基因和表观及联合治疗对喉癌荷瘤鼠肿瘤的抑制作用研究

Inhibition of human laryngeal carcinoma growth by gene therapy and epigenetic therapy

目的观察基因和表观治疗及其联合应用对喉癌Hep-2细胞荷瘤鼠肿瘤细胞生长的影响，并探讨其相关机制。方法将Hep-2细胞注射到裸鼠的右腋皮下建立肿瘤模型，数字表法随机分为四组，即p53治疗组（重组人p53腺病毒注射液，rAd-p53）、表观治疗组（5-氮-2-脱氧胞苷，5-aza—dC）、联合治疗组（rAd—p53＋5-aza—dC）和空白组。当肿瘤长径约4～5mm时将不同的治疗方法分别作用于荷瘤鼠，并记录肿瘤的自然生长情况，动物处死后免疫组化和实时荧光定量聚合酶链式反应检测肿瘤组织分子标记物（p53、E—cadherin）的蛋白和基因表达。两组均数比较采用t检验，多组均数比较采用单因素方差分析，各组的交互作用采用析因设计方差分析。结果在首次注射药物后的第20天，p53治疗组肿瘤平均体积（面±s，下同）为（106．09±24．40）mm3、表观治疗组为（166．554-40．11）mm3、联合治疗组为（126．11±22．49）mm3、空白组为（252．83±54．09）mm3；基因治疗与表观治疗存在交互作用（F=22．01，P〈0．05），且基因治疗（F=37．30，P〈0．05）与表观治疗（F：4．79，P〈0．05）对肿瘤的生长均有一定的抑制作用。p53治疗组p53蛋白的表达的积分光密度为628．07±95．16，明显高于联合治疗组的494．764-100．22，差异有统计学意义（t=8．72，P〈0．05）。p53治疗组E．cadherin蛋白表达的积分光密度为558．89±97．58，表观治疗组为380．414-90．60，联合治疗组494．76±102．88，空白组为162．60±40．38；基因治疗与表观治疗存在交互作用（F=21．82，P〈0．05），且基因治疗（F=45．24，P〈0．05）与表观治疗（F=5．73，P〈0．05）对E—cadherin蛋白表达均有一定的增强作用。p53治疗组p53基因相对表达量为4．43-4-0．12、表观治疗组为1．064-0．11、联合治疗组为3．51±4-0．10、空白组为1．09±0．11，基因治疗与表观治疗存在交互作用（F=298．11，P〈0．05）。p53治疗组E-eadherin基因表达为4．50±0．34、表观治疗组为2．02±0．16、联合治疗组为2．99±0．12、空白组为1．004-0．11；基因治疗与表观治疗存在交互作用（F=122．17，P〈0．05），且基因治疗（F=329．12，P〈0．05）与表观治疗（F=88．57，P〈0．05）对该基因的表达均有一定的增强作用。结论基因和表观治疗及联合治疗对荷瘤鼠喉癌均具有一定的抑制效果，且基因治疗疗效明显优于联合治疗和表观治疗。其相关机制考虑为p53与5-aza—dC具有拮抗作用。

Objective To observe the effects of gene therapy and epigenetic therapy on the tumor growth of laryngeal carcinoma and the underlying mechanisms. Methods The animal model of human laryngeal carcinoma was established by the subcutaneous inoculation of Hep-2 cells at the right armpit of BALB/c nu/nu mice. The tumor-bearing mice were randomized into 4 groups, p53 therapy group （rAd- 1353 ）, epigenetie therapy group （ 5-aza-dC ）, combination therapy group （ rAd-p53 ± 5-aza-dC ） and control group. The gene and protein expressions of molecular markers p53 and E-cadherin were detected by FQ-PCRand immunohistochemistry. Results By the day 20 of the treatments, the mean tumor volumes were （106. 09-± 24. 40）mm3 in p53 therapy group, （166. 55 ± 40. 11 ）mm3 in epigenetic therapy group, （ 126.11 ± 22.49 ） mm3 in combination therapy group, and （ 252.83 ± 54.09 ） mm3 in control group. Both gene therapy（ F = 37.30, P 〈 0.05 ） and epigenetic therapy （ F = 4. 79, P 〈 0.05 ） inhibited the growth of xenografted tumors, with an interaction effect （ F = 22.01, P 〈 0.05 ） between the two groups. The integral optical density value of p53 protein expression of p53 therapy group （ 628.07 ± 95. 16 ） was significantly higher than that of combination therapy group （494.76 ± 100.22） , （ t = 8.72, P 〈 0.05 ）. The integral optical density values of E-eadherin protein expression were 558. 89 ± 97. 58 in p53 therapy group, 380.41 ± 90.60 in epigenetic therapy group, 494.76 ± 102.88 in combination therapy group, and 162.60 ± 40.38 in control group respectively, indicating the enhancements of E-cadherin protein expression by gene therapy（ F = 45.24 ,P 〈 0.05 ） or epigenetic therapy （ F = 5.73, P 〈 0.05 ） and the existence of interaction effect（F = 21.82, P 〈 0.05 ） between gene therapy and epigenetic therapy. The expression levels of p53 gene were 4.43 ±0.12 in p53 therapy group, 1.06 ±0. 11 in epigenetic therapy group, 3.51 ±0. 10 in combination therapy group, and 1.09 ± 0.11 in control group, respectively, showing an interaction effect between gene therapy and epigenetic therapy（ F = 298.11, P 〈 0.05 ）. The expression levels of E-cadherin gene were 4.50 ±0.34 in p53 therapy group, 2.02 ±0.16 in epigenetie therapy group, 2.99 ± 0. 12 in combination therapy group, and 1.00 ± 0.11 in control group, respectively. The expression of E-cadherin gene was enhanced by gene therapy （ F = 329.12, P 〈 0.05 ） or epigenetic therapy （ F = 88.57, P 〈 0.05 ）, with an interaction effect between the two therapies （ F = 122. 17, P 〈 0.05 ）. Conclusions Xenografted tumors of human laryngeal carcinoma cells are inhibited by gene therapy, the epigenetie therapy and the combination therapy. The gene therapy was significantly better than the epigenetic therapy or the combination theraov. There might be antagonistic effect between p53 and 5-aza-dC.