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目的:探讨生物钟基因Period2对卵巢癌裸鼠移植瘤生长转移和血管生成的作用及可能机制。方法:利用卵巢癌细胞株构建裸鼠卵巢癌皮下移植瘤,利用基因转染技术,外源性导入重组基因Period2,使之在肿瘤组织成功稳定表达,分别采用Real-time定量PCR和Western blot检测移植瘤中Period2表达,治疗期间测量移植瘤体积,治疗2周后处死裸鼠,称取瘤重,免疫组化检测肿瘤组织中血管内皮生长因子/血管通透性因子(VEGF/VPF)、血管内皮生长因子受体1(VEGFR1)、微血管密度(MVD)(CD34标记)的表达情况,Western blot检测肿瘤转移相关基因(MTA1)、基质金属蛋白酶-9(MMP-9),以及PI3K/Akt信号通路的表达。结果:(1)外源性导入Period2基因在裸鼠移植瘤肿瘤组织中成功稳定表达。(2)Period2组移植瘤体积与其他两组相比较,差异有统计学意义(F=23.469,P<0.001)。转染后2周,Period2组移植瘤重量明显低于空质粒组和对照组(P<0.05),Period2组抑瘤率达到38.9%。(3)免疫组化结果表明,Period2组的VEGF/VPF、VEGFR1表达下降(F=46.80/48.09,P<0.001),MVD计数(CD34标记)显著减少(F=138.4,P<0.001)。(4)Western blot结果表明,Period2组MTA-1和MMP-9表达明显少于其他组(P<0.05),自噬与凋亡相关信号通路PI3K/Akt中标志性蛋白PI3K、Akt表达明显下调(P<0.05)。结论:(1)外源性导入Period2过表达可使卵巢癌生长速度减慢,抑瘤率明显提高。(2)Period2可能通过抑制VEGF/VPF、MTA-1、MMP-9表达而抑制卵巢癌的血管新生和浸润转移。(3)Period2可能通过干扰PI3K/Akt信号通路影响凋亡,抑制肿瘤血管形成来发挥抑瘤作用。
Objective: To investigate the effect and possible mechanism of circadian clock gene Period2 on the growth, metastasis and angiogenesis of ovarian cancer xenografts in nude mice. METHODS: The ovarian cancer cell lines were used to construct subcutaneous xenografts of ovarian cancer in nude mice. The gene transfection technique was used to introduce the recombinant gene Period2 exogenously and successfully and stably expressed in tumor tissues. Real-time quantitative PCR and Western blot were used respectively The expression of Period2 in the xenograft tumor was measured during the treatment. After 2 weeks of treatment, the nude mice were sacrificed and the tumor weights were weighed. The expression of VEGF / VPF and VEGF The expression of VEGFR1 and MVD (CD34) was detected by Western blot. The expressions of MTA1, MMP-9 and PI3K / Akt Path of expression. Results: (1) Exogenous introduction of Period2 gene was successfully and stably expressed in tumor tissue of nude mice. (2) Compared with the other two groups, the tumor volume in Period2 group was significantly different (F = 23.469, P <0.001). Two weeks after transfection, the weight of tumor in Period2 group was significantly lower than that in empty plasmid group and control group (P <0.05). The inhibition rate of tumor in Period2 group was 38.9%. (3) The results of immunohistochemistry showed that the expression of VEGF / VPF and VEGFR1 in Period2 group decreased (F = 46.80 / 48.09, P <0.001) and MVD count (CD34 mark) decreased significantly. (4) Western blot results showed that the expression of MTA-1 and MMP-9 in Period2 group was significantly lower than that in other groups (P <0.05), and the expression of PI3K / Akt in PI3K / Akt pathway was significantly down-regulated (P <0.05). Conclusion: (1) Exogenous introduction of Period2 overexpression can slow down the growth of ovarian cancer, tumor inhibition rate was significantly improved. (2) Period2 may inhibit angiogenesis and invasion and metastasis of ovarian cancer by inhibiting the expression of VEGF / VPF, MTA-1 and MMP-9. (3) Period2 may exert anti-tumor effect by interfering with PI3K / Akt signaling pathway and affecting the apoptosis and inhibiting tumor angiogenesis.