The purpose of this study is to investigate the intracellular transporters effect and the cytotoxicity of carboxyl nanodiamond (CND) - podophyllotoxin (PPT). Nanodiamond (ND) was treated with mixed carboxylic acid and finally got 64 nm CND by centrifugation, and then it was reacted with PPT to form CND-PPT. UV spectrophotometry was used to calculate the content of PPT in CND-PPT, the particle size distribution and zeta potential were measured by Dynamic laser scattering instrument. CND, PPT, CND-PPT and CND + PPT (physical mixture of CND and PPT) were characterized by Fourier transform infrared spectroscopy, at the same time, thermal analysis and element analysis were used to estimate the content of the PPT in CND-PPT. The affect of CND, PPT, CND-PPT on HeLa cell was measured with MTT assay. The results showed that content of PPT combined with CND accounted for about 10%. MTT assay showed that CND has low cytotoxicity and CND-PPT can increase the water soluble of PPT. As a conclusion, CND as a hydrophilic pharmaceutical carrier combined with PPT is able to increase the water solubility of PPT, at low concentration, CND-PPT can enhance the antitumor activity in comparison with PPT, so CND can be used as a potential anticancer drug carrier.

Objective To explore the TCM pathogenesis regularity of nonalcoholic fatty liver disease (NAFLD);To provide basis for clinical syndrome differentiation and treatment. Methods A cross-sectional study was used. The clinical observation form of NAFLD was used to collect the patient data. Factor analysis and singular value decomposition methods were used to study the internal factors of the four diagnostic characteristics. The differences between the various analytical methods were compared. Results Factor analysis used the α factor analysis method, the principal component method, and the image factor method. The results of α-factor analysis were the best. The first three factors had the highest explanatory rates of data, namely spleen-dampness factor, kidney deficiency factor and qi deficiency factor, which constituted the basic pathogenesis factor of NAFLD. The singular value decomposition method obtained 3 singular values, namely spleen and kidney qi deficiency, liver and kidney yin deficiency, and qi and yin deficiency, also highlighted the spleen and kidney deficiency in the pathogenesis of NAFLD. Conclusion Spleen-kidney deficiency plays an important role in the pathogenesis of NAFLD. Treatment based on deficiency for nonalcoholic fatty liver disease has great clinical significance.

The study was to express prME protein of Japanese encephalitis virus (JEV) in Pichia pastoris and then to evaluate the immunological properties of the recombinant protein in mice, so as to explore a new way for subunit vaccine development of JEV. The JEV prME gene was amplified by RT-PCR with genome RNA of JEV vaccine strain SA14-14-2 and subcloned into pPICZa-A vector, designated as pPICZα-prME. pPICZα-SprME was constructed same as pPICZα-prME besides with the additional 19 Aa signal peptides coding gene of the JEV cap protein C terminal. The linearized expression vector was integrated into the genome of Pichia pastoris X33 under the control of the alcohol oxidase (AOX1) promoter and induced with methanol during fermentation expression. The expression of JEV prME protein was identified by SDS-PAGE and Western blotting, and then it was purified by S-400 High Resolution HiPrep 16/60 Sephacry. The expressed products of Pichia pastoris were visualized by electron microscopy. In the immunization test, four groups of four-week old female mice were immunized subcutaneously with different doses purified JEV prME protein with complete Freund's adjuvant at a volumetric ratio of 1:1 and a control group was injected with sterile PBS. 10 μg/dose purified JEV prME protein mixing different doses nucleic acid adjuvant (Naa) was vaccinated in mice as the same mode. SDS-PAGE and Western blotting indicate that JEV prME was not cleaved between prM and E during secreted expression in Pichia pastoris. The purified recombinant prME was eluted in the first eluting peak which indicated that its molecular weight about 1×10⁶ Da to 20×10⁶ Da and may form a multimeric. Both the culture supernatant and the purified protein, examined by electron microscopy, we found to contain JEV virus like particles (VLPs) with diameters of 30-50 nm. The anti-JEV VLPs antibody titration reached peak at 3 wpi and still maintained in mice at 7 wpi inoculated with 10 μg and 15 μg prME. The strong antibody response was observed when the mice immunized with prME mixing nucleic acid adjuvant, which elicited high neutralizing antibody titer among 1:80 to 1:160. In conclusion, although JEV prME protein expressed in Pichia pastoris was not cleaved, which formed VLPs and showed efficient immunological properties in mice experiments.