Construction and functional analysis of EGFRvIII CAR-T cells co-expressing IL-15 and CCL19 / 生物工程学报

The aim of this study was to investigate the functional characteristics and in vitro specific killing effect of EGFRvIII CAR-T cells co-expressing interleukin-15 and chemokine CCL19, in order to optimize the multiple functions of CAR-T cells and improve the therapeutic effect of CAR-T cells targeting EGFRvIII on glioblastoma (GBM). The recombinant lentivirus plasmid was obtained by genetic engineering, transfected into 293T cells to obtain lentivirus and infected T cells to obtain the fourth generation CAR-T cells targeting EGFRvIII (EGFRvIII-IL-15-CCL19 CAR-T). The expression rate of CAR molecules, proliferation, chemotactic ability, in vitro specific killing ability and anti-apoptotic ability of the fourth and second generation CAR-T cells (EGFRvIII CAR-T) were detected by flow cytometry, cell counter, chemotaxis chamber and apoptosis kit. The results showed that compared with EGFRvIII CAR-T cells, EGFRvIII-IL-15-CCL19 CAR-T cells successfully secreted IL-15 and CCL19, and had stronger proliferation, chemotactic ability and anti-apoptosis ability in vitro (all P < 0.05), while there was no significant difference in killing ability in vitro. Therefore, CAR-T cells targeting EGFRvIII and secreting IL-15 and CCL19 are expected to improve the therapeutic effect of glioblastoma and provide an experimental basis for clinical trials.

The clinical relevance of MBL2 gene polymorphism and sepsis

Objective:To detect the clinical relevance of mannose-binding lectin 2 (MBL2) gene polymorphism and sepsis in Chinese lived in Hainan island.Methods:Blood samples from 57 patients with sepsis and 69 patients without sepsis were collected in the ICU of several large hospitals in Hainan province. Genomic DNA was extracted from whole blood and then PCR purification product was sequenced and typed by 3730 sequencing analyzer. The concentration of MBL2 in serum was detected by ELISA.Results:We found that genotype and allele distributions in two groups were in accordance with the Hardy-Weinberg Equilibrium. The frequency of GA genotype was significantly higher than that in non-sepsis group (P=0.013). A allele frequency in sepsis group was also much higher than that in non-sepsis group (P=0.028). Logister regression analysis showed that the patients who carried A allele were more prone to get sepsis than G allele carrier (P=0.014, 0R=2.550, 95%CI=1.207-5.386). The MBL2 level in serum of sepsis patients with genotype GG and GA was significantly lower than that in non-sepsis group (P<0.05). In sepsis group, the MBL2 serum level of patients with genotype GA was obviously lower than that in patients with genotype GG (P<0.05).Conclusions:The variation of rs1800450 G→A increased the incidence of sepsis and decreased the level of MBL2 in serum.

Effect of lung protection ventilation on postoperative lung complications in patients with esophageal cancer / 中国内镜杂志

Objective?To investigate the effect of lung protection ventilation on postoperative pulmonary complications in patients with esophageal cancer.?Methods?98 patients underwent thoracoscopic laparoscopic esophagectomy for radical resection from January 2014 to January 2017 were enrolled in this study. Patients were divided into observation group (n = 49) and control group (n = 49) randomly. Two groups of patients underwent intraoperative single lumen tracheal catheter supplemented with CO2 pneumothorax. The control group received conventional ventilation. The protective group of patients in the observation group were given protective ventilation. The time of tracheal intubation after 10 min (T1); single lung ventilation 1 h (T2); at the end of surgery (T3); 24 h (T4) after pulmonary function parameters, inflammatory response, blood gas were analyzed. The patients were divided into recurrence group (n = 24) and non-recurrence group (n = 74), pulmonary complications and its related factors were analyzed according to whether the patient had recurrent or not pulmonary complications after recurrent (n = 24) or non-recurrent (n = 74).?Results?Pplat, airway peak pressure (Ppeak), airway resistance (Raw), interleukin-6 (IL-6), and interleukin-8 (in the two groups) at time T1. Interleukin-8, (IL-8), soluble intercellular adhesion molecule-1 (Sicam-1), partial pressure of carbon dioxide in artery (PaCO2), partial pressure of oxygen There was no significant difference in the index of oxygen, PaO2, and oxygenation index (PaO2/FiO2) (P > 0.05); the changes in Pplat, Ppeak, and Raw in the control group between T1 and T3 were significantly greater than those in the observation group. The changes of IL-6, IL-8, and Sicam-1 in the control group from T1 to T4 were significantly greater than those in the observation group (P < 0.05). T1 to T4 The change of PaO2 in the control group was significantly greater than that in the observation group, and the difference was statistically significant (P < 0.05). There were multiple pulmonary complications in some patients in both groups. There were 18.36% in the observation group and 30.61% in the control group combined with various types of pulmonary complications. The difference was statistically significant (P < 0.05). Preoperative smoking, operation time, degree of radicalization, clinical pathological stage, lymphatic infiltration, microvascular infiltration, postoperative adjuvant chemotherapy, and pulmonary complications in patients undergoing radical surgery for esophageal cancer were closely related (P < 0.05).?Conclusion?Lung protection ventilation can relieve the thoracoscopic laryngeal esophageal cancer surgery in the airway resistance, pressure and inflammation, increased oxygen saturation, should strengthen the high risk of pulmonary complications in patients with monitoring, targeted prevention, reduce postoperative the incidence of pulmonary complications.

The clinical relevance of MBL2 gene polymorphism and sepsis

Objective: To detect the clinical relevance of mannose-binding lectin 2 (MBL2) gene polymorphism and sepsis in Chinese lived in Hainan island. Methods: Blood samples from 57 patients with sepsis and 69 patients without sepsis were collected in the ICU of several large hospitals in Hainan province. Genomic DNA was extracted from whole blood and then PCR purification product was sequenced and typed by 3730 sequencing analyzer. The concentration of MBL2 in serum was detected by ELISA. Results: We found that genotype and allele distributions in two groups were in accordance with the Hardy-Weinberg Equilibrium. The frequency of GA genotype was significantly higher than that in non-sepsis group (P=0.013). A allele frequency in sepsis group was also much higher than that in non-sepsis group (P=0.028). Logister regression analysis showed that the patients who carried A allele were more prone to get sepsis than G allele carrier (P=0.014, 0R=2.550, 95%CI=1.207-5.386). The MBL2 level in serum of sepsis patients with genotype GG and GA was significantly lower than that in non-sepsis group (P<0.05). In sepsis group, the MBL2 serum level of patients with genotype GA was obviously lower than that in patients with genotype GG (P<0.05). Conclusions: The variation of rs1800450 G→A increased the incidence of sepsis and decreased the level of MBL2 in serum.

Clinical significance of dynamic detection for serum levels of MCP-1, TNF-α and IL-8 in patients with acute pancreatitis

OBJECTIVE@#To observe dynamic changes of levels of monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in patients with acute pancreatitis and to investigate its evaluation value on the severity of acute pancreatitis.@*METHODS@#A total of 109 patients with acute pancreatitis admitted were divided into mild acute pancreatitis group (MAP group, 42 cases), moderately severe acute pancreatitis (MSAP group, 35 cases) and severe acute pancreatitis (SAP group, 32 cases). ELISA was used to detect the serum levels of MCP-1, TNF-α and IL-8 of patients at day 1, day 4 and day 7 of admission to hospital.@*RESULTS@#The serum levels of MCP-1, TNF-α and IL-8 from MAP group, MSAP group and SAP group at day 1 of admission to hospital all significantly increased. There was a significant difference between MAP group and control group, MSAP group and MAP group, SAP group and MSAP group (P  0.05); The serum concentrations of MCP-1, TNF-α and IL-8 from MAP group all reached the highest level at day 4, which were significantly higher than the detection levels at day 1. In MSAP group and SAP group, the serum concentrations of MCP-1, TNF-α and IL-8 were the highest at day 1, which were significantly higher than the detection levels at day 4 and 7. At each detecting timing, the serum concentrations of MCP-1, TNF-α and IL-8 from MSAP group and SAP group were all higher than those of MAP group and MSAP group, respectively.@*CONCLUSIONS@#The dynamic changes of serum levels of MCP-1, TNF-α and IL-8 in patients with acute pancreatitis have their rules, and the change rule of MAP group was different with that of MSAP and SAP group, which showed the reference value for the diagnosis and illness severity evaluation of acute pancreatitis.

Clinical significance of dynamic detection for serum levels of MCP-1, TNF-α and IL-8 in patients with acute pancreatitis

Objective To observe dynamic changes of levels of monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in patients with acute pancreatitis and to investigate its evaluation value on the severity of acute pancreatitis. Methods A total of 109 patients with acute pancreatitis admitted were divided into mild acute pancreatitis group (MAP group, 42 cases), moderately severe acute pancreatitis (MSAP group, 35 cases) and severe acute pancreatitis (SAP group, 32 cases). ELISA was used to detect the serum levels of MCP-1, TNF-α and IL-8 of patients at day 1, day 4 and day 7 of admission to hospital. Results The serum levels of MCP-1, TNF-α and IL-8 from MAP group, MSAP group and SAP group at day 1 of admission to hospital all significantly increased. There was a significant difference between MAP group and control group, MSAP group and MAP group, SAP group and MSAP group (P 0.05); The serum concentrations of MCP-1, TNF-α and IL-8 from MAP group all reached the highest level at day 4, which were significantly higher than the detection levels at day 1. In MSAP group and SAP group, the serum concentrations of MCP-1, TNF-α and IL-8 were the highest at day 1, which were significantly higher than the detection levels at day 4 and 7. At each detecting timing, the serum concentrations of MCP-1, TNF-α and IL-8 from MSAP group and SAP group were all higher than those of MAP group and MSAP group, respectively. Conclusions The dynamic changes of serum levels of MCP-1, TNF-α and IL-8 in patients with acute pancreatitis have their rules, and the change rule of MAP group was different with that of MSAP and SAP group, which showed the reference value for the diagnosis and illness severity evaluation of acute pancreatitis.

Notch1 regulates stemness and chemotherapeutic sensitivity of human gli-oma U251 cells / 中国病理生理杂志

AIM:To investigate whether Notch1 changes stemness and chemotherapeutic sensitivity in human glioma U251 cells.METHODS: The lentiviral vectors, which expressed Notch1-shRNA or Notch1 intracellular domain ( NICD) , were transfected into U251 cells .Western blot and immunofluorescence staining were applied to monitor the va-lidity of the cells, down-regulation of Notch1 expression or over-expression of NICD.The proportion of CD133 +cells was analyzed by flow cytometry.The expression of nestin and GFAP was identified by immunofluorescence staining.The forma-tion rate of tumor cell spheres and the implanted tumor growth in SCID mice were observed.MTT assay was performed to e-valuate the chemotherapeutic sensitivity to VM-26 and BCNU of the cells with different treatments.RESULTS:Stemness was significantly enhanced in the cells over-expressing NICD.For example, the proportion of CD133 +cells was increased, the expression of nestin was up-regulated, the expression of GFAP was down-regulated, and the formation rate of tumor cell spheres and implanted tumor growth were increased.The chemotherapeutic sensitivity to VM-26 and BCNU of the cells was decreased.In the cells with Notch1 gene down-regulation by RNAi, the stemness was inhibited and chemotherapeutic sensi-tivity was increased.CONCLUSION:Notch1, which leads to the change of stemness and chemotherapeutic sensitivity in human glioma U251 cells, is likely to be a potential molecular target for treatment of glioma.

Correlation of the expressions of advanced glycation end products and its receptor in serum and ;placenta with the pathogenesis of preeclampsia / 中华妇产科杂志

Objective To investigate the correlation of the expressions of advanced glycation end products(AGE) and the receptor for advanced glycation end products(RAGE) in serum and placenta with the pathogenesis of preeclampsia. Methods From December 2013 to June 2014, 32 women with severe preeclampsia who received cesarean section in the Affiliated Hospital of Qingdao University were recruited in the study, defined as the severe preeclampsia group. 30 healthy pregnant women who received cesarean section in the same hospital were recruited as the control group. ELISA was used to measure the maternal serum AGE, soluble receptor for advanced glycation end products (sRAGE) and tumor necrosis factor-α(TNF-α) in these women. Furthermore, ELISA was also used to measure AGE and TNF-α in the placenta. The localizations of AGE and RAGE protein in placentas were detected by immunohistochemical SP method. RAGE and TNF-α mRNA expression in placentas were measured by real-time quantitative PCR. AGE, RAGE and TNF-αprotein expression in placentas were measured by western blot, respectively. Results (1) The serum levels of AGE,sRAGE and TNF-αin the severe preeclampsia group were (538 ± 75),(367 ± 86) and (322 ± 40) ng/L,respectively. They were significantly higher than those in the control group[(454 ± 50), (286 ± 35) and (270 ± 35) ng/L, respectively](P0.05). (2) In the severe preeclampsia group, the levels of AGE and TNF-αin placentas were (500 ± 82) and (334 ± 57) ng/L, which were higher than those in the control group [(431 ± 74) and (263 ± 46) ng/L, respectively](P<0.05). The levels of AGE showed positive correlation with the levels of TNF-ɑ(r=0.406,P<0.05). (3)AGE and RAGE protein mainly located in the syncytiotrophoblasts, macrophages and vascular endothelial cells in the placentas of the two groups. AGE expressed mainly in the cytoplasm, and RAGE expressed in the cytoplasm and cell membranes.(4)RAGE and TNF-αmRNA expression in the placentas of the severe preeclampsia group were 12.6 ± 4.6 and 10.4 ± 2.4, which were significantly higher than those in the control group (0.9 ± 0.4 and 3.5 ± 0.9,P<0.01). (5) The expressions of AGE、RAGE and TNF-αprotein in placentas of the severe preeclampsia group were 0.68 ± 0.06, 0.82 ± 0.08 and 0.76 ± 0.08. All were significantly higher than those of the control group (0.46 ± 0.05,0.42 ± 0.09 and 0.52 ± 0.07;P<0.01). Conclusions The levels of AGE and RAGE in serum and placentas elevated in the severe preeclampsia group, and the expression of TNF-αalso elevated. These indicated that AGE and RAGE might be involved in the systemic inflammatory response and local inflammatory response in placentas, and then caused the preeclampsia.

One new ent-kaurane glycoside from stems of Acanthopanax gracilistylus / 中草药

Objective: To study the chemical constituents from the stems of Acanthopanax gracilistylus. Methods: The chemical constituents of the plant were isolated and purified by column chromatography and their structures were elucidated on the basis of physico-chemical properties and spectral data. Results: A new ent-kaurane glycoside, named kaurane acid glycoside A (16α, 17-dihydroxy-ent-kauran-19-oic 19-[β-D-glucopyranosyl-(1→2)-β-D- glucopyranosyl] ester) (1), was isolated from the n-butanol part. Conclusion: Compound 1 is a new one.