Objective To generate and identify the Itgbl1(integrin beta-like)promoter-driven Cre knock-in mouse line.Methods Itgbll-Cre knock-in mice were generated using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene editing.The Itgbl1-Cre mice were crossed with the Cre reporter ROSALSL-tdTomato)mice to detect the expression profile of Cre activity.The tdTomato expression pattern across tissues and cell-specific markers were used to identify the cell types of Itgbl1-expressing cells and their progeny.Results and Conclusion tdTomato was specifically expressed in mucous acinar cells of the sublingual gland,pancreatic islet cells,and gastric endocrine cells.In addition,tdTomato expression was also found in some of the neurons of the retina and brain,as well as in a few cells in the serosal layer of the intestine,articular cartilage,periosteum,and bone marrow.The first Itgbl1-Cre recombinase transgenic mouse line was established,which can specifically label the mucous acinar cells of the sublingual gland.

In order to deal with the shortage of medical talents in Tibet, medical group assistance to Tibet is started as an innovative move in a new era, not only providing high-quality medical resources to Tibet, but also training local medical personnel by drawing on the "Master-Apprentice" model in traditional Chinese medicine. During the past three-year, the Shanghai Medical Team took advantage of medical group assistance to Tibet, enriched the types, methods and contents of teaching and mentoring tasks, and highlighted the role of "experts leading the backbone" and "team leading the team" in the "Master-Apprentice" model. The total amount and quality of local medical talents have thus been significantly improved. On the basis of summarizing experience, this study proposes a number of measures to optimize the current "Master-Apprentice" training model, evaluate the implementation process, and improve the feedback and quality management, so as to speed up the construction of the Tibetan medical talent team.

Objective:To report the surgical techniques and clinical outcomes of multi-dimensional fixation of patellar multi-fragmentary fractures with locking plates.Methods:A retrospective study was performed in the 26 patients with patellar multi-fragmentary fracture who had undergone open reduction and 3-D internal fixation with locking plates from November 2016 to July 2020 at Department of Orthopaedic Surgery, The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University. There were 17 males and 9 females, with an average age of 62.6 years (from 31 to 90 years). The patellar fractures were exposed and reduced via the longitudinal anterior midline incision of the knee. After the reduction was initially maintained with a cerclage wire, a trimmed and pre-contoured 3.5 mm locking plate was applied onto the patellar surface. After-wards, locking screws were inserted from the lower pole to the upper pole of the patella, from the anterior to the posterior and from the lateral to the medial, respectively, to complete the multi-planar fixation. Follow-ups assessed the B?stman score, knee pain visual analogue scale (VAS), radiographic image and fracture healing, range of motion of the knee, and complications.Results:All the 26 patients were followed up for 12 to 56 months (average, 28 months). Crutches were used while walking until an average of 1.6 months (from 1 to 3 months) after operation in all patients. At the last follow-up, the B?stman score averaged 27.5 points (from 17 to 30 points), yielding 12 excellent, 13 good and 1 poor case with an excellent to good rate of 96.2% (25/26); the knee pain VAS averaged 1.2 points (from 0 to 5 points); the active knee flexion averaged 125° (from 100° to 150°). No breakage, loosening or displacement of the patellar plates or screws was observed during follow-up, but cerclage wire breakage occurred without any symptom in 11 cases. Four patients complained of hardware irritation, and 4 patients underwent hardware removal after fracture union.Conclusion:Multi-dimensional fixation with locking plates is a viable and safe surgical option for patellar multi-fragmentary fractures, due to its satisfactory therapeutic outcomes.

Objective: To explore an optimal method for granulocyte cell production from umbilical cord blood mononuclear cells. Methods: Erythrocytes were precipitated by hydroxyethyl starch. Mononuclear cells were isolated through Ficoll density gradient centrifugation. Different media, additives and cultivation model were chosen for granulocyte induction. Cell morphology was observed by microscopy, and cell phenotype was detected by flow cytometry. The CD18 expression of granulocytes was tested by immunofluorescence assay, and phagocytosis test was executed as well. Results: Compared to fetal bovine serum (FBS) treatment group, cell viability, counts and differentiation rate of granulocytes induced by X-VIVO^TM 15 combined with TPO, SCF, G-CSF but without FBS were superior. And X-VIVO^TM15 medium was better than SCGM medium at effectiveness and cost. Using two-stage mode of hematopoietic stem cell expansion followed by granulocyte induction with X-VIVO^TM15 combining TPO, SCF and G-CSF, cell proliferation was nearly 132 times at day 21. Flow cytometry showed that the differentiation was lagged in 2-stage mode than in direct induction mode, CD15 expression was (69.60± 1.06) % vs (97.73±0.39) %; Wright-Giemsa staining demonstrated mature granulocytes; immunofluorescence showed the expression of lysosomal proteins CD18. A strong phagocytic function of mature granulocytes was demonstrated by phagotrophic efficiency of (51.43±0.05) %. And granulocyte had chemotaxis ability under the role of chemotactic factor IL-8. Conclusion Optimized culture media and cultivation mode are achieved for functional granulocytes induction in vitro.

OBJECTIVETo discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs).

METHODSUCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing.

RESULTSWith the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective.

CONCLUSIONSThis non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.

Cell Culture Techniques ; Cell Differentiation ; Cell Survival ; Cells, Cultured ; Cryopreservation ; methods ; Erythroblasts ; cytology ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Umbilical Cord

Objectives To explore different factors (clinical presentations and laboratory investigations ) between the severe and mild Guillain-Barré syndrome (GBS) in southeast China ,and to find the predictors of severe GBS. Meth?ods Retrospective analysis was conducted on 101 cases of patients with GBS admitted to our Hospital from Jan. 2006 to Nov. 2015, who were divided into mild and severe groups according to Hughes scale. The different factors were compared between these two groups such as age, sex, precursor infection factors, the initial symptoms, bulbar dysfunction, cranial nerves involvement, autonomic nervous dysfunction, peripheral nerve axonal damage to find the predictors for the severe GBS. Results Severe GBS more frequently presented with non-paresthesia as initial symptom (P<0.001) , bulbar dysfunc?tion (P<0.001), cranial nerves involvement (P=0.025), autonomic nervous dysfunction (P=0.018), motion system involve?ment (P = 0.004) and peripheral nerve axonal damage (P<0.001). After multivariable logistic regression analysis, we found that the axon damage(P=0.008, OR=4.632), bulbar dysfunction(P=0.010, OR=10.420), and cranial nerves in?volvement(P=0.047, OR=0.076)were the independent risk factors for sever GBS. Conclusion Axon damage, bulbar dys?function, and cranial nerves involvement might be significant predictors of sever GBS.

OBJECTIVETo establish a new glioma cell line and analyze its biological characteristics, and to provide a useful cellular tool with new features for cancer research.

METHODSGlioma tissue was taken from surgical specimen clinical of a clinical patient. Primary culture was carried out, and a cell line (SHG139) was established after 10 passages. Immunofluorescence staining was performed to detect the expression of proteins, and cell proliferation and cycle were detected by flow cytometry method (FCM). The biological characteristics of SHG139 cells were detected by chromosome karyotype analysis. SHG139s glioma cells derived from SHG139 glioma cell line were cultured with neural stem cell medium. Then stem cell markers were determined. SHG139s cells were induced with serum-containing medium, and their expression of A2B5, GFAP, β-III tubulin, and GalC was detected. Intracranial xenograft tumor of both SHG139 glioma cells and SHG139s glioma stem cell spheres was generated in rats.

RESULTSThe expressions of A2B5, GalC, GFAP, S-100, and vimentin in the 20 and 60 passages of SHG139 cells were positive, consistent with the immunohistochemical results and pathological features. SHG139 cells proliferated significantly within 24 h after subculture, and their total number of chromosomes was 68 and mostly multiploid. They were positive for A2B5 (84.12±9.96)%, nestin (73.86±5.01)%, and NG2 (73.37±2.09)%. SHG139s cells were induced, and the ratio of positive cells of GFAP, β-III tubulin and GalC was (92.89±2.24)%, (64.85±4.09)% and (33.57±4.14)%, respectively.

CONCLUSIONSSHG139 is an astroglioma cell line, from which SHG139s cells can be successfully obtained by culture with NSCM. SHG139s cells are of A2B5(+)/CD133(-) GSCs subgroup cells, with potentials of self-renewal and multi-directional differentiation. Compared with the intracranial SHG139 xenograft tumor, the intracranial SHG139s xenograft tumor is more malignant and aggressive.

Animals ; Astrocytoma ; Brain Neoplasms ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Flow Cytometry ; Glioma ; Humans ; Neoplastic Stem Cells ; Nestin ; Rats ; Transplantation, Heterologous ; Vimentin

Objective To establish a new glioma cell line and analyze its biological characteristics, and to provide a useful cellular tool with new features for cancer research. Methods Glioma tissue was taken from surgical specimen clinical of a clinical patient. Primary culture was carried out, and a cell line ( SHG139 ) was established after 10 passages. Immunofluorescence staining was performed to detect the expression of proteins, and cell proliferation and cycle were detected by flow cytometry method ( FCM) . The biological characteristics of SHG139 cells were detected by chromosome karyotype analysis. SHG139s glioma cells derived from SHG139 glioma cell line were cultured with neural stem cell medium. Then stem cell markers were determined. SHG139s cells were induced with serum-containing medium, and their expression of A2B5, GFAP,β-Ⅲtubulin, and GalC was detected. Intracranial xenograft tumor of both SHG139 glioma cells and SHG139s glioma stem cell spheres was generated in rats. Results The expressions of A2B5, GalC, GFAP, S-100, and vimentin in the 20 and 60 passages of SHG139 cells were positive, consistent with the immunohistochemical results and pathological features. SHG139 cells proliferated significantly within 24 h after subculture, and their total number of chromosomes was 68 and mostly multiploid. They were positive for A2B5 (84. 12 ± 9. 96)%, nestin (73. 86 ± 5. 01)%, and NG2 (73. 37 ± 2. 09)%. SHG139s cells were induced, and the ratio of positive cells of GFAP, β-Ⅲ tubulin and GalC was (92. 89 ± 2. 24)%,(64. 85 ± 4. 09)% and (33. 57 ± 4. 14)%, respectively. Conclusions SHG139 is an astroglioma cell line, from which SHG139s cells can be successfully obtained by culture with NSCM. SHG139s cells are of A2B5+/CD133-GSCs subgroup cells, with potentials of self-renewal and multi-directional differentiation. Compared with the intracranial SHG139 xenograft tumor, the intracranial SHG139s xenograft tumor is more malignant and aggressive.

Objective To establish a new glioma cell line and analyze its biological characteristics, and to provide a useful cellular tool with new features for cancer research. Methods Glioma tissue was taken from surgical specimen clinical of a clinical patient. Primary culture was carried out, and a cell line ( SHG139 ) was established after 10 passages. Immunofluorescence staining was performed to detect the expression of proteins, and cell proliferation and cycle were detected by flow cytometry method ( FCM) . The biological characteristics of SHG139 cells were detected by chromosome karyotype analysis. SHG139s glioma cells derived from SHG139 glioma cell line were cultured with neural stem cell medium. Then stem cell markers were determined. SHG139s cells were induced with serum-containing medium, and their expression of A2B5, GFAP,β-Ⅲtubulin, and GalC was detected. Intracranial xenograft tumor of both SHG139 glioma cells and SHG139s glioma stem cell spheres was generated in rats. Results The expressions of A2B5, GalC, GFAP, S-100, and vimentin in the 20 and 60 passages of SHG139 cells were positive, consistent with the immunohistochemical results and pathological features. SHG139 cells proliferated significantly within 24 h after subculture, and their total number of chromosomes was 68 and mostly multiploid. They were positive for A2B5 (84. 12 ± 9. 96)%, nestin (73. 86 ± 5. 01)%, and NG2 (73. 37 ± 2. 09)%. SHG139s cells were induced, and the ratio of positive cells of GFAP, β-Ⅲ tubulin and GalC was (92. 89 ± 2. 24)%,(64. 85 ± 4. 09)% and (33. 57 ± 4. 14)%, respectively. Conclusions SHG139 is an astroglioma cell line, from which SHG139s cells can be successfully obtained by culture with NSCM. SHG139s cells are of A2B5+/CD133-GSCs subgroup cells, with potentials of self-renewal and multi-directional differentiation. Compared with the intracranial SHG139 xenograft tumor, the intracranial SHG139s xenograft tumor is more malignant and aggressive.

OBJECTIVETo build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells.

METHODSRed blood cells were precipitated by hydroxyethyl starch (HES). Mononuclear cells were obtained by density gradient centrifugation with Ficoll. The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed.

RESULTSThe best choice for erythrocyte sedimentation and high efficiency of nucleated cells retrieving were obtained by using of 1.5% HES. The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with 116t (IL-11, IL-6, TPO), st36(SCF, TPO, IL-3, IL-6), pt36 (PDGF,TPO,IL-3,IL-6) or pst36 for 7 days. St36 group (50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3 and 50 ng/ml IL-6) yielded the most CD41/CD61 positive [(6.79±1.97)×10⁴]. The cell viability [(82.85 ± 0.64)%] of st36 group by using of imported serum-free medium was better than [(60.90±6.93)%] that in domestic medium on day 7 after induction, and CD41/CD61 positive cells count [(18.60±1.97)×10⁴] were more than domestic serum-free medium group. Therefore, we chose imported serum-free medium containing st36 to induce cord blood mononuclear cells. After a prolonged culture, the total cell numbers increased accompanied with an elevated percentage of CD41/CD61 positive cells, which reached (54.27 ± 6.31)% on day 14. Wright-Giemsa staining showed that different phase cells, such as megakaryoblast, promegakaryocyte and granular megakaryocyte, occurred after 10 days'culture. Clone forming unit-megakarocytes (CFU-MK) assay showed that the colonies count increased with the prolonged incubation. CFU-MK colonies were [1 236.0±32.9] on day 14, which was higher than that in medium without induction (P<0.01). Platelets from megakaryocytes showed agglutination function after 10 days'culture.

CONCLUSION1.5% HES was the best solution to precipitate erythrocytes. The combination of an imported serum-free medium with IL-3, IL-6, SCF and TPO showed better induction efficiency than domestic medium or other cytokine cocktails. Meanwhile, induced megakaryocytes produced functional platelets.

Cell Culture Techniques ; Cell Differentiation ; Cell Division ; Cell Separation ; methods ; Cells, Cultured ; Culture Media, Serum-Free ; Fetal Blood ; cytology ; Humans ; Megakaryocyte Progenitor Cells ; cytology