Establishment of a novel co-cultured liver cancer model based on tumor microenvironment / 中华肿瘤杂志

Objective: To construct a new co-cultured liver cancer research model composed of activated hepatic stellate cells (aHSC) and liver cancer cells, explore the efficacy difference between it and traditional model, so as to establish a liver cancer research model in vitro and in vivo that can reflect the real clinical efficacy. Methods: A new co-culture model of liver cancer consisting of aHSC and liver cancer cells was constructed. The differences in efficacy between the new co-culture model and the traditional single cell model were compared by cytotoxicity test, cell migration test, drug retention test and in vivo tumor inhibition test. Western blot was used to detect the drug-resistant protein P-gp and epithelial-mesenchymal transition-related proteins. Masson staining was used to observe the deposition of collagen fibers in tumor tissues of tumor-bearing mice. CD31 immunohistochemical staining was used to observe the microvessel density in tumor tissues of tumor-bearing mice. Results: The cytotoxicity of single cell model and co-culture model was dose-dependent. With the increase of curcumin (CUR) concentration, the cell viability decreased, but the cell viability of single cell model decreased faster than that of co-culture model. When the concentration of CUR was 10 μg/ml, the cell viability of the co-culture model was 62.3% and the migration rate was (28.05±3.68)%, which were higher than those of the single cell model [38.5% and (14.91±5.92)%, both P<0.05]. Western blot analysis showed that the expressions of P-gp and vimentin were up-regulated in the co-culture model, which were 1.55 and 2.04 fold changes of the single cell model, respectively. The expression of E-cadherin was down-regulated, and the expression level of E-cadherin in the single cell model was 1.17 fold changes of the co-culture model. Drug retention experiment showed that the co-culture model could promote drug efflux and reduce drug retention. In vivo tumor inhibition experiment showed that the m-HSC+ H22 co-transplantation model had faster tumor growth and larger tumor volume than those of the H22 single cell transplantation model. After CUR treatment, the tumor growths of m-HSC+ H22 co-transplantation model and H22 single cell transplantation model were inhibited. Masson staining showed that the deposition of collagen fibers in tumor tissues of m-HSC+ H22 co-transplantation model mice was more than that of H22 single cell transplantation model. CD31 immunohistochemical staining showed that the microvessel density in tumor tissue of m-HSC+ H22 co-transplantation model was higher than that of H22 single cell transplantation model. Conclusions: The aHSC+ liver cancer cell co-culture model has strong proliferation and metastasis ability and is easy to be resistant to drugs. It is a new type of liver cancer treatment research model superior to the traditional single cell model.

Immunogenicity and safety of a severe acute respiratory syndrome coronavirus 2 inactivated vaccine in healthy adults: randomized, double-blind, and placebo-controlled phase 1 and phase 2 clinical trials / 中华医学杂志(英文版)

BACKGROUND@#The significant morbidity and mortality resulted from the infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for urgent development of effective and safe vaccines. We report the immunogenicity and safety of an inactivated SARS-CoV-2 vaccine, KCONVAC, in healthy adults.@*METHODS@#Phase 1 and phase 2 randomized, double-blind, and placebo-controlled trials of KCONVAC were conducted in healthy Chinese adults aged 18 to 59 years. The participants in the phase 1 trial were randomized to receive two doses, one each on Days 0 and 14, of either KCONVAC (5 or 10 μg/dose) or placebo. The participants in the phase 2 trial were randomized to receive either KCONVAC (at 5 or 10 μg/dose) or placebo on Days 0 and 14 (0/14 regimen) or Days 0 and 28 (0/28 regimen). In the phase 1 trial, the primary safety endpoint was the proportion of participants experiencing adverse reactions/events within 28 days following the administration of each dose. In the phase 2 trial, the primary immunogenicity endpoints were neutralization antibody seroconversion and titer and anti-receptor-binding domain immunoglobulin G seroconversion at 28 days after the second dose.@*RESULTS@#In the phase 1 trial, 60 participants were enrolled and received at least one dose of 5-μg vaccine (n = 24), 10-μg vaccine (n = 24), or placebo (n = 12). In the phase 2 trial, 500 participants were enrolled and received at least one dose of 5-μg vaccine (n = 100 for 0/14 or 0/28 regimens), 10-μg vaccine (n = 100 for each regimen), or placebo (n = 50 for each regimen). In the phase 1 trial, 13 (54%), 11 (46%), and seven (7/12) participants reported at least one adverse event (AE) after receiving 5-, 10-μg vaccine, or placebo, respectively. In the phase 2 trial, 16 (16%), 19 (19%), and nine (18%) 0/14-regimen participants reported at least one AE after receiving 5-, 10-μg vaccine, or placebo, respectively. Similar AE incidences were observed in the three 0/28-regimen treatment groups. No AEs with an intensity of grade 3+ were reported, expect for one vaccine-unrelated serious AE (foot fracture) reported in the phase 1 trial. KCONVAC induced significant antibody responses; 0/28 regimen showed a higher immune responses than that did 0/14 regimen after receiving two vaccine doses.@*CONCLUSIONS@#Both doses of KCONVAC are well tolerated and able to induce robust immune responses in healthy adults. These results support testing 5-μg vaccine in the 0/28 regimen in an upcoming phase 3 efficacy trial.@*TRIAL REGISTRATION@#http://www.chictr.org.cn/index.aspx (No. ChiCTR2000038804, http://www.chictr.org.cn/showproj.aspx?proj=62350; No. ChiCTR2000039462, http://www.chictr.org.cn/showproj.aspx?proj=63353).

Preparation and proliferation effect on hepatoma cells of adenine loaded glycyrrhetinic acid modified hyaluronic acid nanoparticles / 中国药理学通报

Aim To prepare hyaluronic acid nanoparti-cles(Ade/GA-HA) using glycyrrhetinic acid modified hyaluronic acid as the carrier and adenine as a model drug, and analyze their physicochemical property and proliferation effect on Bel-7402 cells. Methods Gly-cyrrhetinic acid and hyaluronic acid were combined by chemical cross-linking method, dialysis and freeze-dr-ying,based on which Ade/GA-HA was prepared using ultrasonic method, and the particle size and Zeta po-tential were determined by Malvern laser particle analy-zer,and the morphology was observed by transmission electron microscopy, and the absorbance was deter-mined by ultraviolet-visible spectrophotometer, high performance liquid chromatograph and microplate read-er to caculate drug load, encapsulation efficiency and in vitro release. MTT assays were utilized to determine the proliferation of nanoparticles treated Bel-7402 cells. Results GA-HA nanoparticles had spherical shape with a good dispersion, at diameters of 398.1 nm, of which Zeta potential was - 34.2 mV, and presented good short term stability. The drug load and encapsulation efficiency of Ade/GA-HA nanoparticles were (22.5 ± 5.8)% and (87.27 ± 0.33) %, re-spectively. Burst release was observed in Ade/GA-HA nanoparticles within 4 h, while controlled release 4 h later. Compared with free adenine,Ade/GA-HA nano-particles had a stronger inhibitory effect on cell prolif-eration with statistically significant difference. Conclu-sion GA-HA nanoparticles has excellent physico-chemical properties and meet the design requirement.