Objective To establish a method to determine six kinds of PAEs in human serum simultaneously,and on this basis to understand the level of PAEs in human body.Methods Ultrasonic extraction,gas chromatography-mass spectrometry-selected ion monitoring(GC-MS-SIM) detection and quantitative analysis based on internal standard were used to detect six kinds of PAEs in human serum simultaneously.Finally this method was used in fifty human serum samples.Results The results showed that this method had a good linear relation in the range from 10 to 1 000 ng/ml,and the correlation coefficients of standard curve were higher than 0.999.The average recovery rate and the relative standard deviation(RSD,n=6) of the target compounds in standard-addition blank was 94.5% and 4.4% respectively,the lower limits of detection for DEP,DMP,DBP,BBP,DEHP,DNOP were 0.31,0.85,0.90,0.63,0.88,0.41 ng/ml respectively,and the recovery rates of all samples ranged from 67.98% to 110.8%.Conclusion This method has good recovery rate,reproducibility,lower limit of detection and can be used for the determination of many kinds of PAEs substances in human serum.

Objective To evaluate the efficacy and adverse effects of low doses gemcitabine chemotherapy combined with synchronous high-low oxygen radiotherapy in patients with locally advanced pancreatic cancer.Methods Fifty-six patients with locally advanced pancreatic cancer were randomly divided into two groups by envelop method:radio-chemotherapy group or chemotherapy group.Patients in radio-chmotherapy group were treated with low doses of gemcitabine chemotherapy ( 600 mg/m2 ) combined with high-low oxygen radiotherapy synchronously,paients in chmotherapy group were treated with full doses of gemcitabine chemotherapy ( 1000 mg/m2).The short-term effect,distant metastasis rate,clinical benefit rate,survival rate and adverse events of two groups were observed.Results There was one patient achievedcomplete relief and 15 patients achieved partial relief in radio-chemotherapy group with an overall effective rate of 66.7％ (16/24) ; there were 9 patients achieved partial relief in chemotherapy group with an overall effective rate of 36.0％ (9/25),the difference between the two groups was statistically significant (X2 =4.6082,P =0.0318 ).The clinical benefit rates were 83.3 ％ ( 20/24 ) and 60％ ( 15/25 ),respectively,the difference between the two groups was not statistically significant ( P =0.070).The distant metastasis rates were 66.7％(16/24) and 72％ (18/25),respectively,the difference between the two groups was not statistically significant (P =0.6855).The 12,24 months survival rates were 62.5％ vs 32％,37.5％ vs 12％,the difference between the two groups was statistically significant ( P =0.0325,0.0380).The incidence of serious adverse events was 45.8％ and 4 0 ％ without statistical difference.Conclusions Low doses of gemcitabine chemotherapy combined with high-low oxygen radiotherapy synchronously is better than full doses of gemcitabine chemotherapy with regard to total effective rates and 12,24 months survival rates,with no obvious increase in the incidence of serious adverse events.

Objective:To screen the best extraction process of four medicinal materials in Simiao Junyi ointment by analgesic ex-periments. Methods:The analgesic experiments were performed by the hot-plate test and writhing test in mice to select the best extrac-tion process of four medicinal materials in Simiao Junyi ointment. Results:According to the results of level consistency check, the data of hot-plate test in mice in different groups had no significant difference, and the pesticide effect of Simiao Junyi ointment prepared by different extraction process was similar. However, the data of writhing test had significant difference. The extraction process of the oint-ment with the best analgesic activity was as follows:pepper and rhizoma corydalis were extracted by SFE-CO2 , myrrha and pseudo-gin-seng was extracted by 95% and 50% ethanol, respectively. Conclusion: The optimal extraction process tested by analgesic experi-ments is scientific, reasonable and feasible, and suitable for the research and development of modern traditional Chinese medicine preparations.

Objective To explore influence of thoracic duct ligation on lipid metabolism after esophagectomy. Methods Seventy-four patients with esophageal cancer received esophagectomy were divided into two groups by their thoracic duct ligation, 39 in ligation group and 35 in non-ligation group. All the patients were fed with nutrients through nasal-duodenal tube placed during operation from the 1 st day to the 8th day after surgery, and started taking liquid diet on the 6th day. Blood specimens were collected from the patients on the 1st day after admission, the 9th day, one month and three months after surgery,respectively for biochemical analysis,including determinations of total cholesterol(TC),triglyeeride(TG),high-density lipoprotein-cholesterol ( HDL-C) and low-density lipoprotein-cholesterol ( LDL-C) . Databetween the two groups were compared with statistical analysis. Results No significant difference in plasma levels of TC, TG and HDI.-C was found between the two groups at varied time points. Plasma level of LDL-C in the ligation group significantly decreased on the 9th day after surgery, as compared with that in the nonligation group ( P ＜ 0. 05), and went down to the lowest level one month after surgery. Conclusions Chylomicron was blocked to enter blood stream and production of LDL-C decreased by thoracic duct ligation,which affect lipid absorption and metabolism leading to poor early nutrition in patients with esophagealsurgery due to slower establishment of collateral circulation.

Objective·To investigate the clinicopathologic characteristics and prognosis of adult rhabdomyosarcoma (RMS).Methods·The clinical and pathological data,immunohistochemical test results of 9 RMS were analyzed retrospectively.Results·The information of 6 males and 3 females were collected in this research,whose average age was (46.89+20.09) years old.The tumors had a wide range distribution.The subtypes of histology included 6 cases of embryonal RMS,1 case of alveolar RMS,1 case of spindle cell RMS and 1 case of pleomorphic RMS.Immunohistochemically study showed that 6 cases were myogenin positive,4 cases were MyoDl positive and 8 cases were desmin positive.During the follow-up period of the 7 cases,only 2 patients survived free of tumors,2 patients presented with tumor recurrence and metastasis after the operations,and 3 patients died of tumor.Conclusion·In this study,embryonaI RMS is the most common type,and immunohistochemistry test is helpful to diagnosis of RMS.

Objective To establish a universal stem loop primer (USLP) based real-time PCR method to scan mature miR profile and quantify it's expression.Methods The common universal stem-loop primer pairs were re-designed; 8 random nucleotides were introduced at 3 ' end for reverse transcription of the mature miR,establishing a miR scanning and quantifying system based on SYBR Green Ⅰ PCR (improved USLP method).10-fold gradient diluted standard miRNA-155 cDNA ( 1 ～ 109 copies/μ1) were utilized to evaluate the sensitivity of this method.The specificity was verified by melting curve assay; the precision was assessed by intra-assay coefficient of variation (ICV) of threshold cycle (Ct value) through 20 repeated detections of the standard miR-155 cDNA (2 × 105,2 × 106,2 × 107 copies/μl) ; cost of the primers and time were evaluated,compared with that of the conventional USLP method.Peripheral blood samples were cultured with phytohaemagglutinin (PHA) for0 h,16 h,24 h,48 h and 72 h,and 87 candidate miR that may be associated with human immunity from PubMed data were scanned and quantified from the cultured T cells.Results The sensitivity of the improved USLP method was 103 copies/μl of standard miR-155 cDNA.Melting curve assay showed a single melting peak at 80 ℃,suggesting the excellent PCR specificity of miR-155.Precision of our method quantifying miR-155 was acceptable (ICV ＜ 2.5％ ).Compared with the traditional stem loop primers,our method saved 75％ cost of primers ( 1 917 bp vs 7 851 bp) and 60％ test time of reverse transcription (85 min vs 205 min).By our method,85 of the 87 miR expression in T cells had no significant difference after the PHA stimulation; the expression of miR-150 (72 h) decreased by 10 times and that of miR-155 (48 h) increased 8 times after culture with PHA (Z =-2.032,P =0.042;Z =- 2.023,P =0.043,respectively ).Conclusions The improved USLP method is fast,precise,sensitive,and cost-effective.It could be used for miR profile scanning and quantifying in T cells.

DCD WeChat ethical review had its necessity and superiority but problems and confusion as well. For instance, WeChat review time was urgent and limited;WeChat review discussed insufficiently even hard to car-ry out;the methods of WeChat review were limited;the authenticity of audit opinion was difficult to grasp;the re-view data privacy existed hidden danger;the review quality was difficult to guarantee;WeChat conference content file archive was difficult, and so on. As to this, the author put forward the following countermeasures:giving war-ranty on the ethical review time as far as possible;initiating the committee review by the referee and uploading the review information to WeChat group, then ethical reviewers expressing their opinions in real names to ensure data confidentiality and privacy protection; strengthening the ethics committee education for organ transplantation; im-proving the quality of the review committee members and moral cultivation;archiving the WeChat conference infor-mation.

AIM:To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transfor-mation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E.METHODS:NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at con-centrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10μmol/L).After cultured for 24 h, the mRNA expression of Ptch1, Smo,α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR.The protein levels of Shh and TGF-β1 were detected by ELISA .Immunofluorescence staining was used to evaluate the expression of Ptch 1, Smo,α-SMA, E-cadherin and type III collagen in the NRK-52E cells.RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling , and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells.Treatment with cyclopamine inhibited HH signaling by decrea-sing Smo expression and increasing Ptch 1 expression.Moreover, cyclopamine also down-regulated the expression of TGF-β1,α-SMA, type I collagen and III collagen , and up-regulated the expression of BMP-7, ZO-1 and E-cadherin.CON-CLUSION:AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells , which can be inhibited by cyclopamine treatment .The possible mechanism is that cyclopamine suppresses the activation of HH signaling , resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition .

OBJECTIVE To investigate the effect ofγ-secretase inhibitor N-[N-(3,5-difluorophen?acetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)on phenotypic transformation and matrix accu?mulation induced by aristolochic acid(AA) in renal tubular epithelial cells(NRK-52E)and explore the mechanism. METHODS NRK-52E cells were divided stochastically into normal cell control group,AA 10 mg·L-1 group and AA 10 mg·L-1+DAPT 1 and 10μmol·L-1 group. After 24 h,the mRNA expressions of Notch1,Jagged1,Numb,E-cadherin,transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),bone morphogenic protein 7 (Bmp7),typeⅠ a1 (Col1a1) and Ⅲ collagens a1 (Col3a1)were quantified by quantitative real-time RT-PCR. The protein expressions of Notch1,Jagged1,α-SMA,and Col3a1 in NRK-52E cel s were detected by immunofluorescence staining. RESULTS In NRK-52E cells,AA enhanced the expression of TGF-β1,α-SMA and Col3a1 mRNA(P<0.05),reduced the expression of E-cadherin mRNA(P<0.05),up-regulated the mRNA expression of Notch1 mRNA(P<0.01)and Jagged1(P<0.05),and down-regulated the mRNA expression of Numb mRNA(P<0.05) compared with normal cell control group,indicating that phenotypic transformation and matrix accumu?lation occurred in AA-treated NRK-52E cells,accompanied by activated Notch signaling. Treatment with DAPT inhibited Notch signaling by decreasing the expression of Notch1 and Jagged1 (P<0.05),and increasing the expression of Numb mRNA(P<0.05). Furthermore, DAPT also down-regulated the expression levels of TGF-β1,α-SMA,Col1a1 and Col3a1 mRNA(P<0.05), and up-regulated the expression level of Bmp7 and E-cadherin mRNA(P<0.05) compared with AA group,suggesting that DAPT inhibited phenotypic transformation and matrix accumulation in AA-treated NRK-52E cells. CONCLUSION AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells,which is inhibited by DAPT treatment. The possible mechanism is that DAPT suppresses the activation of Notch signaling,resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.

Objective To investigate the expression of monocyte-macrophage-related factors and interstitial fibrosis in kidney tissues of rats with ureter obstruction and recanalization .Methods Forty-eight male Spragur-Dawley rats were divided randomly into the obstructive group:sham (n=6), unilateral ureteral obstruction(UUO)3 days (n=6), UUO 7 days (n=6), and UUO 14 days (n=6) and recanalization group:bilateral ureteral obstruction(RBUO)0 day (n=6), 3 days after RBUO (n=6), 7 days after RBUO (n=6), and 14 days after RBUO (n=6).The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fibers in kidney was detected with HE and Masson staining . Immunohistochemical analysis was performed to evaluate the protein expressions of monocyte chemoattractant protein -1 (MCP-1), macrophage colony-stimulating factor (M-CSF) and activated-macrophage marker CD68.Real-time PCR was used to detect the mRNA expressions of MCP-1 and M-CSF.TGF-β1 levels were determined by ELISA .Results Fibrosis observed with HE and Masson staining was obviously increased in kidney tissue of UUO rats , and aggravated as time prolonged, but alleviated in rats with recanalization .TGF-β1 levels were increased obviously in the UUO group , but decreased in rats with recanalization compared with those in BUO rats .In UUO rats, mRNA and protein expression levels of MCP-1 and M-CSF were increased .MCP-1 and M-CSF expression was gradually decreased in rats with recanalization compared with those in BUO rats .The dynamic change in expression of MCP-1 and M-CSF in both UUO rats and recanalization rats was consistent with the change in expression of CD 68. Conclusion Dynamic change in expression of MCP-1 and M-CSF in kidney tissues reflects change of activated and accumulated monocyte -macrophages , which may be one of the major mechanisms contributing to fibrosis induced by ureter obstruction .Renal fibrosis is alleviated by down-regulated expression of monocyte-macrophages factors with recanalization operation .