inhibition rate of U937 cells [(43.9±0.7)%] increased in the interference group (P<0.05). Wright's staining showed that the ratio of karyon to cytoplasm was reduced and mitotic phase was rare in the interference group. Apoptosis rate in the interference group [(27.1±1.4)%] was significantly higher than in the negative [(19.4±1.9)%] and untreated groups [(5.5±1.3)%] (P<0.05).</p><p><b>CONCLUSIONS</b>Lentivirus mediated RNAi can reduce the expression level of HOXA10, effectively inhibit proliferation and promote apoptosis of U937 cells. HOXA10 gene is expected to become a new target for the treatment of leukemia at gene level.</p>

Systematic study on QAMS method for quality control of Panax notoginseng / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a quantitative method of multi-components by single marker (QAMS) for determining ginsenoside Rg1, Rb1, Rd, Re and notoginsenoside R1 for the purpose of the quality control of Panax notoginseng.</p><p><b>METHOD</b>The relative correction factors (RCFs) between the five active saponins were determined by HPLC-DAD. With any of the five consituents as reference, a QAMS method was established for detect the quantitation of the other four consituents. The durability of the method was evaluated with five different HPLC instruments, five different Cis18 chromatographic columns and four detective wavelengths. Subsequently, the new QAMS method was used to determine the contents of five saponins contained in 43 batches of notoginseng samples, and compare with external standard methods, in order to evaluate the accuracy of the QAMS method.</p><p><b>RESULT</b>When the five saponins were taken for reference, there was no significant difference between the contents of Rg1, Rb1, Rd, Re and R1 contained in the 43 batches of medicines calculated by the QAMS method (Wf) and the content determination result of the external standard method (Ws). The ratio of their results was (Ws/Wr) (94.02 +/- 2.11)%-(99.75 +/- 0.79)%, suggesting that the method was highly accurate. Their relative correction factors showed good durability, ranging between 0.42%-3.7%, 0.52%-3.5% and 0.79%-4.9%, respectively, with different chromatographic columns, different instruments and different detective wavelengths. The relative retention value method could be adopted for accurately position the chromatographic peak of the five consituents, with their values ranging between 0.18%-13%.</p><p><b>CONCLUSION</b>An accurate, rapid and highly durable QAMS method is established for simultaneous determination and location of five saponins, so as to provide reliable basis for the application of the QAMS method in quality control of traditional Chinese medicines.</p>

Reversion of multidrug resistance in leukemia K562 cells by RNA interference targeting homeobox A10 gene / 肿瘤

Objective: To investigate a new strategy to reverse multidrug resistance of chronic myeloid leukemia cell line K562 by RNA interference technique through constructing an eukaryotic vector of short hairpin RNA (shRNA) targeting homeobox A10 (HOXA10) gene. Methods: The eukaryotic vector pGPHI/GFP/Neo-HOXA10 with shRNA targeting HOXA10 gene was constructed and then transfected into K562 cells by positive ion liposome. The stable transfectants were vertificated by reverse transcriptase PCR (RT-PCR) 4 weeks after G418 pressure selection. The changes of sensitivity of K562 cells to leurocristine (VCR) and etoposide (VP-16) after transfection with shRNA-HOXA10 were detected by MTT method, and the apoptosis rate was detected by flow cytometry. Results: After selection with G418, the cell clones stably transfected with pGPHI/GFP/Neo-HOXA10 were successfully constructed and verificated by RTPCR. The half inhibitory concentration (IC50) values of VCR and VP-16 for K562 cells transfected with shRNA-HOXA10 were significantly reduced and the apoptosis rate of K562 cells after HOXA10 gene interference combining with chemotherapy was increased significantly as compared with those of shRNA-negative control group and the normal control group (P0.05). Conclusion: The eukaryotic vector of short hairpin RNA (shRNA) targeting HOXA10 gene can enhance the abilities of VCR and VP-16 to inhibit the cell proliferation and induce the apoptosis of K562 cells, namely an increase of sensitivity to these chemotherapeutics in K562 cells. It is hinted that RNA interference targeting HOXA10 gene may reverse the multidrug resistance of leukemia cells in some degree. Copyright© 2011 by TUMOR.

Effects of bone marrow stromal cells and VLA-4 antibody on apoptosis of childhood leukemia cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the protective effect of bone marrow stromal cells (BMSCs) upon childhood leukemia cells and the influence of VLA-4 antibody in vitro on leukemia cell apoptosis.</p><p><b>METHODS</b>BMSCs from children with acute leukemia-were isolated by human lymphocyte separation medium. BMSCs (adherent) and leukemia cells (suspended) were cultured in vitro. This study included four groups: leukemia cells alone (control), leukemia cells+BMSCs, leukemia cells+BMSCs supernatant and leukemia cells+BMSCs+VLA-4 antibody. The apoptosis rate of leukemia cells in the four groups was determined by Annexin Ⅴ-FITC double-labeled flow cytometry. The expression of survivin and bcl-2 genes in leukemia cells was ascertained by RT-PCR.</p><p><b>RESULTS</b>The apoptosis rate of leukemia cells in the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups was lower than that in the control group (P<0.05). Compared with the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups, the apoptosis rate of leukemia cells in the VLA-4 antibody group increased significantly (P<0.05). In the VLA-4 antibody group, the apoptosis rate of leukemia cells increased with prolonged culture time. There were significant differences in the apoptosis rate between 12 hrs and 24 hrs after VLA-4 antibody treatment (P<0.01). The expression of survivin and bcl-2 genes in leukemia cells from the VLA-4 antibody groups was reduced compared with that from the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups (P<0.05).</p><p><b>CONCLUSIONS</b>BMSCs play protective roles on leukemia cells. VLA-4 antibody can block the adhesion between BMSCs and leukemia cells and promote leukemia cell apoptosis.</p>

Effect of Pulmonary Surfactant on Respiratory Function of Newborn Infants with Respiratory Distress Syndrome in the Advanced Stage / 实用儿科临床杂志

Objective To observe the effect of pulmonary surfactant(PS) on lung function and ventilator parameters of neonatal respiratory distress syndrome(NRDS) in the advanced stage.Methods Twenty-eight infants with NRDS were given PS in one dose by endotracheal intubation on the left side,right side,feet high with head low,and level decub respectively.The dose of PS was 100-150 mg/kg each time,each posture slow note of the drug were required 1/4,out of the straw,hand-controlled ventilation,to reduce fluid loss,with the exception of a clear airway obstruction,within 6 hours after the administration not to shoot back suction,to give mechanical ventilation after the injection.Lung function parameters were also measured:pressure of oxygen in artery[p_a(O_2)],carbon dioxide partial pressure[p_a(CO_2)],the ratio of pressure of oxygen in artery and alveolar oxygen partial pressure[a/Ap(O_2)] and oxygenation index(OI) were determined.Ventilator parameters were determined:oxygen concentration(FiO_2),oxygen peak(PIP),end-expiratory positive pressure(PEEP) and mean airway pressure(MAP) were determined.These numerical data were analyzed and compared before and after treatment with PS.Clinical manifestations,thoracic X-ray changes,survival rate and incidence rate of complications were also analyzed and compared before and after PS therapy.Results p_a(O_2),a/Ap(O_2) showed significant upgrade and OI had a decrease after PS administration in comparison with those before PS therapy.The ventilator parameters(except for PEEP) acquired were also lower after drug administration than those in before drug therapy.There were significant differences in both stages(P_a90%,respiratory sound in 24 cases enhanced,the observation of chest film after 24 h indicated that,lesions in 21 cases improved significantly,5 cases took a favorable turn.The survival rate was 85.7%.The incidence rate of complication was as follows:pneumonia was 25%,patent ductus arteriosus was 10.7%,pneumorrhagia was 7.1% and intraventricular hemorrhage was 3.6%,respectively.Conclusion Respiratory function of NRDS is significantly improved by using PS in the advanced stage,and therapeutic effect is apparent.