Objective To knock out p21 gene in human malignant rhab doid tumor(MRT)cell line G401 by using CRISPR/Cas9 genome engineering technology. Methods The expression of p21 was detected by reverse transcription quantitative PCR (RT-qPCR) and Western blot assay in several MRT cell lines. The guide RNA was designed by targeting the third exon of p21 gene,which encoded its home domains, and then subcloned into lentiCRISPR v2 vector and validated sequencing. The validated plasmids were further used to package and produce the lentivirus in 293T cells, and the G401 cells were infected, then puromycin was used to screen positive cells, and the clusters of G401 monoclonal cells, were obtained by selecting monoclonal cells and culturing under the microscope. The RNA and protein of new clonal cell line were extracted, and RT-qPCR and Western blot assay were applied to confirm whether p21 was successfully knocked out. Results The p21 was highly expressed in MRT tumor cells. The CRISPR/Cas9 lentivirus plasmids, targeted p21 gene were successfully constructed. Compared with negative control group,the expression of p21 was not detected in G401 monoclonal cells, which were successfully screened. Conclusion In view of the difficult transfection of cells such as G401, p21 knockout stable cell line has been successfully constructed by using CRISPR/Cas9 system, which lays the foundation for further study of the mechanism of p21 in MRT tumors .

Objective Implement etimicin polymethylmethacrylat (PMMA) bone cement implantation treatment for the postoperative patients suitable with intramedullary infection after open fracture surgery,and observe the efficacy,provide new ideas for clinical treatment.Methods Selected 60 patients in Second People's Hospital of Kunshan from Jan.2009 to Dec.2016 whose infection was confined to the fracture caused by trauma after the internal fixation.Thirty cases of etimicin bone cement implantation were treated group,30 cases of CEMEX GEN antibiotic bone cement (containing gentamicin) implantation were control group.The internal fixation was taken out of 9 cases of autogenous PMMA bone graft with bone cement cover in the treatment group,debrided and taken first phase of bone grafting internal fixation combined with etimicin PMMA bone cement,21 patients with direct bone cement filling underwent surgery to remove the internal fixation devices,debridement cavity filling etimicin bone cement.Six months later when the infection was controlled,the second phase of autogenous bone graft fixation was done.Ten cases of autogenous bone graft with bone cement cover in the control group,debrided and taken first phaes of bone grafting internal fixation combined with CEMEX GEN antibiotic bone cement (containing gentamicin).Twenty patients with direct bone cement filling underwent surgery to remove the internal fixation devices,debridement cavity filling CEMEX GEN antibiotic bone cement (containing gcntamicin).Six months later when the infection was controlled,the second phase of autogenous bone graft fixation was done.SPSS 19.0 software was used for statistical analysis.The t test was used for measurement data,and the counting data were checked by chi-square.Results The pathogens were mainly staphylococcus aureus and coagulase-negative staphylococci.Thirty-seven strains of pathogenic bacteria were detected with MIC ≤ 2 mg/L in the treatment group,which was in the safe and effective drug concentration range of etimicin(MIC ≤ 12 mg/L).Thirty strains of pathogenic bacteria were detected with MIC ≤ 8 mg/L in the control group,which was in the safe and effective drug concentration range of gentamicin(MIC ≤ 12 mg/L).The average follow-up period of the treatment group was 11.2 months (range 8 to 24 months),29 cases were controlled.The average follow-up period of the control group was 15.6 months (range 6-35 months),24 cases were controlled.The difference of infection control rate between treatment group and control group was statistically significant (x2 =4.043,P =0.044).Conclusion The complete debridement,a lot of washing,and etimicin bone cement implantation can effectively control the infection after the internal fixation of fracture and reduce the recurrence,which can be used in clinical practice.

Objective:To analyze the cause of varicocele (VC) recurrence and investigate the efficacy of sub-inguinal microsurgical varicocelectomy (MV) for recurrent VC.Methods:All of 16 inpatients diagnosed as recurrent VC, in the Department of Andrology of the Affiliated Hospital of Qingdao University from 2015 April to 2019 April, were performed sub-inguinal MV. The age of the inpatients was 18-36 years old, median 27 years old.5 cases were originally performed retroperitoneal high ligation of spermatic vein and other 11 cases were originally performed laparoscopic varicocelectomy. During the review one to three years after the previous operation, all of 16 patients were diagnosed as VC recurrence. The complains of these patients during the review included male subfertility (10 cases) and scrotal pain (12 cases), in which 6 cases’ complains were male subfertility with scrotal pain. After admission, 13 patients were classified as Grade Ⅲ (left in 8 cases, bilateral in 5 cases) and 3 patients as Grade Ⅱ (all left). The median of their visual analogue scale (VAS) was 2.5. Color doppler flow imaging (CDFI) grading showed: Grade Ⅲ in 12 cases (left in 7 cases, bilateral in 5 cases), Grade Ⅱ in 4 cases (all left). Particularly, 12 of them were Graded as Ⅲ simultaneously accompanying with Nut-cracker Phenomenon (NCP). Preoperative tests showed that the average serum testosterone was (16.2±4.9)nmol/ml, the average sperm concentration was (11.8±3.9)×10 6/ml and the progressive motility rate (PR) was (24.4±4.2)%. All of the patients were performed sub-inguinal MV using general anesthesia and supine position. The spermatic cords were clearly exposed and padded up by inserting gauze strips under them. During the operation, the field was magnified 4-6 times with the microscope. Then all of the dilated external and internal spermatic veins were ligated, at the same time the internal spermatic artery and lymph vessels were well preserved. During these operations, 11 patients underwent left-side MV, while other 5 did bilateral MVs. During these MVs, we found twisted and dilated external and internal spermatic veins in all cases and well preserved the internal spermatic arteries and lymph vessels. The number of ligated left and right external spermatic veins were(2.1±0.6) and (1.4±0.5)respectively and the number of ligated left and right internal spermatic veins were (10.1±1.1) and (6.6±0.5) respectively. We also found out(1.3±0.5) internal spermatic arteries and (3.0±1.0)lymph-vessels on left side. On right side, there were (1.4±0.5) internal spermatic arteries and (2.6±0.5) lymph-vessels respectively. At last, we summarily analyzed the pre-operative and post-operative VAS, serum testosterone, CDFI and semen analysis data. Results:All of the 16 sub-inguinal MVs were successfully performed. All patients were reviewed comprehensively 6 months after MV. The reviewed results showed that the post-operative VAS was significantly reduced ( Z=-2.994, P<0.05), palpable scrotal vessels disappeared and Valsalva tests were negative. No obvious reflux of internal spermatic veins were detected by CDFI. Interestingly, the sperm concentration and motility were both significantly improved 6 months after MV ( P<0.05), while there was not remarkable increase of the serum testosterone after MV ( P>0.05). During the follow up, no testicular atrophy, hydrocele and other complications were found. Up to submission, five of the ten patients who presented for male subfertility have impregnated their wives. Conclusions:The most possible cause of VC recurrence could be the omission of the external and internal spermatic veins, particularly in the grade Ⅲ VC patients or VC accompanied with NCP. The sub-inguinal MV, which can discover more twisted spermatic veins and at the same time preserve the spermatic artery and lymph-vessels, shows better clinical efficacy than other procedures.

Objective:To investigate the characteristics of anxiety and depression of postgraduates in military medical university under public health emergencies.Methods:By means of self-rating anxiety scale (SAS) and self-rating depression scale (SDS), 944 postgraduate students of a military medical university were investigated to analyze their characteristics of anxiety and depression. SPSS 25.0 was used to statistically analyze the collected data.Results:①The positive rates of anxiety and depression were respectively 8.03% and 8.25%. ②The positive rate of anxiety of male postgraduates was significantly higher than that of female postgraduates ( P=0.035). ③The positive rate of anxiety of serviceman students was significantly higher than that of civilian students ( P=0.022). ④There was no statistical difference in the positive rates of anxiety and depression among postgraduates in different areas ( P > 0.05). ⑤The levels of anxiety and depression of left-behind children were significantly higher than those of unleft-behind children ( P=0.000, P=0.018). ⑥The levels of anxiety and depression of postgraduate students were lower than those of undergraduate students ( P=0.000, P=0.002). Conclusion:Under the outbreak of public health emergencies, anxiety and depression occur in the postgraduates. To strengthen the mental health maintenance of postgraduates in military medical university during the outbreak, it is necessary to pay more attention on the mental health of military students, males and who were left-behind children before.

OBJECTIVE：To provide th e ideas a nd for individualized anti-infective treatment of infection after surgery for infants and young children with intussusception and enterobrosis ，and to provide reference for clinical pharmacists participating in the clinical treatment. METHODS ：Clinical pharmacists optimized the anti-infection program for an 11-month-old infant patient infected after surgery with intussusception and enterobrosis in Ordos Central Hospital ；they put forward medication suggestions in respects of the selection of initial anti-infection treatment program ，drug replacement ，the selection of anti-infection treatment program after blood culture showed Enterococcus coli and Enterococcus faecium ，and dosage adjustment. RESULTS ：According to the judgment of the common pathogens and the hospital or community infections in the infant patient with intussusception and enterobrosis，cefoperazone sulbactam 1.0 g，q12 h was adjusted to cefoperazone sulbactam 0.5 g，q8 h combined with Metronidazole chloride sodium injection 20 mL，q8 h；when the blood culture showed E. coli （ESBL-）and E. faecium ，it was recommended to add vancomycin 0.15 g，q12 h. After poor treatment ，it was recommended to adjust the vancomycin dose to 0.2 g，q8 h. All the above suggestions were adopted by doctors. And the child ’s body temperature dropped after treatment ，the blood culture turned negative and laboratory indicators returned to normal. The child was discharged smoothly. CONCLUSIONS ：Infants and young children are special groups. Therefore ，before using antibiotics ，clinical pharmacists should evaluate the age ，body weight ，liver and kidney functions of infants and young children. They should also help doctors select and adjust drugs ，frequency and dosage on the basis of pharmacokinetic characteristics and safety ，so as to avoid adverse drug reactions while ensure curative effect.

OBJECTIVE： To investigate the formulation of therapy plan and pharmaceutical care for a penicillin allergic patient with necrotizing fasciitis combined with diabetes mellitus. METHODS： A penicillin allergic patient with necrotizing fasciitis combined with diabetes mellitus was admitted to Ordos central hospital on March 30， 2018. According to the disease characteristics of the patient， clinical pharmacists conducted pharmaceutical care during the treatment process from the aspects of antiinfective drugs selection， monitoring points of necrotizing fasciitis， blood glucose monitoring， effects of hypoproteinemia on antibiotic pharmacokinetics-pharmacodynamics， and assisted the physician to develop a more reasonable and effective individualized therapentic regimen. RESULTS： For diabetes mellitus， the patient adjusted Insulin lispro injection （insulin pump）， Acarbose tablets， Metformin hydrochloride tablets and Glimepiride tablets successively. For rapid atrial fibrillation with intraventricular differential conduction and ST-T changes， Metoprolol tartrate tablets were used to reduce heart rate. For necrotizing fasciitis， when choosing drugs to ensure the anti-infective effect， considering that the patient had penicillin allergic and hypoproteinemia， clinical pharmacists chose drugs with low protein binding rates and different from the mother nucleus and side chain of penicillin. Imipenem cilastatin for injection， Aztreonam for injection， Levofloxacin lactate and sodium chloride injection were used successively. Voriconazole for injection was adopted for anti-fungal treatment. Physicians adopted the suggestions of clinical pharmacists. Debridement through early thorough drainage and rational drug use， the patient’s condition was effectively controlled. CONCLUSIONS： Clinical pharmacists assist physicians to formulate and optimize whole course therapentic regimen so as to improve the safety and effectiveness of drug treatment.