Isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma

AIM： To explore the method of isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma cell line PANC-1. METHODS： The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 （1：1 volume） （DMEM-F12） supplemented with 20% fetal bovine serum （FBS）. Subpopulation cells with properties of tumor stem cells were isolated from pancreatic adenocarcinoma cell line PANC-1 according to the cell surface markers CD44 and CD24 by flow cytometry. The proliferative capability of these cells in vitro were estimated by 3-[4,5-dimehyl-2-thiazolyl]-2, 5-diphenyl- 2H-tetrazolium bromide （MTT） method. And the tumor growth of different subpopulation cells which were injected into the hypodermisof right and left armpit of nude mice was studied, and expression of CD44 and CD24 of the CD44^＋CD24^＋ cell-formed nodules and PANC-1 cells were detected by avidin-biotin-peroxidase complex （ABC） immunohistochemical staining. RESULTS： The 5.1%-17.5% of sorted PANC-1 cells expressed the cell surface marker CD44, 57.8% -70.1% expressed CD24, only 2.1%-3.5% of cells were CD44^＋ CD24^＋. Compared with CD44-CD24- cells, CD44^＋CD24^＋ cells had a lower growth rate in vitro. Implantation of 104 CD44 CD24 cells in nude mice showed no evidenttumor growth at wk 12. In contrast, large tumors were found in nude mice implanted with 103 CD44^＋CD24^＋ cells at wk 4 （2/8）, a 20-fold increase in tumorigenic potential （P 〈 0.05 or P 〈 0.01）. There was no obvious histological difference between the cells of the CD44^＋CD24^＋ cell-formed nodules and PANC-1 cells. CONCLUSION： CD44 and CD24 may be used as the cell surface markers for isolation of pancreatic cancer stem cells from pancreatic adenocarcinoma cell line PANC-1. Subpopulation cells CD44^＋CD24^＋ have properties of tumor stem cells. Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy, it may be a very promising target for new dr     

Cost-Effective Isolation of a Process Impurity of Pregabalin

Cost-effective isolation methods were developed on preparative HPLC, flash LC, and simulated moving bed (SMB) to prepare the process impurity, 3-(aminomethyl)-5-methylhex-4-enoic acid (4-ene impurity), of pregabalin. By a thorough experimental study on the different isolation techniques available, it was concluded that SMB was the most cost-effective. Hence, it was a continuous chromatography that utilized the advantage of SMB so that a high quantity of the impurity was generated in a short period of time. SMB was equipped with eight reversed-phased columns and was used to separate the process impurity of pregabalin. The effects of flow rate in zone 2 (Q2) and 3 (Q3), as well as switching time, on the operating performance parameters like purity, productivity, and desorbent consumption were studied. Operating conditions leading to more than 90% purity in the raffinate outlet stream were identified, together with those achieving optimal performance. All of these developed methods are novel, cost-effective, and can be applied to the isolation of other process- and stability-related impurities of pregabalin.     

Isolation and characterization of beta 1-adrenergic receptors from adult, rat cardiac myocytes

The beta-receptors were isolated from rat cardiac myocytes and characterized. Isolated myocytes were prepared from adult rat hearts and characterized for viability. Membrane proteins were solubilized from myocytes with 1% Triton X-102. The solubilized membrane proteins were fractionated by DEAE-Sephacel ion exchange column chromatography. Two major protein peaks were obtained. The second protein peak sample was found to contain beta-receptors to which 125I-15-(4'-azido-3'-iodobenzyl)-carazolol (125I-ABC) was specifically bound. This sample was labeled covalently with 125I-ABC by UV irradiation. The radiolabeled sample was applied to a Sepharose CL-6B gel column. Two radiolabeled protein peaks, one with a molecular weight of approximately 570,000 and the other with a molecular weight of approximately 95,000 were found. When the 570,000-dalton complex was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, it was dissociated into a component with a molecular weight of 66,000. The 95,000-dalton complex was dissociated into a 58,000-dalton component upon SDS-PAGE under reducing conditions. An excess amount of isoproterenol and propranolol decreased photolabeling of the beta-receptors with 125I-ABC by 60% and 40%, respectively.     

Linear lesions for treating the recurrence of persistent atrial tachyarrhythmia after cryoballoon ablation of atrial fibrillation

Overall, 133 patients underwent 170 procedures for the treatment of persistent ATa following an index cryoballoon pulmonary vein isolation (n = 715). After all the procedures, > 90% of the patients had a roof line, a mitral isthmus and/or septal line, and a cavotricuspid isthmus line. Ninety-two patients (69.2%) were in sinus rhythm after a median of 36 months since the index cryoballoon PVI. ATa: atrial tachyarrhythmia; cryo: cryoballoon; CTI: cavotricuspid isthmus; LSPV: left superior pulmonary vein; LIPV: left inferior pulmonary vein; PVI: pulmonary vein isolation; RF: radiofrequency; RSPV: right superior pulmonary vein; RIPV: right inferior pulmonary vein.

     

Enzymatic Digestion of Adult Human Articular Cartilage Yields a Small Fraction of the Total Available Cells

We investigated whether different protocols for the digestion of adult human articular cartilage influence the cell yield and capacity to attach and proliferate in culture dishes. Chondrocyte yields were expressed as a percentage of the total number of cells in the tissue, determined both histologically (using the dissector method) and biochemically (measuring the DNA content of tissue digests). Human cartilage specimens ( n = 79) were digested using different protocols based on combinations of collagenase II (CGN), trypsin/EDTA, hyaluronidase, and tosyllysylchloromethane (TLCM). Yields of viable chondrocytes were the highest within a specific range of CGN concentrations and digestion times, but always < 22% of the total available cells. The combination of CGN with trypsin/EDTA or TLCM accelerated the digestion process but did not significantly increase cell yields. The percentage of viable cells that attached to culture dishes ranged 75-85% (< 19% of the total) and was reduced by TLCM. Doubling times of attached cells were comparable in all experimental groups. Our results indicate that chondrocyte yields and capacity to attach and proliferate are not highly sensitive to the specific isolation protocol used. However, typically used cartilage digestion protocols yield only a small fraction of the total available cells, possibly introducing an uncontrolled selection of certain chondrocyte subpopulations.     

硝酸稀土促进布鲁氏菌生长试验

我们用硝酸稀土培养基做了猪种、羊种布鲁氏菌促进生长试验,结果表明,猪种布鲁氏菌菌体细胞每分裂一次其时间可提前22分钟,羊种布鲁氏菌则可提前12.7～17.5分钟,说明硝酸稀土有促进布鲁氏菌生长作用。     

COVID-19疫情下医院服务区分布与隔离点设置方法的初步探讨

目的 从空间设施布局优化的角度探讨疫情下的医院服务区分布与基于服务区设置集中隔离点的方法。 方法 基于Huff模型描绘了上海市三级医院的服务区空间分布;以最大化覆盖率为目标,采用位置分配模型预测医院选址的优化布局,设置潜在的候选位置。 结果 在上海市可收治病例的三级医院中,中心地区医院密度高;城乡医院服务区空间分布差异明显,郊区医院的服务区面积大;优化配置布局的结果显示,可在松江区中心新增1家医院,在浦东新区中间区域增加适量医院。 结论 结合医院服务区分布,隔离点可以设置于各街道中与所属医院相近的位置,明确出现病例时各个隔离点对应送往的医院。优化布局建议在松江区和浦东新区增加三级医院。