Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, esta...Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, establishment of a suspension cell line, and isolation of protoplasts from cell suspensions. Murashige and Skoog (MS) medium was used for callus induction from mature seeds of N. sibirica. Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mg L-1 6-benzylaminopurine (6-BA) and 1.0 mg L-1 2,4-dichlorophenoxy (2,4-D) acetic acid. Suspension cultures of N. sibirica were initiated by transferring friable calli to the same liquid multiplication medium. Characterization of the suspension culture was assessed based on fresh mass, dry mass, cell viability and pH value of the culture. A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium, including a lag phase, an exponential growth phase, a stationary phase, and a negative acceleration phase. The effect of factors such as pre-plasmolysis, enzyme combination, enzymolysis time and mannitol concentration, on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures. The maximum yield (9.79 x 10(6) cells/g) and highest viability (79.97%) of protoplast were reached when approximately 1 g of cell suspension (cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 mol L-1 mannitol mixture solution, cellulose onozuka R-10 2% (w/v), hemicellulose 0.2%, macerozyme R-10 1%, and pectolyase Y-23 0.5%. Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10 (P < 0.05).展开更多
Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A ...Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of 17.5 ℃) in cell suspension at 45 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of 12.5 ℃ in nonacclimated cells to LT50 of 17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P. euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.展开更多
Orthopedic conditions have emerged as global health concerns,impacting approximately 1.7 billion individuals worldwide.However,the limited understanding of the underlying pathological processes at the cellular and mol...Orthopedic conditions have emerged as global health concerns,impacting approximately 1.7 billion individuals worldwide.However,the limited understanding of the underlying pathological processes at the cellular and molecular level has hindered the development of comprehensive treatment options for these disorders.The advent of single-cell RNA sequencing(scRNA-seq)technology has revolutionized biomedical research by enabling detailed examination of cellular and molecular diversity.Nevertheless,investigating mechanisms at the single-cell level in highly mineralized skeletal tissue poses technical challenges.In this comprehensive review,we present a streamlined approach to obtaining high-quality single cells from skeletal tissue and provide an overview of existing scRNA-seq technologies employed in skeletal studies along with practical bioinformatic analysis pipelines.By utilizing these methodologies,crucial insights into the developmental dynamics,maintenance of homeostasis,and pathological processes involved in spine,joint,bone,muscle,and tendon disorders have been uncovered.Specifically focusing on the joint diseases of degenerative disc disease,osteoarthritis,and rheumatoid arthritis using scRNA-seq has provided novel insights and a more nuanced comprehension.These findings have paved the way for discovering novel therapeutic targets that offer potential benefits to patients suffering from diverse skeletal disorders.展开更多
为建立鸡新城疫病毒La Sota株在LMH细胞上的全悬浮培养工艺,以获得高滴度的新城疫疫苗抗原,采用LMH贴壁细胞悬浮培养驯化方法,获得了形态良好、能稳定传代的LMH悬浮细胞株;以LMH悬浮细胞在摇瓶培养NDV La Sota株为基础,对接毒细胞密度...为建立鸡新城疫病毒La Sota株在LMH细胞上的全悬浮培养工艺,以获得高滴度的新城疫疫苗抗原,采用LMH贴壁细胞悬浮培养驯化方法,获得了形态良好、能稳定传代的LMH悬浮细胞株;以LMH悬浮细胞在摇瓶培养NDV La Sota株为基础,对接毒细胞密度、接毒剂量、胰酶添加浓度、收获时间等工艺参数进行了摸索和优化,并在14 L生物反应器中进一步进行3个批次的培养验证。结果显示:LMH悬浮细胞以初始密度1.5×10^(6)/mL左右接种,培养72 h可增殖到8.0×10^(6)~9.0×10^(6)/mL,细胞活率达97%以上。确定NDV La Sota株在LMH悬浮细胞株上的培养工艺:LMH细胞悬浮培养至不低于4.5×10^(6)/mL按照感染复数不低于0.2接种病毒,并添加终浓度为5μg/mL的胰酶,于37℃培养72 h左右,细胞活率在30%左右收获病毒液,NDV HA均可达1∶2048~1∶4096,病毒含量≥10^(7.63)EID50/0.1 mL。结果表明成功建立了LMH细胞全悬浮培养工艺,并能在生物反应器扩大培养,为ND相关疫苗研发提供了技术支撑。展开更多
基金supported by the National Science&Technology Pillar Program during the 12th Five-year Plan Period(2011AA10020102)
文摘Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, establishment of a suspension cell line, and isolation of protoplasts from cell suspensions. Murashige and Skoog (MS) medium was used for callus induction from mature seeds of N. sibirica. Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mg L-1 6-benzylaminopurine (6-BA) and 1.0 mg L-1 2,4-dichlorophenoxy (2,4-D) acetic acid. Suspension cultures of N. sibirica were initiated by transferring friable calli to the same liquid multiplication medium. Characterization of the suspension culture was assessed based on fresh mass, dry mass, cell viability and pH value of the culture. A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium, including a lag phase, an exponential growth phase, a stationary phase, and a negative acceleration phase. The effect of factors such as pre-plasmolysis, enzyme combination, enzymolysis time and mannitol concentration, on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures. The maximum yield (9.79 x 10(6) cells/g) and highest viability (79.97%) of protoplast were reached when approximately 1 g of cell suspension (cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 mol L-1 mannitol mixture solution, cellulose onozuka R-10 2% (w/v), hemicellulose 0.2%, macerozyme R-10 1%, and pectolyase Y-23 0.5%. Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10 (P < 0.05).
基金the National Natural Science Foundation of China (Grant No. 30271067)Fok Ying Tung Education Foundation (71030)+1 种基金 Key Teachers Foundation of the Educational Ministry of China and the State Key Basic Research and Development Plan of China (G199901600
文摘Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of 17.5 ℃) in cell suspension at 45 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of 12.5 ℃ in nonacclimated cells to LT50 of 17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P. euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.
基金National Key Research and Development Program of China(2022YFA1103202)National Natural Science Foundation of China(82272507,32270887,and 32200654)+6 种基金Natural Science Foundation of Chongqing(CSTB2023NSCQ-ZDJO008)Postdoctoral Innovative Talent Support Program(BX20220397)Independent Research Project of State Key Laboratory of Trauma and Chemical Poisoning(SFLKF202201)Project for Enhancing Innovation of Army Medical University(2023X1839)Talent Innovation Training Program at the Army Medical Center(ZXZYTSYS09)General Hospital of Western Theater Command Research Project(2021-XZYG-B10)University Grants Committee,Research Grants Council of Hong Kong,China(14113723,N_CUHK472/22,C7030-18G,T13-402/17-N,and AoE/M-402/20)。
文摘Orthopedic conditions have emerged as global health concerns,impacting approximately 1.7 billion individuals worldwide.However,the limited understanding of the underlying pathological processes at the cellular and molecular level has hindered the development of comprehensive treatment options for these disorders.The advent of single-cell RNA sequencing(scRNA-seq)technology has revolutionized biomedical research by enabling detailed examination of cellular and molecular diversity.Nevertheless,investigating mechanisms at the single-cell level in highly mineralized skeletal tissue poses technical challenges.In this comprehensive review,we present a streamlined approach to obtaining high-quality single cells from skeletal tissue and provide an overview of existing scRNA-seq technologies employed in skeletal studies along with practical bioinformatic analysis pipelines.By utilizing these methodologies,crucial insights into the developmental dynamics,maintenance of homeostasis,and pathological processes involved in spine,joint,bone,muscle,and tendon disorders have been uncovered.Specifically focusing on the joint diseases of degenerative disc disease,osteoarthritis,and rheumatoid arthritis using scRNA-seq has provided novel insights and a more nuanced comprehension.These findings have paved the way for discovering novel therapeutic targets that offer potential benefits to patients suffering from diverse skeletal disorders.
