Objective: To construct a Family with sequence similarity 50 member A(FAM50A) gene knockout mouse insulinoma pancreatic β-cell line Beta-TC-6 using CRISPR/Cas9 gene editing technology and to prepare polyclonal antibodies specifically recognizing FAM50A. Methods: Two guide RNAs(sgRNAs) targeting the FAM50A gene were designed,and a recombinant plasmid expressing blue fluorescent protein(BFP) was constructed for gene knockout.The successfully constructed plasmid was transfected into Beta-TC-6 cells,and BFP-positive single cells were isolated for clonal expansion.The expanded monoclonal cell lines were genotyped by Sanger sequencing,and FAM50A protein expression was assessed by Western blot.Purified human recombinant FAM50A protein was used to immunize New Zealand rabbits for the preparation of a polyclonal antibody.The specificity of the prepared antibody was then validated using the successfully established FAM50A knockout cell line. Results: A monoclonal cell line with a successful knockout of the FAM50A gene was identified.Sanger sequencing confirmed base deletions at the target site.Western blot analysis showed a complete absence of FAM50A protein expression in this cell line.The prepared polyclonal antibody successfully recognized endogenous murine FAM50A protein in wild-type Beta-TC-6 cells and in hTERT-RPE1 cells overexpressing human FAM50A-GFP fusion protein,while no signal was detected in the FAM50A knockout cells. Conclusion This study successfully established a FAM50A gene knockout Beta-TC-6 cell model and generated a FAM50A polyclonal antibody,providing powerful tools for future research.

Objective:To optimize the formula and preparation of exendin W /O/W multiple emulsion , and study the main proper-ties in vitro to lay foundation for the development of exendin prolonged action preparation .Methods:Using multiple emulsion yield and centrifugal separation time as the comprehensive index , the formula and preparation of exendin multiple emulsion were optimized by or-thogonal tests, and the morphology, particle size, stability, encapsulation efficiency and drug release in vitro were studied.Results:The optimal formula was as follows:the volume ratio of W1 phase to O phase was 1:1.2, the quality percentage of emulsifierⅠin the O phase was 10％, poloxamer 407 in W2 phase was 10％, and the quantity ratio of W1/O to W2 phase was 1:1.The optimal prepara-tion conditions were as follows:the stirring speed for W1/O was 10000 r· min-1 , and the stirring time was 10 min, and that for W1/O/W2 was 2500 r· min-1 with the stirring time of 3 min.The multiple emulsion was spherical with the mean size of (46.3 ±2.6)μm, and the centrifugal and viscosity stability were both promising .The drug encapsulation efficiency was above 90％, and the drug release in vitro accorded with a Higuchi equation: Y=13.7930X+5.5981(r=0.9883), showing notable sustained and prolonged property .Conclusion:The optimal formula and preparation process of exendin multiple emulsion are simple with high drug content , good stability and notable sustained release property in vitro.

Objective:To improve the stability of exendin W/O/W multiple emulsion.Methods:The amount of electrolyte sodium chloride in the internal water phase and that of thickener PVP in the external water phase were both screened for exendin W/O/W mul-tiple emulsion,and the cryoprotectant in the freeze-drying process was optimized in order to further optimize the formula and preparation of exendin W/O/W multiple emulsion. Verification experiment was carried out to study the main properties of the optimized exendin W/O/W multiple emulsion in vitro. Results: The optimal quality volume percentage concentration of sodium chloride in the internal water phase was 0.10%,and that of PVP in the external water phase was 0.15%,and the best cryoprotectant was 10% mycose. The main in vitro properties of exendin W/O/W multiple emulsion showed no significant changes before the freeze-drying and after the rehy-dration. Conclusion:Through the optimization of the internal and external water phase of exendin W/O/W multiple emulsion,the sta-bility is enhanced,which lays foundation for the in vivo studies.

Objective: To study the hypoglycemic effect and the hypodermic injection irritation of exendin multiple emulsion in tes-ting animals. Methods: Using normal saline and blank multiple emulsion as the blank controls and the marketed exendin injection as the positive control, the hypoglycemic effects of exendin multiple emulsion in healthy mice and mice with gastric administration of 10% glucose solution were studied. Type Ⅱ diabetes rat model was established, and the hypoglycemic effect of exendin multiple emulsion was investigated. The injection of local skin was observed and the slides of injection tissue were studied after HE staining to evaluate the irritation of exendin multiple emulsion. Results: In the healthy mice, the blood glucose reached the lowest point at 90min for ex-endin injection and rose gradually at 120 min, while the blood glucose maintained low levels during the testing period for exendin multi-ple emulsion. After the gastric administration of glucose solution, the blood glucose increased at 60min for exendin injection, while that kept low levels for exendin multiple emulsion. In type Ⅱ diabetes rats, the blood glucose increased gradually after 7-day withdrawal (P<0. 05), while that kept low levels for exendin multiple emulsion. The hypodermic injection of exendin multiple emulsion showed no significant or irreversible irritation to the local skin and tissue. Conclusion: Compared with exendin injection, exendin multiple emul-sion exhibits stable and sustained hypodermic effects with high local use safety.

Objective To explore the influence of drug dosage, solvent and other main influencing factors on the successful establishment of alloxan-induced hyperglycemia mouse model and the effect on the stability of this model. Methods 160 6-8-week-old Kunming mice ofSPF grade, (male:female=1:1) were used in this study.The influences of different dosages of alloxan and solvent combinations on the successful establishment rate of the model, survival rate, body weight, fasting blood glucose, blood glucose area under curve, serum insulin level and their stabilities were dynamically observed for six weeks.Results By single intraperitoneal injection of 160 mg/kg bw alloxan ( pH 4.5 citrate sodium as solvent) , we were able to obtain a stable experimental hyperglycemic mouse model with higher levels of successful establishment rate (70%), survival rate (75%), fasting blood glucose (15-20 mmol/L), glucose area under the curve (55-65 mmol/L) and a lower but not loss of serum insulin levels (21 mIU/L).Conclusions In the present study we have carefully considered the influence of main factors such as drug dosages, solvent, etc., on the alloxan-induced experimental hyperglycemic mouse model, and successfully established this model after 6-week period observation of its stability.This model may provide a useful tool in the research of experimental diabetes and hypoglycemic functional studies.