Effect of dura mater on enhancement of cranial osteogenesis in rats / 中国组织工程研究

BACKGROUND:The dura mater and skull are physically and functionally related,although there have been few investigations on primary extraction of dura mater and cranial cells,as well as co-culture of the two.The use of primary cells to investigate the influence of the dura mater on the skull is novel,and it is hoped that it may give a theoretical foundation for therapeutic therapy. OBJECTIVE:Rat dura mater and cranial bone cells were retrieved in situ to observe the influence of dura mater on cranial bone proliferation and differentiation,as well as to get a basic knowledge of the involvement of Twist1 in this process. METHODS:The enzyme digestion method was used in conjunction with the tissue block method to extract dural cells and cranial osteoblasts from rats within three days of birth.Immunofluorescence staining was used to identify the extracted cells,and alizarin red staining was used to identify and evaluate cranial osteoblasts and their mineralization ability.After co-culturing dural cells and cranial osteoblasts,real-time PCR was utilized to identify the expression of genes associated to cranial osteoblast proliferation and osteogenesis,as well as Twist1. RESULTS AND CONCLUSION:(1)Morphology:The retrieved dural cells had morphological traits similar to fibroblasts,while the osteoblasts were spindle-shaped.(2)Cell identification:immunofluorescence staining revealed that extracted dural cells expressed high levels of vimentin and cranial osteoblasts expressed high levels of alkaline phosphatase;cranial osteoblasts were stained with alizarin red 28 days after osteogenic induction,and obvious mineralized nodules were observed.(3)Real time PCR detection showed that the co-culture group had higher levels of PCNA,alkaline phosphatase,and RUNX2 mRNA expression than the control group(P＜0.01);however,Twist1 mRNA expression was lower(P＜0.01).(4)The findings showed that the primary extracted cranial osteoblasts had a high mineralization capacity,and that the dura mater was a key factor in promoting cranial growth and development and osteogenic differentiation,with Twist1 playing a key role in this process.

Comparison and optimization of isolation and extraction methods for Kupffer and hepatic stellate cells from mouse liver / 中国比较医学杂志

Objective To explore the extraction and purification method of Kupffer and hepatic stellate cells from mouse liver and provide references and suggestions for the separation and extraction method ology of primary non-parenchymal cells from mouse liver.Methods After in vivo collagenase perfusion digestion,various reagents and method,such as Percoll and OptiPrep,were used to extract C57BL/6 mice Kupffer and hepatic stellate cells,and evaluate their purity by flow cytometry and immunofluorescence.Results The two-layer Percoll method to extract Kupffer cells and the two-layer OptiPrep method to extract hepatic stellate cells were feasible,and purity reached>90%.The cell yield was 1～2×107/liver,and the cell survival rate was>90%.After 48 hours of primary cell culture,the number of F4/80-positive Kupffer cells and α-SMA-positive hepatic stellate cells reached>90%.Conclusions The separation and extraction method of Kupffer and hepatic stellate cells from mouse liver are perfect,reliable,cost-effective,and reproducible.

Replication of Rocio virus in primary cultures of mouse neural cells

ABSTRACT Objective This study verified the replication efficiency of the Rocio virus in a primary culture of mouse neural cells. Methods Mixed primary cultures (neurons/glia) obtained from the brains of newborn isogenic BALB/c mice were inoculated with Rocio virus on the 7 th day of culture, and the development of cytopathogenic effects was monitored. The infection was confirmed via immunocytochemistry (anti-ROCV), while viral replication was quantified in infected primary cultures. The titration method used depended on the infection period. Results Rocio virus efficiently infected primary cultured neural cells, with the highest viral titer causing cytopathic changes was observed at 2 days post infection. The virus-infected primary culture survived for up to 7 days post infection, and viral load quantitation showed viral replication kinetics compatible with the cell death kinetics of cultures. Conclusion The findings of this study suggest that mouse neural cell primary cultures support Rocio virus replication and could be used as an alternative system for studying Flavivirus infection in the central nervous system.

Effects of excessive fluoride exposure on astrocytes and the expression of glial fibrillary acidic protein in vitro and in vivo / 中华地方病学杂志

Objective:To investigate the effects of excessive fluoride exposure on astrocytes and the expression of glial fibrillary acidic protein (GFAP), in vitro and in vivo. Methods:(1) In vivo experiment: 24 SPF SD rats, half male and half female, were randomly divided into control and fluoride exposed groups according to sex and body weight, 12 rats in each group. Rats were fed with < 1 mg/L and 50 mg/L sodium fluoride solution prepared by tap water for 6 months, respectively. The expression levels of GFAP protein in rat brain tissue were measured by immunofluorescence, immunohistochemistry and Western blotting. (2) In vitro experiment: adult (6-month-old) rat cortical astrocytes were extracted and cultured in primary culture (4 mmol/L sodium fluoride solution for 24 h), and the astrocytes were identified by immunofluorescence, and GFAP mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR and Western blotting, respectively, and astrocytes apoptosis and calcium ion content were detected by flow cytometry. Results:(1) In vivo experiment: the results of immunofluorescence, immunohistochemistry and Western blotting showed that the GFAP protein expression level in brain tissue of rats exposed to fluoride was higher than that of control group (0.440 ± 0.200 vs 0.250 ± 0.120, t =-5.93, P = 0.027; 0.270 ± 0.020 vs 0.240 ± 0.050, t =-4.87, P = 0.040; 1.017 ± 0.001 vs 0.486 ± 0.006, t =-52.48, P = 0.001). (2) In vitro experiment: GFAP positive cells were identified as astrocytes by immunofluorescence; GFAP mRNA expression level was higher in fluoride exposed group than that of control group by real-time fluorescence quantitative PCR (2.780 ± 0.120 vs 0.134 ± 0.005, t =-37.84, P = 0.001). The Western blotting results showed that the GFAP protein expression level was higher in fluoride exposed group than that of control group (2.76 ± 0.10 vs 1.38 ± 0.05, t =-20.44, P = 0.002). Flow cytometry results showed that the apoptosis rate of astrocytes was higher in fluoride exposed group than that of control group (%: 55.0 ± 1.0 vs 3.5 ± 0.6, t =-10.28, P = 0.009) and the calcium ion content was lower than that of control group (%: 54 ± 9 vs 72 ± 13, t = 4.64, P = 0.043). Conclusion:Excessive fluoride exposure causes increased GFAP expression in astrocytes in vitro and in vivo, promotes apoptosis, and affects calcium signaling pathways.

In vitro culture of melanocytes from segmental vitiligo-like nevus depigmentosus lesions and its clinical significance / 中华皮肤科杂志

Objective:To assess the value of culture of epidermal melanocytes from negative-pressure suction blisters in the auxiliary diagnosis of segmental vitiligo-like nevus depigmentosus.Methods:Between June 2019 and March 2020, 8 patients with segmental vitiligo-like nevus depigmentosus, who met the Coupe′s clinical diagnostic criteria, were enrolled from Department of Dermatology, Hangzhou Third People′s Hospital. All patients were evaluated by the Wood′s lamp, reflectance confocal microscopy (RCM) , 308-nm excimer laser radiation, and in vitro culture of epidermal melanocytes from negative-pressure suction blisters. Results:Among the 8 patients, fluorescence was observed in 6 under the Wood′s lamp, dermal papillary rings were incomplete or absent in 4 as shown by RCM, and 5 experienced no repigmentation after 308-nm excimer laser radiation. Among the 8 patients, in vitro cultured lesional melanocytes were all positive for ferrous sulfate staining, yellowish-white precipitates were obtained after digestion and centrifugation of the melanocytes, and stage Ⅰ-Ⅲ melanosomes were observed in the cytoplasm of melanocytes under the electron microscope; however, the precipitates were black in color after digestion and centrifugation of the melanocytes collected from the normal skin tissues at the contralateral anatomical site, and stageⅠ-Ⅳ melanosomes were seen in the cytoplasm of the melanocytes under the electron microscope. Conclusion:Culture of epidermal melanocytes from negative-pressure suction blisters may facilitate the diagnosis of segmental vitiligo-like nevus depigmentosus.

Cultivation and characterization of primary human parathyroid cells from patients with severe secondary hyperparathyroidism / 南方医科大学学报

OBJECTIVE@#To establish an cell model of hyperparathyroidism by isolation, in vitro culture, and identification of parathyroid cells from patients with secondary hyperparathyroidism (SHPT).@*METHODS@#The parathyroid gland tissues obtained from 10 patients with SHPT were dissociated by collagenase digestion for primary culture of the parathyroid cells. Morphological changes and growth characteristics of the cells were assessed by microscopic imaging and cell counting. The mRNA and protein expression levels of parathyroid hormone (PTH), calcium-sensing receptor (CaSR), and glial cells missing 2 (GCM2) in the primary and passaged cells were determined by immunofluorescence, qRT-PCR, and Western blotting.@*RESULTS@#Primary cultures of parathyroid cells were successfully obtained. The cells exhibited a high expression of PTH shown by immunofluorescence assay and had a population doubling time of approximately 71.61 h. PTH secretion in the second-passage (P2) cells was significantly lower than that in the primary (P0) and first-passage (P1) cells (P < 0.001). Despite a significant downregulation of CaSR mRNA (P=0.017) and protein (P=0.006) in P1 cells as compared with P0 cells, no significant differences were found in mRNA and protein expressions of PTH or GCM2 between the two cell generations.@*CONCLUSION@#Primary cultures of parathyroid cells isolated from SHPT patients by collagenase digestion show similar biological properties to the cells in vivo.

Regulation effect of endoplasmic reticulum stress and unfolded protein response activation on skeletal muscle fiber immune behavior / 解剖学报

Objective To investigate the regulatory effects of activated endoplasmic reticulum stress（ERS） and unfolded protein response（UPR） on the immune behavior of the stressed muscle fibers in inflammatory environments induced by interferon-γ（IFN-γ）. Methods The myogenic precursor cells（MPCs） of C57 BL/6 mice cultured in vitro were differentiated into multinucleated myogenic tubes by horse serum and then to set up: 1. Control group; 2. IFN-γ group; 3. Tunicamycin group; 4. Thapsigargin group; 5. IFN-γ and 4-phenylbutyrate（4-PBA） combined treatment group; 6. IFN-γ, TG and 4-PBA combined treatment group; 7. IFN-γ and 4μ8 c combined treatment group; 8. IFN-γ, TG and 4μ8 c combined treatment group; 9. IFN-γ and GSK2606414 combined treatment group; 10. IFN-γ, TG and GSK2606414 combined treatment group. The level of myokines gene was detected by Real-time PCR. The expression of UPR key molecules including eukaryotic intiatio factor 2α（eIF2α）, inositol requrring enzyme 1α（IRE1α） and activating transcription factor 6（ATF6） in muscle fibers was observed by immunofluorescence. Western blotting was used to detect immune molecules related to muscle cells, myokines and key molecules of UPR. Luminex analyzed the levels of pro-inflammatory myokines in muscle fibers. Results The expression of H-2 Kb, H2-Ea, Toll like receptor 3（TLR3）, p-eIF2α and p-IRE1α were up-regulated in IFN-γ induced inflammatory environment. The expression of H-2 Kb, H2-Ea, TLR3 and myokines in the group with UPR inhibitor 4-PBA was down-regulated compared with IFN-γ group, and the expression of these molecules in the group with IRE1α specific inhibitor 4μ8 c was down-regulated compared with the IFN-γ group. The addition of protein kinase R-like endoplasmic eticulum（PERK） specific inhibitor GSK2606414 showed no significant change. Conclusion In IFN-γ induced inflammatory environment, the UPR-IRE1α pathway activates and inhibits the synthesis of muscle fiber immune-related molecules, which further inhibits the muscle fiber mediated immune response and facilitates muscle regeneration.

Comparison of primary keloid fibroblast cultivation methods and the characteristics of fibroblasts cultured from keloids, keloid-surrounding tissues, and normal skin tissues / Comparación de los métodos de cultivo de fibroblastos queloides primarios y las características de los fibroblastos cultivados a partir de queloides, tejidos circundantes queloides y tejidos cutáneos normales

SUMMARY: The establishment of primary keloid fibroblast culture has always been a fundamental measure for studying mechanisms of keloid disease. The quality of the primary cell culture can directly affect the results of further experiments. This study was performed to investigate the optimal growth conditions, including the optimal storage time and collagenase treatment time, for in vitro cell culture models and the suitable methods for epidermis-dermis separation in different tissues. Keloid tissues, keloid-surrounding tissues, and normal skin tissues were collected from patients, for primary fibroblast culture. Two methods, tissue explant and collagenase digestion, were deployed and compared. Expression levels of the keloid-related genes α -SMA, Col1, and Col3 were assessed in cells cultured using both methods, to verify the qualities of the primary cells. A comparative analysis was conducted between the two methods and among the three different tissues used. Bacterial and lipid contamination was immediately minimized after the samples were processed. Different methods of epidermis removal and different durations of collagenase digestion were required in different tissues to generate optimal results. Real-time PCR results showed that the mRNA expression levels of keloid-related genes in cultured fibroblasts correlated to their in vivo expression profile, as previously reported in other studies. The results of this study have revealed several key points in the culture of primary keloid fibroblasts and demonstrated the correlation in gene expression between in vivo keloid fibroblasts and in vitro primary keloid fibroblasts.

RESUMEN: La identificación de un cultivo de fibroblastos queloides primarios, siempre ha sido una medida fundamental para estudiar los mecanismos de la enfermedad queloide. La calidad del cultivo de células primarias puede afectar directamente los resultados de otros experimentos. Este estudio se realizó para investigar las condiciones óptimas de crecimiento, incluido el tiempo óptimo de almacenamiento y el tiempo de tratamiento con colagenasa, para modelos de cultivo celular in vitro y los métodos adecuados para la separación epidermis-dermis en diferentes tejidos. Se recogieron de los pacientes tejidos queloides, tejidos circundantes queloides y tejidos cutáneos normales, para cultivo primario de fibroblastos. Se implementaron y compararon dos métodos, explante de tejido y digestión con colagenasa. Los niveles de expresión de los genes relacionados con queloides α -SMA, Col1 y Col3 se evaluaron en células cultivadas usando ambos métodos, para verificar las cualidades de las células primarias. Se realizó un análisis comparativo entre los dos métodos y entre los tres tejidos diferentes utilizados. La contaminación de bacterias y lípidos se minimizó inmediatamente después de que se procesaron las muestras. Se requirieron varios métodos de eliminación de la epidermis y diferentes tiempos de digestión con colagenasa en los tejidos para generar resultados óptimos. Los resultados de la PCR en tiempo real mostraron que los niveles de expresión de ARNm de genes relacionados con queloides en fibroblastos cultivados se correlacionaban con su perfil de expresión in vivo, como se informó en estudios anteriores. Los resultados de este studio indicaron varios puntos clave en el cultivo de fibroblastos queloides primarios y han demostrado la correlación en la expresión génica entre fibroblastos queloides in vivo y fibroblastos queloides primarios in vitro.

Procedimiento para el cultivo e identificación de células madre obtenidas de lipoaspirado humano con fines de investigación / Procedure for culture and identification of stem cells from human lipoaspirate for research purposes

RESUMEN Las células madre humanas nacen con la creación de la vida misma y algunas de estas permanecen durante toda la vida. Por consiguiente, se pueden hallar en tejidos adultos y utilizarlas para investigaciones a nivel básico y aplicado. Actualmente, en nuestro país existe un creciente interés en el estudio y aplicación de células madre; sin embargo, existe poco conocimiento acerca del procedimiento para su identificación. Es por ello que este artículo tiene como objetivo dar a conocer, desde un punto de vista práctico, un procedimiento para el cultivo e identificación de células madre/estromales obtenidas de lipoaspirado humano (Adipose Stem Cells) con fines de investigación, el cual incluye la caracterización a nivel de inmunofenotipo, el potencial de diferenciación celular, la expresión génica y el control de calidad del cultivo celular, que sirva de apoyo para los profesionales de la comunidad científica peruana que deseen desarrollar esta línea de investigación.

ABSTRACT Human stem cells are born with the creation of life itself and some of them remain throughout life. Therefore, they can be found in adult tissues and used for basic and applied research. Currently, in our country there is a growing interest in the study and application of stem cells; however, little is known about the identification procedure. For this reason, this study aims to present, from a practical point of view, a procedure for the culture and identification of stem/stromal cells obtained from human lipoaspirate (Adipose Stem Cells), for research purposes. This procedure includes the immunophenotype characterization, cell differentiation potential, gene expression and cell culture quality control; and will serve as support for Peruvian scientific community professionals who wish to develop this line of research.

Effects of different culture systems on the culture of prepuberal buffalo (Bubalus bubalis) spermatogonial stem cell-like cells in vitro

AHVAC- inhibits the proliferation in human primary gastric cancer cells / 中国临床药理学与治疗学

AIM: To explore the effect of inhibiting proliferation and inducing apoptosis in human primary gastric tumor by the Agkis-trodon halys venom anti-tumor component (AHVAC-). METHODS: Human primary gastric cancer cells were isolated by trypsin digestion, serum-free culture, and purified by differential adherence method, and cells were identified by immunohistochemistry. Cell proliferation and toxicity assay (CCK-8) was used to detect the inhibition rate of AHVAC- in different concentrations of primary gastric cancer cells. Immunohistochemistry was used to verify the apoptosis of human primary gastric cancer cells induced by AHVAC- and the morphological changes were observed by Hematoxylin-Eosin staining (HE staining). AHVAC--induced primary gastric cancer cell transformation rate was detected by flow cytometry Annexin V/PI double staining.RESULTS: Seven human primary gastric cancer cells were successfully isolated and purified, and 11 cases failed. Immunohistochemical identifications of carcinoembryonic antigen (CEA) and broad-spectrum keratin protein (AE1/AE3) were positive for both antibodies. AHVAC- inhibited the proliferation of human primary gastric cancer cells and showed a dose-dependent effect (P<0.01). Immunohistochemistry showed that the expression level of cysteine aspartic protease-3 (Caspase-3) up-regulated with the increase of AHVAC- concentration. HE staining showed that with the increase of AHVAC- concentration, the cell gap increased, nuclear pyknosis, and apoptosis cells increased. Flow cytometry showed that the apoptosis rate of human primary gastric cancer cells up-regulated with the increase of AHVAC- concentration (P<0.05). CONCLUSION: AHVAC- can inhibit the proliferation and induce apoptosis in human primary gastric cancer cells in a dose-dependent manner.

Interaction of Biomphalaria cells in primary cultures with Schistosoma mansoni sporocysts

Abstract INTRODUCTION: Biomphalaria snails may display varying levels of susceptibility to Schistosoma mansoni infection. We have been developing an in vitro model to study the interaction between the snail and the parasite, using tissue-derived cell cultures from Biomphalaria. METHODS: The digestive gland- and kidney-derived cells from primary cultures of resistant (B. tenagophila Taim) and susceptible (B. tenagophila HM and B. glabrata BH) strains of Biomphalaria were exposed to S. mansoni sporocysts. RESULTS: S. mansoni sporocysts were surrounded and encapsulated exclusively by cells derived from the digestive gland (DG) of B. tenagophila Taim. The process was followed by a marked decrease in the number of free sporocysts in the culture medium. The morphological characteristics of DG-derived cells in culture have been described. CONCLUSIONS: Cells derived from DG (but not SK) primary cultures of B. tenagophila Taim may participate in S. mansoni sporocyst control.

Collection and culture of human connective tissue cells from gingival explant technique for oral tissue bioengineering / Colección de células de tejido conectivo humano a partir de la técnica de explante gingival para bioingeniería de tejidos orales

SUMMARY: Cell culture is an important tool in medical, odontological and biological research laboratories, supporting cell therapies and tissue bioengineering strategies. Gingival fibroblasts present structural function, being able to modulate their metabolic capacity, which is reflected in the tissue morphology. The possibility of culturing fibroblasts in vitro, in monolayer or on three-dimensional scaffolds, for subsequent transplants in vivo opens important perspectives for the periodontal surgical clinic. The objective of the present article is to present a method of obtaining and cultivating viable human gingival fibroblasts for in vitro research. Explants derived from periodontal surgical discards were used, grown in 25 cm2 bottles to obtain a primary cell culture. After observing the proliferation and growth of the fibroblasts that interconnected and formed a monolayer network, involving the periphery of the explants, it was possible to remove the explants, to make the passage and the new subcultures were obtained in a ratio of 1:1. After 7 days, the amount of viable cells was analyzed in triplicate, using the Neubauer chamber technique, in cell culture bottles of 25 mm2 (T25) and 75 mm2 (T75). Fibroblasts were described and subclassified morphologically. The results showed a growth pattern in both bottles, but with a larger number in bottles of 75 cm2. Cells with fibroblastic morphology were subclassified into reticular and fusiform, being predominant those with fusiform morphology. In conclusion, culture of explant of human gingival connective tissue is a viable method for obtaining gingival connective tissue cells suitable for laboratory tests in cell culture, aiming at obtaining constructs for gingival tissue engineering.

RESUMEN: El cultivo celular es una herramienta importante en los laboratorios de investigación médica, odontológica y biológica, que apoyan las terapias celulares y las estrategias de bioingeniería de tejidos. Los fibroblastos gingivales presentan una función estructural, pudiendo modular su capacidad metabólica, que se refleja en la morfología tisular. La posibilidad de cultivar fibroblastos in vitro, en monocapa o en andamios tridimensionales, para trasplantes posteriores in vivo abre perspectivas importantes para la clínica de cirugía periodontal. El objetivo del presente artículo es presentar un método para obtener y cultivar fibroblastos gingivales humanos viables para investigación in vitro. Se utilizaron explantes derivados de los descartes quirúrgicos periodontales, crecidos en frascos de 25 cm2 para obtener un cultivo de células primarias. Después de observar la proliferación y el crecimiento de los fibroblastos que se interconectaron y formaron una red de monocapa, que involucraba la periferia de los explantes, fue posible eliminar los explantes, hacer el pasaje y los nuevos subcultivos se obtuvieron en una proporción de 1:1. Después de 7 días, la cantidad de células viables se analizó por triplicado, utilizando la técnica de cámara de Neubauer, en botellas de cultivo celular de 25 mm2 (T25) y 75 mm2 (T75). Los fibroblastos fueron descritos y sub-clasificados morfológicamente. Los resultados mostraron un patrón de crecimiento en ambas botellas, pero con un número mayor en botellas de 75 cm2. Las células con morfología fibroblástica se subclasificaron en reticulares y fusiformes, predominando aquellas con morfología fusiforme. En conclusión, el cultivo de explante de tejido conectivo gingival humano es un método viable para obtener células de tejido conectivo gingival adecuadas para pruebas de laboratorio en cultivos celulares, con el objetivo de obtener construcciones para la ingeniería del tejido gingival.

Establishment of a culture method for primary human nail matrix cells in serum-free media / 中华皮肤科杂志

Objective To establish a culture method for primary human nail matrix cells in serumfree media.Methods Nail matrix tissues were collected from 9 patients,who received nail or toe amputation and nail bed repair in Peking University Shenzhen Hospital between January 2016 and December 2016,and cultured in the serum-free DEME/F-12 media at a 37℃ incubator with an atmosphere of 5％ CO2 in air for 2-3 days.Then,primary human nail matrix cells were cultured in keratinocyte serumfree media (CnT-07),and the morphology of human nail matrix cells was observed by microscopy during the culture process.Immunofluorescence cytochemistry with anti-keratin 5 (K5) and K10 was performed to identify the acquired cells,and flow cytometry to analyze the cell purity.Results After 2 or 3 days of the culture,some cells began to crawl out from the tissue.On day 10,large cell masses were formed,some cells were morphologically similar to epithelioid cells arranged in a paving stone-like pattern,and some were flat giving a spindle-shaped or star-shaped appearance.Immunofluorescence cytochemistry showed that some cells could express both K5 and K10,which proved the existence of nail matrix cells,and 37.6％ of the cells expressed K10.Conclusion Human primary nail matrix cells could be successfully cultured by using the tissue culture method with serum-free culture media,and the nail matrix cells cultured in vitro can express both K5 and K10.

Guanabenz Acetate Induces Endoplasmic Reticulum Stress–Related Cell Death in Hepatocellular Carcinoma Cells

BACKGROUND: Development of chemotherapeutics for the treatment of advanced hepatocellular carcinoma (HCC) has been lagging. Screening of candidate therapeutic agents by using patient-derived preclinical models may facilitate drug discovery for HCC patients. METHODS: Four primary cultured HCC cells from surgically resected tumor tissues and six HCC cell lines were used for high-throughput screening of 252 drugs from the Prestwick Chemical Library. The efficacy and mechanisms of action of the candidate anti-cancer drug were analyzed via cell viability, cell cycle assays, and western blotting. RESULTS: Guanabenz acetate, which has been used as an antihypertensive drug, was screened as a candidate anti-cancer agent for HCC through a drug sensitivity assay by using the primary cultured HCC cells and HCC cell lines. Guanabenz acetate reduced HCC cell viability through apoptosis and autophagy. This occurred via inhibition of growth arrest and DNA damage-inducible protein 34, increased phosphorylation of eukaryotic initiation factor 2α, increased activating transcription factor 4, and cell cycle arrest. CONCLUSIONS: Guanabenz acetate induces endoplasmic reticulum stress–related cell death in HCC and may be repositioned as an anti-cancer therapeutic agent for HCC patients.

Establishment and genomic characterization of a novel retinoblastoma cell line without RB1 mutationderived from a Han Chinese donor / 上海交通大学学报（医学版）

Objective: To develop and characterize a novel retinoblastoma cell line from a Han Chinese patient. Methods: Cells were dispersed from tumor tissue harvested from an enucleated eyeball harbouring retinoblastoma under sterile conditions. The dissociated cells were cultured, purified and passaged in vitro. Morphologic and genetic analysis and detection of surface biomarkers were performed on the cell line and primary tumor by immunofluorescence, immunohistochemical staining, DNA short tandem repeat (STR) analysis, karyotype analysis, and exome sequencing. Results: This human retinoblastoma cell line was designated as SNPH-Rb-C24. It expressed neural cell adhesion molecule 1 and synaptophysin, which confirmed its neuronal derivation. DNA STR analysis showed an identical match between SNPH-Rb-C24 and primary tumor. Karyotype analysis showed complex chromosomal abnormalities in SNPH-Rb-C24, while no alteration in 13q was observed. Comparative exome sequencing identified common mutated genes and RB1+/+ in both SNPH-Rb-C24 and primary tumor. Orthotopic xenograft tumors derived from early passage cells were established. Conclusion: A human retinoblastoma cell line (SNPH-Rb-C24) derived from a Han Chinese patient with RB1+/+ retinoblastoma is developed, which retains critical biological and genomic features of the donor tumor.

A simplified method for isolating murine primary pancreatic acinar cells / 中华胰腺病杂志

Objective To explore a simplified and economical method to isolate the murine primary pancreatic acinar cells.Methods The collagenase and trypsin inhibitor dissolving in DMEM solution were used to digest the murine pancreas,and 4％ BSA dissolving in DMEM solution was used to purify and isolate primary pancreatic acinar cells from pancreas.CCK-8 method was applied to check the ability of pancreatic acinar cells to secret amylase.Results After digestion,shaking in the water bath,resuspension,filtration and precipitation,murine primary pancreatic acinar cells could be obtained within 2 hours.Pancreatic acinar cells in good conditions appeared in clusters,and their basolateral domains were round and devoid of blebs,and the cytoplasm appeared clear.Their apical domain were surrounded by hundreds of zymogen granules which looked darker.The nucleus was located in the basal area of the vesicular region.The basal level of amylase release as a percent of total release from pancreatic acinar cells was around 2.5％ in CCK8-unstimulated group.This rate started to increase after CCK-8 stimulation and reached its peak [(12.83 ± 1.04) ％] at a concentration of 50 pmol/L of CCK-8,but the ratio of the amylase level secreted by the pancreatic acinar cells to the total amylase level displayed a decreasing trend with the increase of CCK-8 concentration.Conclusions This optimized method had the advantage of being fast and simple,low technical difficulty and good repetition.It was a new simplified and cheap method for isolating murine pancreatic acinar cells.

Modified primary culture method of mouse olfactory sensory neurons and its identification / 重庆医学

Objective To establish a convenient method for primary culture of olfactory sensory neurons(OSNs).Methods The C57BL/6 mouse embryos olfactory mucosa in the mouse with gestation of 15? 20 d was collected.Then the OSNs primary culture was conducted by adopting the routine and modified separation procedure.The cellular morphology was observed on 1,3,5,7,9 d after plating by using inverted microscope.The OSNs specific β-tubulin Ⅲ antigen and olfactory marker protein(OMP) in the cultured cells were identified by using the routine immunofluorescence method.Results According to modified experimental process,partial survival cells demonstrated the morphology of typical bipolar neuron,the cells on 5 d were verified as OSNs by β-tubulin Ⅲ and OMP immunofluorescence labeling.By adopting the common process,the fibrocytes began to grow on day 3 and gradually covered the culture dish.Conclusion This research successfully establishes a relatively simple and convenient method for primary culture of OSNs.

Effect of aminolevulinic acid-based photodynamic therapy on the expression of protein kinase D1 and its phosphorylation sites in a cutaneous squamous cell carcinoma cell line A431 / 中华皮肤科杂志

Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the expression of protein kinase D1 (PKD1) in a cutaneous squamous cell carcinoma cell line A431,and to explore the mechanism underlying ALA-PDT-induced apoptosis of A431 ceils.Methods A431 cells were cultured in vitro,and cell counting kit-8 (CCK-8) assay was performed to select the optimal combination of ALA concentration and PDT dose with the strongest proliferation inhibitory effect.A431 ceils at exponential growth phase were randomly divided into 4 groups:control group receiving no treatment,ALA group treated with ALA solution alone,PDT group treated with PDT alone,and ALA-PDT group treated firstly with ALA solution and then with PDT.After 12-,24-,36-and 48-hour additional culture,CCK-8 assay was conducted to evaluate the cellular proliferation inhibition,and the apoptosis rate at the time point of the strongest proliferation inhibitory effect was measured by flow cytometry.RT-PCR was performed to determine the expression of protein kinase D1 gene (PRKD1) in A431 cells at different time points after the ALA-PDT treatment,and Western blot analysis to measure protein expression of PKD 1 and its phosphorylation at Tyr463 (pTyr463) and Ser916 (pSer916) in A431 cells.Results The combination of ALA at the concentration of 1.5 mmol/L with PDT at an irradiation dose of 2 J/cm2 was optimal due to its strongest proliferation inhibitory effect.After 12-,24-,36-and 48-hour additional culture,there were significant differences in the proliferation inhibition rate among the 4 groups (F =39.56,P ＜ 0.05).At 24 hours after the treatment,the ALA-PDT group showed significantly higher proliferation inhibition rate (46.26％ ± 1.25％) compared with the ALA group (14.65％ ± 0.33％,P ＜ 0.05),PDT group (14.96％ ± 0.68％,P ＜ 0.05) and control group (11.98％ ± 0.32％,P ＜ 0.05),as well as compared with that at 12 hours (P ＜ 0.05).At 24 hours after the treatment,the apoptosis rate significantly differed among the 4 groups (F =16.32,P ＜ 0.05),and the ALA-PDT group showed a significantly higher apoptosis rate (41.92％ ± 3.23％) compared with the control group (4.67％ ± 0.88％,P ＜ 0.05),ALA group (7.02％ ± 1.52％,P ＜ 0.05) and PDT group (8.37％ ± 0.59％,P ＜ 0.05).At 0,6,12,24,36 and 48 hours after the treatment,there were significant differences in the mRNA expression of PRKD 1 among the 4 groups (F =22.24,P ＜ 0.05),and the mRNA expression of PRKD1 at 24 hours was significantly lower than that at 0,6,12 hours (all P ＜ 0.05),but was not significantly different from that at 36 and 48 hours (both P ＞ 0.05).No significant difference in the Ser916-phosphorylated PKD1 expression was found among the 4 groups (F =1.53,P ＞ 0.05),while there were significant differences in the expression of PKD1 and Tyr463-phosphorylated PKD 1 among the 4 groups (F =10.04,8.27,both P ＜ 0.05).Additionally,the ALA-PDT group showed significantly lower expression of PKD 1 and Tyr463-phosphorylated PKD 1 compared with the control group,ALA group and PDT group (all P ＜ 0.05).Conclusion PKD1 may be involved in the photochemical process of A431 cell apoptosis induced by ALA-PDT,and may promote the occurrence of squamous cell carcinoma by Tyr463 phosphorylation.

Isolation and culture of mouse bone marrow mesenchymal stem cells and induced differentiation into alveolar epithelial cells / 医学研究生学报

Objective It has traditionally been difficult to isolate and culture mouse bone marrow mesenchymal stem cells (BMSC),which has low success rate.And thus restricts the development of related research to some extent.We aimed to optimize the whole bone marrow adherent method for isolation and culture of mouse bone marrow mesenchymal stem cells and search for an effective method of inducing BMSCs to differentiate into alveolar epithelial cells.Methods Bone marrow contents harvested from the tibia and femur of C57BL/6 mice were cultured based on the whole bone marrow adherent method.The timing and split ratios of passage were determined according to the size and number of cell colonies.After 6 passages,cells were counted to detect cell proliferation ability,surface markers were examined by flow cytometry and Small Airway Epithelial Cell Medium (SAEpiCM) was used to induce the differentiation of BMSCs.Results With the increase of passages and the purity of BMSCs,the proliferation of cells at passages 6 tended to be stable.Flow cytometry showed that they were strongly positive for bone marrow mesenchymal stem cell surface markers CD29 and Sca-1 (99.1％,88.5％),but almost negative for the surface marker of hematopoietic stem cells CD117 (0.008 2％).BMSCs cultured in SA-EpiCM showed an epithelium-like morphological change and expressed surfactant associated protein C,a specific marker of alveolar epithelial cells.Conclusion It is effective to isolate and culture mouse bone marrow mesenchymal stem cells by adjusting the timing and split ratios of passage according to the size and number of the clonal cell colonies,which possessed the potential to differentiate into alveolar epithelial cells.