Objective:To extract rat brain microvascular endothelial cells(BMECs)using thin-layer cell culture method.Methods:The brain cortex of four-week-old SD rats was obtained,which was chopped,sieved,and digested with type II collagenase to obtain microvascular segments.The amount of culture medium was strictly controlled,and it was inoculated into culture flasks for primary culture.The target cells were morphologically observed by using an invert-ed phase contrast microscope,and the factor Ⅷ related antigen(FⅧRag)was identified by using immunocytochemi-cal staining.Results:The target cells cultured in the inverted phase contrast microscope showed typical endothelial-like monolayer cobble stone-like mosaic growth;FⅧRag positive cells accounted for more than 99％of the total cells.Conclusion:Thin-layer cell culture method can successfully isolate and cultivate high-purity rat BMECs.

Objective:To investigate the changes of neuron-specific enolase (NSE) in serum and cerebrospinal fluid of patients with subacute 1, 2-dichloroethane (DCE) poisoning.Methods:Ten patients with subacute 1, 2-DCE poisoning hospitalized in Guangzhou 12th Municipal People's Hospital from December 2014 to March 2019 were taken as the subacute 1, 2-DCE poisoning group, 34 typical acute toxic encephalopathy patients hospitalized at the same time as typical acute toxic encephalopathy group, 40 healthy physical examinees as normal control group. The levels of serum NSE in patients of subacute 1, 2-DCE poisoning and typical acute toxic encephalopathy group during onset and improvement were detected by chemiluminescence method, and the results were analyzed statistically. The level of NSE in cerebrospinal fluid of subacute 1, 2-DCE poisoning group was detected and analyzed its correlation with the level of NSE in serum. Using receiver operator characteristic (ROC) curve to analyze the diagnostic efficacy of NSE in subacute 1, 2-DCE poisoning and typical acute toxic encephalopathy (area under curve, AUC) .Results:There was no significant difference between the serum NSE level of the patients with subacute 1, 2-DCE poisoning in the onset group and the normal control group and the improvement group ( P>0.05) . The serum NSE level of subacute 1, 2-DCE poisoning in the improvement group was lower than those in the normal control group ( P<0.01) . The serum NSE level of the subacute 1, 2-DCE poisoning in the onset group was lower than those in the typical acute toxic encephalopathy in the onset group ( P<0.01) . There was no linear correlation between cerebrospinal fluid NSE and serum NSE in patients with subacute 1, 2-DCE poisoning ( r=-0.183, P=0.52) . ROC curve showed that the AUC of serum NSE in diagnosing subacute 1, 2-DCE poisoning and typical acute toxic encephalopathy were 0.661 and 0.726, respectively. Conclusion:There is no significant change in serum NSE in patients with subacute 1, 2-DCE poisoning.

Objective:To analyze the levels of T lymphocyte subsets (CD3, CD4, CD8 and CD4/CD8) in patients with paraquat poisoning, and to explore the relationship between the changes of T lymphocyte subsets and the prognosis of pulmonary fibrosis.Methods:In October 2019, a total of 47 patients with oral 20% paraquat low water solvent poisoning in Guangzhou 12th people's Hospital from June 2018 to June 2019 were selected as the research objects. Patients were divided into early death group (16 cases died within 2 weeks) and non early death group (31 cases survived more than 2 weeks) . The non early death group was divided into pulmonary fibrosis group (23 cases) and normal lung group (8 cases) . 20 healthy people in the same period were randomly selected as the control group. The neutrophils (N) , C reaction protein (CRP) , alanine aminotransferase (ALT) , creatinine (Cr) , amylase (aAMY) , creatine kinase isoenzyme (CKMB) , pH, HCO 3-, blood oxygen saturation (SO 2) and lactic acid (Lac) of patients poisoned within 3 d were examined every day. Independent sample t-test was used for inter group comparison, and paired sample t-test was used for intra group comparison. Results:Compared with non early death group, the levels of N, CRP, ALT, Cr, aAMY, CKMB and Lac in early death group increased ( P<0.05) , while pH and HCO 3- decreased ( P<0.05) . Compared with the control group, the levels of CD3, CD4 and CD4/CD8 were decreased on the first day in the early death group and non early death group ( P<0.05) , and the levels of CD3, CD4 and CD4/CD8 were decreased on the 15th day in the pulmonary fibrosis group ( P<0.05) . Compared with the normal lung group, the levels of CD3, CD4 and CD4/CD8 in the pulmonary fibrosis group decreased on the 15th day ( P<0.05) . Conclusion:The persistent low cellular immune function in patients with paraquat poisoning is related to the progress of pulmonary fibrosis, which is an important factor affecting the prognosis of patients with pulmonary fibrosis.

Objective:To investigate the changes of neuron-specific enolase (NSE) in serum and cerebrospinal fluid of patients with subacute 1, 2-dichloroethane (DCE) poisoning.Methods:Ten patients with subacute 1, 2-DCE poisoning hospitalized in Guangzhou 12th Municipal People's Hospital from December 2014 to March 2019 were taken as the subacute 1, 2-DCE poisoning group, 34 typical acute toxic encephalopathy patients hospitalized at the same time as typical acute toxic encephalopathy group, 40 healthy physical examinees as normal control group. The levels of serum NSE in patients of subacute 1, 2-DCE poisoning and typical acute toxic encephalopathy group during onset and improvement were detected by chemiluminescence method, and the results were analyzed statistically. The level of NSE in cerebrospinal fluid of subacute 1, 2-DCE poisoning group was detected and analyzed its correlation with the level of NSE in serum. Using receiver operator characteristic (ROC) curve to analyze the diagnostic efficacy of NSE in subacute 1, 2-DCE poisoning and typical acute toxic encephalopathy (area under curve, AUC) .Results:There was no significant difference between the serum NSE level of the patients with subacute 1, 2-DCE poisoning in the onset group and the normal control group and the improvement group ( P>0.05) . The serum NSE level of subacute 1, 2-DCE poisoning in the improvement group was lower than those in the normal control group ( P<0.01) . The serum NSE level of the subacute 1, 2-DCE poisoning in the onset group was lower than those in the typical acute toxic encephalopathy in the onset group ( P<0.01) . There was no linear correlation between cerebrospinal fluid NSE and serum NSE in patients with subacute 1, 2-DCE poisoning ( r=-0.183, P=0.52) . ROC curve showed that the AUC of serum NSE in diagnosing subacute 1, 2-DCE poisoning and typical acute toxic encephalopathy were 0.661 and 0.726, respectively. Conclusion:There is no significant change in serum NSE in patients with subacute 1, 2-DCE poisoning.

Objective:To analyze the levels of T lymphocyte subsets (CD3, CD4, CD8 and CD4/CD8) in patients with paraquat poisoning, and to explore the relationship between the changes of T lymphocyte subsets and the prognosis of pulmonary fibrosis.Methods:In October 2019, a total of 47 patients with oral 20% paraquat low water solvent poisoning in Guangzhou 12th people's Hospital from June 2018 to June 2019 were selected as the research objects. Patients were divided into early death group (16 cases died within 2 weeks) and non early death group (31 cases survived more than 2 weeks) . The non early death group was divided into pulmonary fibrosis group (23 cases) and normal lung group (8 cases) . 20 healthy people in the same period were randomly selected as the control group. The neutrophils (N) , C reaction protein (CRP) , alanine aminotransferase (ALT) , creatinine (Cr) , amylase (aAMY) , creatine kinase isoenzyme (CKMB) , pH, HCO 3-, blood oxygen saturation (SO 2) and lactic acid (Lac) of patients poisoned within 3 d were examined every day. Independent sample t-test was used for inter group comparison, and paired sample t-test was used for intra group comparison. Results:Compared with non early death group, the levels of N, CRP, ALT, Cr, aAMY, CKMB and Lac in early death group increased ( P<0.05) , while pH and HCO 3- decreased ( P<0.05) . Compared with the control group, the levels of CD3, CD4 and CD4/CD8 were decreased on the first day in the early death group and non early death group ( P<0.05) , and the levels of CD3, CD4 and CD4/CD8 were decreased on the 15th day in the pulmonary fibrosis group ( P<0.05) . Compared with the normal lung group, the levels of CD3, CD4 and CD4/CD8 in the pulmonary fibrosis group decreased on the 15th day ( P<0.05) . Conclusion:The persistent low cellular immune function in patients with paraquat poisoning is related to the progress of pulmonary fibrosis, which is an important factor affecting the prognosis of patients with pulmonary fibrosis.

Objective : To investigate the brain microvascular tissue block culture method for extracting primary rat brain microvascular endothelial cells and identify its effect. Methods : Brain tissue from 4-week-old Sprague Dawley rats was screened, pre-digested and solidified to obtain brain microvascular segments. These segments were subsequently placed in a CO2incubator for primary culture. The target cells were identified by cell morphology and immunocytochemical staining for factor Ⅷ-related antigen. Results : After a 48-hour culture periodin vitro, the short spindle cells crawled out from around the brain microvascular segments. After 72 hours, island-like cell culsters formed. After 96 hours the clusters fused and the cells formed a typical monolayer, cobble stone-like, and mosaic arrangement. Factor Ⅷ-related antigen immunocytochemical staining showed that the cytoplasm of the cells appeared brown-red, indicating positive expression; DAB stained the nucleus, showing blue-dark. Conclusion The brain microvascular tissue block culture method can isolate and culture primary rat brain microvascular endothelial cells.

Immunotherapy has revolutionized the landscape of cancer treatment. However, single immunotherapy only works well in a small subset of patients. Combined immunotherapy with antitumor synergism holds considerable potential to boost the therapeutic outcome. Nevertheless, the synergistic, additive or antagonistic antitumor effects of combined immunotherapies have been rarely explored. Herein, we established a novel combined cancer treatment modality by synergizing p21-activated kinase 4 (PAK4) silencing with immunogenic phototherapy in engineered extracellular vesicles (EVs) that were fabricated by coating M1 macrophage-derived EVs on the surface of the nano-complex cores assembled with siRNA against PAK4 and a photoactivatable polyethyleneimine. The engineered EVs induced potent PAK4 silencing and robust immunogenic phototherapy, thus contributing to effective antitumor effects in vitro and in vivo. Moreover, the antitumor synergism of the combined treatment was quantitatively determined by the CompuSyn method. The combination index (CI) and isobologram results confirmed that there was an antitumor synergism for the combined treatment. Furthermore, the dose reduction index (DRI) showed favorable dose reduction, revealing lower toxicity and higher biocompatibility of the engineered EVs. Collectively, the study presents a synergistically potentiated cancer treatment modality by combining PAK4 silencing with immunogenic phototherapy in engineered EVs, which is promising for boosting the therapeutic outcome of cancer immunotherapy.