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 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 employ adeno-associated viruses as vectors to design and prepare pseudoviruses comprising multiple DNA viral gene fragments. These were used as positive controls for the detection of human viruses in the field of molecular testing, thereby compensating for the absence of wild-type viral controls in viral molecular testing methods.

The adeno-associated viral vectors served as the backbone of the pseudoviruses, with a total of four target gene fragments from three viruses incorporated: human papillomavirus type 16 (HPV16), type 18 (including HPV18-1 and HPV18-2) and human polyomavirus (HPyV) were, respectively, inserted into one viral vector, and the adeno-associated viral pseudoviruses were packaged by multiple plasmid cotransfection. The infectious activity of the pseudoviruses was confirmed by a cell infection assay, and a quantitative analysis of the pseudoviruses genome was conducted using fluorescence quantitative PCR.

The titers of HPV16, HPV18-1, HPV18-2, and HPyV genomes in the pseudoviruses were 7.77×10⁸ copies·mL^-1,6.77×10⁸ copies·mL^-1, 7.04×10⁸ copies·mL^-1, and 1.24×10⁹copies·mL^-1, respectively, as determined by fluorescence quantitative PCR. Following a 48 h period of infection in 293T/17 cells, the viral gene sequences of HPV16, HPV18-1, HPV18-2, and HPyV were successfully identified using fluorescence quantitative PCR with the intracellular pseudoviruses. The copy numbers were 1.30×10⁶ copies·mL^-1, 6.59×10⁵ copies·mL^-1,6.27×10⁵ copies·mL^-1, and 4.17×10⁶ copies·mL^-1. Following the infection of 293T/17 cells with the reporter gene pseudoviruses for a period of 48 hours, a distinct green fluorescence was evident under a fluorescence microscope, thereby confirming the infectious activity of the pseudoviruses. The pseudoviruses was employed as a positive control for the verification of applicability in human mesenchymal stem cells, and the recoveries of the four viral gene fragments by fluorescence quantitative PCR for nucleic acid extraction were 94.4%, 70.7%, 83.1%and 90.9%, respectively.

The present study demonstrates that the adeno-associated virus packaging method can be employed to produce a multiviral gene pseudoviruses with infectious activity. The pseudoviruses can be employed as a positive control for the replacement of multiple wild-type viruses in the viral fluorescence quantitative polymerase chain reaction (PCR) of stem cell samples.

This method not only reduces the financial burden associated with the preparation of positive controls, but it also offers enhanced biosafety and improves the efficiency and quality control of the qPCR method, from the extraction of genes to their amplification.

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 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.

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 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.