Objective:To prepare the anti-programmed death-ligand 1 (PD-L1) nanoantibody P3C8-C3Fab by ligating with C3Fab and to investigate its role in plasma half-life.Methods:The C3Fab peptide derived from protein G was molecularly fused with the nanobody P3C8 by DNA recombination technology. The nanoantibody P3C8-C3Fab was inducibly expressed and purified in the E. coli BL21 strain, and the binding of it to PD-L1 protein, mouse IgG, and PD-L1-expressing tumor cells was detected by enzyme-linked immunosorbent assay (ELISA). The residual P3C8-C3Fab was detected in mouse serum at different times using double-antibody sandwich ELISA to assess the prolongation of the plasma half-life of PD-L1 nanobodies by C3Fab. Results:The nanoantibody P3C8-C3Fab was successfully constructed, and it could efficiently express itself in soluble form in BL21. The purified NbP3C8-C3Fab protein was obtained with a mass fraction of about 90% at a yield of 7.18 mg/L. The affinity of P3C8-C3Fab for PD-L1 protein and mouse IgG gradually increased with increasing mass concentration and showed a concentration correlation. The binding of P3C8-C3Fab to lung cancer A549 cells showed a concentration correlation. The concentration standard curve of P3C8-C3Fab in mouse serum showed a typical S-shape with a concentration correlation. The plasma half-life of P3C8 was only 0.44 h, while the plasma half-life of P3C8-C3Fab was 21.27-fold higher, up to 9.36 h.Conclusions:The linkage of C3Fab to the nanobodies of P3C8 can significantly prolong the plasma half-life of P3C8, which is valuable for the improvement of in vivo nanobody effects.

Objective:To analyze the pathological findings of ultrasound-guided transplant kidney puncture after renal transplantation and the pathogenesis of different types of diseases.Methods:A retrospective study was conducted to select 257 patients who underwent ultrasound-guided transplant kidney puncture pathology biopsy due to abnormal tests or uncomfortable symptoms at Beijing Friendship Hospital, Capital Medical University from June 2020 to April 2022, and to analyze the pathological results of puncture and the pathogenesis of different types of diseases and puncture-related complications in the post-transplantation patients after transplant kidney puncture biopsy. Measurement data conforming to normal distribution were expressed as mean ± standard deviation ( ± s), and independent sample t-test was used to compare different types of diseases; measurement data did not conform to normal distribution were expressed as median (interquartile distance) [ M( Q1, Q3)], and the comparison between different types of diseases was conducted by non-parametric test. The count data were compared among different types of diseases using Chi-squre test. Results:Among the 257 patients who underwent transplant renal puncture, 93 cases (36.2%) suffered from antibody-mediated rejection (ABMR), 76 cases (29.6%) suffered from IgA nephropathy, 63 cases (24.5%) suffered from T cell-mediated rejection (TCMR), 21 cases (8.2%) suffered from polyomavirus-associated nephropathy (PVAN), and 4 cases (1.6%) suffered from thrombotic microangiopathy (TMA), 16 cases (6.2%) suffered from diabetic nephropathy, and 12 cases (4.7%) suffered from calcineurin inhibitor (CNI) nephropathy. TCMR, TMA and PVAN occurred significantly in the early post-transplantation period (within about 4 years) ( P<0.001), and ABMR occurred significantly in the late post-transplantation period (after about 8 years) ( P<0.001). In terms of time distribution, creatinine abnormality and proteinuria were the main reasons for puncture. Among those diagnosed with PVAN, the time to transplantation was significantly shorter in those who underwent puncture for creatinine abnormality than in those who underwent puncture for proteinuria ( P=0.011). In terms of puncture-related complications, a total of 8 cases were found to have arteriovenous fistulae at the time of review, 2 cases had perinephric hematomas, and 1 case had both of these two puncture-related complications. Conclusions:Transplant renal complications in renal transplant patients mainly include ABMR, IgA nephropathy, TCMR, PVAN, diabetic nephropathy, CNI nephropathy and TMA. In terms of the pathogenesis of different types of diseases after transplantation, post-transplantation PVAN, TMA, and TCMR mostly occur in the early post-transplantation period, while ABMR occurs at a later time. However, it is worth noting that the clinical symptoms of different types of transplantation kidney-related diseases are similar and not typical.

Objective To observe the effect of Latexin treatment on the chemoresistance in gemcitabine-resistant pancreatic cancer cell line SW1990, and explore the potential mechanism.Methods Gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was induced and established by increasing gemcitabine dosage intermittently.IC50 of gemcitabine in SW1990 cells and SWl990/GZ cells pre and post Latexin treatment at the dosage of 10, 20 and 40 ng/μl for 48 h was evaluated using CCK-8 assay.The mRNA and protein expression of Latexin gene in SW1990 and SW1990/GZ cells were evaluated using qRT-PCR and Western blot, and the expression of Shh and Gli1 in 40 ng/μl Latexin treated SW1990 and SW1990/GZ cells for 48 h.Results A gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was obtained successfully, which can grow stably and passage in the media containing 150 μmol/L gemcitabine.The IC50 values of gemcitabine in SW1990 cells and SWl990/GZ cells were (3.8±0.4)μmol/L and(226.52±13.61)μmol/L, respectively, and the later was greatly higher than the former, which was statistically different (P=0.000).The drug resistance indexes (RI) was 59.6.After treated with different concentrations of Latexin(10,20,40 ng/μl), the IC50 of SW1990 cells was (3.0±0.4)μmol/L, (2.5±0.3)μmol/L and (1.8±0.3)μmol/L, respectively,and the IC50 of SW1990/GZ cells was(113.08±5.01)μmol/L,(70.26±2.31)μmol/L and (42.12±1.31)μmol/L, respectively.Compared with the untreated cells,the IC50 of gemcitabine in 20,40 ng/μl Latexin treated cells was obviously decreased, and the differences were statistically significant (P<0.05).Compared with the SW1990 cells,the expression of Latexin in SW1990/GZ cells was obviously decreased.RI were 37.7, 28.1 and 23.1,respectively.mRNA relative expression of Latexin in SW1990 and SW1990/GZ cells were 0.85±0.08 and 0.31±0.07, and protein relative expression were 0.49±0.09 and 0.13±0.05, and Latexin expression in SW1990/GZ was obviously lower than that in SW1990 cells and the difference was statistically significant (P<0.05).After being treated by 40 ng/μl Latexin, SHH mRNA in SW1990/GZ cells decreased from 0.89±0.09 (control cells) to 0.53±0.06, Gli1 mRNA decreased from 0.58±0.06 to 0.35±0.05, Shh protein decreased from 0.72±0.09 to 0.35±0.06,Gli1 protein level decreased from 0.78±0.08 to 0.28±0.03, and all the differences were statistically significant (P<0.05 or 0.01).Conclusions Latexin can significantly improve the chemosensitivity in gemcitabine-resistant pancreatic cancer cells, and the potential mechanism may be related to the inhibition of sonic hedgehog pathway activation.

Objective To investigate the effect of astragalus polysaccharides injection (APS) on autophagy of human nasopharyngeal cancer CNE-2 cells.Methods The inhibitory effect of APS on proliferation of CNE-2 cells was measured by CCK8 assay.Morphological changes of autophagy of APS was detected by acridine orange (AO) staining.Transmission electron microscopy was perform to observe the morphological alterations in the autophagic cells.The expression of Beclin-1 protein was detected by Western blot assay.Results APS markedly inhibited cell growth in a dose-and time-dependent manner.Increases of the number of large vacuoles and double layered membrane structure were observed by transmission electron microscopy.Results of AO staining revealed more bright red cytoplasm or nucleus in the ceradime group,which proved the existence of acidic vesicular organelles.Western blotting showed that Beclin-1 protein level was up-regulated.Conclusion APS may induce autophagy of human nasopharyngeal cancer CNE-2 cells via up-regulating the expression of apoptosis-related gene of beclin1 and inhibit the proliferation of CNE-2 cells.

Objective To study the effects of Lxn on CD133 + PANC-1 pancreatic cancer cells.Methods CD133 + PANC-1 cell were isolated by magnetic activated cell sorting (MACS).The properties of the CD133 + PANC-1 cells and Lxn effects on CD133 + PANC-1 cell proliferation in transplanted tumor in nude mice were determined by floating spheres test and tumor xenograft assays.Cell proliferation was assayed by Cell Counting Kit-8 (CCK-8).The Bcl-2,Bax protein and mRNA expression of CD133 + PANC-1 cells treated by Lxn were analyzed by Western blot and Quantitative real-time PCR (qRT-PCR).Results We successfully isolated the CD133 + PANC-1 cells and cultured in serum free medium,CD133 + PANC-1 cells formed sphere,while CD133-PANC-1 cells grew with adherence slowly and then underwent apoptotic process.CD133 + PANC-1 cells showed high tumorigenic in athymic BALB/c mice.Lxn suppressed the growth of transplanted tumor obviously.Compared with control group [(225.52 ± 34.09) mm3],tumor volume decreased significantly (P ＜ 0.05).Significant reduction in cell proliferation was observed in response to Lxn in PANC-1 CD133 + cells by CCK-8 assay with concentration and time dependent manners (P ＜ 0.05).Treated by Lxn,Bcl-2 expression decreased,Bax expression increased.Conclusions Lxn inhibits the proliferation of CD133 + PANC-1 cells probably through a mechanism down-regualting Bcl-2 and up-regulating Bax.

Objective To explore the effect of Latexin (Lxn) gene transfection on proliferation of CD13;MIAPaca-2 pancreatic cancer stem-like cells.Methods CD133+ MIAPaca-2 cells were isolated and sorted by magnetic activated cell sorting from pancreatic cancer MIAPaca-2 celt line.CD133+ MIAPaca-2 cells were cultured in serum-free medium and the capacity for proliferation,and tumorigenicity of CD133+ MIAPaca-2 cells was determined by the floating spheres test and tumor xenograft assays.The CD133+ MIAPaca-2 cells were transfected with Lxn plasmid (1,3,5 μg).After transfection,the protein and mRNA expression of Lxn in CD133+ and CD133+-MIAPaca-2 cells were detected by Western blotting and RT-PCR,respectively.Cell proliferation was assayed by CCK-8.Results CD133+ MIAPaca-2 cells were successfully isolated,and it grew into a ball-suspended way,the tumorigenicity rate in nude mice with subcutaneous injection 1 × 105 cancer cells was 100％.After Lxn plasmid transfection,the expression of Lxn in CD133+ MIAPaca-2 cells was increased in a dose dependent manner,the Lxn protein and mRNA expression of tumor cells transfected with 5 μg plasmid was 20.80 ±0.98,16.80± 2.73,which was significantly higher than that in non-transfected cells (1.02 ± 0.01,1.01 ± 0.01),and the difference between the two groups was statistically significant (P ＜ 0.05).After transfection,cellular proliferation activity also showed a transfection dose and culture time-dependent decrease,the inhibition rate of tumor cells transfected with 0.4 μg plasmid was 36.2％,which was significantly different from that in non-transfected cells (P ＜ 0.05).Conclusions CD133+ MIAPaca-2 pancreatic cancer cells have some characteristics of cancer stem cells.Lxn gene transfection can inhibit the proliferation of CD133+ MIAPaca-2 cells.

BACKGROUND/AIMS: To investigate the effects of esomeprazole and rebamipide combination therapy on symptomatic improvement in patients with reflux esophagitis. METHODS: A total of 501 patients with reflux esophagitis were randomized into one of the following two treatment regimens: 40 mg esomeprazole plus 300 mg rebamipide daily (combination therapy group) or 40 mg esomeprazole daily (monotherapy group). We used a symptom questionnaire that evaluated heartburn, acid regurgitation, and four upper gastrointestinal symptoms. The primary efficacy end point was the mean decrease in the total symptom score. RESULTS: The mean decreases in the total symptom score at 4 weeks were estimated to be −18.1±13.8 in the combination therapy group and −15.1±11.9 in the monotherapy group (p=0.011). Changes in reflux symptoms from baseline after 4 weeks of treatment were −8.4±6.6 in the combination therapy group and −6.8±5.9 in the monotherapy group (p=0.009). CONCLUSIONS: Over a 4-week treatment course, esomeprazole and rebamipide combination therapy was more effective in decreasing the symptoms of reflux esophagitis than esomeprazole monotherapy.
Esomeprazole* ; Esophagitis, Peptic* ; Heartburn ; Humans

BACKGROUND/AIMS: To investigate the effects of esomeprazole and rebamipide combination therapy on symptomatic improvement in patients with reflux esophagitis. METHODS: A total of 501 patients with reflux esophagitis were randomized into one of the following two treatment regimens: 40 mg esomeprazole plus 300 mg rebamipide daily (combination therapy group) or 40 mg esomeprazole daily (monotherapy group). We used a symptom questionnaire that evaluated heartburn, acid regurgitation, and four upper gastrointestinal symptoms. The primary efficacy end point was the mean decrease in the total symptom score. RESULTS: The mean decreases in the total symptom score at 4 weeks were estimated to be −18.1±13.8 in the combination therapy group and −15.1±11.9 in the monotherapy group (p=0.011). Changes in reflux symptoms from baseline after 4 weeks of treatment were −8.4±6.6 in the combination therapy group and −6.8±5.9 in the monotherapy group (p=0.009). CONCLUSIONS: Over a 4-week treatment course, esomeprazole and rebamipide combination therapy was more effective in decreasing the symptoms of reflux esophagitis than esomeprazole monotherapy.
Esomeprazole* ; Esophagitis, Peptic* ; Heartburn ; Humans

Objective To investigate the application value of Photoshop in grading invasive risk of gastric stromal tumors (GSTs). Methods EUS image of 97 cases of GSTs confirmed by pathological and immunohistochemical examination were collected. GSTs were divided into four groups (very low risk, low risk, intermediate risk, high risk) by tumor size, mitotic count and rupture of tumor. Mean gray value (intensity of echo) and gray value standard deviation (uniformity of echo) of EUS images of the lesions were determined by Photoshop and then the differences of each group were found by statistical analysis. Results It is difficult to differentiate EUS images of GSTs from each group by visual observation. The mean gray value of EUS image of very low risk group,low risk group, intermediate risk group and high risk group of GSTs respectively were (56.54 ± 6.10), (59.20 ± 7.51), (77.77 ± 10.90) and (83.43 ± 12.47). There was no significant difference between very low risk group and low risk group (P > 0.05). There was no significant difference between intermediate risk group and high risk group (P > 0.05). In addition, the others all had significantly different from that of each group (P < 0.05). The mean gray value standard deviation of EUS image of very low risk group, low risk group, intermediate risk group and high risk group of GSTs respectively were (8.46 ± 2.59), (12.57 ± 5.89), (12.84 ± 4.15) and (16.69 ± 4.69). There was no significant difference between low risk group and intermediate risk group (P > 0.05). In addition, the others all had significantly different from that of each group (P < 0.05). Conclusions The higher risk of GSTs, the higher of echo intensity and the worse of echo uniformity under EUS. Photoshop combined with EUS is helpful for differentiating different risk of GSTs by analyzing mean gray value and gray value standard deviation of the lesions.

OBJECTIVE:To compare the clinical efficacy and safety of amiodarone and propafenone in the treatment of chron-ic atrial fibrillation cardioversion. METHODS:60 patients with chronic atrial fibrillation cardioversion were randomly divided into propafenone group and amiodarone group. All patients were given conventional treatment,including treating primary disease,con-trolling symptoms,orally giving aspirin,intravenous infusion of GIK,monitoring heart rate,QT interval,P-R interval,QRS time,serum potassium and serum magnesium,etc. On this basis,propafenone group was orally given propafenone 450 mg for con-tinuous 3 months,3 times a day,and then the dose was decreased to 300 mg to maintain the sinus rhythm;amiodarone group was orally given amiodarone 200 mg for continuous 7 d a month,twice a day,and then the dose was decreased to 200 mg or 100 mg to maintain the sinus rhythm. The clinic data in 2 groups was observed,including clinical efficacy,simple drug cardioversion,elec-trical cardioversion,electrical cardioversion power,hospitalization time and incidence of adverse reactions,and the recurrence rate in 48 months was followed up. RESULTS:There were no significant differences in the clinical efficacy,simple drug cardiover-sion,electrical cardioversion,electrical cardioversion power,hospitalization time,incidence of adverse reactions and recurrence rate between 2 groups(P>0.05). CONCLUSIONS:Based on the conventinal treatment,amiodarone and propafenone have similar clinical efficacy and safety in the treatment of chronic atrial fibrillation cardioversion,and both of them can be used as the conven-tional drugs for treating chronic atrial fibrillation cardioversion.