AIM: To construct engineered E.coli strains which can express nattokinase with fibrinolysis activity using gene engineering technology. METHODS: The pro-nattokinase (pro-NK) gene was amplified by PCR and inserted into expression vector pET3c. The recombined plasmid pENK which expressed the fusion protein of pro-NK and 22 amino acid peptide was then transferred into lysogenic host strains BL21(DE3)pLysS - and BL21(DE3)pLysS +. Both SDS-PAGE and the fibrin plate assay were used to examine the expression and the activity of the target protein. RESULTS: SDS-PAGE assay showed the fused gene encoding 42 kD fusion protein was expressed in both expression strains pENK-(DE3)pLysS - and pENK-(DE3)pLysS +, and the fibrin plate assay indicated that the expression product had fibrinolysis activity. pENK-(DE3)pLysS - exhibited the basal expression of the target gene, while fusion protein was only induced by IPTG in pENK-(DE3)pLysS +. Basal expression of the fused toxic gene in pENK-(DE3)pLysS - led to bacteriolysis and hollow lawns. CONCLUSION: A pro-NK fusion protein with fibrinolysis activity is successfully expressed in E.coli , which lay a foundation for the exploitation of nattokinase.

Objective:To explore the microRNA profile changes in the development of NKT cells.Methods: Differently developmental stage of NKT cells in mouse thymus were sorted by flow cytometry.Total RNA were extracted,reversely transcribed and pre-amplified.TaqMan low density microRNA assay and single real-time PCR were applied to detect the expression changes of microRNAs in the developmental process of NKT cells.Results: There were total 92 microRNAs whose expression changed significantly during the development and maturation of NKT cells.Among them,increasly expressed microRNAs were 71,including 36 microRNAs whose expression continuously increased;decreasly expressed microRNAs were 21,including 12 microRNAs whose expression continuously decreased.In addition,single real-time PCR analysis showed that the expression of Let-7f,miR-150,miR-24,miR-29 increased,while the expression of miR-223 and miR-155 decreased during the development and maturation of NKT cells.Conclusion: NKT development and maturation is accompanied by expression changes of large amount of microRNAs,indicating that specific microRNA regulates NKT development and function.

AIM: To construct a recombinant hEGF-hbFGF(78-154aa)fusion protein, which not only has the heparin-binding ability, but also promotes the growth of the cells, and to express the fusion protein in E. coli expression system with high expression level.METHODS: hEGF gene was joined with 231 bp fragment coding hbFGF(78-154aa) and expressed in E. coli. The fusion protein was purified using affinity chromatography of heparin-Hyper D and analyzed with western blot. The pI value and the biological activity were both assayed.RESULTS: The fusion protein was expressed in a high expression level of about 30% of the total cell protein, as estimated by SDS-PAGE. Western analysis results showed that the antigenicity of fusion protein was similar to hEGF. Fusion protein could not only bind heparin but also promote the growth of 3T3 cell. The pI value of fusion protein was 5.2.CONCLUSION: The recombinant hEGF-hbFGF(78-154aa) fusion protein possessed the characteristics of both hEGF and hbFGF. This new-designed protein would become a good object for the research on the relationship between the structure and the function of the growth factor.

Objective To explore the clinical efficacy of Da Vinci robot-assisted iliofemoral vein bypass grafting.Methods The retrospective descriptive study was conducted.The clinical data of one 66-year-old male patient who underwent Da Vinci robot-assisted iliofemoral vein bypass grafting in the First Affiliated Hospital of Army Military Medical University in March 2019 were collected.The patient was failed to recanalize iliofemoral vein stent thrombosis by endovascular measures and underwent Da Vinci robot-assisted iliofemoral vein bypass grafting after balloon occlusion preset in the common iliac vein.Observation indicators:(1) intra-and postoperative situations;(2) follow-up and survival situations.Follow-up using outpatient examination was performed to detect the patient's postoperative survival and swelling reduction of affected extremity up to April 2019.Results (1) Intra-and post-operative situations:the patient underwent Da Vinci robot-assisted iliofemoral vein bypass grafting successfully.The operation time of balloon occlusion preset by digital subtraction angiography was 35 minutes.The operation time of Da Vinci robot-assisted iliofemoral vein bypass grafting was 502 minutes (50 minutes of exposure time of femoral vein,80 minutes of exposure time of iliac vein,40 minutes of great saphenous vein harvesting time,70 minutes of end to side anastomosis between autogenous great saphenous vein and femoral vein,10 minutes of subcutaneous tunnel construction,90 minutes of end to side anastomosis between autogenous great saphenous vein and iliac vein,60 minutes of suturing except vessel closure,102 minutes of preparation time,check and washing time).The volume of intraoperative blood loss was 500 mL and no intraoperative complications occurred.The autogenous great saphenous vein graft was well filled and no bleeding was found at both proximal and distal anastomoses after iliofemoral vein bypass grafting.There were 4 abdominal Trocar holes including 2 of 1.2 cm and 2 of 0.8 cm.The incisional length of right groin and left great saphenous vein harvesting region was 5.0 cm and 15.0 cm,respectively.At the discharge time,the patient had swelling subsided partially at right lower extremity and skin tesion reduced significantly compared with the admission.The perimeters at 15 cm above right knee joint and left knee joint were 53.5 cm and 48.0 cm.The maximum perimeters of right calf and left calf were 41.0 cm and 38.0 cm.No postoperative complications occurred.Duration of hospital stay after surgery was 3 days.(2) Follow-up and survival situations:the patient was followed up for 1 month,with good survival.The patient had swelling subsided of affected extremity.The perimeters at 15 cm above right and left knee joint were 52.0 cm and 48.0 cm.The maximum perimeters of right calf and left calf were 40.0 cm and 38.0 cm.Conclusion The Da Vinci robot-assisted iliofemoral vein bypass grafting is safe and feasible,with good short-term outcomes.

Objective:To investigate the clinical efficacy of endovascular interventional therapy for spontaneous isolated superior mesenteric artery dissection(SISMAD).Methods:The retrospective cohort study was conducted. The clinical data of 87 patients with SISMAD who were admitted to The First Affiliated Hospital of Army Medical University from March 2012 to March 2023 were collected. There were 80 males and 7 femals, aged 54(49,61)years. Of 87 patients, 55 cases undergoing conservative therapy were allocated into conservative therapy group and 32 cases under-going endovascular interventional therapy were allocated into endovascular interventional therapy group. Observation indicators: (1) clinical characteristics; (2) treatment; (3) follow-up. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the t test. Measurement data with skewed distribution were represented as M( Q1, Q3), and comparison between groups was conducted using the rank sum test. Count data were represented as absolute numbers and comparison between groups was conducted using the chi-square test or Fisher exact probability. Results:(1) clinical characteristics. There were significant differences in the cases with symptoms, percentage of neutrophils between the conservative therapy group and the endovascular interventional therapy group ( P<0.05). There was no significant difference in the proportion of Yun classification between the two groups ( P>0.05). (2)Treatment. There were significant differences in the complete vascular remodeling, duration of hospital stay, and total expenses between the conservative therapy group and the endovascular interventional therapy group ( χ2=23.752, t=-4.213, -16.421, P<0.05). There were 34 patients in the conservative therapy group and 24 patients in the endovascular interventional therapy group with relieved abdominal pain, respectively, showing no significant difference between the two groups ( P>0.05). For symptomatic patients in the conservative therapy group, symptoms including abdominal pain, nausea, vomiting, diarrhea, hematochezia were relieved or disappeared, and no intestinal ischemia or rupture occurred. For patients in the endovascular interventional therapy group, 30 cases were implanted stents, the operation time was 115(86,155)minutes, volume of intraoperative blood loss was 5(5,10)mL, dose of contrast media was (200±51)mL. There were 23, 8 and 1 cases with the contrast medium as Iodoxanol, Ioprosamide, Iodohexanol, respectively. About the surgical methods, 14 patients received single bare stent implantation, 3 cases received bare stent-assisted coil embolization, 10 cases received multiple bare stent implantation, 3 cases received covered stent implantation, 2 cases received angiography alone. A total of 39 self-expandable bare metal stents and 3 self-expandable covered stents were implanted. The diameter and length of the stents were (6.5±1.0)mm and (69±23)mm, respectively. Two asymptomatic patients had failure in endovascular interventional therapy and underwent superior mesenteric artery angiography. For the endovascular interventional therapy group, 92.3%(24/26) of patients were relieved abdominal pain and 2 patients with abdominal pain were improved after symptomatic treatment. (3) Follow-up. All the 87 patients were followed up for 12(4,24)months, without recurrent abdominal pain or secondary intervention. During the follow-up, 82 patients underwent computed tomography angiography or ultrasonography, and 5 patients had no available results. There was no SISMAD related death or superior mesenteric artery rupture. Eight patients in the conservative therapy group achieved complete vascular remodeling, versus 21 cases in the endovascular interventional therapy group, showing a significant difference between the two groups ( χ2=23.752, P<0.05). Conclusions:Compared with conservative therapy, patients undergoing endovascular interventional therapy for SISMAD has loner hospital stay, higher total costs, higher complete vascular remodeling rate. There is no recurrent abdominal pain in two methods.

Objective To explore the impact of exercise intervention on the reading abilities of chil-dren with Chinese developmental dyslexia(CDD).Methods Twenty-seven CDD children were recruited from patients of Peking University Sixth Hospital,and randomly divided into a CDD exercise interven-tion group of 14 and a CDD control group of 13.Another 14 age-matched normal children were select-ed into a normal control group.All groups kept daily physical activities in school,while the CDD ex-ercise intervention group additionally underwent 8-week individualized gross and fine motor interven-tion.The literacy,reading fluency and reading comprehension of all groups were evaluated by using"Elementary School Students'Literacy Test and Evaluation Scale",one-minute word reading and ele-mentary school students'reading ability assessment.Results Before the intervention,there were no sig-nificant differences between the exercise intervention and control groups in the literacy level,reading fluency,and reading comprehension(P>0.05),which were all significantly lower than the normal con-trol group(P<0.05).However,after the intervention,significant improvement was observed in the aver-age literacy level,reading fluency,and reading comprehension of the CDD exercise intervention group,and the literacy and reading fluency of the CDD control group,as well as the average literacy level and reading comprehension of the normal control group.Moreover,there were no significant differ-ences between the CDD exercise intervention and control groups in the average literacy level and read-ing fluency score(P>0.05),both significantly lower than the normal control group(P<0.05).Mean-while,the reading comprehension score of the CDD exercise intervention group was significantly higher than the CDD control group(P<0.05),but significantly lower than the normal control group(P<0.05).Conclusion Eight-week targeted gross and fine motor intervention can effectively improve reading abili-ty of DD children,especially their reading comprehension.