Objective To observe three-dimensional structure and biological features of rat acellular spinal cord scaffold prepared by sonic oscillation and chemical extraction in order to offer an ideal scaffold for spinal cord tissue engineering research.Methods Rat spinal cord underwent acellular treatment with sonic oscillation and chemical extraction (Triton X-100 at volume fracture of 2％ and sodium deoxycholate at volume fracture of 2％) (acellular spinal cord group).In contrast with spinal cord tissue of normal rats (control group),general morphology,histology and ultramicro three-dimensional structure of acellular spinal cord scaffold were observed and aperture size,factor of porosity,water ratio,enzymolysis ratio and stability in water solution of the scaffold were also detected.Results Acellular spinal cord group showed effective removal of original cell components with factor of porosity for (94.57 ±3.45) ％ and water content for (88.62 ± 1.0) ％,and satisfactory three-dimensional structure with average aperture of 46 μm.Scaffold showed gradual degradation in enzymolysis solution and enzymolysis rate reached (69.03 ± 2.19)％ at 20 hours.Besides,scaffold showed stepwise disintegration in double distilled water and hydrolysis rate was (62.55 ± 1.70) ％ at 8 days.While,normal spinal cord showed close structure,generous neurons and myelin sheath with factor of porosity for (0.04 ± 0.02) ％ and water content for (62.4 ± 1.5) ％,and unobvious pore structure under scanning electron microscope.Normal spinal cord were degraded gradually in enzymolysis solution and enzymolysis rate was (37.62 ± 0.9)％ at 20hours.In the meantime,normal spinal cord were disintegrated gradually in double distilled water and hydrolysis rate was (40.97 ± 0.81) ％ at 8 days.Conclusions Acellular spinal cord scaffold prepared by sonic oscillation plus chemical extraction achieves complete removal of cell components,intact extracellular matrix,and satisfactory results in three-dimensional network structures,factor of porosity and water content.Also,the scaffold meets theoretical demands of tissue-engineered spinal cord scaffold and is an ideal alterative for tissue-engineered spinal cord scaffold.

Objective To construct genipin-crosslinked rat acellular spinal cord scaffolds and evaluate their enzymatic degradation rate,biomechanical properties and cytotoxicity.Methods Rat spinal cord scaffolds were decellularized by chemical extraction and chemically crosslinked with 5 g/L genipin solution.Micro-structure of the uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds were observed by HE staining and scanning electron microscopy and properties of pore size,porosity,water ratio,and degradation rate in 2.5 g/L trypsin enzyme solution were examined.Ultimate tensile strength and elastic modulus of normal rat thoracic spinal cord,uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds were determined on Instron mechanical testing instrument.Rat bone marrow mesenchymal stem cells were cultured in lixivium of uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds and MTT assay for relative cell growth rate was test to evaluate the cytotoxicity of scaffolds.Results The uncrosslinked and the genipin-crosslinked acellular spinal cord scaffolds possessed a similar three-dimensional mesh-porous structure with a mean pore diameter about 30 μm and a porosity over 80％,but there was a statistical difference between the two groups(P ＞ 0.05).Water ratio of genipincrosslinked scaffolds was (229.7 ± 12.5) ％,far lower than (283.4 ± 11.2) ％ of uncrosslinked scaffolds (P ＜ O.05) ; genipin-crosslinked acellular spinal cord scaffolds had lower weight loss at each time point than the uncrosslinked acellular spinal cord scaffolds (P ＜ 0.05),but the stability in trypsin,ultimate tensile strength and elastic modulus of acellular spinal cord scaffolds were significantly enhanced by genipin-crosslinking (P ＜ 0.05).Furthermore,no obvious cytotoxicity was observed in the uncroslinked and genipin-crosslinked scaffolds.Conclusions Rat acellular spinal cord scaffolds present no obvious change in structure after genipin-crosslinking,but there is significant improvement in the biomechanical properties and ability against enzymatic degradation and no marked cytotoxicity.Hence,the genipincrosslinked scaffolds are promising in tissue engineering for spinal injury.

OBJECTIVETo construct full-length human bladder cancer-specific antibody libraries for efficient display of full-length antibodies on the surface of mammalian cells.

METHODSThe total RNA was isolated from peripheral blood mononuclear cells from patients with bladder cancer. The repertoires of IgG1 heavy chain variable region (VH) and Kappa light chain were amplified by RT-PCR using specific primers. The antibody genes were inserted into the vector pDGB-HC-TM to construct the bladder-cancer-specific antibody libraries of heavy chains and light chains. Ten clones from each library were randomly picked for gene sequencing and transient transfection into FCHO cells to analyze antibody display on mammalian cell surface by flow cytometry after staining with corresponding fluorescent labeled antibodies.

RESULTSThe libraries of bladder-cancer-specific antibody heavy chain (IgG1) and light chain (LCk) were successfully constructed. Seven out of the 10 clones randomly selected from the heavy chain library and 9 out of the 10 clones from the light chain library showed correct open reading frame, coding for 7 unique VH and 9 unique LCk. The combinatory library size reached 3.32×10(11).

CONCLUSIONWe have successfully constructed a full-length human bladder-cancer-specific antibody library with a combinatory diversity of 3.32×10(11) based on mammalian display technology, which can be used for screening monoclonal antibodies against bladder-cancer-associated antigens.

Amino Acid Sequence ; Animals ; Antibodies ; genetics ; Cell Surface Display Techniques ; Gene Library ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin kappa-Chains ; genetics ; Peptide Library ; Urinary Bladder Neoplasms ; genetics ; immunology

Objective: To explore whether human adipose-derived stem cells (ADSCs) express PD-L1 and Gal-9 and its potential influence factors. Methods: ADSCs isolated from 28 healthy female donors who underwent liposuction of the abdomen or breast tissue were cultured and characterized. The expression of PD-L1 and Gal-9 were detected using flow cytometry. The impact of donor age, body mass index (BMI), donor site and interferon-γ (IFN-γ) on the expression of PD-L1 and Gal-9 was analyzed using multivariate linear regression analysis. Results: The cultured cells fulfilled the criteria for defining MSCs according to the international standards and expressed PD-L1 and Gal-9. Breast-derived ADSCs had higher expression levels of PD-L1 (37.24%±8.20%) and Gal-9 ( 4.41%±2.65%) than those in abdomen-derived ADSCs (28.80%±8.59% and 2.51%±1.39%, respectively) (P=0.018, P=0.039). Multivariate linear regression analysis showed that donor age and site were independent risk factors affecting PD-L1 expression (P=0.009, P=0.006). The expression of PD-L1 decreased by 0.385% for every one year of age increase, and it was 8.58% higher in the breast-derived ADSCs than in the abdomen-derived ADSCs. Donor site was an independent risk factor for Gal-9 expression (P=0.041). Gal-9 positive expression rate in the breast-derived ADSCs was 1.898% higher than that in the abdomen-derived ADSCs. BMI was not a risk factor affecting PD-L1 or Gal-9 expression on ADSCs. The expression of PD-L1 and Gal-9 on ADSCs were significantly upregulated after 48 hours stimulation with 20 ng/ml IFN-γ in vitro. Conclusions Human ADSCs expressed PD-L1 and Gal-9. Donor age, site and IFN-γ treatment were independent risk factors affecting the expression of PD-L1 and Gal-9 on ADSCs.

Objective: To evaluate the clinical effect of subcutaneous pedicle skin flap for repairing skin and soft tissue defect in the philtrum area. Methods: From November 2015 to July 2016, 6 patients with pigmented nevus and basal cell carcinoma were treated with regional subcutaneous pedicled flaps. The functional and aesthetical outcomes were evaluated. Results: Follow-up was performed at 6-12 months postoperatively. All 6 flaps survived. The wounds healing was good, and the postoperative appearance was satisfactory. Conclusions The kite subcutaneous pedicled flap is an ideal method for repairing the skin and soft tissue defects in philtrum area, and it can produce satisfactory clinical results.

OBJECTIVETo construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique.

METHODSTotal RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry.

RESULTSUsing 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)].

CONCLUSIONUsing 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

Animals ; Antibodies, Viral ; CHO Cells ; Cell Surface Display Techniques ; Cricetinae ; Cricetulus ; Dengue Virus ; immunology ; Gene Library ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; immunology ; Transfection

Objective To acquire the basic information on diagnostic radiology and radiotherapy institutions and the frequency of medical radiation of Hebei province in 2016.Methods A survey was made using questionnaires for basic information on medical X-ray diagnosis,interventional radiology,radiotherapy and clinical nuclear medicine.Values of frequencies were derived for each type of medical X-ray procedure by dividing the total population.Results In 2016 there were 2 951 institutions at province level and 6 966 pieces of equipment involved in diagnostic radiology and radiotherapy treatments.Frequency values were estimated to be 379.25 examinations per 1 000 population in medical X-ay diagnosis,2.31 per 1 000 in interventional radiology,0.55 per 1 000 in radiotherapy and 1.65 per 1 000 in clinical nuclear medicine.The frequency for CT examination was 136.39 per 1 000,accounting for 36％ of the total frequencies in the province,with average annual growth rate of 12.4％.The highest value of frequency in clinical nuclear medicine was from Shijiazhuang among all types of medical procedures in different regions,11 times higher than the lowest value found in Hengshui city.Although the number of tertiary hospitals only accounted for 2.3％ of the total number of institutions,they contributed 25.5％,by examination number,of the medical X-ray diagnosis and 35.3％ of the CT examination.Interventional radiology and radiotherapy contributed more than 70％ of the total frequencies in the province,and clinical nuclear medicine was as high as 97.7％.Conclusions The basic information on diagnostic radiology and radiotherapy institutions and the frequency of medical procedures in Hebei in 2016 have been preliminarily made clear.Medical exposure could maintain a long-term growth trend.The frequency distribution of medical procedures in various regions and medical institutions is highly unbalanced.It is desirable to enhance the macroeconomic regulation and the management of medical radiation protection in order to rationally perform all types of diagnostic radiology and radiotherapy treatments to protect the health and safety of the patients and the examined individuals,and to promote the sustainable development of the health care services.

Objective:To investigate the factors affecting the prognosis of stage Ⅰa2-Ⅱa2 cervical cancer after laparoscopic radical hysterectomy (LRH), and to compare the prognosis and recurrence sites of patients with different colpotomy paths.Methods:The clinical data of 965 patients with stage Ⅰa2-Ⅱa2 cervical cancer who underwent LRH in the First Affiliated Hospital of Army Medical University from January 2015 to December 2018 were collected. The median age was 47.0 years of all patients with a median follow-up of 62 months (48-74 months). Cox regression was used to perform the univariate and multivariate analysis of the clinicopathological factors associated with the prognosis that included disease-free survival (DFS) and overall survival (OS). Patients were categorized into LRH through vaginal colpotomy (VC group, n=475) and LRH through intracorporeal colpotomy (IC group, n=490) according to the colpotomic approaches. The prognosis and recurrence sites of patients in each group were compared. Results:(1) During the follow-up period, 137 cases recurred (14.2%, 137/965) and 98 cases died (10.2%, 98/965). The 5-year DFS and OS were 85.8% and 89.9%, respectively. In univariate analysis, positive vaginal margin (PVM) was significantly affected the 5-year OS of patients with cervical cancer ( P=0.023), while clinical stage, maximum diameter of tumor, degree of pathological differentiation, lymph node metastasis (LNM), depth of cervical stromal invasion, parametrium involvement, and uterine corpus invasion (UCI) were significantly associated with 5-year DFS and OS in patients with cervical cancer (all P<0.05). In multivariate analysis, clinical stage ( HR=1.882, 95% CI: 1.305-2.716), LNM ( HR=2.178, 95% CI: 1.483-3.200) and UCI ( HR=3.650, 95% CI: 1.906-6.988) were independent risk factors of 5-year DFS (all P<0.001). Clinical stage ( HR=2.500, 95% CI: 1.580-3.956), LNM ( HR=2.053, 95% CI: 1.309-3.218), UCI ( HR=3.984, 95%C I: 1.917-8.280), PVM ( HR=3.235, 95% CI: 1.021-10.244) were independent risk factors of 5-year OS (all P<0.05). (2) Different colpotomy paths did not significantly affect the 5-year DFS and OS of patients with stage Ⅰa2-Ⅱa2 cervical cancer. The 5-year DFS in VC group and IC group were 85.9% and 85.6% ( P=0.794), and the 5-year OS were 90.8% and 89.3% ( P=0.966), respectively. Recurrence patterns consisted of intraperitoneal recurrence, pelvic recurrence, vaginal stump recurrence, and lymph node and distant metastasis. The intraperitoneal recurrence rate of VC group was significantly lower than that of IC group [0.6%(3/468) vs 2.3% (11/485), P=0.037], while the rates of pelvic recurrence, vaginal stump recurrence, lymph node and distant metastasis and overall recurrence were not significantly different between two groups (all P>0.05). Subgroup analysis of patients with different clinical stages, LNM and UCI showed that statistical differences of the intraperitoneal recurrence rates between two groups were only in patients without LNM (0.5% vs 2.3%, P=0.030) or without UCI (0.7% vs 2.3%, P=0.037). Conclusions:Clinical stage, LNM, PVM and UCI are independent risk factors for the prognosis of patients with stage Ⅰa2-Ⅱa2 cervical cancer. For patients without LNM or UCI, LRH through VC could reduce the intraperitoneal recurrence rate, while it is not enough to improve 5-year DFS and OS of patients. Low proportion of intraperitoneal recurrence, intra-operative tumor cells spillage to vagina stump and pelvic cavity might be the explanation.

Objective To investigate the number, distribution, and types of radiation of non-medical radiation institutions in Hebei Province, China, and to explore the current radiation protection in the employing units and occupational health management of radiation workers in 2022. Methods A questionnaire survey was conducted in the non-medical institutions engaged in nuclear technology application in Hebei Province, and different types of employing units were selected to monitor the radioactivity level in the workplace. Results A total of 681 non-medical institutions engaged in radiation technology application completed the survey, covering all cities with subordinate districts in the province, including 1605 radioactive devices, 2960 active devices, 45 non-uranium metal mines, and 14 non-sealed workplaces. A total of 8617 radiation workers were surveyed, with a personal dose monitoring rate of 70.9%, a radiation protection training rate of 61.1%, and an occupational health examination rate for radiation workers of 59.3%. A total of 614 radiation protection monitoring instruments were provided, with a personal protective equipment allocation rate of 51.1% and a personal dose alarm device allocation rate of 51.8%. The radiation occupational hazardous factor testing was completed for 54 workplaces, and the results were all qualified. Conclusion There are still significant deficiencies in personal dose monitoring in the radiation work units in non-medical institutions and occupational health examination in the radiation work units in our province. The health administrative departments should strengthen health supervision and law enforcement, enhance radiation protection and skill training for employers, and more effectively control the impact of radiation hazards on personnel health.