ObjectiveA HPLC-FLD method was developed to determine the levels of serum AAA in CRIpatients, and to studythe variationof serum AAAinCRI patientsanditsclinical significances. MethodsSerumsampleswerecollected from100healthcontrolsand 80CRI patients. According to 2002 National Kidney Foundation (NKF) staging diagnosis, CRI patients included 4 of stage 2, 12 of stage 3, 12 of stage 4, 52 of stage 5. According to pathogenesis, CRI patients were also divided into 3 groups :chronic nephritis group ( n = 32), DM group ( n = 36), hypertension group ( n = 12 ).Serums were deproteinized by equal volume of 5％ (v/v) PCA and supernate were analyzed direcdy. External standard method was used as quantitative method. The analytical column was Megres C18. 10％ acetonitrile in water was used as mobile phase. Flow rote was 1.0 ml/min. The wavelengths of fluorescence excitation and emission were changed with specific time. The levels of Tyr, Phe and Trp in CRI groups, different CKD stages and different pathogenesis were compared with healthy control groups to evaluated the sensitivity and specificity of serum AAA for CRI diagnosis. ResultsThe linear ranges of the method were 0. 550 -275.000, 3. 050 - 1220. 000 and 0. 049 -49. 000 pμmol/L for Tyr, Phe and Trp, respectively. The limit of detection (LOD) was 0.014 μmol/L for Tyr, 0.500μmol/L for Phe, and 0.005 μmol/L for Trp. The average recovery was 100. 9％, 101.3％ and 98. 5％ for Tyr, Phe and Trp, respectively. Intra-day CVwas 3. 18％ -4. 20％ ( mean was 3. 13％ )and inter-day CV was 3. 18％ -4. 20％ ( mean was 3. 58％ ). The concentration of serum AAA, Tyr and Trp and the ratio of Tyr/Phe in CRI patients were( 135.74 ±23.23 )μmol/L, (52.27 +8.25) μmol/L, (21.49 ±4.25) μmol/L and[0.87(0.68 - 1.05)]μmol/L. which were lower than that in healthy groups (t value was -14. 709, 4.452, 22. 100, U value was 266.000,respectively, P＜0. 05). The concentration of serum AAA, Tyr and Trp and the ratio of Tyr/Phe in healthy groups were ( 174. 47 ± 11.57 ) μmol/L, ( 63.53 ± 4. 68 ) μmol/L, (44. 22 ± 3. 67 ) μmol/L and[0. 97(0. 94 - 1.00)]μmoL/L. There were no statistically significant difference between the different stage of CRI. Compared with the concentration of Tyr, Phe and Trp among chronic nephritis group, DM group,hypertension group, the concentration of Tyr had no significant changes among these three kinds of diseases (P ＞ 0. 05 ). The concentration of Phe had significant changes between Chronic nephritis group and DM group, Chronic nephritis group and hypertension group ( U = 395.00, 114. 00, P ＜ 0. 05 ) ; the concentration of Trp haad significant changes between Chronic nephritis group and DM group ( U = 349.00, P ＜ 0. 05 ).The diagnostic sensitivity and specificity of serum AAA for CRI were 90％ (72/80) and 100. 0％ (100/100).ConclusionsThe method of high-performance liquid chromatography with fluorescence detection ( HPLC-FLD) is simple, rapid, sensitive and specific. Simultaneous determination of serum AAA was benefit to the diagnosis and evaluation of CRI patients.

Objective To establish a HPLC method and fingerprints study for the simultaneous determination of loganic acid,gentiopicroside,paeoniflorin,vitexin,liquiritin,and ammonium glycyrrhizinatein,and other components in the alcohol extract of Mongolian medicine Qinggan Manu-4.Methods The Agilent Eclipse XDB-C18 column(250 mm×4.6 mm,5 μm)was used,with the detection wavelength of 230 nm for paeoniflorin,vitexin and liquiritin,237 nm for loganic acid,250 nm for glycyrrhizic acid,and 270 nm for gentiopicroside.The mobile phase was 0.1%phosphoric acid aqueous solution-acetonitrile with gradient elution.The flow rate was 1.0 mL/min,the column temperature was 30℃,and the injection volume was 10 μL.Results The mass concentrations of loganic acid,gentiopicroside,paeoniflorin,vitexin,liquiritin and ammonium gentiopicroside in the alcohol extract of Mongolian medicine Qinggan Manu-4 showed good linear relationships with the chromatographic peak area in the range of 0.020 3-0.121 0,0.053 3-0.317 7,0.021 3-0.127 0,0.011 5-0.069 0,0.014 1-0.083 8 and 0.035 1-0.209 1 mg/mL,respectively(r≥0.999 8).The average recovery rates of the six components were 94.75%,99.49%,92.32%,95.82%,101.29%and 98.04%,with the RSDs of 1.11%,0.76%,0.99%,2.75%,1.09%and 2.43%,respectively(n=6).The HPLC fingerprint of the alcoholic extract of Mongolian medicine Qinggan Manu-4 was established,using the chromatogram of sample S1 as the reference chromatogram,the control fingerprints were generated through multi-point correction and full-spectrum peak matching.A total of ten common peaks were identified,and after comparison with the reference substance,eight components were identified.Conclusion The established method and fingerprints are accurate,reliable,reproducible and specific,which provide a basis for the quality control and subsequent development of Mongolian medicine Qinggan Manu-4.

Objective To investigate the effect of knockdown of EpCAM by siRNA on invasion,migration,and colony abilities in hypopharyngeal carcinoma FaDu cells.Methods A siRNA against EpCAM was employed to inhibit the expression of EpCAM in FaDu cells.Measurements included the Transwell assay for invasion and migration,plate colony formation assay for cell colony ability,Western blot assay for EpCAM,E-cadherin,and β-catenin expressions in total protein,cytoplasm,and cytoskeleton,respectively.Results mRNA and protein expressions of EpCAM were suppressed significantly in FaDu cells transfected by EpCAM siRNA (t =6.46,P ＜ 0.05 ; t =10.25,P ＜ 0.05).Transwell assay showed in transwell assay,the average invasive cells in EpCAM siRNA cells (26.33 ± 3.71) was less than that in FaDu cells (61.47 ± 6.70 ; t =7.95,P ＜ 0.05) and control cells (54.13 ± 6.51 ; t =6.42,P ＜ 0.05) ; the average number of migration cells in EpCAM siRNA cells (79.87 ± 8.44) was lower than that in FaDu (167.53 ± 11.49 ; t =10.90,P ＜ 0.05) cells and control cells (162.13 ± 13.45 ; t =8.97,P ＜ 0.05).In plate colony formation assay,the average colony number of EpCAM siRNA cells was (78.00 ± 5.57),which was less than that of FaDu cells(177.30 ± 16.50; t =9.78,P ＜0.05) and control cells (173.67 ± 13.50; t =11.35,P ＜0.05).Western blot assays showed,silencing of EpCAM increased the expressions of E-cadherin (t =4.58,P =0.01) and β-catenin (t =3.76,P =0.02) in cytoskeleton,and decreased the expressions of E-cadherin (t =6.60,P ＜ 0.05) and β-catenin (t =8.20,P ＜ 0.05) in cytoplasm.Conclusions The knockdown of EpCAM inhibits the invasion,migration,and colony formation abilities of FaDu cells,which is probably related to the regulation of E-cadherin and β-catenin in cytoplasm and cytoskeleton,and EpCAM may be a promising gene therapy target for hypopharyngeal carcinoma.

Objective To investigate the expression of microRNA-214 (miR-214) in advanced hypopharyngeal carcinoma tissues and its effects on the invasion,migration and colone formation of FaDu cells.Methods miR-214 expression in 30 cases of advanced hypopharyngeal carcinoma tissues and normal hypopharyngeal mucosa tissues was detected by real-time quantitative polymerase chain reaction (RT-qPCR).miR-214 was upregulated through transfecting the overexpression vector hsa-mir-214 into FaDu cells.The influences of miR-214 upregulation on the invasion,migration,clone formation and Twist expression were measured by Transwell invasion,Transwell migration,plate clone formation and Western blot assays,respectively.Results The expression of miR-214 in advanced hypopharyngeal carcinoma tissues (0.31 1 ±0.206) was significantly less than normal hypopharyngeal mucosa tissues(1.620 ± 1.394;t =5.09,P ＜ 0.05).The expression of miR-214 was notably upregulated after tranfected with hsa-mir-214 compared with the negative control group (t =6.347,P ＜ 0.05).The migration and invasion ability of FaDu cells transfeced with hsa-mir-214 was decreased by comparison with negative control cells (t =1 1.6,P ＜0.01; t =6.499,P ＜ 0.05).There was no significant difference of the average clony number and the cloning efficiency between the experimental and negative control groups (t =0.592,P ＞ 0.05).Results of Western blot assay showed that,Twist expression in the miR-214-overexpressed group was apparently decreased compared with that in the control group (t =6.545,P ＜ 0.05).Conclusions miR-214 is expressed at a low level in advanced hypopharyngeal carcinoma tissues,and can obviously inhibit the invasion and migration abilities of FaDu cells,possibly because of its inhibiting effect on Twist expression.Additionally,miR-214 plays no significant role in the proliferation of FaDu cells.

AIM:To explore the influence factors of the treatment zone diameter(TZD)and its correlation with axial length growth(ALG)after Paragon CRT orthokeratology.METHODS: Retrospective clinical study. The data of 226 myopic patients(226 eyes)wearing Paragon CRT orthokeratology from April 2020 to September 2022 were collect. The correlated factors of TZD after wearing lens for 1mo, and the relationship between the overlapping treatment zone/ pupil area ratio and the ALG after wearing lens for 1a were analyzed.RESULTS: After wearing lens for 1mo, the TZD was negatively correlated with central corneal thickness(CCT)and positively correlated with the flat corneal eccentricity. After wearing lens for 1a, the ALG of the small TZD group(0.25±0.18mm)was significantly smaller than that of the large TZD group(0.34±0.24mm, P=0.002), and the ALG of the small area ratio group(0.24±0.19mm)was significantly smaller than that of the large area ratio group(0.35±0.23mm,P<0.001). Age and overlapping treatment zone area/pupil area ratio were significantly associated with the ALG in multivariate linear regression(all P<0.05).CONCLUSION: The wearers with thicker CCT and smaller flat corneal eccentricity usually had smaller TZD, and both the TZD and the overlapping treatment zone area/pupil area ratio were correlated with the ALG.