不同类型胶原酶消化大鼠椎间盘髓核的细胞活力比较

Comparison of cell viability between different types of collagenases digestion in rat nucleus pulposus

目的:比较经Ⅰ型和Ⅱ型胶原酶消化后得到的细胞总数和细胞活力,并观察髓核细胞的形态学及生物学特点。方法:取相同质量的大鼠髓核组织,经0.25%EDTA-胰酶消化40 min后,分别加入0.2%Ⅰ型胶原酶(A组)和Ⅱ型胶原酶(B组)消化1 h,chamber计数板计算各组的细胞总量,使用0.4%台盼蓝染色计算细胞活力。培养3 d后,倒置相差显微镜下观察髓核细胞的形态学特点。甲苯胺蓝、番红O及阿利新兰染色,对髓核分泌基质初步定性。实时无标记动态分析技术(RTCA)检测第2代髓核细胞的增殖生长曲线。结果:经胶原酶消化1 h后,细胞计数发现,B组得到的细胞总数为(62.58±2.95)个,多于A组的(13.92±1.31)个(t=15.100,P=0.000);A组细胞活力(71.26±6.79)%,高于B组的(55.94±5.38)%(t=7.597,P=0.017);倒置显微镜下观察,髓核原代细胞形态不规则,呈椭圆形、三角形及多边形,细胞内部含有较多空泡,可以形成细胞集落,其子代细胞内部不含有空泡结构;甲苯胺蓝、番红O及阿利新兰染色,能分别被染成蓝紫色、红色和淡蓝色;RTCA检测,细胞在接种培养40~70 h内,生长最快。结论:髓核组织经Ⅰ型胶原酶消化后得到的细胞活性较高,后期培养更容易贴壁。

Objective:To compare the total number of cells and cell viability after digestion with type I and type II collagenase and observe the morphological and biological characteristics of nucleus pulposus cells.Methods:The same mass of rat nucleus pulposus were taken,after 0.25%EDTA-trypsin digestion for 40 minutes added 0.2%typeⅠcollagenase(group A)and typeⅡcollagenase(group B)and digested for 1 hour.Chamber counting plate was used to calculate the total cell’s number and 0.4%trypan blue staining was used to calculate the cell viability in each group.After 3 days of culturing,the morphological features of the nucleus pulposus were observed under the inverted phase contrast microscope.The primary quality of nucleus pulposus was determined by the toluidine blue,safranin O and Alcanblue staining.Real-time cell analysis(RTCA)was made to detect the proliferation growth curve of the 2nd generation of nucleus pulposus cells.Results:After collagenase digestion for 1 hour,the cell’s counting found that the total number of cells in group B(62.58±2.95)was much more than that in group A(13.92±1.31)(t=15.100,P=0.000);Cell viability in group A(71.26±6.79)%was higher than that in group B(55.94±5.38)%(t=7.597,P=0.017).Under inverted microscope,the nucleus pulposus primary cells were irregular in shape,oval,triangular or polygonal,and contained more"vacuoles"and could form cell colonies,whose subgeneration cells did not contain"vacuole"structure.Toluidine blue,safranin O and Alcanblue staining can be dyed blue-purple,red and light blue.RTCA detected that cells grew fastest in the 40 to 70 hours of inoculation.Conclusion:The nucleus pulposus after type I collagenase digestion is more active and the culture later is easier to attach.