多聚D-赖氨酸在辅助悬浮细胞转染及集落形成实验中的应用  

Application of poly-D-lysine in assisting suspension cells transfection and colony formation experiments

作　　者：李佳[1] 周建鹏[2] 白鸥[1] Li Jia;Zhou Jianpeng;Bai Ou(Department of Hematology,the First Hospital of Jilin University,Changchun 130000,China;Department of Hepatobiliary and Pancreatic Surgery 1,General Surgery Center,the First Hospital of Jilin University,Changchun 130000,China)

机构地区：[1]吉林大学第一医院血液科,长春130000 [2]吉林大学第一医院普通外科中心肝胆胰外一科,长春130000

出　　处：《白血病．淋巴瘤》2024年第9期528-533,共6页Journal of Leukemia & Lymphoma

基　　金：吉林大学自然科学纵向科研项目(3D4233129428)。

摘　　要：目的:探讨多聚D-赖氨酸(PDL)在辅助悬浮细胞转染及集落形成实验中的应用。方法:选择人源弥漫大B细胞淋巴瘤(DLBCL)细胞株SU-DHL-4。采用CCK-8法检测不同浓度(0.01、0.1、1.0、10.0 mg/ml)PDL溶液对SU-DHL-4细胞的毒性,计算细胞存活率。将形态正常、处于对数生长期的SU-DHL-4细胞分别接种于包被PDL和未包被PDL的96孔板中,选择感染复数(MOI)为50、100的病毒液分别感染细胞,观察细胞形态。采用慢病毒感染法构建稳定转染荧光素酶基因的SU-DHL-4细胞模型,将感染后的SU-DHL-4细胞分为对照组(感染对照组病毒液)和实验组(感染携带荧光素酶基因的病毒液);转染后的细胞持续在含嘌呤霉素的RPMI 1640完全培养液中培养;采用双荧光素酶报告基因试剂盒检测荧光强度。使用PDL包被的培养皿进行悬浮细胞的集落形成实验,分别使用含0.1%二甲基亚砜(DMSO)(对照组)、0.1 μmol/L依托泊苷(A组)和0.2 μmol/L依托泊苷(B组)的RPMI 1640完全培养液培养,观察集落形成情况并计算集落形成率。结果:SU-DHL-4细胞接种于0.01、0.1、1.0、10.0 mg/ml PDL包被的96孔板48 h后,细胞存活率分别为(98.1±1.6)%、(97.1±0.7)%、(91.7±1.5)%、(83.3±2.0)%,对照组(不加PDL溶液)为(100.0±2.7)%;0.01、0.1 mg/ml PDL包被组分别与对照组比较,差异均无统计学意义(均 P>0.05);1.0、10.0 mg/ml PDL包被组分别与对照组比较,差异均有统计学意义(均 P<0.001);10.0 mg/ml PDL包被组与1.0 mg/ml PDL包被组比较,差异有统计学意义( t=5.80, P=0.004);故后续实验采用对SU-DHL-4细胞生长无明显毒性的0.1 mg/ml PDL包被培养皿。接种于未包被PDL培养皿的SU-DHL-4细胞悬浮生长;接种于包被0.1 mg/ml PDL培养皿的SU-DHL-4细胞贴于皿底呈单细胞平铺生长模式。将SU-DHL-4细胞接种于未包被PDL的96孔板,使用MOI为50、100的病毒液感染细胞,转染失败;将SU-DHL-4细胞接种于PDL包被的96孔板,原本悬浮生长细胞变为贴ObjectiveTo investigate the application of poly-D-lysine(PDL)in assisting suspension cells transfection and colony formation experiments.MethodsThe human-derived diffuse large B-cell lymphoma(DLBCL)cell line SU-DHL-4 was selected.The cytotoxicity of different concentrations(0.01,0.1,1.0,10.0 mg/ml)of PDL solution on SU-DHL-4 cells was assessed by using the CCK-8 assay,and cell viability was calculated.SU-DHL-4 cells with normal morphology in logarithmic growth phase were seeded into 96-well plates coated and uncoated with PDL.Cells were then infected with viral suspensions at a multiplicity of infection(MOI)of 50 or 100,and cell morphology was observed.A stable SU-DHL-4 cell model transfected with the luciferase gene was constructed by using lentiviral infection method.The infected SU-DHL-4 cells were divided into the control group(infected with control virus fluid)and the experimental group(infected with virus fluid carrying luciferase gene).The transfected cells were continuously cultured in RPMI 1640 complete medium containing puromycin.A dual-luciferase gene reporter assay kit was used to detect fluorescence intensity.Colony formation experiments with suspension cells were conducted by using PDL-coated dishes.The cells were cultured in RPMI 1640 complete medium containing 0.1%dimethyl sulfoxide(DMSO)(the control group),0.1µmol/L etoposide(group A),and 0.2µmol/L etoposide(group B),and colony formation was observed and colony formation rate was calculated.ResultsAfter 48 h of seeding SU-DHL-4 cells into 96-well plates coated with 0.01,0.1,1.0,and 10.0 mg/ml PDL,cell viability was(98.1±1.6)%,(97.1±0.7)%,(91.7±1.5)%,and(83.3±2.0)%,respectively,compared to(100.0±2.7)%in the control group(not adding PDL solution).There were no statistically significant differences between 0.01 and 0.1 mg/ml PDL-coated groups and the control group(both P>0.05);there were statistically significant differences between 1.0 and 10.0 mg/ml PDL-coated groups and the control group(both P<0.001).There were statistically significant d

关 键 词：聚赖氨酸 悬浮细胞 转染 克隆形成 

分 类 号：R457.1[医药卫生—治疗学]