喉癌中自噬基因Beclin1的表达及对顺铂药敏的实验研究

发布时间：2018-02-24 00:41

  本文关键词： 喉癌 自噬 Beclin1 凋亡 顺铂　出处：《山西医科大学》2013年博士论文　论文类型：学位论文

【摘要】：背景与研究目的 喉癌是头颈部常见的恶性肿瘤之一,严重威胁着患者的健康,寻找有效的治疗方法一直是临床的迫切需求,在喉癌的治疗中,基因治疗已成为手术、化疗放疗三大治疗外新的治疗手段。 自噬是细胞内成分被溶酶体降解过程的统称,在清除损伤或多余的细胞器,维持细胞内环境稳定方面起着重要作用。自噬可清除损伤的细胞器,避免如自由基等有毒性物质的产生,从而使突变率降低,抑制肿瘤的形成,而自噬的缺陷可导致肿瘤的发生。Beclin1基因是第一个被发现的哺乳动物的自噬基因,在多种肿瘤中表现为表达降低和缺失,Beclin1表达缺失明显增加肝癌、肺癌和淋巴瘤的发生率,但自噬基因Beclin1与喉癌的关系尚不清楚。 本研究通过检测喉癌组织中自噬基因Beclin1的表达,探讨自噬基因Beclin1与喉癌发生发展的关系；研究自噬基因Beclin1在喉癌Hep-2细胞中表达增强和缺失对细胞体外增殖、自噬、凋亡的作用及对喉癌Hep-2细胞顺铂敏感性的影响,探讨自噬诱导在喉癌治疗中的可行性,为喉癌的基因治疗提供新的思路。 方法： 1.采用免疫组化SP法对77例喉癌组织和22例正常喉组织进行Beclin1蛋白表达水平进行检测,分析Beclin1与喉癌临床分型、T分期、组织分化程度、淋巴结转移等临床病理因素的相关性； 2.通过RT-PCR法从人正常喉组织中获得目的基因Beclin1,将其插入真核表达载体pcDNA3.1中,构建真核表达载体pcDNA3.1/Beclin1;设计针对Beclin1的特异性RNA干扰序列,构建Beclin1基因的小发夹状RNA (shRNA)真核表达质粒pSUPER/Beclin1; 3.脂质体法将重组质粒pcDNA3.1/Beclinl和pSUPER/Beclin1转染人喉癌细胞株Hep-2,筛选稳定表达株,采用荧光定量RT-PCR及Western Blot分别在mRNA和蛋白质水平检测转染前后Beclin1、LC3和p62蛋白的表达变化；流式细胞仪检测自噬情况；Hoechs检测细胞凋亡；MTT法检测细胞的增殖变化； 4.顺铂处理Hep-2细胞后,MDC染色检测自噬情况；将重组质粒pcDNA3.1/Beclin1和pSUPER/Beclin1转染人喉癌细胞株Hep-2后,顺铂处理,MTT法检测细胞的增殖变化。 结果： 1.喉癌组织中Beclin1的蛋白表达阳性率低于正常喉组织(P0.05),喉癌中常见Beclin1表达下调和缺失；Beclin1表达与喉癌的临床分型无关(P0.05),与喉癌的T分期、分化程度及淋巴结转移相关(P0.05) 2.利用PCR分析,酶切鉴定及DNA测序证实Beclin1基因的表达质粒pcDNA3.1/Beclin1(?)PRNA干扰质粒pSUPER/Beclin1构建成功； 3. pcDNA3.1/Beclin1转染Hep-2细胞后,Beclin1、LC3Ⅱ mRNA和蛋白的表达显著提高,p62mRNA和蛋白的表达显著降低(P0.05),与未转染组相比,凋亡细胞和自噬细胞明显增加,细胞生长活性降低； 4. pSUPER/Beclin1转染Hep-2细胞后,Beclin1 LC3Ⅱ mRNA和蛋白的表达明显降低,p62mRNA和蛋白的表达显著增高(P0.05),与未转染组相比,凋亡细胞和自噬细胞明显减少,细胞生长活性增强； 5.顺铂处理Hep-2细胞后,细胞内自噬增加,且呈浓度依赖关系,重组质粒pSUPER/Beclin1可以显著提高Hep-2细胞对顺铂的敏感性。 结论： 自噬基因Beclin1与喉癌的发生、发展有关,自噬基因Beclin1可有效抑制喉癌细胞的增殖,提高肿瘤细胞的自噬和凋亡能力,同时抑制自噬基因Beclin1表达能提高喉癌Hep-2细胞对顺铂的敏感性。
[Abstract]:Background and research purpose
Laryngeal cancer is one of the most common malignant tumors in head and neck. It seriously threatens the health of patients. Finding effective treatment is always an urgent need. In the treatment of laryngeal cancer, gene therapy has become a new treatment for surgery, chemotherapy and radiotherapy, the three major treatment.
Autophagy is a cellular constituents known lysosomal degradation process, in the clearance of damaged or superfluous organelles, plays an important role in maintaining cell homeostasis. Autophagy may remove cell injury, to avoid such as free radicals, toxic substances, so that the mutation rate, inhibit the formation of tumors,.Beclin1 gene and autophagy defects can lead to tumor is the autophagy gene first discovered in mammals, in a variety of tumors in reduced expression and deletion, loss of Beclin1 expression significantly increased the incidence of liver cancer, lung cancer and lymphoma, but the relationship between autophagy gene Beclin1 and laryngeal cancer is not clear.
This study through the detection of expression of autophagy gene Beclin1 in laryngeal squamous cell carcinoma, and explore the relationship between autophagy gene Beclin1 and the occurrence and development of laryngeal carcinoma; and the lack of enhanced expression of autophagy on cell proliferation in vitro, study of autophagy gene Beclin1 in laryngeal carcinoma Hep-2 cells, apoptosis and sensitivity to cisplatin of Hep-2 laryngeal carcinoma cells, to explore the feasibility of autophagy induction in the treatment of laryngeal cancer in, to provide new ideas for gene therapy of laryngeal carcinoma.
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