Objective To construct a stable synovial cell line MH7A from rheumatoid arthritis(RA)patients using lentiviral vectors that interfere with the expression of tumor necrosis factor receptor associated factor 2(TRAF2),and to study the role of TNF-α-TRAF2 signaling in MH7A abnormal proliferation.Methods Based on the design principles of human TRAF2 gene sequence and shRNA sequence,three pairs of TRAF2 shRNA interference se-quences were designed and synthesized.The primers were annealed by PCR,and a linear vector was obtained by double enzyme digestion PLKO.1-puro.The linearized vector was connected to the annealed primers through Solu-tion I,and the connected products were introduced into receptive cells.The plates were coated,and positive colo-nies were selected for sequencing.Three different recombinant plasmids of PLKO.1-TRAF2-shRNA lentivirus were constructed,and lentivirus packaging plasmids was used to package logarithmic growth phase HEK 293T cells.Vi-rus solution was collected to infect MH7A cells.At the same time,puromycin was used to screen MH7A stable transgenic strains with low TRAF2 expression.CCK-8 method,Western blot,and qPCR were used to detect the proliferation function of MH7A induced by TNF-α and low expression of TRAF2,as well as downstream signal TRAF2,P65 protein expression and mRNA levels.Results PLKO.1-TRAF2-shRNA(1),PLKO.1-TRAF2-shR-NA(2),and PLKO.1-TRAF2-shRNA(3)lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were successfully constructed.The three TRAF2-shRNA lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were respectively introduced into the lentivirus packaging plasmid of HEK 293T to obtain virus solution.After infecting MH7A cells with the virus solution,they were treated with puromycin(2.00 μ G/mL)screening and obtaining MH7A stable transgenic plants after 2 days.Through qPCR and Western blot results,it was found that the expression of TRAF2 mRNA and protein in PLKO.1-TRAF2-shRNA(1)MH7A stably transfected cells was significantly reduced compared to the negative control group.The results of CCK-8 and Western blot showed that after knocking down TRAF2 in MH7A,the proliferation of MH7A cells with low TRAF2 expression induced by TNF-α and the phosphorylation level of P65 were significantly reduced.Conclusion A sta-ble transgenic strain of PLKO.1-TRAF2-shRNA(1)MH7A cells was successfully constructed to investigate the role of TNF-α-TRAF2 signal activation in mediating abnormal proliferation of RA synovial cells.

OBJECTIVE To analyze the situation and causes of the shortage of drugs in centralized volume-based procurement， and to put forward countermeasures and suggestions for optimizing centralized procurement rules and improving the drug shortage supply guarantee system. METHODS The shortage data of the first five batches of centralized purchasing drugs during January 2019 and December 2021 were collected from the Shortage Database of Chinese medicine economic information； descriptive statistical analysis was performed for shortage frequency， varieties and reasons. RESULTS & CONCLUSIONS After the first five batches of centralized volumetric procurement policy landed， the frequency of drug shortage reported for selected specifications and selected enterprises increased significantly， mainly drug shortage of selected enterprises. The shortage of some varieties from selected enterprises in the short term was obvious， and most of them were commonly used clinical drugs. The occurrence of drug shortages was affected by various links such as the production， circulation and use， and the main reasons were shortage or monopoly of raw materials. In order to ensure the drugs supply， the government should scientifically assess the risk of production interruption of enterprises， further optimize centralized procurement rules， improve the reporting system for drug shortages， and attach importance to the supply of key drugs； in addition， hospitals should take a variety of measures within the scopes of policies to flexibly respond to drug shortages so as to avoid delaying the treatment of patients.

Objective: To construct a human G protein-coupled receptor kinase 2 ( GRK2) eukaryotic expression system． Methods: The primers were designed ，and the His-GRK2 target gene was amplified by PCR using the Pans-EGFP-GrK2 (full-length) gene as the template．The His-GRK2 target gene was connected to the pcDNA3．1EGFP eukaryotic expression vector． The pcDNA3. 1-EGFP-His-GRK2 plasmid was transfected into HEK 293T cells．48 h later，the expression of GRK2 protein was detected by Western blot，and the GRK2 protein was purified by nickel chelated magnetic bead method．The purification of GRK2 protein was detected by Coomassie bright blue staining and Western blot，and the activity of GRK2 protein was detected by His pull down. Results : The results of double enzyme digestion and sequencing showed that pcDNA3. 1-EGFP-His-GRK2 eukaryotic expression plasmid was successfully constructed．Western blot analysis showed that the molecular weight of GRK2 protein was about 80 ku，indicating that GRK2 protein was successfully expressed in HEK 293T cells (t = 6. 433，P = 0. 003) ．GRK2 protein was purified by nickel chelated magnetic beads．His pull down experiment results showed that GRK2 was bound to prostaglandin E2 receptor 4 (EP4) ，suggesting that GRK2 protein had biological activity (t = 13. 5，P = 0. 000 2) ． Conclusion The pcDNA3．1-EGFP-His-GRK2 eukaryotic expression plasmid was correctly sequenced and the GRK2 recombinant plasmid was successfully constructed．The GRK2 recombinant plasmid was successfully expressed in eukaryotic cells HEK 293T and the protein expressed was biologically active.

OBJECTIVE To explore the impact of the adjustment of the national reimbursement drug list on rare disease drugs in hospitals， and to provide reference for improving the drug security of patients with rare diseases in China.METHODS The monthly procurement data of rare disease drugs from 789 medical institutions that continuously reported data from January 2016 to December 2018 were extracted from the Chinese Medicine Economic Information. The single-group interrupted time series model was used to compare drug varieties， procurement amount， average defined daily cost （DDDc） and defined daily doses （DDDs） of rare disease drugs before and after the adjustment of national reimbursement drug list. RESULTS In 2017， a total of 9 rare disease drugs were newly included in the national reimbursement drug list， including pirfenidone， carbidopa/levodopa， riluzole， ropinirole， droxidopa， ezetimibe， everolimus， coagulation factor Ⅸ human recombinant and coagulation factor Ⅶa human recombinant. After the adjustment of the national reimbursement drug list， the average DDDc of 9 rare disease drugs was significantly decreased， the upward trend of DDDs and the procurement amount was significantly increased （P＜0.001）. CONCLUSIONS The number of newly included rare disease drugs in national reimbursement drug list keeps increasing， the coverage of medical security keeps expanding， the price of rare disease drugs is significantly decreased， the economic burden of patients is further decreased， and the consumption of rare disease drugs is significantly increased， benefiting more patients with rare diseases； but at the same time， it also increases the procurement amount of rare disease drugs in hospitals. National medical security departments need to fully consider how to balance the affordability of medical insurance funds with the demand for rare disease drug coverage.

OBJECTIVE:To construct the evaluation index system for the effects of rational drug use policy in the military hos-pital. METHODS:Based on methods like literature analysis,expert consultation,modified Delphi method,analytic hierarchy pro-cess(AHP),the evaluation index system was constructed and the weight of indicators was calculated. The feasibility of the index system was verified by experimental evaluation. Through comparing drug use level between military patients and local insurance for medical care,the effects of rational drug use policy were evaluated in military hospital. RESULTS & CONCLUSIONS:The index system includes 3 first-level indexes as rationality,accessibility,economics,10 second-level indexes and 20 third-level indexes. The results of the experimental evaluation in 176 military hospitals show that the economic indexes scoring rate is lower than the reasonable indexes and the availability indexes,and the difference value are 3.12%and 4.06%;the first-level indexes average scor-ing rate of divisional hospital is higher than that of regimental hospital,and the difference value is 21.6%. The index system solves the problems of false data,incomplete evaluation,inadequate check,improves informatization level of evaluation,and realizes long-term dynamic monitoring and remote evaluation.

Objective To analyze the impact of the declining balance rate in military hospitals during new healthcare reform.Methods A military medical expense model was established,the parameters of the model were changed and the impact of the declining balance rate was analyzed.Results and Conclusion Military hospitals have to face up to the negative influence caused by the declining balance rate,actively promote expenditure compensation mechanism reforms,increase funding,enhance supervision,and separate revenues from expenditures.

Objective To explore the application of a system dynamics(SD) model to military medical expenses in PLA hospitals.Methods According to relevant theories of SD, the study has selected some important variables and defined the primary function relations in order to establish a military medical expenses SD model.Results and Conclusion The SD model is suited to modeling the PLA medical expenses and can serve as a theoretical basis for the policy innovation of the PLA medical system.

OBJECTIVE:To know about the implementation and existing problems of current Reasonable Medicine List for Mil-itary(Medicine List for short)(2014 edition). METHODS:The data of field investigation were summarized and analyzed. Compar-ative analysis was made to the data of medicines used by military patients and local insured patients in the general hospitals,hospi-tals affiliated to the military medical university,central hospitals,authorities'clinics and cadre's sanitariums'clinics (a total of 20)of 4 military regions in Nanjing,Lanzhou,Chengdu and Shenyang before and after the modification of the Medicine List. RE-SULTS & CONCLUSIONS:After the modification of the Medicine List,all the above-mentioned medical institutions of the 4 mili-tary regions used more varieties of medicines as set forth in the Medicine List,where there was the highest average increase up to 12.2% in the varieties of the medicines used by central hospitals,and the lowest average increase of 4.8% in those by general hos-pitals. Central hospitals used the largest number of varieties of medicines as set forth in the Medicine List,accounting for 83.3% of all varieties in the Medicine List on average;followed by hospitals affiliated to the military medical university,accounting for 80.6% on average;and the cadre's sanitariums'clinics used a smaller number of varieties thereof,only accounting for 63.9%. For military inpatients and outpatients,the medicine expense per capita increased by 15.0% and 18.0% respectively,which was differ-ent from that for local insured patients in the same period. It is suggested that the implementation of the Medicine List should be monitored and evaluated on a regular basis pursuant to the survey,to provide a reference for future adjustment of the Medicine List.

Objective To assess differences between the medicine treatment level of military inpatients in military hospi-tal in 2009 and 2011 ,provide references for reasonable adjustments of health policy in future .Methods Medicine treatment da-ta from acute simple appendicitis ,coronary heart disease ,chronic bronchitis and type 2 diabetes from 16 military hospitals were excavated and analyzed by statistical methods to provide a comparative study of the medicine treatment level in 2009 and 2011 . Results 3748 cases from 16 hospitals showed the significant difference in the level of the medicine treatment in 2009 and 2011 , such as medicine cost ,medicine varieties ,primary medicine costs ,hospital daily medication cost ,and the date in 2011 is higher than 2009 .Conclusion It was showed that the medicine treatment level in four common multiple diseases in the military in 2009 and 2011 was changed .A long-term stability mechanism should be established for the evaluation of military medicine sup-port level ,which could provide strong evidence for future medicine policy adjustment .

The book of 68W Advanced Field Craft was introduced in this paper .The characteristics of the teaching pro-gram and details of combat medic of the US Army were summarized .Some suggestions were raised about the development and training of field first-aid skills of the PLA .