人乳腺癌原代细胞组织块培养方法的改良及其鉴定

Improvement and characterization of the primary cell culture of human breast cancer tissue block

目的通过对人乳腺癌原代细胞组织块培养方法进行改良,建立一种快速高效可获得高纯度的人乳腺癌细胞原代培养方法,为乳腺癌深入研究提供实验基础。方法取经临床病理诊断确诊为乳腺导管原位癌患者的癌组织标本,所有样本随机分为改良组(全程正置培养瓶进行培养,原代细胞铺满瓶底50%左右,用延长酶消化时间差法一次性纯化原代细胞)和传统组(翻转培养瓶倒置孵箱1~2 h后,再正置培养瓶培养,原代细胞铺满瓶底80%~100%时,用反复酶消化时间差法纯化原代细胞)。在倒置显微镜下观察,比较两组原代细胞爬出时间、爬出数量和贴壁生长情况;细胞计数比较两组组织块培养7 d后获得贴壁细胞总数;免疫组化和流式细胞术鉴定、比较两组纯化后原代细胞的纯度;MTT法检测两组纯化培养48 h后贴壁细胞的活性。结果经改良组织块培养法2 d后,见大量细胞从组织块四周爬出,培养3 d后见爬出原代细胞贴壁生长,胞质展开,培养5 d后见细胞融合贴壁生长,铺满瓶底50%左右;而传统组织块培养法培养2 d后见少量细胞爬出,培养5 d才见少量细胞贴壁生长,培养10d后原代细胞方可铺满瓶底50%左右;人乳腺癌原代细胞培养7 d贴壁细胞总量,改良组为(9.88±0.20)×10^5个,传统组为(4.88±0.21)×10^5个;纯化后改良组细胞纯度为(99.13±0.06)%,传统组为(75.72±4.96)%;纯化培养48 h后贴壁原代细胞活性改良组为(0.86±0.03),传统组为(0.47±0.01),以上观察指标均明显高于传统组织块培养方法,有统计学意义(P<0.05)。获得的贴壁人乳腺癌原代细胞胞质展开,多呈扁平多边形,细胞体积较大,细胞核多位于细胞中央,相互衔接后呈典型的“铺路石”状,细胞结合紧密,生长分裂旺盛。结论本实验成功建立了一种高效快速获得高纯度的人原代乳腺癌细胞原代培养的方法,为进一步的实验研究奠定基础。

Objective To establish a rapid and efficient method of the primary cell culture of human breast cancer by improving the primary tissue culture,this provided an experimental basis for further research on breast cancer.Methods The cancerous tissue samples were taken in situ breast cancer from the patients who were diagnosed as ductal carcinoma.All samples were randomly divided into two groups:the improved group(The flask was cultured uprightly in the whole process.The primary cells covered about 50%of the bottom,and were purified by the extended enzyme digestion time difference method.),and the traditional group(The culture flask was inverted for 1~2 hours,then placed uprightly.The primary cells covered 80%~100%of the bottom,and purified by repeated enzymatic digestion time difference method.).Under an inverted microscope,the climb time,climb number and adherent growth of the primary cells in the two groups were observed.By cell count,the total number of patch cells was obtained after 7 days of tissue culture.Immunohistochemistry and flow cytometry were used to identify and compare the purity of the primary cells.The activity of adherent cells was detected by MTT assay after 48 hours of purification.Results After 2 days of culture,a large number of cells climbed out from the tissue block in the improved group.After 3 days of culture,the primary cells were climbed out and the cytoplasm was developed.After 5 days of culture,the cell fusion was attached and the bottom was covered about 50%.In the traditional group,a small number of cells climbed out after 2 days.Only a small amount of cells were adhered to the wall after 5 days.After 10 days of culture,the primary cells could cover about 50%of the bottom.The total number of adherent cells in 7 days was(9.88±0.20)×10^5 in the improved group and(4.88±0.21)×10^5 in the traditional group.After purification,the purity was(99.13±0.06)%in the improved group,and(75.72±4.96)%in the traditional group.After 48 hours of purification,the activity of the adherent primary cells was(0.86±0.03)in the improved group,and(0.47±0.01)in the traditional group.The above indexes were significantly higher in the improved group than in the traditional group(P<0.05).The cytoplasm of the adhered primary cells from human breast cancer was mostly flat polygonal,the cell volume was large,and the nucleus was mostly located in the center of the cell.After being connected to each other,the cells were typical"paving stone",and tightly bound.The growth and division were vigorous.Conclusion This study successfully established an efficient and rapid culture method to obtain high purity primary breast cancer cell,which laid a foundation for further experimental research.