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.

Objective: To investigate the effects and mechanisms of Xuebijing injection (XBJ) on Chimeric antigen receptor-T (CAR-T) cell function and its therapeutic potential against CAR-T therapy-associated cytokine storms (CRS). Methods: Anti-CD19 CAR-T cells were established based on FMC63 antibodies. Different doses of XBJ (1 and 10 mg/mL) were added to the culture system. Untreated anti-CD19 CAR-T cells served as negative controls. After 48-h co-culture, the effects of XBJ on CAR-T cell function were assessed. Carboxyfluorescein diacetate succinimidyl ester staining was used to assess the effect of XBJ on CAR-T cell proliferation. Flow cytometry, luciferase reporter gene assays, and real time cellular analysis were employed to evaluate the effects of XBJ on CAR-T cell cytotoxicity in vitro. RNA-sequencing was performed to analyze the effects of XBJ on CAR-T cell gene expression. Network pharmacology predicted potential XBJ therapeutic targets for CRS, which were verified in a THP-1 macrophage inflammation model. Results: XBJ enhanced both the proliferation and tumor killing capacities of CAR-T cells. Transcriptome analysis showed that XBJ treatment affects multiple genes and pathways in CAR-T cells, with differential gene enrichment in multiple cell proliferation and growth factor pathways. Potential targets for CRS control by XBJ were predicted using network pharmacology, and the inhibitory effect of XBJ on the expression of relevant genes was verified using a macrophage model. Conclusion The results of this study indicate that XBJ can enhance the killing effect of CAR-T cells on tumor cells and that the mechanism is related to the regulation of T cell proliferation and activation. Moreover, XBJ inhibited excessive inflammation associated with CAR-T therapy. However, the current findings remain to be further validated through in vivo experiments.

Objective:To explore the establishment of radiation-induced heart damage (RIDH) SD rat models caused by irradiation of 15Gy/3f and the changes in early detection indicators, and evaluate the effect of irradiation combined with recombinant human endostatin (Endostar).Methods:75 adult male SD rats were randomly divided into the blank control group (C group), Endostar group (E group), 25Gy irradiation group (MHD 25 group), 15Gy irradiation group (MHD 15 group) and 15Gy irradiation combined with Endostar group (MHD 15+ E group), respectively. Blood sample was taken to measure the CK, CK-MB, LDH and CRP at 24h, 48h and 15d after corresponding interventions. After cardiac echocardiography at 1, 3 and 6 months, 5 rats in each group were randomly sacrificed and myocardial tissues were collected for HE and Masson staining. Two-way ANOVA was employed for statistical analysis. Results:Compared with group C, myocardial fibrosis were observed in the MHD 15 group at 6 months ( P<0.05), which occurred later than that in the MHD 25 group. Ejection fraction (EF) and fractional shortening (FS) were significantly decreased after 3 months in each irradiation group (all P<0.05), whereas the degree of decrease was similar among all groups (all P>0.05). The expression levels of myocardial enzymes and inflammatory cytokines did not significantly differ among different groups (all P>0.05). Conclusions:In the early stage, exposure to 15Gy/3f irradiation can cause cardiac function damage in SD rat hearts, such as the reduction of EF and FS, and even lead to myocardial fibrosis in the late stage, which is delayed and less severe than high-dose irradiation. Irradiation combined with Endostar has no significant effect on radiation myocardial injury in rats.

Objective:To construct a rat model of hypothalamic obesity by two point electrical damage to the ventromedial hypothalamus and arcuate nucleus.Methods:Twenty adult male SD rats of SPF grade were randomly divided into experimental group and sham operation group.A 25GA (0.45 mm) solid iron needle was used, the needle was coated with an insulating layer, and the tip exposed a 0.5 mm conductive area.With reference to The Rat Brain in Stereotaxic Coordinates and using the stereotactic instrument (AP: -2.6 mm, ML: ± 0.6 mm, DV: -9.6 mm) as the coordinate, 1.5 mA current was continuously applied for 25 s, the ventromedial nucleus (VMH) and arcuate nucleus (ARC) of bilateral brain in SD rats was damaged.During the experiment, the body weight(BW), food intake(FI) and water intake(WI) of the two groups were recorded regularly.The rats were sacrificed on the 28th day after the operation, and the changes of periprenal fat mass and body length were measured.The changes of liver and adipose tissue were detected by HE staining method, leptin by ELISA, leptin receptor(LEPR) by Western blot.Results:(1) The body weight of rats in the experimental group ((427.5±17.7)g) and weight gain ((208.5±14.8)g) were significantly increased compared with the rats in the control group((349.2±17.7)g), ((136.2±21.4)g)on the 28th day after operation ( t=7.661, 6.806, both P<0.001). (2) The daily food intake of rats in the experimental group ((44.2±6.6)g) on the 28th day after surgery was significantly higher than that in the control group ((23.0±3.6)g) ( t=6.918, P<0.001). There was no significant difference of the daily drinking water of rats between experimental group((37.5±12.1)ml) and the control group ((35.0±11.8)ml) ( t=0.361, P=0.726). (3) Perikidney fat mass of experimental group rats ((13.4±2.7)g) significantly increased 28 days after operation compared with control group rats((6.3±0.9)g)( t=4.250, P<0.05). The naso-anal length of experimental group((21.8±0.4)cm) was significantly decreased compared with the control group ((23.4±0.2)cm) ( t=-6.788, P<0.01). The Lee index of the experimental group (348.9±8.5) was significantly higher than that of the control group(305.5±4.3)( t=7.898, P<0.01). (4) The serum leptin content ((8 324.10±159.00)μg/L) of the experimental group rats at 28 days after surgery was significantly higher than that of the control group((2 705.31±407.10)μg/L) ( t=25.712, P<0.001). The lateral hypothalamus area (LHA) LEPR protein expression (1.3±0.1) in the experimental group was significantly higher than that in the control group (0.9±0.1) ( t=4.932, P<0.01). Conclusion:Two-point electrical damage to bilateral VMH and ARC of rats can establish hypothalamic obese rat model.

Objective To observe the effects of endoscopic drug therap on hemostasis, re-bleeding and the risk of occurrence of complication in patients with acute esophageal and gastric variceal bleeding (EGVB). Methods A retrospective method was conducted, and 100 patients with EGVB who were admitted to the Affiliated Hospital of Yan'an University from June 2015 to June 2017 were enrolled. According to the difference in treatment methods, they were divided into an endoscopy Sclerotherapy and Ligation group and transjugular intrahepatic portosystemic shunt (TIPS) group, 50 cases in each group. The TIPS group was treated with TIPS; the endoscopy Sclerotherapy and Ligation group underwent endoscopic variceal sclerotherapy, endoscopic esophageal variceal ligation and postoperative non-selective β blockers oral administration. After 2 years of follow-up, the patients' hemostasis, re-bleeding at acute stage, survival situation and the incidences of complications were recorded. Before treatment and 2 weeks after treatment, the levels of serum albumin (Alb), total bilirubin (TBil) and platelet count (PLT) were measured by Hitachi automatic biochemical analyzer in the two groups. Results The success rate of hemostasis in the endoscopy Sclerotherapy and Ligation group was significantly higher than that in the TIPS group [98.0% (49/50) vs. 82.0% (41/50) ], and the recurrence rate of varices, during 1- and 2-year follow-up, the recurrence rate of bleeding and the incidences of complications were significantly lower than those in TIPS group [the recurrence rate of varicose veins: 6.0% (3/50) vs. 24.0 (12/50), 1-year re-bleeding rate: 12.0% (6/50) vs. 30.0% (15/50), 2-year re-bleeding rate: 42.0% (21/50) vs. 66.0% (33/50), esophageal ulcer: 2.0% (1/50) vs. 14.0% (7/50), upper abdominal discomfort: 2.0% (1/50) vs. 14.0% (7/50), hepatic encephalopathy:4.0% (2/50) vs. 16.0% (8/50), chest pain: 6.0% (3/50) vs. 20.0% (10/50), all P < 0.05]. After treatment, the levels of Alb and PLT in the endoscopy Sclerotherapy and Ligation group were higher than those in the TIPS group [Alb (g/L):43.84±4.34 vs. 40.83±3.21, PLT (×109/L): 26.33±3.37 vs. 21.12±3.89, both P < 0.05], and the TBil was lower than that in the TIPS group (μmol/L: 13.82±4.32 vs. 19.33±4.59). Conclusion Endoscopic Sclerotherapy and Ligation can significantly improve the effect of hemostasis of patients with acute EGVB, the rate of re-bleeding does not increase compared with that of western medicine group using TIPS, and the incidences of complications are significantly lower than those of applying TIPS.

OBJECTIVE: To investigate the mechanism by which doublecortin promotes the recovery of cytoskeleton in arginine vasopressin (AVP) neurons in rats with electrical lesions of the pituitary stalk (PEL). METHODS: Thirty-two SD rats were randomized into PEL group with electrical lesions of the pituitary stalk through the floor of the skull base (=25) and sham operation group (=7), and the daily water consumption (DWC), daily urine volume (DUV) and urine specific gravity (USG) of the rats were recorded. Four rats on day 1 and 7 rats on each of days 3, 7 and 14 after PEL as well as the sham-operated rats were sacrificed for detection of the expressions of β-Tubulin (Tuj1), doublecortin and caspase- 3 in the AVP neurons of the supraoptic nucleus using immunofluorescence assay and Western blotting. RESULTS: After PEL, the rats exhibited a typical triphasic pattern of diabetes insipidus, with the postoperative days 1-2 as the phase one, days 3-5 as the phase two, and days 6-14 as the phase three. Immunofluorescent results indicated the repair of the AVP neurons evidenced by significantly increased doublecortin expressions in the AVP neurons following PEL; similarly, the expression of Tuj1 also increased progressively after PEL, reaching the peak level on day 7 after PEL. The apoptotic rates of the AVP neurons exhibited a reverse pattern of variation, peaking on postoperative day 3 followed by progressive reduction till day 14. Western blotting showed that the expressions of c-Jun and p-c-Jun were up-regulated significantly on day 3 ( < 0.05) and 7 ( < 0.01) after PEL, while an upregulated p-JNK expression was detected only on day 3 ( < 0.05), as was consistent with the time-courses of neuronal recovery and apoptosis after PEL. CONCLUSIONS JNK/c-Jun pathway is activated after PEL to induce apoptosis of AVP neurons in the acute phase and to promote the repair of neuronal cytoskeleton by up-regulation of doublecortin and Tuj1 expressions.
Animals ; Apoptosis ; Arginine Vasopressin ; pharmacology ; Cytoskeleton ; metabolism ; MAP Kinase Signaling System ; Neurons ; cytology ; Pituitary Gland ; cytology ; injuries ; Proto-Oncogene Proteins c-jun ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Regeneration ; Tubulin ; metabolism

Objective: To analyze the risk factors of cervical anastomotic leakage after thoracoscopic-lapacoscopic esophagectomy. Methods: 530 patients with esophageal cancer underwent thoracoscopic-lapacoscopic esophagectomy at the Cancer Hospital, Chinese Academy of Medical Sciences from Jan 2011 to Dec 2015. The demographic, surgical and clinical data of patients were retrospectively analyzed. Multivariate logistic regression was used to evaluate risk factors of cervical anastomotic leakage in these patients. Results: A total of 530 patients undergoing thoracoscopic-lapacoscopic esophagectomy were enrolled in this study. There were 421 males and 109 females. The mean age was (59.40±8.08) years old, and 91 patients with cervical anastomotic leakage. Sigle factor analysis revealed that the risk grading by American Society of Aneshesiologists, previous history of chest surgery, respiratory comorbidity, diffusion capacity for carbon monoxide of the lung, operation time, anastomosis, average days of postoperative hospitalization, death within 30 days after surgery, respiratory complications, pleural effusion or empyema, and poor healing of the incision were statistically associated with cervical anastomotic leakage (all P<0.05). Multivariate analysis showed that previous history of chest surgery, hepatic insufficiency, manual anastomosis, prolonged postoperative hospitalization, and poor healing of the incision were independent risk factors for cervical anastomotic leakage after thoracoscopic-lapacoscopic esophagectomy (all P<0.05). Conclusions Previous history of chest surgery, hepatic insufficiency, poor healing of the incision, manual anastomosis and prolonged postoperative hospitalization were significantly associated with cervical anastomotic leakage after thoracoscopic-lapacoscopic esophagectomy. It′s important to strengthen perioperative nursing and surgical techniques to prevent anastomotic leakage after thoracoscopic-lapacoscopic esophagectomy.

Objective To analyze the risk factors of cervical anastomotic leakage after thoracoscopic?lapacoscopic esophagectomy. Methods 530 patients with esophageal cancer underwent thoracoscopic?lapacoscopic esophagectomy at the Cancer Hospital, Chinese Academy of Medical Sciences from Jan 2011 to Dec 2015. The demographic, surgical and clinical data of patients were retrospectively analyzed. Multivariate logistic regression was used to evaluate risk factors of cervical anastomotic leakage in these patients. Results A total of 530 patients undergoing thoracoscopic?lapacoscopic esophagectomy were enrolled in this study. There were 421 males and 109 females. The mean age was (59.40±8.08) years old, and 91 patients with cervical anastomotic leakage. Sigle factor analysis revealed that the risk grading by American Society of Aneshesiologists, previous history of chest surgery, respiratory comorbidity, diffusion capacity for carbon monoxide of the lung, operation time, anastomosis, average days of postoperative hospitalization, death within 30 days after surgery, respiratory complications, pleural effusion or empyema, and poor healing of the incision were statistically associated with cervical anastomotic leakage (all P＜0.05). Multivariate analysis showed that previous history of chest surgery, hepatic insufficiency, manual anastomosis, prolonged postoperative hospitalization, and poor healing of the incision were independent risk factors for cervical anastomotic leakage after thoracoscopic?lapacoscopic esophagectomy ( all P＜0.05 ). Conclusions Previous history of chest surgery, hepatic insufficiency, poor healing of the incision, manual anastomosis and prolonged postoperative hospitalization were significantly associated with cervical anastomotic leakage after thoracoscopic?lapacoscopic esophagectomy. It′s important to strengthen perioperative nursing and surgical techniques to prevent anastomotic leakage after thoracoscopic?lapacoscopic esophagectomy.

Objective To analyze the risk factors of cervical anastomotic leakage after thoracoscopic?lapacoscopic esophagectomy. Methods 530 patients with esophageal cancer underwent thoracoscopic?lapacoscopic esophagectomy at the Cancer Hospital, Chinese Academy of Medical Sciences from Jan 2011 to Dec 2015. The demographic, surgical and clinical data of patients were retrospectively analyzed. Multivariate logistic regression was used to evaluate risk factors of cervical anastomotic leakage in these patients. Results A total of 530 patients undergoing thoracoscopic?lapacoscopic esophagectomy were enrolled in this study. There were 421 males and 109 females. The mean age was (59.40±8.08) years old, and 91 patients with cervical anastomotic leakage. Sigle factor analysis revealed that the risk grading by American Society of Aneshesiologists, previous history of chest surgery, respiratory comorbidity, diffusion capacity for carbon monoxide of the lung, operation time, anastomosis, average days of postoperative hospitalization, death within 30 days after surgery, respiratory complications, pleural effusion or empyema, and poor healing of the incision were statistically associated with cervical anastomotic leakage (all P＜0.05). Multivariate analysis showed that previous history of chest surgery, hepatic insufficiency, manual anastomosis, prolonged postoperative hospitalization, and poor healing of the incision were independent risk factors for cervical anastomotic leakage after thoracoscopic?lapacoscopic esophagectomy ( all P＜0.05 ). Conclusions Previous history of chest surgery, hepatic insufficiency, poor healing of the incision, manual anastomosis and prolonged postoperative hospitalization were significantly associated with cervical anastomotic leakage after thoracoscopic?lapacoscopic esophagectomy. It′s important to strengthen perioperative nursing and surgical techniques to prevent anastomotic leakage after thoracoscopic?lapacoscopic esophagectomy.

Objective To compare the transparency efficiency of six different optical clearing method on the rat brain tissues. Methods Brain tissue blocks of 14 SD rats were processed with iDISCO, SeeDB, CUBIC, SCALEVIEW-A2,CLARITY-CUBIC, Passive-CLARITY clearing method, respectively. Results The gray value of PBS group was 13.031 ± 0.586,that of iDISCO,SeeDB,CUBIC,SCALEVIEW-A2,CLARITY-CUBIC,passive-CLARITY clearing were 6.447 ± 0.574,11.690 ± 0.909,2.318 ± 0.986,8.118 ± 1.026,8.591 ± 0.384,4.198 ± 0.182, respectively. Except the SeeDB group(P=0.185),the rest groups showed significant differences compared with the PBS group(P< 0.01), and there were significant differences between CUBIC and other groups(P < 0.01). After the clearing treatment, the changes of tissue area ratio in the iDISCO, SeeDB, CUBIC, SCALEVIEW-A2, CLARITY-CUBIC, Passive-CLARITY method were(-30.02 ± 2.39)%,(19.74 ± 4.09)%,(14.7 ± 3.92)%,(10.7 ± 5.55)%,(23.01 ± 4.19)%,(66.51 ± 5.68)%,respectively. Each group showed a significant difference compared with the groups iDISCO and the Passive-CLARITY,P< 0.01. Conclusions Except the SeeDB method,all the clearing methods can achieve a transparent effect, while CUBIC is better than the other groups applied for rat brain tissues. The tissue block volume is shrunken after iDISCO clearing,and expanded after Passive-CLARITY processing.