| Objective :To establish transplantation tumor model of nasopharyngeal carcinoma(NPC) well-differentiated cell line (CNE1)in the muscles of babl/c mice, in order to investigate the capability of growth and invasion of CNE1 in vivo and make the basement of geting the message of Clinical target volum.This model have no peplos that the tumor cells invasion easier.And it is easy for us to investigate the single tumor cell's biological behaviour.Materials : Human nasopharyngeal carcinoma cell line CNE1(LMP1 positive expression); fetal bovine serum (FCS); RPMI-1640 medium; trypsinase; LMP1( CS1-4)Mab;SABC kit; pEGFP-C1; Shuohua-SofastTM transfection agentia; plasmid extract kit(V-gene); four or six mounth-old balb/c nude mice; TJ-TY-500C auto- image analysis system;super-clean Lab and SPF animal Lab.Methods Cultivated CNE1 in vitro, and detected the expression of LMP1 by immunohistochemical method. CNE1 cell line were transferred with a plasmid vector(pEGFP-C1) containing the EGFP gene. Stable EGFP-expressing clones were isolated and cultured to amplify them.Establish transplantable tumor model by muscular injection of cell suspension of CNE1 in the gluteus maximus muscle and subcutaneous injection on the back of babl/c mice after amplification in vitro. Recorded the size of the tumors every day and sacrificed all the mice once one was dying and then dissected out the transplantation tumors, and fixed them with 4% Polyoxymethylene and embedded with paraffin, and then made into sections. The immunohistochemical method and the hematoxylin– eosin stain were used to detect the invasion and metastasis of tumor cells.Results : Transplantation tumors which have peplos grew slowly and have smaller gross tumor volume.Comparing with the models of subcutaneous injection, the CNE1 cells which were injected in muscles infiltrated into adjacent tissue obviously ,and cancer nest could be seen in the muscle tissue and cancer cellular invasion to muscle cells also could be found from the histological sections under HP; The latency period(11.92±2.02d) and doubling time(2.32±0.24d)of transplantable tumors injected in muscles were both significantly shorten(P<0.01);The average weight of the tumors increasedobviously(1.32±0.17g,P<0.05);And the metastasis rate of lymph nodes and livers was increased significantly(41.7%/100%,P <0.05) while there was no significant difference on the tumor production rate (83.3%/100%,P<0.05).EGFP was expressed stablely in vivo of nude mouse ,It didn't affect the growth of the transplantable tumors.The latency period(10.98±2.21d) and doubling time(2.29±0.62d)of transplantable tumors injected in muscles were both significantly shorten(P>0.05);The average weight of the tumors increased obviously(1.33±0.11g,P>0.05).Tt was easy to discriminate tumor and non-tumor margin and even the single cancer cell by fluorescence microscopy. It is more superior than hematoxylin-eosin stain and immunohistochemical method on sensitivity and specificity.Conclusion: Transplantation tumor model in muscles of babl/c mice was a more economical and effective method to investigate the growth , invasion and delimitation of clinical target volum of the NPC differentiated cell line (CNE1) in vivo.It is available for visualization of clinical tumor volume investigation further.In a word,it is a ideal animal model this cell line. CNE1 cell line transfected with the enhanced green fluorescence protein gene in vitro and establishment of transplantation tumor model were significant to investigate the invasion of nasopharyngeal carcinoma in vivo. |
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