Objective: To obtain chimeric antigen receptor macrophages ( CAR-M) targeting HER2 stably transfected． Methods : CAR lentivirus vector targeting HER2 was constructed and infected with human monocytic leukemia cell line (THP-1) ．CAR THP-1 cells with green fluorescent labeling were selected by sorting flow cytometry and continued to be cultured in vitro．The CAR THP-1 cells targeting HER2 were co-cultured with the endometrial cancer cell line Ishikawa with negative and positive HER2 expression，and their targeted phagocytosis of CAR-M to HER2 positive tumor cells was detected by imaging flow cytometry ，and the targeted phagocytosis efficiency of CAR-M to HER2 positive tumor cells was detected by flow cytometry. Results : CAR lentivirus infection with THP- 1 cells was less efficient ; After co-culture with cancer cells，flow cytometry and imaging flow cytometry showed that CAR THP-1 cells had enhanced phagocytosis of HER2 positive Ishikawa cells compared with the empty body group (P＜0. 01) ． Conclusion In this experiment，CAR THP-1 cell line targeting HER2 was established by constructing CAR lentivirus vector and transfecting THP-1 cells ，and it was proved that CAR THP-1 could phagocytize HER2 positive Ishikawa cells through specific targeting.

Objective To investigate the effects of N-acetyl-D-glucosamine(GlcNAc)on acute pancreatitis in rats.Methods Twenty male SD rats were randomly divided into a control group,AP group,low GlcNAc + AP group and high GlcNAc + AP group,with five rats in each group.Acute pancreatitis was induced in AP group,low GlcNAc + AP group and high GlcNAc + AP group by two intraperitoneal injections of 2.5 g/kg L-arginine with a 1 hour interval.Among them,low GlcNAc + AP group and high GlcNAc + AP group were administered 50 and 200 mg/kg GlcNAc,respectively,by intraperitoneal injection at 24 hours before the first intraperitoneal injection of L-arginine.Group control and AP were intraperitoneally injected with the same volume of normal saline.After 24 h,the rats were sacrificed,and serum and pancreatic tissues were collected.Pancreatic tissue morphology was observed by HE staining,and serum levels of amylase(AMY),lipase(LPS),interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),superoxide dismutase(SOD)and malondialdehyde(MDA)were measured by enzyme-linked immunosorbent assay.Protein expression of nuclear factor E2-related factor 2(NRF2),heme oxygenase-1(HO-1),and peroxisome proliferator-activated receptor-γ(PPAR-γ)in pancreatic tissue was detected by Western Blot.Cluster of differentiation(CD)86,CD206 and macrophage markers(F4/80)were detected by immunofluorescence.Expression of CD86 and CD206 in pancreatic tissue was detected by immunohistochemistry.Results(1)Compared with control group,AMY,LPS,IL-1β,IL-6,TNF-α,and MDA levels and pancreatic CD86 expression in AP group were significantly increased(P<0.05),while SOD activity,protein expression levels of NRF2,HO-1,and PPAR-γ,and pancreatic CD206 expression were significantly decreased(P<0.05).(2)Compared with AP group,serum IL-1β,IL-6,TNF-α,MDA,and LPS and the pancreatic CD86 expression in low GlcNAc + AP group were significantly decreased(P<0.05).The PPAR-γ protein level in the pancreas was significantly increased(P<0.05).(3)Compared with AP group,serum AMY,LPS,IL-1β,IL-6,TNF-α,and MDA and pancreatic CD86 expression in high GlcNAc + AP group were significantly decreased(P<0.05),while serum SOD,and NRF2,HO-1,PPAR-γ,and pancreatic CD206 expression were significantly increased(P<0.05).(4)Compared with low GlcNAc + AP group,serum LPS,IL-1β and IL-6 in high GlcNAc + AP group were significantly decreased(P<0.05).Pancreatic expression of HO-1,PPAR-γ,and pancreatic CD206 were significantly increased(P<0.05).Conclusion GlcNAc treatment attenuates acute pancreatitis injury in AP rats,possibly by activating the NRF2/HO-1 signaling pathway to inhibit oxidative stress and promoting M2 macrophage polarization to attenuate pancreatic injury in AP rats.

Objective:To establish a lectin enzyme-linked immunosorbent assay (lectin-ELISA) for the dection of sialylated fetuin-A and to explore the clinical diagnostic value of sialylated fetuin-A in hepatocellular carcinoma (HCC).Methods:From January 2017 to December 2020, 300 HCC patients and 160 disease controls, including 36 liver cirrhosis subgroups and 124 chronic hepatitis B subgroups, were collected from Shanghai Eastern Hepatobiliary Surgery Hospital. At the same time, 100 healthy subjects were collected as healthy controls. Lectin-ELISA method for detecting sialylated fetuin A was established based on the principle that Sambucus nigra lectin (SNA) can recognize the structure of α-2, 6-linked sialic acid residues. Differences between groups were compared using t-test or analysis of variance. Logistic regression method was used to establish the multi-index joint detection model, and receiver operating characteristic curve (ROC) was used to evaluate the efficacy of single index and joint detection model in the diagnosis of HCC.Results:A lectin-ELISA method for the detection of serum Sia-fetuin A was established. The linear regression coefficient of the system was 0.978 5, and the precision evaluation and interference experiments were in line with the clinical detection requirements. Using this method to detect serum Sia-fetuin A levels in each group, the levels of HCC group, disease control group and healthy control group were 1.362±0.310, 1.199±0.370, 1.086±0.420, respectively, and the three groups decreased in turn. The areas under the curve of Sia-fetuin A, α-fetoprotein, and their combined detection models for differential diagnosis of HCC were 0.790, 0.809, and 0.860, respectively. The diagnostic model had a sensitivity of 79.3% (238/300) and a specificity of 95.0% (247/260). Among the 300 patients in the HCC group, 138 (46%) patients were negative for serum AFP (<20 μg/L), and their serum Sia-fetuin A level was 1.364±0.305. Combining the disease control group and the healthy control group into the non-Cancer group, the serum Sia-fetuin A level was 1.146±0.381. The serum level of Sia-fetuin A in AFP-negative HCC patients was higher than that in non-HCC group ( t=6.134, P<0.001). The areas under the curve of Sia-fetuin A and the combined diagnostic model for the diagnosis of AFP-negative HCC were 0.776 and 0.919, respectively. The combined diagnostic model had a sensitivity of 93.4% (129/138) and a specificity of 77.3% (201/260). Conclusion:Serum Sia-fetuin A and combined determination model can provide a new auxiliary diagnostic index for AFP-negative HCC.

Objective : The live cell application was used to observe the process of phagocytosis of endometrial cancer cells by macrophages, and flow cytometry was used to detect the effects of macrophages engulfing tumor cells on activation of cytotoxic T cell. Methods : Ishikawa cells and THP1-induced macrophages were labeled with CFSE fluorescent probe and CD11 b respectively, and then mixed and seeded on a glass imaging dish.The live cell application was performed to record the phagocytosis of Ishikawa cells by macrophages within 120 minutes.Flow cytometry was used to detect the effect of macrophages engulfing tumor cells on activation of cytotoxic T cell. Results : The green fluorescence of Ishikawa cells was taken up by macrophages after the co-cultured two types of cells were in contact with each other, and macrophages were able to engulf multiple Ishikawa cells continuously.Macrophages that engulfed Ishikawa cells could induce activation of cytotoxic T cell. Conclusion The live cell application was successfully conducted to construct an in vitro model of phagocytosis of tumor cells by macrophages, which provided a feasible experimental method for detecting the dual killing process of macrophages and T cells on tumor cells.

To establish a simple, quick and effective method to get a large amount of spider toxin JZTX-26 (35 aa) and JZTX-51 (27 aa) with 3 disulfide bonds each, the mature peptides coding gene fragments were constructed and fused with maltose-binding protein (MBP) tag in an Escherichia coli expression vector pMAL-p2x. The recombinant constructs pMAL-jz26 and pMAL-jz51 were transformed and cultured in E. coli TB1 and BL21 (DE3). After being induced by isopropyl-β-d-thiogalactoside (IPTG), the periplasmic proteins were purified by amylose affinity chromatography and analyzed by SDS-PAGE. The fusion proteins were digested with factor X, and purified by Sizes-Exclusion chromatography and Reversed Phase HPLC. Molecular weights of the purified peptides were obtained by using a MALDI-TOF-TOF mass spectrometer, which were consistent with the theoretical molecular weights. Five milligram of target protein could be purified from 1 L of culture medium. The results indicate that the peptides with three disulfide bonds can be expressed by using the prokaryotic expression system with MBP tag. Our findings suggest the possibility of genetic engineering to obtain large amount of spider peptide toxins.

Objective To investigate the application value of pancreaticojejunostomy with double-layer continuous suture in total laparoscopic pancreaticoduodenectomy (TLPD).Methods The retrospective crosssectional study was conducted.The clinicopathological data of 21 patients who underwent TLPD with pancreaticojejunostomy using double-layer continuous suture in the Second Hospital of Jilin University between January and December 2017 were collected.The anastomosis used Child method,and pancreaticojejunostomy,choledochojejunostomy and gastroenteric anastomosis in turn were done.Observation indicators:(1) surgical and postoperative recovery;(2) postoperative pathological examination;(3) follow-up and survival situations.Follow-up using outpatient examination and telephone interview was performed to detect postoperative survival and tumor recurrence or metastasis up to February 2018.Measurement data with normal distribution were represented as x±s.Measurement data with skewed distribution were described as M (P25,P75).Results (1) Surgical and postoperative recovery:21 patients underwent successful TLPD with pancreaticojejunostomy with double-layer continuous suture.The operation time,time of pancreaticojejunostomy and volume of intraoperative blood loss were respectively (352±25)minutes,(46±8)minutes and (168±34) mL.There was no intraoperative blood transfusion.The time of postoperative abdominal drainage-tube removal was (10.1±4.4)days.Of 21 patients,12 were complicated with biochemical fistula,and 3 with grading B of pancreatic fistula (pancreatic duct in type Ⅱ),and they were improved by inhibiting pancreatic secretion and drainage patency.There was no occurrence of biliary fistula,chylous fistula,postoperative bleeding,abdominal infection and delayed gastric emptying.The duration of postoperative hospital stay of 21 patients was (11.3± 2.0) days.(2) Postoperative pathological examination:surgical margins of 21 patients were negative.The pathological type:8,6,4,2 and 1 patients were diagnosed as distal bile duct cancer,ampulla cancer,duodenal papilla and duodenal cancer,pancreatic head cancer and neuroendocrine cancer of ampulla,respectively.(3) Follow-up and survival situations:21 patients were followed up for 3-12 months,with a median time of 7 months.During the follow-up,all the patients survived,and there was no tumor recurrence and metastasis.Conclusion Pancreaticojejunostomy with double-layer continuous suture is safe and feasible for TLPD,with advantages of exact anastomosis effect and good application value.

Objective To investigate the clinical, neuroimaging and serum risk factors of cerebral microbleeds (CMBs) in patients with ischemic stroke and find the associations between these risk factors and the location and num?bers of CMBs were also analyzed. Methods One hundred and fifty-three patients with acute ischemic stroke were re?cruited in this study and their data werewas retrospectively analyzed. All of the patients underwent MRI- susceptibility weighted imaging (SWI). The location and numbers of CMBs were recordedexamined. The severity of WMLs was assessed using the Fazekas scale. Logistic regressions were performed to find the predictors of the presence of CMBs. The relation?ships between these risk factors and the location and numbers of CMBs were also analyzed. Results Fifty-nine(38.6%) cases had at least one CMB. The frequency of cortical-subcortical, deep and infratentorial CMBs were 34.0%, 24.8%and 27.5%, respectively. Multivariate logistic regression showed that male sex, hypertension and moderate-to-severe deep white matter hyperintensities (DWMH) were independent risk factors of the presence of CMBs. Adjusted with age and sex, partial correlation showed that hypertension only correlated with the numbers of deep CMBs significantly (r=0.174, P=0.032). Moderate-to-severe DWMH significantly correlated with the numbers of cortical-subcortical and deep CMBs (r=0.285, P<0.001 and r=0.258, P=0.001, respectively). Conclusion Male sex, hypertension and moderate-to-severe DWMH were are independent risk factors of CMBs in patients with ischemic stroke. Hypertension correlates with Deep deep CMBs, while Moderatemoderate-to-severe DWMH correlates with cortical-subcortical and deep CMBs.

Objective To study the influence of qiqishen granules on the cellular and humoral immune functions in model mice. Methods Six-week-old mice were divided into control group and qiqishen granule (high, medium and low dose) groups. The suspension of chicken red blood cells was injected into the mouse abdominal cavity. The influence of qiq-ishen granules on the phagocytic function of the macrophages in mouse abdominal cavity was observed. The sheep red blood cells (SRBC) were prepared. The blood corpuscle coagulation was observed, and the serum hemolysin was detected. The ac-tivity of the mouse natural killer (NK) cells were detected by the interaction between the target cell (YAC-1) and spleen cell (the response cell). The influence of qiqishen granules on the cellular immunity was detected by the lymphocyte transforming assay. The influence of qiqishen granules on organ/body weight ratio was measured by calculating the thymus/body and spleen/body ratio. Results Compared with the control,qiqishen granules significantly improved phagocytic function of the macrophages in mouse abdominal cavity. The humoral immune function was also improved in mice. The activity of NK cell was enhanced by qiqishen granules, which also enhanced the lymphocyte transforming induced by the concanavalin (Con) A. There was no significant influence in the organ/body weight ratio. Conclusion The qiqishen granules could increase the im-munomodulating effect, indicating it would have a good using prospect.

BACKGROUND: Amnion has been widely used in ophthalmology. Numerous studies have suggested that amnion transplantation did not induce acute immunologic rejection. These indicated that amnion transplantation can be used as a safe material for repair of dural defects.OBJECTIVE: To study the probability of freeze-dried amniotic membrane (FDAM) as a dural substitue. METHODS: Each of the guinea pigs underwent bilateral parietal craniectomy behind the coronal suture and beside the midline to expose the dura. On the right side, a piece of dura mater was removed. The dural defect was covered with a piece of FDAM. The exposed dura on the left was cut and sutured itself as control. The animals in each group were sacrificed at 15, 30, 60 and 90 days after operation, respectively. The implants were harvested and stained with hematoxylin-eosin, and histologically analyzed. RESULTS AND CONCLUSION: After operation, the behavior of all guinea pigs remained completely normal. The wound healing was achieved in all cases. No wound infection, subcutaneous effusion or cerebrospinal fluid (CSF) leakage occurred. The graft was degraded gradually and covered with a sheet of connective tissue. Dural defects repaired with FDAM showed no adhesions to the brain surface. 15 days after operation, plenty of scattered fibroblasts appeared in the dural substitute. 30 days alter dural graft implantation, parts of the implant disappeared; meanwhile the hyperplasia of fibrous connective tissue took place in the center part of the dural substitute, without the infiltration of inflammatory cells. 60 days after implantation, a majority of the dural graft was degraded, substituted by fibrous connective tissue which was of hyperplasia and low-grade degeneration, surrounded by a small quantity of giant cells. 90 days after operation, colloidal degeneration happened in the dural substitute, surrounded by ossification tissue and the degenerated fibrous connective tissue. The inflammatory cells were not discovered. The animal experiment proves FDAM to be a safe and applicable dural substitute.