To investigate the mechanism of drug resistance occurring in hepatocellular carcinoma treated with sorafeinib from epigenetic perspective, and examine the effect of sensitivity of sorafeinib on hepatocellular carcinoma after in vita combination of epigenetic drug decitabine (DAC), so as to provide new ideas and methods for clinical treatment of hepatocellular carcinoma.

The GEPIA 2 database was used to retrieve information of 508 primary hepatocellular liver cancer patients, and the correlation between the expression of OATP1B3 and survival time was analyzed by Kaplan-Meier method. The methylation rate of SLCO1B3 promoter was detected by bisulfite methylation method. RT-qPCR and Western blot were used to detect the expression changes of cancer cell lines OATP1B3 before and after liver DAC treatment. RTCA-eSight experiment was performed to monitor the effect of sorafeinib in combination with DAC on the proliferation of hepatocellular carcinoma cells. Changes in the uptake of sorafeinib by hepatocellular carcinoma cells after the combination of the two drugs were detected by experimental LC-MS/MS.

The results of GEPIA2 database analysis showed that the overall survival rate of patients with hepatocellular carcinoma with high OATP1B3 expression was significantly higher than those with low expression. Bisulfite methylation sequencing showed that the promoter methylation rate of SLCO1B3 was higher in Hep3B and HepG2. RT-qPCR and Western blot showed that the mRNA and protein expressions of OATP1B3 in hepatocellular carcinoma cell lines Hep3B and HepG2 were relatively low, and the expression of OATP1B3 was upregulated after incubation with DAC. RTCA-eSight experiment showed that DAC combination treatment significantly enhanced the effect of sorafeinib on Hep3B and HepG2 inhibition. LC-MS/MS determination showed that the uptake of HEK293-OATP1B3 on sorafeinib was 2.10 times higher than that of HEK293-Wild. The uptake of sorafeinib by Hep3B and HepG2 was increased by 1.87-fold and 2.47-fold after being combined with DAC.

DAC can inhibit SLCO1B3 DNA methylation, up-regulate the expression of OATP1B3, improve the capacity of sorafenib transport, increase the accumulation of sorafenib in liver cancer cells, and enhance the sensitivity of liver cancer cells, so as to reverse the resistance of sorafenib.

To develop the national reference standards of 1,1,1,2-tetrafluoroethane as the excipient of medicinal aerosol and its impurities thus to improve the standard of quality control.

The national reference standards of 1,1,1,2-tetrafluoroethane and its main impurities 1,1,2,2-tetrafluoroethane, pentafluoroethane, pentafluorochloroethane and 1,1,1-trifluoroethane were sub-packaged in liquid state directly. The structures were confirmed by IR, MS and MR spectroscopy, the moisture was determined by Coulomb method, the tightness of the package was confirmed by immersion in water. The content of each substance was calculated by mass balance method．Homogeneity and stability were investigated by F test and linear fitting respectively, and the content of 1,1,1,2-tetrafluoroethane was confirmed by multiple laboratories simultaneously.

The contents of 1,1,1,2-tetrafluoroethane, 1,1,2,2-tetrafluoroethane, pentafluoroethane, pentafluorochloroethane and 1,1,1-trifluoroethane were 100.0%, 99.9%, 99.7%, 99.2%, and 99.9%, respectively, with good homogeneity and stability.

In this study, the national reference standards of 1,1,1,2-tetrafluoroethane and its main impurities were developed for the first time, where 1,1,1,2-tetrafluoroethane can be applied for content determination, and the other substances can be applied for the inspection of related substances.

To develop a highly sensitive, efficient, and stable cell-based platform for detection of anti-adeno-associated virus 8 (AAV8) neutralizing antibody titer in monkey plasma and perform preliminary validation of the relevant performance of the method.

In vitro, an equal volume of diluted monkey plasma was incubated with AAV8-Luciferase and then the mixture was added to a 96-well plate lined with HEK293 cells and incubated overnight to infect HEK293 cells; after cell lysis, the cells were added to a firefly luciferase reporter gene assay and the fluorescence signal (RLU) was detected using ELISA to assess the plasma levels of anti-AAV8 neutralising antibodies and subsequently obtain the corresponding antibody titres. The assay's sensitivity (LOD) and titer reproducibility, precision, method robustness and sample stability were preliminary were preliminary validated.

A highly sensitive, efficient and stable method for the detection of anti-AAV8 neutralizing antibody titer in monkey plasma at the cellular level was developed and preliminarily validated. The LOD value of the method was 10.36 ng·mL^-1. The repeatability, precision and method robustness of the titer assay were good, and the positive control samples were stable after being placed at room temperature and 2 ℃~8 ℃ for 24 h and after freeze-thawing for 6 times at -65 ℃~-90 ℃/room temperature.

It has been preliminarily validated that the method can be used for the analysis and detection of anti-AAV8 neutralizing antibody titer in monkey plasma, which is useful for grasping the process of drug treatment and adjusting the treatment protocol in time and is suggestive for the development of AAV8 serotype gene therapy drugs and individualized treatment.

To prepare the β-cyclodextrin inclusion complexes of volatile oil in Qizhu granules.

The technology parameters like inclusion time, inclusion temperature, and the ratio of volatile oil to β-cyclodextrin in the preparation process of inclusion complexes were investigated by single factor experiment with inclusion rate as the index. With inclusion rate and inclusion yield as indexes, Box-Behnken response surface method was used to optimize the preparation process of inclusion complexes. Thin-layer chromatography, infrared spectroscopy and differential scanning calorimetry were utilized to characterize the inclusion complexes.

The optimal conditions were as follows: 9∶1 for volatile oils-(β-cyclodextrin) ratio, 64 ℃ for inclusion temperature, and 1 h for inclusion time. The inclusion rate and yield of the obtained inclusion complexes were 90.68% and 80.40%, respectively. All characterization results revealed the formation of inclusion complexes.

The established method is stable and feasible, which can be used for preparing the β-cyclodextrin inclusion complexes of volatile oil in Qizhu granules．

To establish a method of accelerated release of brexpiprazole sustained release microspheres for injection in vitro and discuss the in vivo and in vitro relationship, so as to provide reference for prescription screening and quality control of this preparation.

The effects of surfactant type, salt type and temperature on the release behavior were investigated by the method of constant temperature water bath. The in vitro accelerated release method was finally determined by the analysis of distinguishing ability and in vivo and in vitro relationship (IVIVR).

A method for accelerated release of pH 7.4 HEPES (containing 0.2% cetyltrimethylammonium bromide) at 45 ℃ in vitro was established, which showed a good correlation with in vivo release (r²>0.99).

The method of accelerated release in vitro established in this paper can guide prescription screening and process modification in all stages of drug development, and it is of great significance for product quality control.

To isolate and prepare iridin monomer from traditional Chinese medicine Iridis tectori Rhizoma, and to explore the regulatory effect of iridin on the isolated intestinal muscle in vitro.

The multi-solvent extraction method was used to prepare Iridin. The effect of iridin on acetylcholinesterase (AChE) activity was studied by modified Ellman colorimetry. The isolated intestinal muscle experiment was used for in vitro evaluation. In this study, the mouse small intestinal smooth muscle was obtained, from which the small intestinal smooth muscle ring was prepared in K-H solution. The change of the tension was measured by the BL-420S biological function experimental system. The resting tension and its effects on the contraction of small intestinal smooth muscle induced by neostigmine and pralidoxime iodide were investigated.

The iridin monomer obtained by the multi-solvent extraction method has a purity of over 98% calculated by the HPLC peak area normalization method. Iridin had inhibitory effect on AChE activity with a dose-dependent manner, and its half-inhibitory concentration (IC₅₀) is 4.09 mg·mL^-1. Iridin (0.014~4.604 mg·mL^-1) had obvious excitatory effect on the contraction of isolated small intestine in normal mice, and the inhibitory effect was dose-dependent. Iridin had a significant antagonistic effect on the inhibition of small intestinal contraction caused by the cholinesterase revitalizer iodopyridine (P<0.05), but had no significant synergistic effect on promoting the small intestinal motility by the cholinesterase inhibitor neostigmine (P>0.05).

Multi-solvent extraction method is a simple and efficient method for the isolation and preparation of iridin in Iridis tectori Rhizoma, and iridin has the cholinergic effect on inhibiting AChE. It can promote the contraction of the small intestine, and is expected to be a candidate for ache inhibitor.