人胰腺癌细胞系与大鼠背根神经节体外共培养的研究

In vitro interaction of human pancreatic cancer cells and rat dorsal root ganglia： a co-culture model

目的建立一种新的体外胰腺癌神经浸润（PNI）研究模型，并观察PNI过程中神经突的生长状况，以及胰腺癌细胞的增殖、凋亡和迁移情况。方法取人胰腺癌细胞系MIAPaCa-2和大鼠背根神经节（DRG），于Matrigel胶中共培养，建立研究模型。倒置显微镜下观察胰腺癌细胞集落形成和DRG神经突生长情况以及胰腺癌细胞的迁移情况，并用Image．ProPlus5．0图像分析软件进行图像分析，计算神经突和胰腺癌细胞集落所占的面积。免疫细胞化学法检测胰腺癌细胞增殖细胞核抗原Ki-67的表达情况，计算胰腺癌细胞增殖指数，四甲基偶氮唑蓝（Mrrr）法检测胰腺癌细胞的增殖情况。利用流式细胞仪检测胰腺癌细胞的凋亡率。结果共培养24和72h，MIAPaCa-2／DRG共培养组的细胞集落面积分别为176．67±5．57和242．83±4．92，明显大于MIAPaCa-2细胞单独培养组（分别为151．17±5．98和182．50±5．39，均P〈0．01）；MIAPaCa-2／DRG共培养组的神经突面积分别为129．33±2．73和202．50±8．04，明显大于DRG单独培养组（分别为63．674-5．17和78．674-3．61，均P〈0．01）。神经突有明显向胰腺癌细胞集落方向生长的趋势，而神经突与胰腺癌细胞集落接触后，胰腺癌细胞则沿神经突生长的方向逆向迁移，最终到达DRG周围，出现类似PNI的表现。MTY与免疫细胞化学实验结果均表明，上清液培养组胰腺癌细胞的增殖远比普通培养基培养组活跃。流式细胞仪检测结果表明，上清液培养组胰腺癌细胞凋亡率为2．46％，明显低于普通培养基培养组（4．89％，P〈0．01）。结论MIAPaCa-2／DRG共培养模型成功构建，有助于胰腺癌PNI的研究。神经细胞与胰腺癌细胞的相互作用在胰腺癌的PNI过程中发挥重要作用。神经细胞与胰腺癌细胞可以相互促进对方的生长，并且胰腺癌细胞有沿神经突生长的方向逆向迁移的趋势。

Objective To establish an in vitro model of perineural invasion （PNI） with co-cuhure of human pancreatic cancer cells and rat root ganglion, to observe the neurite outgrowth and pancreatic cancer cell proliferation and migration, and to explore the molecular basis of perineural invasion （PNI） of pancreatic cancer. Methods Human pancreatic cancer cell line （ MIA PaCa-2） and rat dorsal root ganglion （DRG） were co-cultured in Matrigel matrix to generate the PNI model. The neurite outgrowth, pancreatic cancer cell colony formation, neurite-colony contact and retrograde migration were observed under an inverted microscope. The data were analyzed with the Image-Pro Plus 5.0 system. The proliferative index （PI） was measured by immunohistochemical staining with the Ki-67 antibody. In order to determine the absorbance （A） of the pancreatic cancer cells, MrIT assay was used. The apoptotic index （AI） was evaluated by flow cytometry. Results Neurite outgrowth was stimulated in the presence of pancreatic cancer cells. After 72 hours of the co-cuhure, MIA PaCa colonies co-cultured with DRG exhibited a significantly larger colony area （242.83 ±4.92） than that of the control （ 182.50 ±5.39, P 〈 0. 001 ）. In the MIA PaCa-2/DRG coculture system, the neurites exhibited a trend of growing towards the pancreatic cancer cell colony. However, the pancreatic cancer cells showed a trend of retrogradely migrating to the DRG along the neurite outgrowth, when MIA PaCa-2 colonies touched the DRG. The positive rate of Ki-67 nuclear antigen was significantlyhigher than in the co-culture group. The PI value was higher in the experimental group （ 12.80% ） than that in the control group （6.81%, P 〈 0.01 ）. The MTY assay showed that proliferation of the pancreatic cancer cells was more active than that in the control group. Flow cytometry analysis showed that the apoptosis rate of the pancreatic cancer cell was 2.46%, significantly lower than that of the control group （4.89%, P 〈 0. 001 ）. Conclusions An in vitro co-culture model of rat dorsal root ganglion and human pancreatic cancer cell line is successfully established in this study. This MIA PaCa-2/DRG co-culture system demonstrates that the neural-pancreatic carcinoma cell interaction is a mutually beneficial process for the growth of neurites and pancreatic carcinoma cells. The pancreatic cancer cells show a trend of migrating to the DRG along the neurite outarowth.