利用细胞膜片技术构建新型前列腺癌皮下移植瘤动物模型

Developing a new animal model of subcutaneous transplanted prostate cancer with cell sheet technology

背景与目的:传统前列腺癌皮下移植瘤模型的建立采取细胞悬液注射方式,但该方式存在较大局限性。探索利用细胞膜片技术构建新型前列腺癌皮下移植瘤动物模型的可行性。方法:将人前列腺癌细胞系DU145接种至35 mm温度敏感性细胞培养皿中,连续培养制备前列腺癌细胞膜片,并采用H-E染色和免疫组织化学方法鉴定。根据研究目的,设置细胞悬液组和细胞膜片组,细胞悬液组在BALB/c裸小鼠皮下注射DU145细胞悬液,细胞膜片组在裸小鼠皮下注射DU145细胞膜片。移植后4周处死裸小鼠,剥离两组移植瘤作进一步组织学分析,包括胶原纤维含量、局部浸润及血管生成情况。同时,解剖前列腺癌常见的转移脏器,如骨、肺和肝脏等,评估两组的全身转移情况。结果:培养1周后可制备前列腺癌DU145细胞膜片,大体观察呈现半透明胶质薄片,具备较好的组织学强度。组织学染色见膜片状结构,平均厚度约为(32.6±7.5)μm。DU145细胞标志物vimentin高表达,E-cadherin低表达,胶原纤维含量丰富,在膜片中高表达。皮下移植4周后,H-E染色显示,细胞膜片组肿瘤结构致密,有明显周围肌肉浸润,而细胞悬液组肿瘤存在较多空泡,与周围组织边界清楚。皮下移植4周后,细胞膜片组移植瘤体积明显大于细胞悬液组,两组测量肿瘤体积分别为(0.967±0.129)和(0.437±0.054)cm^(3)(t=3.774,P=0.0195)。马松染色显示,细胞膜片组蓝色胶原纤维含量明显高于细胞悬液组,两组吸光度(D)值分别为0.0230±0.0011和0.0140±0.0007(t=7.022,P=0.0001)。CD31免疫组织化学染色表明,在200倍显微镜视野下细胞膜片组和细胞悬液组移植瘤组织中微血管密度分别为53.20±3.56和32.40±4.98(t=3.392,P=0.0095)。皮下移植4周后,两组通过大体解剖和H-E染色均未见到明显的全身转移病灶。结论:利用细胞膜片技术构建前列腺癌皮下移植瘤动物模型具备可行性和一定优势,与传统细胞悬液注射方式相比,成瘤体积更大,与周围组织浸润程度更深,新生微血管密度更高,胶原含量丰富,更有助于揭示前列腺癌真实的生物学特征,有望应用于前列腺癌分子遗传学研究、肿瘤耐药机制及新药研发等领域。

Background and purpose:Traditional preparation of subcutaneous transplanted tumor of prostate cancer relies on cell suspension injection.However,there are great limitations.The purpose of this study was to explore the feasibility of developing a new animal model of subcutaneous transplanted prostate cancer with cell sheet technology.Methods:Human prostate cancer DU145 cells were inoculated into a 35 mm temperature sensitive cell culture dish,and the prostate cancer cell sheet was prepared by continuous culture and identified by H-E staining and immunohistochemistry.According to the research purpose,cell suspension group and cell membrane group were set up.DU145 cell suspension was subcutaneously injected into nude mice in cell suspension group,and DU145 cell sheet was subcutaneously injected into BALB/c nude mice in cell sheet group.The BALB/c nude mice were sacrificed 4 weeks after transplantation,and the grafted tumors in both groups were dissected for further histological analysis,including collagen fiber content,local infiltration and angiogenesis.Meanwhile,the common organs of prostate cancer metastasis,such as bone,lung and liver,were dissected to evaluate the systemic metastasis in both groups.Results:DU145 prostate cancer cell sheet could be prepared at 1 week after continuous culture.Histological staining revealed a membrane structure with an average thickness of(32.6±7.5)μm.The DU145 cell sheet was rich in collagen fibrin with high vimentin expression and low E-cadherin expression.H-E staining showed that the tumor structure was dense with obvious infiltration of surrounding muscles in the cell sheet group,while the tumor had more vacuoles and a clear boundary with surrounding tissues in the cell suspension group 4 weeks after subcutaneous transplantation.Moreover,the transplanted tumor volume was significantly higher in the cell sheet group than in the cell suspension group,and the measured tumor volume in the two groups were(0.967±0.129)and(0.437±0.054)cm^(3)(t=3.774,P=0.0195),respectively.Masson staining showed that the content of blue collagen fiber was significantly higher in the cell sheet group than in the cell suspension group,and the optical densities(D)value of the two groups were 0.0230±0.0011 and 0.0140±0.0007(t=7.022,P=0.0001),respectively.Immunohistochemical staining of CD31 showed that the microvascular density per×200 field in the cell sheet group and the cell suspension group were 53.20±3.56 and 32.40±4.98(t=3.392,P=0.0095),respectively.Gross anatomy and H-E staining showed no obvious systemic metastasis in the two groups 4 weeks after subcutaneous transplantation.Conclusion:It is feasible and advantageous to develop the animal model of subcutaneous transplanted prostate cancer using cell sheet technology.Compared with traditional cell suspension injection method,the tumor volum was larger,the infiltration degree was deeper with surrounding tissues,the density of new microvessels was higher,and the collagen content was richer,which is more helpful to reveal the real biological characteristics of prostate cancer.It is expected to be applied in the research of molecular genetics of prostate cancer,drug resistance mechanism and drug discovery.