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Objective:To construct an expression system of lentivirus vector encoding epidermal growth factor receptor-specific chimeric antigen receptor (EGFR-CAR) and programmed cell death ligand-1 (PD-L1) antibody.Methods:Human PD-L1-Fc protein was used to immunize BALB/c mice. Cell-fusion and subcloning were performed to screen stable hybridoma strains with high secretion of PD-L1-specific antibodies, which were identified by both ELISA and Western blot. The activity of the antibodies in blocking the binding of programmed cell death-1 (PD-1) to PD-L1 was determined by fluorescence-activated cell sorting (FACS). Antibody affinity was analyzed by Fortebio Octet96. A single-chain variable fragment (scFv) was further constructed after antibody full-length sequencing and humanization using CDR grafting method. Meanwhile, the genes encoding the light and heavy chain variable regions (VL and VH) were cloned from a hybridoma secreting antibody against human EGFR by 5′ RACE technology to construct scFv gene. The expression of scFv was confirmed using pcDNA3.1 vector. EGFR-CAR containing CD137 intracellular function domain and PD-L1-scFv was ligated using 2A gene. The synthetic single molecule was cloned into pLVX-EF1a-IRES-ZsGreen1 lentivirus expression vector, and then transfected into 293T cells using Lenti-X Packaging Single Shots (VSV-G) to prepare infectious virus. Expression of CAR on cell surface and the soluble form of PD-L1-scFv in the supernatant of transfected 293V cells were detected by FACS and ELISA.Results:A PD-L1 antibody named 11E3 with high ligand-receptor blocking performance was obtained. The humanized antibody showed a stable affinity (2.67×10 -10 mol/L) after directly grafting the mouse CDRs (CDR1, CDR2 and CDR3) to human frameworks. EGFR-scFv was effectively expressed in a form of Fc-fusion. Secretory CAR (CTZ0431-1) and membrane CAR (CTZ0431-2) expression plasmids were constructed using lentivirus vector containing EGFR-CAR and PD-L1-scFv. The infection efficiency in 293V cells was around 10%. EGFR-scFv on the cell membranes and PD-L1-scfv in the culture supernatants were detected after 293V cells were infected with CTZ0431-1. EGFR-scFv and PD-L1-scfv were expressed on the cell membranes of 293V cells infected with CTZ0431-2. The expression rate of CAR in LV-CART46407-1-transfected activated T cells was 39.3%. Conclusions:The lentivirus vectors co-expressing EGFR-CAR with moderate binding affinity and PD-L1-scFv with high binding affinity were successful constructed, which provided an essential tool for investing EGFR- and PD-L1 double targeted CAR-T cell therapy against solid tumor.
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Objective To investigate the epidemiological characteristics and spatial distribution of hepatitis B in children in Kaizhou District of Chongqing from 2018 to 2019, and to provide a theoretical basis for the prevention and control of hepatitis B. Methods The prevalence of hepatitis B in Kaizhou District of Chongqing from 2018 to 2019 was calculated using Excel2003, and the spatial data was processed by SaTScan9.6. Aggregation of incidence of hepatitis B was investigated by calculating Moran's I coefficient. Aggregated distribution of the incidence of hepatitis B was explored by local spatial autocorrelation analysis. Results From 2018 to 2019, the cumulative number of hepatitis B in children in Kaizhou District of Chongqing was 86, with an average annual reported incidence rate of 3.01 per 100,000. Among 86 children with hepatitis B, 54 were male and 32 were female, with a male to female ratio of 1.69:1. The incidence rate of hepatitis B in 2019 was lower than that in 2018 ( P's I=0.4736, P's I=0.3915, P<0.05). The high-high (H-H) cluster areas of hepatitis B were mainly distributed in Maliu Township, Zishui Township, and Tianhe Town, while the low-low (L-L) cluster areas of hepatitis B were mainly distributed in Zhaojia Street and Changsha Town. Conclusion In 2018-2019, the incidence of hepatitis B among children in Kaizhou District, Chongqing City had a downward trend. The incidence of hepatitis B among males and children aged 10 to 14 years old was relatively high. There was a positive spatial autocorrelation of the incidence. The prevention and treatment of hepatitis B in high-risk population and hot spots should be strengthened.
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Objective To compare a novel mirror-overlap method with the traditional manual measurement and the subjective assessment in assessing breast symmetry after oncoplastic surgery.Methods 59 patients with breast cancer who underwent conservation/reconstruction surgery were recruited.The post-operative assessment for breast symmetry was conducted using manual measurement,subjective assessment by the patient,their family member and a nurse,and the mirror-overlap method respectively.The latter method involved using the Photoshop to assess the area differences in breast outlines and shadows created by a flashlight when bilateral images were overlapped in the axis of midline.Results The distance differences between bilateral breasts measured using manual method were negatively correlated with symmetric coefficients in the mirror-overlap method (P＜0.05).There was a positive correlation between the average scores in the subjective assessment and symmetric coefficients of mirror-overlap method (P＜0.05).Conclusion The mirror-overlap method is an easy,feasible and cheap method which collects 3D breast information and provides an effective approach to assess breast symmetry after oncoplastic surgery.
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Hepatoid adenocarcinoma (HAC) of the gallbladder is a variant of extrahepatic adenocarcinoma with hepatic differentiation.With similar clinicopathological presentation,the main differential diagnoses are hepatocellular carcinoma (HCC) and gallbladder carcinoma (GBC).At present,HAC of the gallbladder have only been reported in a few researches.This article will summarize comprehensive researches on the histological origin,clinical characteristics,histopathological and immunohistochemical characteristics,differential diagnosis,therapy and prognosis of HAC of the gallbladder.