The aim of this study is to establish an gene editing method of Mesomycoplasma hyo-pneumoniae(Mhp)based on the homologous recombination principle.The restriction enzyme di-gestion and ligation method combined with gene synthesis were used to construct a shuttle plasmid to achieve replication in both Mhp and Escherichia coli(E.coli).The pGEM?-T vector was used as the skeleton.The oriC sequence of Mhp which can achieve the replication of the plasmid in Mhp was inserted into the vector.Sequences of the Spiroplasma promoter and puromycin resistance gene were then inserted into the above constructed plasmid to screen recombinant clones.The up-stream and downstream homologous arms of p97 were constructed to initiate homologous recombination.The recA gene of E.coli is inserted to improve the efficiency of homologous recom-bination.The obtained shuttle plasmid was then delivered into Mhp by electro-transformation or chemical transformation.A shuttle plasmid,pGEM?-Mhp-oriC-p 97,which can replicate in both Mhp and E.coli was constructed.With the transformation of this plasmid,the carried puromycin gene and recA gene can be expressed,the p97 gene can be edited.Finally,the genetically unstable p97 gene mutant was initially obtained.In this study,a tool for Mhp gene editing based on the principle of homologous recombination was established,which laid a foundation for the develop-ment of tools for studying the pathogenesis of Mhp.

The aim of this study is to establish an gene editing method of Mesomycoplasma hyo-pneumoniae(Mhp)based on the homologous recombination principle.The restriction enzyme di-gestion and ligation method combined with gene synthesis were used to construct a shuttle plasmid to achieve replication in both Mhp and Escherichia coli(E.coli).The pGEM?-T vector was used as the skeleton.The oriC sequence of Mhp which can achieve the replication of the plasmid in Mhp was inserted into the vector.Sequences of the Spiroplasma promoter and puromycin resistance gene were then inserted into the above constructed plasmid to screen recombinant clones.The up-stream and downstream homologous arms of p97 were constructed to initiate homologous recombination.The recA gene of E.coli is inserted to improve the efficiency of homologous recom-bination.The obtained shuttle plasmid was then delivered into Mhp by electro-transformation or chemical transformation.A shuttle plasmid,pGEM?-Mhp-oriC-p 97,which can replicate in both Mhp and E.coli was constructed.With the transformation of this plasmid,the carried puromycin gene and recA gene can be expressed,the p97 gene can be edited.Finally,the genetically unstable p97 gene mutant was initially obtained.In this study,a tool for Mhp gene editing based on the principle of homologous recombination was established,which laid a foundation for the develop-ment of tools for studying the pathogenesis of Mhp.

Objective To explore the feasibility and rationality of advanced integrated two-stage laparoscopic simulation training course in standardized training of surgical residents.Methods From December 2019 to December 2021,the advanced integrated two-stage laparoscopic simulation training course was carried out among 2019-2020 surgical residents who received standardized training in our hospital.The course was divided into two stages.In the first stage,BEST(best essential surgical technology training)course,adopted Darwin? endoscopic training system,Tianyan? endoscopic training system,Microport? 3D laparoscopic training system and simple simulative models were used.The second stage,BEST PLUS course,same platform as that in BEST course and in vitro animal models were used.The questionnaire survey method(before and after class questionnaire)was adopted to evaluate the curriculum setting,such as curriculum form,simulators,teaching method,time arrangement,curriculum difficulty,training effect,curriculum satisfaction and so on.Results A total of 37 surgical residents completed the two-stage course training and the questionnaire survey.The overall satisfaction rate with the curriculum setting was 100%.There were 32 residents(86.5%)thought that first stage training course could significantly improve their clinical skills,35 residents(94.6%)thought that second stage training course could significantly improve their clinical skills,and 36 resident(97.3%)thought that the first stage curriculum could significantly help them improve performance in the second stage curriculum.Conclusions The trainees had a high degree of recognition and satisfaction for the advanced integrated two-stage laparoscopic simulation training course.The overall design of course was reasonable and feasible,and was attractive to trainees.

Objective:To analyze and summarize the implementation effect of basic essential surgical training (BEST) course of laparoscopic skills over the past 10 years and the practical experience in updating course content and models.Methods:The pre-class assessment questionnaires, basic laparoscopic operation assessment results, and post-class assessment questionnaires of the students who participated in the BEST course of laparoscopic skills were collected. According to the period of the course construction, the students were divided into two groups, namely students who used the course of single training system in the early stage (traditional group) and students who used the course integrating a variety of training systems after the course model was updated in the later stage (test group). The two groups were compared for the scores of track circle moving, tunnel crossing, and high and low columns, as well as their subjective evaluation of course setting and implementation effect. The t-test, Wilcoxon test, or chi-square test was conducted according to the data type using SPSS 13.0. Results:The time for 150 traditional group students to complete track circle moving, tunnel crossing, and high and low columns was 1.08 min (0.81 min, 1.60 min), 2.20 min (1.60 min, 3.27 min), and 4.86 min (3.28 min, 6.36 min), respectively, while the time for 75 test group students to complete the three operations was 1.27 min (0.87 min, 1.83 min), 2.57 min (1.58 min, 4.07 min), and 4.35 min (2.90 min, 6.42 min), respectively, with no significant difference between the two groups ( P>0.05). In terms of students' subjective evaluation of the course, a higher percentage of the test group students were satisfied with classroom environment, teaching method arrangement, training equipment, training opportunities, helping clinical work, and meeting pre-class expectations than those in the traditional group. Conclusion:The constantly updated BEST course can ensure the training quality of trainees and obtain their higher satisfaction. The benefits of this course in clinical practice can be further verified through long-term follow-up of these trainees.

Objective To investigate effect of Nd:YAP laser combined with periodontal basic treatment on periodontal indexes,subgingival microflora,adiponectin,matrix metalloproteinase-13(MMP-13),interleukin-6(IL-6)in gingival crevicular fluid in patients with periodontitis.Methods A total of 162 patients with periodontitis diagnosed and treated in the First People's Hospital of Huzhou from January 2020 to December 2021 were selected and divided into experimental group(n=55),basic group(n=54)and control group(n=53)by random number table method.The basic group received periodontal basic treatment,the control group was given Nd:YAP laser treatment,and the experimental group was given Nd:YAP laser combined with periodontal basic treatment.The clinical efficacy,probing depth(PD),bleeding index(BI),clinical attachment loss(CAL),plaque index(PLI),subgingival microflora,adiponectin,MMP-13 and IL-6 levels in gingival creval fluid and the incidence of complications were compared among three groups.Results At 6 weeks after treatment,there was significant difference in the total effective rate among three groups(P<0.05),and experimental group>control group>basic group.At 6 weeks after treatment,PD,CAL,BI,PLI,bacteroides,clostridium,lactobacillus,MMP-13 and IL-6 in three groups were significantly lower than before treatment,and adiponectin were significantly higher than before treatment(P<0.05).PD,CAL,BI,PLI,bacteroides,clostridium,lactobacillus,MMP-13 and IL-6 in experimental group were significantly lower than those in control group and basic group,and adiponectin was significantly higher than that in control group and basic group(P<0.05).There was no significant difference in the incidence of adverse reactions among three groups(P>0.05).Conclusion Nd:YAP laser combined with periodontal basic treatment has a good therapeutic effect on periodontitis,which can improve periodontal indexes,subgingival microflora,and adiponectin,MMP-13 and IL-6 in gingival crevicular fluid.

Objective:To explore the influence of patient participation-based dietary intervention on nutritional status for patients with severe burn.Methods:From September 2017 to January 2019, 60 severe burn patients hospitalized in the department of burn and plastic surgery of Qingdao Municipal Hospital were recruited and divided into the experimental group ( n=30) and the control group ( n=30) according to the random number table method. The control group received a regular diet. While the experimental group received a patient participation-based dietary intervention(PPDI), The wound healing time and the value of nutritional status index, such as height, body mass index(BMI), serum albumin, serum prealbumin, in the two groups on admission, at 2 weeks after intervention, 4 weeks after intervention were compared. The nutritional knowledge questionnaire of burn patients and "3-day diet diary" were used for investigation. Results:The score of nutritional knowledge showed no significant difference between the two groups before intervention. After intervention, the score of nutritional knowledge in the experimental group was 21.40±2.42, significantly higher than that in the control group (19.00±2.26) ( t value was 3.975, P<0.01). For time effect and between-group effect, there were significant difference in serum albumin between two groups ( F values were 9.232, 4.651, P<0.05); the time effect and between-group effect of serum prealbumin were statistically significant ( F values were 11.592, 6.228, P < 0.05). The wound healing time in the experimental group was significantly lower than that in the control group ( t value was -3.801, P<0.01). Conclusions:Patient participation-based dietary intervention can effectively enhanced the level of nutritional knowledge, improved nutritional status, shortened wound healing time among patients with severe burn.

Objective: To establish a mouse model of psoriasis complicated by bone loss by long-term topical application of imiquimod. Methods: Twelve 10-week-old Kunming mice were randomly and equally divided into 2 groups: experimental group topically treated with 50 mg/d imiquimod cream every day on the shaved back, and control group topically treated with equivalent vaseline ointment every day on the shaved back. Skin manifestations were observed on the mouse back every day. The mice were sacrificed 10 weeks later. Before the sacrifice, the degree of erythema, scaling and skin thickening was evaluated, psoriasis area severity index (PASI) was calculated, mouse weight was measured, and eyeball blood was obtained. After the sacrifice, skin lesions on the back were resected and subjected to hematoxylin-eosin staining, so as to evaluate histological changes. Then, the left tibia was obtained from the mice, immunohistochemical staining was performed to observe the expression and distribution of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-β ligand (RANKL) in bone tissues, and micro-computerized tomography was conducted to determine the bone mass of spongy bone, and the bone volume-to-total volume ratio, number, thickness, spacing and connectivity density of the trabecular bone in the proximal tibia. The left femur was also obtained from the mice, and subjected to three-point bending test for evaluating its biomechanical properties. Enzyme-linked immunosorbent assay (ELISA) was performed to detect serum levels of tumor necrosis factor (TNF) -α and interleukin (IL) -17. RNA was extracted from the right tibia, and real-time PCR was conducted to determine the mRNA expression of OPG and RANKL. Two-independent-sample t test was used to compare the above indices between two groups. Results: Ten-week topical stimulation with imiquimod could lead to psoriasiform dermatitis on the mouse back, presenting as erythema, scales and skin thickening. The PASI score was significantly higher in the experimental group (9.167 ± 1.722) than in the control group (0, t = 13.31, P < 0.001) . Hematoxylin-eosin staining showed thickened spinous layer, extended rete ridges, infiltration of inflammatory cells in the superficial dermis, spongiosis, vasodilatation and increased hair follicles in the experimental group. Immunohistochemical staining revealed that the expression of OPG and RANKL was significantly higher in the experimental group (16 021.33 ± 1 954.61, 35 433.33 ± 1 197.95 respectively) than in the control group (3 307.00 ± 1 158.72, 13 644.67 ± 4 764.61, respectively; t = 9.692, 7.682 respectively, both P < 0.01) . Micro-computerized tomography showed that the bone volume-to-total volume ratio and thickness of trabecular bone in the proximal tibia were significantly lower in the experimental group than in the control group, but the spacing of trabecular bone was significantly higher in the experimental group than in the control group (P < 0.01 or < 0.05) . There were no significant differences in the elastic modulus and fracture energy of the femur between the experimental group and control group (both P > 0.05) . Moreover, no significant differences in the serum levels of TNF-α and IL-17 were observed between the two groups (both P > 0.05) . Real-time PCR revealed that the mRNA expression of OPG and RNAKL was significantly higher in the experimental group than in the control group (P < 0.05, < 0.01 respectively) . Conclusions Long-term topical application of imiquimod can not only induce psoriasiform dermatitis in mice, but also lead to bone loss of spongy bone and micro-architectural deterioration in the proximal tibia. Thus, mouse models of psoriasis complicated by bone loss can be established by long-term imiquimod stimulation.