Objective To investigate the effects of up?regulated miR?206/miR?1 on the proliferation of breast cancer stem cells and the effect mech?anism. Methods Breast cancer stem cells(BCSCs)were isolated from breast cancer cell line MCF?7 by fluorescence?activated cell sorting. Cells in the experiment were divided into the blank control group,the negative control group,the miR?206 group and the miR?1 group. The BCSCs were transfected by negative control mimic,hsa?miR?206mimic and hsa?miR?1mimic in all groups except the blank control group. MiR?206and miR?1 expression levels as well as the transcription factor EVI?1 gene were detected by real time PCR. The expression levels of the transcription factor EVI?1 protein were detected by Western blot. MTT method was used to detect the effects of miR?206 and miR?1 on the proliferation of BCSCs. Results The BCSCs(CD44+/CD24-/low cells)isolated from MCF?7 cell lines were successfully cultured in serum?free medium for subsequent studies. After transfection of hsa?miR?206mimic and hsa?miR?1mimic for 48 hours,miR?206and miR?1relative expression levels increased. EVI?1mRNA ex?pression levels significantly decreased. The results of Western blot and MTT showed that up?regulated expression levels of miR?206 and miR?1 could significantly reduce the expression of EVI?1 protein and inhibited the proliferation of BCSCs. The differences in levels of miR?206,miR?1 and EVI?1 protein were statistically significant(P<0.05). Conclusion Up?regulated miR?206 and miR?1 expression can inhibit the proliferation ability of BCSCs,which may be related to the down?regulation of EVI?1.

In this study, the internal transcribed spacer 2 of nuclear ribosomal DNA (ITS2) sequence was used for i-dentifying A nemone raddeana and its adulterants to ensure the quality of medicines and clinical efficacy. Genomic DNA was extracted from 36 samples using Genomic DNA kit and used as templates for Polymerase Chain Reaction (PCR). Sequence assembly and consensus sequence generation were performed by CodonCode Aligner. The intraspe-cific and interspecific genetic distances were computed and the neighbor-joining tree was constructed by MEGA 5.1 in accordance with the Kimura 2-Parameter (K-2P) model. Results: The length of ITS2 sequence of A nemone rad-deana was 216 bp. The Maximum intraspecific genetic distance was 0.014, the minimum interspecific genetic dis-tance was 0.021. The NJ tree showed that A . raddeana differ from its adulterants obviously. Conclusion: ITS2 se-quence was able to identify A . raddeana and its adulterants correctly stably and correctly, which provides a new tech-nique to its identification.

The aim of this study was to evaluate the safety and efficiency of a novel, oncolytic adenovirus mutant M1 administered in conjunction with immunosuppressive agents. Animal models were established by administering purified M1 either intravenously or retroperitoneally. At different time points, blood samples were taken from the mice for testing of liver and renal function. Microscopic examination of the liver was performed to observe pathological changes. Immunohistochemical analyses were used to evaluate the expression of the adenovirus in the liver. Lymphocyte recruitment to the liver and the activation of adenovirus specific T cells were also analyzed. No signs of general toxicity were observed, but transient increases in ALT and Scr were observed following the administration of M1. Microscopic examination revealed a mild inflammatory response in the liver. Compared to intravenous injection, higher expression levels of adenoviral proteins were observed after retroperitoneal injection. Combined treatment with cyclosporine A resolved the liver and kidney dysfunction and increased the concentration of the adenovirus in the liver. The use of the novel oncolytic adenovirus mutant M1 in vivo is safe, and the combined administration of M1 with immunosuppressive agents was able to enhance the effectiveness and safety profile of M1.

Objective To study the effects of the Notch ligands Dlk1 and recombinant human nucleu factorκB (Jagged1) on the proliferation and transdifferentiation of the type Ⅱ alveolar epithelial cells when the Notch signaling pathway activated.Methods The primary type Ⅱ alveolar epithelial cells (AEC Ⅱ) cultured with recombinant protein Dlk1 and recombinant human nucleu factor-κB (rhNF-κB) (activator of Jagged1),respectively,and then cultured with DMEM (containing 120 mL/L FBS) as controls.Proliferation and differentiation conditions of the AEC Ⅱ were observed at 48 h,72 h,96 h time point by the light microscope and electron microscopes separately.Cell number was counted with hemacytometer; the proliferation rate was measured by methyl thiazolyl tetrazolium (MTT) ; Immunofluorescence double standard method was used to detect the AEC Ⅱ specific surfactant protein C (SP-C) and AEC Ⅰ specific protein aquaporin5 (AQPS) ;the expression of SP-C,AQPS,Dlk1,Jagged1,Notch1 and Hes1 mRNA were detected by real time-PCR.Results The cell population and proliferation:compared with control group,AEC Ⅱ proliferation was promoted in the Dlk1 group [cell numbers (× 109/L) 9.05 ± 0.45 vs 7.95 ± 0.65,11.68 ± 0.43 vs 8.68 ± 0.52,11.55 ± 0.17 vs 8.73 ± 0.48,all P ＜ 0.05 ; MTT results (value A) 0.699 ± 0.050 vs 0.462 ± 0.080,0.912 ± 0.080 vs 0.535 ±0.040,0.726 ±0.050 vs 0.540 ±0.020,all P ＜0.05] and decelerated AEC Ⅱ transdifferentiation into AEC Ⅰ ; while AEC Ⅱ proliferation was inhibited in rhNF-κB group [cell numbers (× 109/L) 4.95 ± 0.33 vs 7.95 ± 0.65,4.73 ±0.71 vs 8.68 ± 0.52,4.04 ± 0.11 vs 8.73 ± 0.48,all P ＜ 0.05; MTT results (value A) 0.398 ± 0.030 vs 0.462 ± 0.080,0.402 ± 0.070 vs 0.535 ± 0.040,0.380 ± 0.110 vs 0.540 ± 0.020,all P ＜ 0.05] and accelerated AEC Ⅱ transdifferentiation into AEC Ⅰ.One-Way ANOVA showed that the difference among the 3 groups had statistical significance (cell numbers:F =486.73,P =0.02; cell proliferation:F =37.16,P =0.02).The mRNA expression:compared with control group,the expression of SP-C mRNA of Dlk1 group was significantly higher (P ＜ 0.05) while the expression of AQP5 mRNA was remarkably lower and delayed (P ＜ 0.05),the expression of Jagged1 mRNA was weak or little,Dlk1 and Notch1 mRNA were up-regulated (P ＜ 0.05),and the Hes1 mRNA was reduced (P ＜ 0.05) ; the expression of SP-C mRNA of rhNF-κB group was significantly reduced (P ＜ 0.05),while the AQP5 mRNA expressed ahead of time and increased (P ＜ 0.05),Jagged1,Hes1 and Notch1 mRNA were higher (P ＜ 0.05),and the Dlk1 mRNA was weak.One-Way ANOVA showed that the difference in the expressions of SP-C,AQP5,D1k1,Jagged1,Hes1 and Notch1 mRNA among the 3 groups had staistical significance (F =96.80,P =0.01 ; F =82.55,P =0.01 ; F =269.80,P=0.00;F =312.34,P =0.00;F =169.17,P =0.01;F =19.85,P =0.02).Conclusions There are varied effects on proliferation and differentiation of the AEC Ⅱ when the Notch signaling is activated by different ligands:Dlk1 promoted proliferation and inhibited differentiation,while Jagged1 inhibited proliferation and promoted transdifferentiation.

This study aimed to design a pair of primers for amplifying internal transcribed spacer 2 region which was used to identify Gastrodia elata through optimizing of DNA extraction and PCR amplification process. Two sequences are used for research object by amplification of universal primers. Design three pairs of specific primers by Primer Premier 5.0 and select the highest specificity through the study of 22 samples. The results showed that identification efficiency of the primer named TM2F-2R is as high as 90.9% when Annealing Temperature is equal to 54 degrees Celsius. Therefore, TM2F-2R can be used as primers ITS2 sequences of G. elata, this article provides a set of accu-rate and stable identification methods for G. elata in the molecular identification.

Objective: This study aimed to discriminate between Eupatorii Herba and its adulterants in order to guarantee the quality and clinical curative effect of this medicinal material. Methods: Genomic DNA extracted from Eupatorii Herba was used as templates. The internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA was amplified. Sequence assembly and consensus sequence generation were performed by CodonCode Aligner. The intraspecific and interspecific genetic distances of Eupatorii Herba and its adulterants were computed by MEGA5 and the phylogenetic tree was constructed using the neighbor-joining (NJ) method. Results: The length of ITS2 sequence of Eupatorii Herba was 218 bp. The maximum intraspecific genetic distance (K2P distance) of Eupatorii Herba was 0.0092. The minimum interspecific genetic distance of Eupatorii Herba and its adulterants was 0.024. The NJ trees showed that the ITS2 sequence would be used to identify Eupatorii Herba and its adulterants. Con-clusion: ITS2 sequence was able to identify Eupatorii Herba and its adulterants correctly and it provided a new technique to ensure clinical safety in utilization of traditional Chinese medicines.

UNLABELLEDOBJECTIVE To investigate an operative method of combined resection of preseptal fat: and partial retro-orbicularis oculus fat (ROOF) for correction of upper eyelid heaviness, and evaluate the efficacy and safety of the method.

METHODSPreseptal fat lies widely under the orbicularis oculi in the upper eyelid, and retro-orbicularis oculus fat (ROOF) lies in the lateral supraorbital area. Combined resection of preseptal fat and partial ROOF was performed in patients selected by examination. The efficacy and safety were evaluated by follow-up study.

RESULTSFrom May 2011 to July 2013, 38 selected patients received the treatment with 3 months to 28 months follow up. The heaviness of upper eyelid improved in all cases. One patient developed postoperative hematoma, and another patient had a transient numbness over the lateral upper brow region. 37 patients were satisfied with the result.

CONCLUSIONSCombined resection of preseptal fat and partial ROOF was effective in reducing the heaviness of upper eyelid, without major complications. The operative method should be an important adjunct for selected patients undergoing blepharoplasty.

Adipose Tissue ; surgery ; Blepharoplasty ; adverse effects ; methods ; Eyelids ; surgery ; Facial Muscles ; Follow-Up Studies ; Forehead ; Humans ; Safety

This study aimed to identify Inulae Herba, Inulae Flos and their closely related species using the ITS2 bar-code, and secure the quality and clinical curative effect of these medicinal materials. DNA was extracted from Inula linariifolia, I. japonica, I. britanica, which are the original species of Inulae Herba and Inulae Flos, together with their closely related species. The ITS2 sequence was amplified by PCR and sequenced bidirectionally. Sequence assembly and generation of consensus sequence were conducted by the CodonCode Aligner. The genetic distances were comput-ed using MEGA 5.0 in accordance with the Kimura-2-parameter (K2P) model, and the phylogenetic tree constructed by the neighbor-joining (NJ) method. The results showed that the ITS2 sequences of the various species have stable variable sites. The intraspecific genetic distances among Inulae Herba and Inulae Flos were obviously lower than the interspecific genetic distance among the above two medicinal materials and its adulterants. The NJ tree based on ITS2 sequences can clearly differ from Inulae Herba, Inulae Flos and their closely related species. It is concluded that ITS2 sequence can be used as DNA barcode to identify Inulae Herba, Inulae Flos and their closely related species.

In this study, the ITS2 sequence was used to identify Pinelliae Rhizoma and its adulterants to ensure its market circulation, clinical effect and safety. All genomic DNA from 59 samples were extracted successfully. The Kimura 2-Parameter (K2P) distances and NJ tree were calculated using software MEGA 6.0. The length of the ITS2 sequence of Pinelliae Rhizoma was 251 bp. The intraspecific genetic distance was smaller than the interspecific ones;The NJ tree indicated that Pinelliae Rhizoma distinguished from its adulterants obviously. The results showed that the ITS2 sequence can be used to distinguish Pinelliae Rhizoma from its adulterants accurately and stably.

In this study,Polygalae radix and its adulterants were identified by psbA-trnH sequence.The genomic DNA was extracted from forty-six samples, the psbA-trnH sequences were amplified and sequenced Bi-directionally, and then assembled sequences by Codoncode Aligner V 3.7.1. The genetic distances were computed by kimura 2-parameter (K2P) model, and the Neighbor-Joining tree was constructed by MEGA 6.0. Results showed that minimum intra-specific K2P distance of Polygala tenuifolia and Polygala sibirica were 0.004 and 0, which were smaller than the maximum intra-specific K2P. The NJ tree showed Polygalae radix can be distinguished from its adulterants by psbA-trnH sequences. Therefore, using psbA-trnH sequences can distinguish Polygalae radix from its adulterants.