摘要
目的：建立稳定的同时分离原代大鼠肝细胞（HC）、肝星状细胞（HSC）和Kupffer细胞（KC）的方法,并初步评估其在体外培养的特征。方法：通过胶原酶原位灌注获得肝脏单细胞悬液,随后行等密度离心结合选择性贴壁纯化肝脏原代细胞,细胞的产量和活力经自动细胞计数仪计算出,纯度经免疫荧光、特殊染色及吞噬等实验进行鉴定。结果：大鼠原代HC产量为（21.0±8.2）×10~6/g肝组织,活力为92.1%±2.1%,纯度为96.5%±2.2%;原代HSC产量为（2.5±0.8）×10~6/g肝组织,活力为90.1%±3.8%,纯度为93.1%±1.5%;原代KC产量为（4.1±1.2）×10~6/g肝组织,活力为96.2%±2.7%,纯度为95.1%±1.4%。分离的HC、HSC和KC适合体外长期培养。HSC在体外培养过程中失去维生素A,逐渐向成纤维细胞表型转变。KC在体外可增殖,至14 d时仍表达CD68,具有较强的吞噬能力。结论：成功建立同时分离和培养大鼠HC、HSC和KC的方法,为进一步研究肝脏病理生理提供细胞基础。
Objective： To investigate the effect of human microRNA212 （miR-212） on the migration and invasion of gastric cancer cell line SGC7901. Methods： The i-miR-212 as the suppressor gene of miR-212 was transfected into SGC7901 cells according to different concentration （25, 50, 75, 100 nmol/L） via lipofectamin 2000. Empty vectors and unrelated fragment were also applied as controls. The expression of mature type miR-212 was detected by real-time PCR. Cell Counting Kit-8 was used to evaluate the effect of miR-212 on the proliferation of SGC-7901 cells, meanwhile Transwell assay for migration and invasion of SGC7901 cells. Results： Real-time PCR showed that cells Transfected with i-miR-212 had significantly low expression of miR-212, with the optimal concentration of miR-212 being 50 nmol/L; the expression of miR-212 was 18 times lower than empty group （P〈0.01）. The Cell Counting Kit-8 showed that the proliferation of cancer cells with i-miR-212 （50 nmol/L） was higher than that of the control group （P〈0.01）. The Transwells showed the migration and invasion of cancer cells with i-miR-212 （50 nmol/L） was higher than that of the control group （P〈0.01）, respectively. Conclusion： The i-miR-212 can effectively reduce miR-212 expression and promote the migration and invasion of SGC7901 cells. miR-212 may be a tumor suppressor gene in gastric cancer tumor cells.
出处
《温州医科大学学报》
CAS
2016年第9期638-643,648,共7页
Journal of Wenzhou Medical University
