Objective To construct Raji-Luc lymphoma cells with CD19 knockout using CRISPR/Cas9 technology and preliminarily validate their immune escape ability.Methods PB-CRISPR-CD19 small guide RNA(sgRNA)plasmids was constructed,the optimal sgRNA sequence was screened,and Raji-Luc cells with pCAG-PBase,PB-CD19 sgRNA,and PB-CRISPR-Cas9 were co-transfected.Stable knockout monoclonal cell lines were screened by flow sorting and limit dilution method and the knockout effect was verified through gene sequence testing.The expression of luciferase on the surface of the cell line was detected by microplate reader,CD19 CAR-T and CD38 CAR-T previously constructed in the laboratory were used as effector cells,and the immune escape ability of Raji-Luc CD19 KO cell line was verified by universal luciferase chemiluminescence method.Results The transfection efficiency of Raji-Luc CD19 KO cells prepared by electro transfection was high,and the knockout efficiency of the two monoclonal cells was more than 99%.There was no significant difference in luciferase expression compared to the original Raji-Luc cells,and CD19 CAR-T cells could not be activated to the kill them.Conclusion Successfully constructed Raji-Luc CD19 KO lymphoma cell line.

ObjectiveTo explore the protective effect and mechanism of Zingiberis Rhizoma Recens alcohol extract on lipopolysaccharide （LPS）-induced acute lung injury in mice. MethodBalb/c mice were randomly divided into normal group， model group， dexamethasone group， and low- and high-dose Zingiberis Rhizoma Recens groups. Mice in the normal group were instilled with normal saline through the nose， and the other groups were instilled with normal saline containing LPS （50 μg）. After 30 minutes of modeling， the dexamethasone group was gavaged with 5 mg·kg^-1 of dexamethasone acetate solution， the low- and high-dose Zingiberis Rhizoma Recens groups were gavaged with different doses of （7， 14 g·kg^-1） of Zingiberis Rhizoma Recens alcohol extract， and the normal group and the model group were gavaged with the same volume of water. After 24 hours of modeling， the total number of white blood cells in bronchoalceolar lavage fluid （BALF） was detected by cell counter， and the levels of the inflammatory factors including tumor necrosis factor （TNF）-α， interleukin （IL）-1β， IL-6， and superoxide dismutase （SOD）， and myeloperoxidase （MPO） was detected by enzyme-linked immunosorbent assay （ELISA）. Haematoxylin-eosin （HE） staining method was used to observe the pathological changes of lung tissue in each group， and the Western blot was used to detect the protein expression of nuclear transcription factor （NF）-κB p65， phosphorylation （p）-NF-κB p65， and Toll-like receptor 4 （TLR4） in lung tissue. ResultCompared with the normal group， the white blood cell count in BALF and the levels of TNF-α， IL-1β， IL-6， and MPO in the model group was increased （P<0.01）， and the level of SOD was decreased （P<0.05）. Pathological damage of lung tissue was obvious， and the protein expression of NF-κB p65， p-NF-κB p65， and TLR4 in lung tissue was increased （P<0.01）. Compared with the model group， the white blood cell count in BALF and the levels of TNF-α， IL-1β， IL-6， and MPO in the treatment group was decreased （P<0.05，P<0.01）， and the level of SOD was increased （P<0.05，P<0.01）. Pathological damage of lung tissue was alleviated， and the protein expression of NF-κB p65， p-NF-κB p65， and TLR4 in lung tissue was decreased （P<0.01）. ConclusionZingiberis Rhizoma Recens alcohol extract may play a protective role in LPS-induced acute lung injury in mice by inhibiting the TLR4/NF-κB signaling pathway.

Traumatic spinal cord injury (TSCI) is a common nerve injury in the world, which has a high incidence and disability rate. Intraspinal pressure (ISP) monitoring is performed by placing a pressure sensing probe under the dura of the most severe part of the spinal cord injury after anterior/posterior laminectomy or vertebral body decompression. ISP value is monitored dynamically and objectively in real time by the pressure sensing system. Recent studies have found that ISP monitoring plays an important role in the clinical management and prognosis evaluation of TSCI. The author reviews the recent advance in ISP monitoring in TSCI in order to provide references for the improvement of clinical diagnosis and treatment of TSCI.

Traumatic spinal cord injury (TSCI) is a common nerve injury in the world, which has a high incidence and disability rate. Intraspinal pressure (ISP) monitoring is performed by placing a pressure sensing probe under the dura of the most severe part of the spinal cord injury after anterior/posterior laminectomy or vertebral body decompression. ISP value is monitored dynamically and objectively in real time by the pressure sensing system. Recent studies have found that ISP monitoring plays an important role in the clinical management and prognosis evaluation of TSCI. The author reviews the recent advance in ISP monitoring in TSCI in order to provide references for the improvement of clinical diagnosis and treatment of TSCI.

Objective:To compare the effects of dexmedetomidine combined with propofol on anesthesia and thromboelastography in patients undergoing radical gastrectomy.Methods:From September 2017 to December 2019, 120 patients undergoing radical gastrectomy in Chaoyang Central Hospital were prospectively selected and divided into control group and observation group according to random number table method, with 60 cases in each group.The two groups used the same drugs before induction and the same way of anesthesia induction. During the maintenance of anesthesia, remifentanil and propofol were injected intravenously in the control group, and dexmedetomidine was injected in the observation group on the basis of the control group. The indexes of thromboelastography, preoperative and postoperative cellular immune function, postoperative analgesic effect [Visual Analogue Scale (VAS)], Ramsay sedation score, and postoperative adverse reactions were compared between the two groups at different times.Results:The reaction time of coagulation factor (R) and fibrinogen (K) in the two groups decreased 3 hours after operation, and those in the observation group were lower than those in the control group (all P<0.05); The maximum thrombus amplitude (MA) of the two groups increased 3 hours after operation, and MA in the observation group was higher than that in the control group (all P<0.05). Compared with that before operation, the VAS scores and Ramsay sedation scores in the control group and the observation group at 24 h and 48 h after operation were significantly lower (all P<0.05), and the VAS scores and Ramsay sedation scores in the observation group at 24 h and 48 h after operation were significantly lower than those in the control group (all P<0.05). Compared with that before operation, the CD4 +, CD8 +, CD4 + /CD8 + in the control group and the observation group were improved at 6 h and 48 h after operation (all P<0.05), and the improvement in the observation group was significantly better than that in the control group at 6 h and 48 h after operation (all P<0.05). The incidence of adverse reactions in the control group was 6.67%(4/60), which was slightly higher than that in the observation group of 5.00%(3/60), but the difference was not statistically significant ( P>0.05). Conclusions:Compared with propofol and remifentanil alone, combined application of dexmedetomidine can help patients undergoing radical gastrectomy for gastric cancer to achieve better analgesic effect, improve the blood coagulation state of patients, and play a better regulatory role on cellular immune function, which is worthy of further promotion in clinic.

Objective: To understand the epidemic status and influencing factors of Mongolian children with ASD in central and eastern Inner Mongolia, so as to provide data support for formulating prevention and intervention strategies and improving the overall epidemiological investigation of ASD in Inner Mongolia. Methods: Sixteen kindergartens and primary schools were selected from Chifeng City, Ulanqab City, Tongliao City, Hulunbuir City and Xilingol League cities in Inner Mongolia by means of random cluster sampling. Firstly, 7 108 children aged 3-14 were initially screened with the Kirschner Autism Behavior Scale(CABS), and then the children with ASD positive were given the autism behavior test scale (ABC). According to the diagnostic criteria, the professionals, including chief physicians and associate chief physicians from the major of child psychiatry, diagnosed ASD with the total score of ABC scale ≥62. Univariate and Logistic regression multivariate analysis were carried out among Mongolian children to find out the influencing factors related to the occurrence of Mongolian ASD in Inner Mongolia. Results: The prevalence of Mongolian children was 0.37%. Mongolian ASD group and Mongolian normal children series in the household register, habitual twitch, hyperactivity, bite lips, families have extreme introverts, mothers age, father s cultural level, cultural degree of mother, father mother mild character, irritable, neonatal diseases, fetal gestational age distribution had statistical significance( χ 2/Z= 12.58 , 16.68, 14.93, 64.43, -3.76, -2.86, 4.57, 11.12, 12.33, 16.66, P <0.05). Conclusion Measures such as shaping a healthy growth environment, adjusting parental style, paying attention to the level of early childhood language development, and preventing neonatal diseases might lower the risk of ASD in children.

Objective: To understand the prevalence and influencing factors of children with ASD in central and eastern Inner Mongolia, and to provide theoretical basis for disease prevention and prevalence of ASD. Methods: Sixteen primary schools and kindergartens were selected from 5 cities in central and eastern Inner Mongolia through random cluster sampling. A total of 15 817 children aged 3-14 years were selected. Children who were positive using Clancy Autism Behavior Scale were further diagnosed according to the teacher s nomination form and the Autism Behavior Checklist, as well as the diagnostic criteria of the fifth edition of the American Diagnostic & Statistical Manual of Mental Disorders by 2 professionals. Results: The prevalence of ASD was 0.27% (42/15 817), with prevalence in urban areas (0.16%, 15/9 231) higher than that of rural areas (0.41%, 27/6 586) ( χ 2=8.89, P <0.01). Logistic regression analysis showed that maternal education and language development were negatively associated with ASD in urban children [ OR =0.29(95% CI =0.12-0.69) and 0.18(95% CI =0.05-0.60), P <0.05]. ASD in rural children were positively associated with enuresis and introverted family members [ OR =7.09(95% CI =1.60-32.27) and 8.63(95% CI =3.10- 24.01 ), P <0.05]. Conclusion High prevalence of ASD is found in urban area of central and eastern Inner Mongolia. Unhealthy habits, neonatal diseases, low parental education, delayed language development and poor exercise performance are primary factors associated with ASD in both urban and rural areas.

To screen the best genotypeⅠJapanese encephalitis virus subunit vaccine candidate antigens, the prMEIII gene, the polytope gene and the prMEIII-polytope fusion gene of the GenotypeⅠJapanese encephalitis virus GS strain were cloned into prokaryotic expression vector pET-30a. The recombinant proteins were obtained after the induction and purification. The prepared recombinant proteins were immunized to mice, and the immunogenicity of the subunit vaccine candidate antigens was evaluated through monitoring the humoral immune response by ELISA, detecting the neutralizing antibody titer by plaque reduction neutralization test, and testing the cell-mediated immune response by lymphocyte proliferation assay and cytokine profiling. The recombinant proteins with the molecular weights of 35 (prMEIII), 28 (polytope antigen) and 57 kDa (prMEIII-polytope) induced strong humoral and cellular immune responses in mice. Compared with prMEIII-polytope and polytope proteins, the prMEIII protein induced a significant expression of IL-2 and IFN-γ (P<0.05) and the significant lymphoproliferation of splenocytes (P<0.05). The neutralizing antibody titer induced by the prMEIII protein was close to that induced by the commercial attenuated vaccine SA14-14-2 (P>0.05). The study suggests that the prMEIII protein can be used for the development of the Japanese encephalitis virus subunit vaccine.
Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; immunology ; Encephalitis Virus, Japanese ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; Immunogenicity, Vaccine ; Mice ; Mice, Inbred BALB C ; Vaccines, Subunit ; immunology ; Viral Vaccines ; immunology

Objective To verify the feasibility of discriminating tissue types in the anterior approach of the cervical spine by the Electrical impedance (EI) and decrease the rate of severe complications.Methods Six New Zealand white rabbits and 6 mini-pigs were performed standard anterior cervical surgery.The esophagus,carotid artery,tracheal cartilage,annular ligaments of trachea,longus colli muscle and anterior longitudinal ligament are classified as the prevertebral group;The cortical bone,cancellous bone,anulus fibrosus and nucleus pulposus are classified as the vertebral group.Once all the tissues were exposed completely,over the frequency range of 200-3 000 kHz (the frequency points were 200 kHz,400 kHz,600 kHz,800 kHz,1 000 kHz,2 000 kHz,3 000 kHz),the in vivo EI of prevertebral group and vertebral group were measured by a probe and a precision inductance-capacitance-resistance (LCR) meter;then the data were analyzed with IBM SPSS Statistics 22 software.At every frequency,Kruskal-Wallis test followed by all pairwise multiple comparisons was applied for all the four groups (the prevertebral group of rabbits,the vertebral group of rabbits,the prevertebral group of mini-pigs,and the vertebral group of mini-pigs),respectively.P values ＜ 0.05 were considered statistically significant.Results At every frequency,the results of Kruskal-Wallis test for all the four groups were significant,and the results of multiple comparisons were as follows.(1) In both miniature experimental animals and large experimental animals,the EI of the vertebral group tissues was significantly different at every investigated frequency (multiple comparisons).(2) In the miniature experimental animals,the EI between longus colli muscle and esophagus,carotid artery and annular ligaments of trachea,and tracheal cartilage and anterior longitudinal ligament had no significant difference at any frequency (multiple comparisons);significant difference could not be detected in the EI between anterior longitudinal ligament and annular ligaments of trachea at the frequencies of 2000-3 000 kHz (multiple comparisonsk) and could be detected at the frequencies of 200-1 000 kHz (multiple comparisons);for comparison of all other paired tissues of prevertebral group there were significant differences at all frequencies (multiple comparisonsk).(3) In the large experimental animals,the EI between longus colli muscle and esophagus,carotid artery and annular ligaments of trachea,and tracheal cartilage and anterior longitudinal ligament had no significant difference at any frequency (multiple comparisons),the same was true for the EI between annular ligaments of trachea and esophagus,carotid artery and anterior longitudinal ligament,and longus colli muscle and annular ligaments of trachea at the frequencies of 800-3 000 kHz,3 000 kHz and 3 000 kHz (multiple comparisons),respectively;for comparison of all other paired tissues of prevertebral group there were significant differences at all frequencies (multiple comparisons).Conclusion At certain frequencies,the EI among tissues was significantly different and could discriminate tissues in the anterior approach of the cervical spine.

Objective The aim of this study was to investigate the distribution and clinical significance of CD8+ T cells in bladder cancer tissues in situ.Methods Immunohistochemistry were used to examine the distribution of CD8+ T cells in bladder cancer tissues,which were obtained from January 2003 to December 2009 from 302 patients.Among all the patients,262 were male while 40 were female;mean age is 60 years;tumor size ≤ 3 cm was in 235 and tumor size ＞ 3 cm was in 67;Unifocal tumor was in 214 and multifocal tumors were in 88.Amount of tumor stage Ta-T1 was 212 and T2-T4 was 90.Sixteen patients have lymph node metastasis.Histological low grade was diagnosed in 175 and histological high grade was diagnosed in 127.According to the differences between anatomic structure and cellular composition,bladder tumor tissues can be classified to two localization patterns:(1) intratumoral regions,defined as tumor cell nests;(2) stromal regions,defined as stromal areas that lack direct contact with tumor cells.Therefore,we divided 302 bladder cancer patients into two groups based on the median frequency of intratumoral CD8+ T cells (median,3/× 400 high resolution) and stromal CD8+ T cells (median,37/× 400 high resolution),respectively.x2 analysis was used to evaluated the correlation between CD8+ T cell density and clinicalpathological variables.Kaplan-Meier analysis and Cox proportional hazards regression models were applied to estimate overall survival (OS).Results CD8+ T cells were predominantly located in the intratumoral regions (mean,14 ± 2/× 400 high resolution) rather than in associated stromal regions (mean,50 ± 3/× 400 high resolution,P ＜ 0.05).The density of intratumoral CD8+ T cells was inversely associated with age (P =0.026),tumor size (P ＜ 0.05) and tumor stage (P ＜ 0.05),and could represent a favorable prognostic predictor of OS (HR =0.427,P =0.003).However,the density of stromal CD8+ T cells was positively associated with age (P =0.004) and histological grade (P ＜ 0.01),and could represent an adverse prognostic predictor of OS (HR =2.206,P =0.009).Conclusions Our findings suggest that intratumoral/ stromal CD8+ T cells could potentially serve as favorable/ adverse prognostic markers for bladder cancer patients,respectively.