To establish a simple and efficient method for detecting telomerase activity for evaluating tumorigenic risk of cell products.

Specific primers and probes were designed for the conserved domain of telomerase catalytic subunit (TERT), and the primers and probes of TERT gene were optimized and screened, and the primers and probes of internal reference genes were set up. Multiple quantitative PCR reaction was performed using two-color fluorescent probes in a reaction system, and RT-qPCR probe method was established. The expression of TERT gene in human mesenchymal stem cells HMSC was used to determine whether telomerase activity existed in the cells indirectly.

TERT gene of 293T/17 positive control cells could be stably and specifically detected by this method, with a mean Ct value of 23.96 and a RSD of 1.5. The internal reference gene GAPDH could be detected successfully, the mean Ct value was 14.13, the RSD was 1.3%. The reference gene GAPDH in MRC-5 could be detected in negative control cells, the mean Ct value was 12.81, the RSD was 0.46%, the TERT gene was not detected, and the telomerase activity was negative. The reference gene GAPDH in HMSC could be detected, the Ct mean value was 13.01, and the RSD was 3.8%, whereas, the telomerase activity of HMSC was negative.

The real-time fluorescent quantitative RT-qPCR probe method established in this study can accurately detect the expression of catalytic subunit mRNA in telomerase positive cells with good repeatability and high specificity. It can be used to analyze telomerase activity of stem cells and indirectly evaluate tumorigenic risk of cell products derived from stem cells.

To establish an HPLC method for simultaneous determination of vancomycin, caffeine and aminophylline in serum of children patients and apply it to clinical detection.

6% perchloric acid was used as protein precipitator and acetanilide was used as internal standard. The determination was performed on Nucifera C₁₈ column (250 mm×4.6 mm, 5 μm) with mobile phase methanol-0.05 mol·L^-1 potassium dihydrogen phosphate (18 ：82, v/v). The detection wavelength was 236 nm, the flow rate was 1 mL·min^-1, the column temperature was 25 ℃, and the sample size was 20 μL.

The serum concentrations of vancomycin and aminophylline were in the range of 1.0-100 μg·mL^-1, and the standard curves were Y=0.010 6X+0.006 9(r=0.998 8), Y=0.012 7X+0.008 4 (r=0.998 9), respectively. The serum concentration of caffeine had a good linear relationship in the range of 1.0-160 μg·mL^-1, and the standard curve was Y=0.015 4X-0.010 1 (r=0.999 1). Quantitative downlines were all 1.0 μg·mL^-1. The average recovery rates of the three compounds ranged from 97.8%to 103.1%, and the relative standard deviations of intraday and daytime precision were less than 10%, indicating good sample stability. Ten cases of children receiving vancomycin, aminophylline or caffeine were collected for method verification, which met the needs of clinical detection.

The method is simple, rapid, accurate, and is suitable for the simultaneous determination of serum vancomycin, caffeine and aminophylline in children.

To establish a new method for in situ visualization of spatial distribution characteristics of various secondary metabolites in the root of Wendang (Codonopsis pilosula Nannf. var. modesta (Nannf.) L. T. Shen), so as to realize the tissular localization of secondary metabolites, and to provide a reference for the in-depth excavation of the Wendang.

Matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) was used for the mass spectrometry imaging analysis of the root metabolites in Wendang. The matrix was DHB⁺/DHB^- (30 mg·mL^-1), the substrate flow rate was 20 μL·min^-1, the nitrogen flow rate was 5 L·min^-1, the nozzle movement speed was 3 mm·s^-1, the nozzle temperature was 60 ℃, and the spraying time of each slice was 50 min. The laser energy intensity in positive ion detection mode was 60%, and that in negative ion detection mode was 40%. The detection mass range was m/z 70-1 050, the mass resolution was 70 000, the spatial resolution was 50 μm, and the pixel size was 420 px×200 px. At the same time, the pathway enrichment analysis was also carried out for the metabolites.

A total of 214 metabolites were detected, and the spatial distribution characteristics of 40 representative metabolites were visualized. The distribution patterns of different kinds of secondary metabolites in the cork - phloem - xylem varied considerably, with flavonoids mainly distributed in xylem, alkaloids, phenolics, and carboxylic acids mainly in the cork and phloem, phenylpropanoids and quinones mainly in the cork, amino acids were abundant in the phloem, and nucleotides were distributed throughout the root tissues, the indicator components atractylenolide Ⅲ and syringoside were distributed in the cork, and lobetyolin was distributed in both the cork and the xylem. Pathway enrichment analysis showed that metabolites were significantly enriched in metabolic pathways, biosynthesis of secondary metabolites, flavonoid biosynthesis, biosynthesis of amino acids, and carbon metabolism pathways.

This study visualizes the spatial distribution characteristics of metabolites in the roots of Wendang. The results of this study can provide certain theoretical support for the quality control, the identification, the extraction and separation of active ingredients, and the metabolic pattern of Wendang.

To confirm the validity of solvent/detergent (S/D) treatment and dry heating for inactivation of virus in C1 esterase inhibitor (C1-INH).

The Sindbis virus in samples with S/D was inactivated by the S/D method, and the virus titer before and after inactivation was detected using the plaque assay. Dry heating method was used to inactivate encephalomyocarditis virus (EMCV) and porcine parvovirus (PPV), and cytopathic assay was used to detect the virus titer before and after inactivation.

After inactivation by the S/D method, the reductions of Sindbis virus in three batches of smaples with S/D were > 4.35 lgPFU·mL^-1, > 4.51 lgPFU·mL^-1,> 4.64 lgPFU·mL^-1, respectively. After dry heating, the reductions of EMCV in three batches of samples without S/D were ≥5.38 lgTCID₅₀/0.1 mL、≥5.12 lgTCID₅₀/0.1 mL、≥5.25 lgTCID₅₀/0.1 mL, respectively, and the reductions of PPV in three batches of samples without S/D were 4.57 lgTCID₅₀/0.1 mL、4.18 lgTCID₅₀/0.1 mL、4.68 lgTCID₅₀/0.1 mL, respectively.

With evaluation on the inactivation capability of the indicator viruses, it is proved that the S/D treatment and dry heating has good inactivation and removal effect on the virus in C1-INH.

To establish a atomic absorption spectroscopy (AAS) method for detecting calcium content in recombinant human coagulation factor Ⅷ for injection, and evaluate its uncertainty.

A calcium hollow cathode lamp light source was used. Air acetylene flame atomizer. Detection wavelength 422.7 nm. 8.9% lanthanum oxide solution was used as an ionization inhibitor. Conduct inspections on specificity, accuracy, precision, quantification limit, linearity and range, stability, etc. in accordance with the validation guidelines for drug quality standard analysis methods in Part 9101 of the 2020 edition of the Chinese Pharmacopoeia. The established method was applyed to detect the content of 6 batches of samples.

There was a good linear relationship between calcium element and absorbance in the concentration range of 0.0-5.0 μg·mL^-1, with r=0.999 8. The detection line was 0.071 6 μg·mL^-1; The quantification limit was 0.216 9 μg·mL^-1. The recovery rate of the spiked sample was 100.8% to 103. 2%. The repeatability RSD was 0.64%, and the solution was stable within 8 hours. The content determination results of 6 batches of samples all complied with the regulations. The uncertainty assessment result was (117.12±4.82) μg·mL^-1, k=2.

AAS method is established for the detection of calcium content in recombinant human coagulation factor Ⅷ for injection. This method has the advantages of high sensitivity, fast analysis speed, good repeatability, high accuracy, and cost savings. At the same time, the uncertainty of this method is evaluated, which can provide quantitative evaluation indicators for the quality control of this variety.

To explore the biological function methods of mesenchymal stem cells(MSCs) for quality analysis.

The surface markers of MSCs were detected by flow cytometry. MSCs osteogenic differentiation was induced by ascorbic acid and β-glycerophosphate sodium, etc., followed by Alizarin Red S staining. MSCs adipogenic differentiation was induced by IBMX, Rosiglitazone, etc., followed by Oil Red O staining. MSCs could differentiate into chondrocytes with treatment of ITS and TGFβ3, etc., followed by Alcian Blue staining. Cell co-culture of THP-1-macrophage with MSCs and ELISA assay were applied to detect the effects of MSCs on macrophage polarization. The expression levels of IL-10 and TNF α in the cell co-culture supernatant were detected by ELISA. To observe the effects of MSCs on lymphocyte proliferation, MSCs cultured with PBMCs, which were labeled with CFSE and activated by CD3/CD28, followed by flow cytometry.

The expression of MSCs surface markers, CD105, CD73, and CD90, was more than 95% respectively, while the expression of CD45, CD34, CD14, CD19, and HLA-DR expression was less than 2%. MSCs osteogenic differentiation assay showed red calcium nodules. Red lipid vacuoles were observed in MSCs adipogenic induction differentiation. Furthermore, MSCs have the differentiation potential to chondrocyte spheroids, and typical cartilage pits were observed. Coculture of MSCs with THP-1 macrophages, an increase in IL-10 expression and downregulate TNFα secretion were observed. MSCs played inhibitory effects on the proliferation of PBMC activated by CD3/CD28, with an inhibition rate of 76.4%.

This study established some of biological activity detection methods for MSCs, including MSCs surface markers, differentiation abilities, promotion of macrophage polarization, and inhibitory effects on lymphocyte proliferation. It provides a potential application for MSCs products quality control.

To study the bioactive components of the hypoglycemic effect of the traditional Chinese medicine Polygoni Cuspidate Rhizoma et Radix through its ability to inhibit α-glucosidase activity.

Four modules: high-performance liquid chromatography system, the nanofraction collector, the channel of bioassay and high-resolution mass spectrometry were integrated to build a high-throughput bioassay profiling analysis platform. The preparative liquid chromatography was introduced to apply segmented enrichment to the extract of Polygoni Cuspidate Rhizoma et Radix, and the rapid screening of α-glucosidase inhibitors from Polygoni Cuspidate Rhizoma et Radix was realized by optimizing the chromatographic separation conditions, microfluidic fractionation collection parameters and bioassaying of enzyme activity in multi-well plates.

28 α-glucosidase inhibitors were identified from Polygoni Cuspidate Rhizoma et Radix. The inhibitory activities of catechin-3-O-galloyl and procyanidin B₂-3''-O-gallate were determined with the IC₅₀ values of (9.49±1.93) μmol·L^-1 and (69.94±8.14)μmol·L^-1, respectively.

This study has enabled high-throughput and high-resolution screening of α-glucosidase inhibitors in traditional Chinese medicine, providing a valuable tool for elucidating the active components and mechanisms of Polygoni Cuspidate Rhizoma et Radix to lower hyperglycemia.

To optimize an LC-Q TOF MS method for the determination of target proteins expressed in recombinant cells from a cell bank.

The recombinant Chinese hamster ovary (CHO) cell line expressing antibodies was selected. The supernatant of recombinant CHO cell suspensions was retained after 12 500 r·min^-1 centrifuging for 10 min and treated with 50 mmol·L^-1 NH₄HCO₃ solution. Chymotrypsin was added for enzymatic digestion, and the comparative analyses were carried out using two LC-Q TOF MS with Advance Bio Peptide(100 mm×2.1 mm, 2.7 μm) column, the Sciex TRIPLE TOF 5600+ and the Agilent Q TOF G6545, respectively. To establish the database, the amino acid sequences of the characteristic peptides of the protein heavy and light chains were entered into SCIEX BioPharmaView Version 3.0 and Agilent MassHunter BioConfirm 12.0 software, respectively. The peptides detected in the samples were searched to identify the target peptides.

The samples were analysed by a Sciex TRIPLE TOF 5600+ instrument to obtain 8 characteristic peptides with 100%amino acid sequence coverage and the samples were analysed by an Agilent Q TOF G6545 instrument to obtain 12 characteristic peptides with 100% amino acid sequence coverage.

The method established in this study is simple to operate, has high coverage and good stability, can be used to identify the expression of target proteins in recombinant CHO cell lines. It also provides a reference for the expression identification of target proteins in similar cell lines.

To establish the ultra performance liquid chromatography (UPLC) characterization profile of Styrax, and to determine the contents of three components (cinnamic acid, cinnamyl cinnamate, 3-phenylpropyl cinnamate) in Styrax, the aim was to study the quality of commercially available sulforaphane of different origins and forms, and to provide a reference for the quality control of imported medicinal herbs Styrax.

The UPLC method was performed on an Acquity UPLC BEH C₁₈ (100 mm×2.1 mm, 1.7 µm) column with acetonitrile-1% acetic acid solution as the mobile phase in a gradient elution. The volume flow rate was 0.3 mL·min^-1. The detection wavelength was 277 nm. and the temperature of the column was 35 ℃. Characteristic chromatograms of styrax were established, and the contents of three major components of Styrax from different sources were determined and the data were statistically analyzed by hierarchical cluster analysis (HCA) and principal component analysis (PCA) with ChemPattern software.

The results showed that the UPLC characteristic chromatograms of Styrax was established with 10 common peaks and 4 identified components. The linear relationship of 3 components in their respective ranges was good (r≥0. 999), and the average recoveries (n=6) were 101.8% (RSD=1.3%), 105.8% (RSD=1.2%), 99.2% (RSD=1.8%), respectively. The content of 3 components were 2.74%-3.69%, 28.21%-30.63%, 16.89%-20.98%, respectively.

The method is simple, accurate and reproducible, and can provide an overall quality control basis for the quality standard of Styrax. Combined with the information of origin research, it has important reference significance for the expansion of the origin of Styrax, as well as the harmonization and improvement of the standards of Styrax in different countries.