局部治疗对残留肝细胞癌恶性生物学特性的影响及KLF6-KLF6-sv (TNF-a)-HSPs通路在其中的触发机制

The triggering mechanism of enhanced difficulty for therapy on the recurrent hepatocellular carcinoma (HCC) is currently not clear. Recent researches show that the malignant biological character (MBC) of tumor tissue is closely linked to the inactivation of antitumor gene KLF6 in the form of its alternatively spliced isoform KLF6-SV. Our former research found that: For HCC, KLF6 can be further inactivatedly expressed into KLF6-SV by local therapy such as local embolization, heat therapy; The expression of some members of heat shock protein (HSPs) and KLF6-SV is closely correlated and co-localized in the same cell; The MBC of HCC can partly be inhibited by tumor necrosis factor (TNF-a). Basing what mentioned above, this study suppose to observe the MBC of post local therapy residual HCC, two-aspectly manifest the role of KLF6-sv, HSPs related pathway and the influence of TNF-a on them through inference, stimulate, quantity, localize their expression. The ultimate purpose is to demonstrate the supposition that the enhanced MBC of residual HCC can be induced by local therapy and its molecular mechanism of KLF6-KLF6-sv (TNF-a)-HSPs pathway. This study is supposed to find a new effective target pathway for thorough HCC therapy.

肝细胞癌 (HCC)治疗复发后再次治疗难度增加，诱发机制不详。新近研究发现肿瘤恶性生物学特性与抑癌基因KLF6表达失活为其剪接异构体KLF6-sv关系密切。我们前期发现：栓塞化疗、热疗等局部治疗可促进残癌细胞内KLF6表达失活为KLF6-sv； KLF6-sv与HSPs（热休克蛋白）部分成员表达高度相关且来源同一细胞；肿瘤坏死因子a （TNF-a）可抑制残癌的部分恶性生物学特性。基于此，本研究拟对体内外HCC模型行热疗等局部治疗，观察残癌细胞转移、多药耐药等多种恶性生物学特性的变化；通过干预、刺激、定量、定位胞内KLF6、KLF6-sv、HSPs主要成员表达及TNF-a对相关通路的影响，正反两方面明确其相互关系。最终论证局部治疗通过对残留HCC恶性生物学特性的影响增加再次治疗的难度及KLF6-KLF6-sv (TNF-a)-HSPs通路在其中的触发作用，为彻底治疗HCC寻找高效靶点。

肝细胞癌 (HCC)治疗复发后再次治疗难度增加，诱发机制不详。本研究首先对体内外肝癌模型进行肿瘤切除、栓塞化疗、热疗三种局部治疗，发现残癌细胞在治疗后表现出更强的平板克隆形成、转移性及血管生成能力，realtime PCR及western blot检测发现残癌细胞内KLF6-sv1和HSP70在mRNA和蛋白水平均显著表达升高，同时KLF6却表达下降；进一步转染真核表达及干扰质粒,正反两反面干预、刺激胞内KLF6、KLF6-sv表达，发现KLF6-sv的高表达或KLF6的低表达均可促进HSP70在细胞内的高聚集；再通过激光共聚焦观察发现：KLF6-sv1、HSP70高表达与KLF6低表达高度相关且来源同一细胞，从而在正反两方面明确KLF6-异构为KLF6-sv1，促进HSP70聚集是局部治疗触发细胞恶性生物学升级的重要原因。最后我们建立了体内外稳定高表达KLF6-sv1的高转移肝癌模型，应用rmhTNF-α（重组改构人肿瘤坏死因子）对其治疗发现：rmhTNF-α对Vx2、 HepG2及HepG2/ KLF6-sv1、Vx2/ KLF6-sv1均可提高其凋亡坏死率，降低其转移及生长作用，但这种作用在HepG2/ KLF6-sv1、Vx2/ KLF6-sv1更为明显；基因定量检测发现：rmhTNF-α并不能改变胞内KLF6或KLF6-sv1的表达，却可降低胞内HSP70的表达，提示了rmhTNF-α可阻断KLF6-sv1对HSP70的上调表达，达到抑制肿瘤生长及转移到作用。总之，本研究发现：① 传统治疗如手术、热疗、栓塞治疗等剧烈治疗手段本身会因为治疗不彻底及对残癌的剧烈刺激，从而可能通过进化筛选的方式激发了肿瘤的恶性潜能，触发肝癌的复发转移。② KLF6—KLF6-sv1—HSP70通路在以上肿瘤恶性生物学升级中有重要触发机制，从而可能成为治疗HCC的高效靶点。③ rmhTNF-α可通过阻断KLF6—KLF6-sv1—HSP70通路降低肿瘤的恶性生物学行为，提示其与传统治疗手段联合，可能会有更好的治疗效果。以上新发现对于防止治疗后肝癌复发转移具有重要意义。

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