Objective To construct a gene knockout cell library based on clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9) system, and construct a cell line stably expressing Cas9 protein using human colon adenocarcinoma cells Caco-2. Methods Caco-2 cells were infected with packaged recombinant Cas9 lentivirus, and then Caco-2/Cas9 monoclonal cells were selected by medium containing blasticidin and twice limited dilution sorting,and identified by PCR and Western blot. Caco-2/Cas9 monoclonal cells were infected with lentivirus expressing both GFP gene and single guide RNA(sgRNA) targeting GFP gene. After puromycin screening, the knockout efficiency of Caco-2 cell line was calculated by flow cytometry, and the proliferation activity was detected by CCK-8 assay. Results Five Caco-2/Cas9 monoclonal cell lines, named Caco-2/Cas9-2, Caco-2/Cas9-3, Caco-2/Cas9-4, Caco-2/Cas9-5 and Caco-2/Cas9-6, were obtained, all of which were amplified for a 392 bp band of Cas9 gene. Cas9 protein was stably expressed in the cells and the knockout efficiency was 91. 27%, 20. 30%, 24. 13%, 11. 33%, 12. 27% and 8. 89%, respectively. The proliferative activity of Caco-2/Cas9-4, Caco-2/Cas9-5, Caco-2/Cas9-6 and uninfected Caco-2 cells was 2. 07, 1. 75, 1. 46 and 1. 40, respectively,with no significant difference between Caco-2/Cas9-5, Caco-2/Cas9-6 and uninfected Caco-2 cells(t = 1. 92 and 0. 37, each P > 0. 05). Conclusion A Caco-2/Cas9 monoclonal cell line, Caco-2/Cas9-6 monoclonal cells with high enzyme digestion activity of Cas9 and no significant change in proliferation activity compared with uninfected Caco-2 cells was screened out,which lays a foundation for further construction of high coverage knockout cell library and provides a platform for screening genes related to virus infection and genes with other specific functions.

Objective:To study behavior problems,family factors an d parental rearing styles in students with poor academic achievement.Meth od:Through stratified cluster sampling,488 primary or middle school studen t s were identified as study group(below 5th percentile in their classes),while th e controls were 599 students whose academic achievement were above 95th percent ile in their classes.All subjects completed EMBU,CBCL(children behavior checkli st).Results:Higher boy/girl ratio was pound in study group(326/162) than in control(291/308,? 2=36.4,p

Objective: To explore the prevalence of depression in junior school students and its relationship with potential psychological and environmental factors. Method:Depressive symptoms, self-esteem, life events, social support, family cohesion and conflict were investigated with self-report questionnaire in 300 junior school students, the relationship between depression and other psychological or environmental factors were analyzed through stepwise regression. Results:The prevalence of depression in junior high school students was 11%. Among the 5 investigated psychological and environmental factors, self-esteem and social support were negatively related with depression (standardized regression coefficient were -0.59 and -0.14), while life events positively related with it(standardized regression coefficient was 0.26). Only "academic pressure" and another factors covering the item "do not want to go to school" showed a negative association with the students' self-esteem among the 6 factors included in the life events questionnaire (standardized regression coefficient were -0.149 and -0.13).Conclusion: Depression is common among junior school students. Poor self-esteem, lack of social support and negative life events are currently related with the development of adolescent depression. The pressure and failing experiences coming from academic activities are the most influential life events to the development of adolescents' self-esteem.

Objective To examine the effects of weight loss on functional state of female judokas. Methods Twelve female judokas belonging to Shanghai female judo team were tested at different periods(first,second,third phases and last phase) during weight loss a month before competition. The changes of physiological and biochemical indexes in different periods were observed and all the data was statistically analyzed. Results Previous three phases,the Fat% in weight loss group was decreased compared with basic data (P

Objective To observe the curative effects of bezafibrate combined medroxyprogesterone acetate in the treatment of AML .Methods 40 cases of AML patients were collected and devided into experimental group (bezafibrate combined medroxypro-gesterone acetate ,6 cases) ,control group (chemotherapy ,28 cases) and non-treatment group (6 cases) ,then through a prospective analysis we studied the curative effects and related results of bezafibrate combined medroxyprogesterone acetate in the treatment of AML .Results The median survival time of the experiment group (accepted bezafibrate combined medroxyprogesterone acetate ) was 15 weeks ;the median survival time of the control group (accepted chemotherapy ) was 24 weeks ;the median survival time of the giving up group(accepted any therapy ) was 8 weeks .Conclusion Bezafibrate combined medroxyprogesterone acetate could prolong the survival time of AML patients and there were no obvious adverse reactions .

Objective To observe the effects of two rehabilitation programs to staff in office with cervical spondylosis,and to explore the appropriate rehabilitation program for this group of people.Methods There were 37 office personnel with cervical spondylosis were selected by the inclusion criteria.They were randomly divided into treatment group A(once a week,n=17) and treatment group B (once a week,n=20) according to the random number table method.The treatment group A took middle frequency electrotherapy and infrared therapy,while the treatment group B took tuina manipulation.The changes of visual analogue scale (VAS) and cervical range of movement (ROM) were observed after 10 weeks of treatment.Results Compared with before treatment,the VAS scores of the two treatment groups decreased with statistically significant difference after 10 weeks of treatment(tA=12.91,tB=13.88,all P<0.05).There was no significant difference in VAS score between the two treatment groups after treatment(t=0.22,P>0.05).Compared with before treatment,the cervical ROM of the two groups increased significantly after treatment(tA anteflexion=-4.59,tA posterior extension=-5.13,tA left flexion=-4.24,tA right flexion=-3.25,tA left circumflexion=-7.13,tA right circumflexion=-7.47,tB anteflexion=-5.85,tB posterior extension=-7.28,tB left flexion=-8.64,tB right flexion=-8.38,tB left circumflexion=-11.72,tB right circumflexion=-11.95,all P<0.05).There were significant differences in the increase range of flexion,left flexion and left circumflexion between the two treatment groups after treatment(tflexion=-2.29,tleft flexion=-2.77,tleft circumflexion=-2.32,all P<0.05),but posterior extension,right flexion and right circumflexion had no statistically significant diffferences(tposterior extension=-1.97,tright flexion=-1.97,tright circumflexion=-1.97,all P>0.05).Conclusion The two kinds of rehabilitation programs can relieve the neck pain of the office personnel,but treatment group B is better than treatment group A in increasing cervical ROM.

51 cases of osteoporosis were put on clinical trial in our OPD and randomly assigned to TCM treatment group (N = 17), TCM control group and blank group. The total effective rate in the TCM treatment group obtained was 94. 1% and the significantly effective rate achieved 88. 2%. After the treatment, the density of the osseous tissue increased by 3-4%, main symptoms improved markedly, the serum calcium increased and the urine pyridine phenol decreased. All the findings were superior than that in the rest two groups. This composite is effective and safe (no side reactions) and worth put into large sale production.

Objective To explore the effect of latissimus dorsi musculocutaneous flap for repairing severe soft tissue defect of the elbow.Methods Seventeen patients with severe soft tissue defect of the elbow were repaired by latissimus dorsi musculocutaneous flap.Ten patients were performed transfer of skin flap for functional reconstruction,9 patients reconstructed elbow flexion,and 1 patient reconstructed elbow extension.Seven patients' tissue defect were repaired by cover type musculocutaneous flap,and the areas of these flaps ranged from 6 cm × 10 cm-12 cm × 27 cm.Results All the musculocutaneous flaps kept alive.After an average postoperative follow-up for 12-40(22.0 ± 9.6) months,the color and luster of the flaps were satisfying.There were no scar contracture of the flap and no bulkiness of the elbow.Ten patients performed transfer of skin flap for functional reconstruction,and the range of motion was 100°-140° of flexion and-20°-0° of extension.Muscle power was M3-M5.Hand function was near normal.Conclusion Latissimus dorsi musculocutaneous flap is an ideal surgical method to repair the severe soft tissue defect of the elbow.

Objective:To evaluate the effects of different denture cleansers on the physical and mechanical properties of heat-cured denture base resin.Methods:Heat-curing denture base resin samples were prepared and respectively immersed in Polident,Stera-dent,0.5% sodium hypochlorite solution and distilled water for 60 h.The flexural strength and elastic modulus were measured(n =10)using 3-point bending test in a universal testing machine.A surface analyzer was used to measure the surface roughness of each specimen(n =10).Results:There were no significant differences of flexural strength and elastic modulus among Polident,Steradent and distilled water(P >0.05).The 0.5% sodium hypochlorite treatment produced lower flexural strength and elastic modulus than the other cleaners(P <0.05).No statistical significance of roughness was found among the solutions(P >0.05).Conclusion:Both Poli-dent and Steradent don't affect the physical and mechanical properties of the heat-cured acrylic resin.0.5% sodium hypochlorite solu-tion doesn't affect the surface roughness,but decreases the flexural strength and elastic modulus of the resin.

AIM: To investigate the role of epigenetic modification in Pdx-1 gene transcription and expression, and to compare the differences between epigenetic modifications of Pdx-1 gene promoter in various cell types of mice. METHODS: The promoter DNA methylation and histone modification status of Pdx-1 and MLH1 genes in NIT-1 cells, NIH3T3 cells and mouse embryonic stem cells were measured by chromatin immunoprecipitation-real time PCR method. The expression levels of these genes in the three cell lines were measured by real time RT-PCR. The relation between epigenetic modifications and gene expression was analyzed. RESULTS: (1) Compared to mES cells, there was lower DNA methylation and higher H3K4m3 modification levels in the promoter of Pdx-1 gene in NIT-1 cells (P<0.05). DNA methylation, H3 acetylation, H3K4m3 and H3K9m3 modification levels in the promoter of Pdx-1 gene in NIH3T3 cells were distinctly increased (P<0.05). (2) Pdx-1 gene transcription expressed only in NIT-1 cells. The Spearman's rho between Pdx-1 gene expression and DNA methylation (r=-0.802,P<0.01) was observed. The Pearson correlation between Pdx-1 gene expression and H3K4m3 modification (r=0.997,P<0.01) was also found. The Spearman's rho between Pdx-1 gene expression and H3K9m3 modification (r=-0.879,P<0.01) was observed. (3) No correlation between housekeeper MLH1 gene expression and epigenetic modification was found. CONCLUSION: DNA methylation, H3K4m3 and H3K9m3 modification coordinated participate to regulate and control the expression of Pdx-1 gene. It is of great significance to the differentiation of β cells from ES cells.