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Establishment of the Tumor Mouse Model of CNE-1 and CNE-1/taxol and Its Biological Characters

Author: ZhangXinLi
Tutor: TanGuoLin
School: Central South University
Course: Otolaryngology Head and Neck Surgery
Keywords: Taxol Cisplatin Nasopharyngeal Resistance Nude mice Bcl-2 VEGF TSP1
CLC: R739.63
Type: Master's thesis
Year: 2011
Downloads: 39
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Abstract

Objective: 1. Establish human nasopharyngeal carcinoma CNE-1 and nude mice xenograft model CNE-1/taxol and its biological characteristics; 2. Research CNE-1 and transplanted into nude mice on CNE-1/taxol Taxol and cisplatin sensitivity in vivo proof of cisplatin can reverse the nasopharyngeal paclitaxel resistant; 3. explore the Bcl-2 VEGF, TSP1 genes in reversal of nasopharyngeal paclitaxel resistance in nasopharyngeal paclitaxel and cisplatin resistance The role of the molecular mechanism of the drug. Method: 1. Cells in culture: human nasopharyngeal carcinoma cell line CNE-1 and its resistant cell subline CNE-1/taxol cultured in serum-free medium containing 14% fetal bovine culture conditions for 37. C, 5? 2, the saturation humidity incubator, containing 0.25% trypsin and 0.02% EDTA completely trypsinized passaged. 2. Establish human nasopharyngeal carcinoma CNE-1 and CNE-1/taxol nude mice xenograft model and grouping: the human nasopharyngeal carcinoma cell lines to a concentration of about 1 × 107/ml cell suspension was prepared, were planted in 4, 6-8 weeks old nude mice axillary subcutaneous. To be tumor grew to a diameter of about 8mm (totaling 45 days, about 50% of the rate of tumor), remove, cut diameter of 1-2mm tumor blocks were planted in 24 6-8 week-old nude mice left right Fupi CNE-1 xenograft tumor inoculation (left side, the right side of the transplanted tumor inoculation CNE.1/taxol) until the tumor grew to a diameter of about 5mm (totaling 14 days, 100% of the rate of tumor formation), were randomly divided into 3 medication group: (1) blank control group: sterile saline; the ② paclitaxel: paclitaxel 10mg/kg; ③ cisplatin group: cisplatin 5mg/kg, are intraperitoneal injection (0.2 ml / / 5 / weekly). The 3. Indicators experimental observation and specimen collection, handling: after injection, daily observation of transplanted tumors in nude mice survival status, measured every other day in nude weight, measured with a vernier caliper transplanted tumors of the longest diameter (a) and the shortest path (b ) calculate the transplanted tumor volume (V, V = 1/2ab2), draw subcutaneous tumor growth curve, calculation the groups transplanted tumor growth multiples and administration groups transplant tumor growth inhibition rates. 4. Withdrawal two days after the mice were sacrificed and the removal of subcutaneously transplanted tumor in 10% formalin fixation the immunohistochemistry assay groups transplanted tumor tissue Bcl-2, VEGF, TSP1 of expression. 5 Statistical analysis: SPSS for windows 17.0 statistical package for data processing, data expressed as mean ± standard deviation (M ± SD), the statistical methods used for the two-sample t-test, and homogeneity of variance analysis of data, if the variance equal variance equal to two-sample t test, if the variance ranging variance ranging situations two-sample t 'test, P lt; 0.05 Differences were considered significant. Results: 1 cell suspension method planting human nasopharyngeal carcinoma CNE-1, and CNE-1/taxol transplanted tumor formation long incubation period, about 30 days, as long as the diameter of about 8mm totaling 45 days, and the high rate of tumor about 50%; almost no incubation period fresh tissue pulomonary method, length to diameter of about 5mm totaling 14 days, and the high rate of tumor formation, up to 100%. Transplanted tumors 2.CNE-1 and CNE-1/taxol by saline treatment after one week of growth, the growth factor were (2.53 ± 0.31), (1.79 ± 0.27), the difference was statistically significant, The prompt CNE-1/taxol tumor growth rate slower than CNE-1 xenograft tumor. After the 3.CNE-1 and CNE-1/taxol transplanted tumors by paclitaxel treatment, the growth of one week, the tumor growth inhibition rates were (67.14 ± 13.03)% (35.84 - 35.3)%, both compared to the difference statistically significant (P lt; 0.05), prompted CNE-1/taxol transplanted tumor resistance to paclitaxel. After 4.CNE-1 and CNE-1/taxol transplanted tumor by cisplatin treatment, one week of growth, the tumor growth inhibition rates were (44.71 ± 14.80)%, (50.73 ± 11.38)% paclitaxel-resistant cells CNE -1/taxol transplanted tumors more sensitive to cisplatin, but the comparison between the two, the difference was not statistically significance (P gt; 0.05). Immunohistochemical detection: ① Bcl-2: the control group CNE-1 xenograft tumor expression of Bcl-2 than CNE-1/taxol; paclitaxel CNE-1 xenograft tumor decreased expression, CNE-1/taxol no significant change; CNE-1 xenograft tumor after cisplatin treatment, no significant difference from, CNE-1/taxol expression was elevated. ② vEGF: CNE-1 xenograft tumor of the control group, the expression of VEGF than CNE-1/taxol; CNE-1 xenograft tumor after paclitaxel treatment reduced expression, CNE-1/taxol expression was elevated; treated with cisplatin CNE-1 and CNE-1/taxol transplanted tumor expression was no significant change; ③ TSP1: CNE-1 xenograft tumor of the control group TSP1 expression than CNE-1/taxol: CNE-1 after paclitaxel and CNE-1/taxo1 transplanted tumor expression reduced; CNE-1 xenograft tumor expression after cisplatin treatment, no significant change CNE-1/taxol expression was significantly increased. Conclusions: 1. Fresh tissue blocks inoculation method is to build human nasopharyngeal carcinoma CNE-1 nude mice xenograft model CNE-1/taxol better way. Paclitaxel, cisplatin on growth of human nasopharyngeal carcinoma CNE-1 tumor growth inhibition, two for nasopharyngeal effective chemotherapy drugs. 3 people the nasopharyngeal CNE-1/taxol planted in nude mice, are still resistant to paclitaxel, cisplatin can reverse the resistance. 4.CNE-1 tumor growth compared CNE-1/taxol faster may be related to high expression of Bcl-2, of VEGF-related. 5. Nasopharyngeal of paclitaxel resistant with low TSP1 expression, nasopharyngeal carcinoma can be reversed by increased expression of TSP1 cisplatin paclitaxel resistant.

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