Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.
Cell Differentiation ; Genetic Vectors ; HEK293 Cells ; Humans ; Nerve Growth Factor ; biosynthesis ; Plasmids ; Recombinant Proteins ; biosynthesis

Objective: To investigate the early clinical effects of tantalum augment assisted with 3D technology in treating acetabular bone defects of Paprosky type III in revision total hip arthroplasty (THA). Methods: From May 2013 to July 2017, a total of 16 patients (18 hips) undergoing revision THA were retrospectively analyzed, including 11 males and 5 females aged 58.06±8.29 years (range 44-69 years). There were 3 cases with infective loosening and 15 cases with aseptic loosening, 13 cases with Paprosky IIIA type bone defects and 5 cases with IIIB type bone defects. 3D technology was used for precise planning before operation. The tantalum augment and cup were used to repair acetabular bone defects during operation. Cup anteversion, abduction angle, ratio of the lateral and contralateral vertical distance of the center of rotation, ratio of the lateral and contralateral horizontal distance of the center of rotation and femoral offset were measured preoperatively and postoperatively. The percentages of hips located in Lewinnek safe zone were calculated preoperatively and postoperatively. Postoperative radiographic evidence of loosening and Harris score were collected at the end of the follow-up. Results: The percentage of the hips located in Lewinnek safe zone increased from 22% (4/18) preoperatively to 61% (11/18) postoperatively. The mean anteversion of the operative side was 11.99°±6.91° (range 1.71°-26.36°) postoperatively. The mean abduction angle of the operative side was 44.91°±5.93° (range 35.6°-56.0°). The mean ratio of the lateral and contralateral vertical distance of the center of rotation was 1.10±0.20 (range 0.87-1.62). The mean ratio of the lateral and contralateral horizontal distance of the center of rotation was 1.00±0.18 (range 0.69-1.46) and the mean ratio of the lateral and contralateral femoral offset was 1.01±0.66 (range 0.51-3.56). All the patients were followed-up for an average of 27.72±12.18 months (range 14-53 months). No complications, such as periprosthetic joint infection, dislocation or aseptic loosening, were observed in all patients. The mean Harris score was 77.28±4.80 (range 65-85) at 6 months postoperatively and 80.9±5.2 (range 69-89) at the end of the follow-up. Conclusion Using tantalum augmentassisted with 3D technology to re construct Paprosky type III severe bone defects of the hip can increase the accuracy of the acetabular cup positioning. The short-term outcomes are satisfying and no early prosthetic loosening was observed.

Objective:To quantify the diagnostic index of probe-based confocal laser endomicroscopy (pCLE) for diagnosing Helicobacter pylori ( HP)-associated chronic atrophic gastritis (HpCAG), and to evaluate the efficacy of the quantified diagnostic index for HpCAG. Methods:The study was divided into two stages. The first stage prospectively included patients undergoing gastroscopy, endoscopic biopsy and 13C breath test from November 2021 to September 2022 at the Second Affiliated Hospital of Baotou Medical College. The capillary diameter (CD), cells spacing (CS), gland spacing (GS), and gland area (GA) in the pCLE field of offline video was measured with Image J. The diagnostic criteria of HpCAG by quantitative indicators under pCLE was established by analyzing the area under the receiver operating characteristic (ROC) curve (AUC). In the second stage, the cases with pCLE examination and 13C breath test at the Second Affiliated Hospital of Baotou Medical College from October 2021 to October 2022 were included. The cases that overlapped with the first stage were excluded. The trial was single-blind, with endoscopists and pathologists blind to each other's diagnoses. The diagnosis of pCLE was conducted according to the criteria obtained in the first stage, and the consistency between pCLE diagnosis and the results of histopathology and 13C breath test was analyzed. Results:The first stage enrolled 191 specimens from 35 patients. According to the pathological results of endoscopic biopsy and 13C breath test results, patients and gastric mucosa samples were divided into 4 groups, HP-positive CAG group ( n=59), HP-positive non-CAG group ( n=52), HP-negative CAG group ( n=40), and HP-negative non-CAG group ( n=40). ROC curve analysis results showed that in HP-positive patients, the optimal critical value of GS to distinguish between CAG and non-CAG gastric mucosa was 29.68 μm, and the AUC was the largest among the 4 parameters. In HP-negative patients, the optimal critical value of GS for distinguishing gastric mucosa from CAG and non-CAG was 23.57 μm, and the AUC was the largest among the 4 parameters. In patients with non-CAG, the optimal critical value for GS to distinguish HP-positive and HP-negative gastric mucosa was 20.57 μm, and the AUC was the largest among the 4 parameters. In patients with CAG, the optimal critical values of CD, CS, GS and GA to distinguish between HP-positive and HP-negative gastric mucosa were 13.23 μm, 1.38 μm, 34.03 μm and 6 066.5 μm 2, respectively, and the AUC were 0.608, 0.888, 0.849 and 0.900, respectively. Finally, GS was selected to distinguish between HpCAG and non-HpCAG gastric mucosa, and the optimal critical value was 31.71 μm. However, considering that it was difficult to measure the distance of 31.71 μm by the ruler below the image, the critical value was changed to 30 μm, so GS>30 μm was used as the diagnostic criteria for HpCAG in pCLE, and the diagnostic sensitivity and the specificity were 91.5% and 76.0%, respectively. In the second phase 224 specimens from 80 patients were observed. The sensitivity, the specificity, the positive predictive value, the negative predictive value and accuracy of pCLE (GS>30 μm) in the diagnosis of HpCAG were 96.5% (164/170), 88.9% (48/54), 96.5% (164/170), 88.9% (48/54) and 94.6% (212/224), respectively, with excellent diagnostic agreement with histopathology and 13C breath test (Kappa=0.854). Conclusion:The quantitative monitoring of gastric mucosal microstructure can be achieved under pCLE, and the quantifying indicators are helpful to improve the accuracy of HpCAG diagnosis.