扶正增效方对S100A9与CyPA在非小细胞肺癌放射增敏作用的调控机制

Genic and molecular targeted radiosensitizer drugs have been a hotspot in radiosensitizer study. Present choices of this kind drug aren’t in accordance with sensitive tissue predictors, so the curative effect varies greatly. Our earlier research verified that the genes S100A9 and CyPA were two radiosensitive targets in treating lung adenocarcinoma. However, more researches need to be done on whether the radiosensitizing effects of these two genes are synergistic or independent of each other and how Chinese herbs regulate radiosensitizing effects of these two genes in non-small cell lung cancer (NSCLC) radiation treatment. In order to investigate the different radiosensitizing effects of no expression and high expression of the two genes, We plan to knock out the related genes(S100A9,CyPA,S100A9 & CyPA) through CRISPR/Cas9 technique and transfect the related genes by retrovirus. Furthermore, we can observe the genes’ expression in NSCLC with different radiosensitivity and determine the radiosensitizing relationships between the two genes. Besides, we plan to observe the radiation biologic changes before and after using Fuzheng Zengxiao Formula in NSCLC of different pathology, which is knocked out the related genes through CRISPR/Cas9 technique, so as to investigate its possible regulation mechanism. This research will establish theoretical basis in pinpointing NSCLC radiosensitive predictors, promoting research and development of targeted radiosensitizers and animal models.

基因和分子靶向放射增敏药物是放射增敏剂研究热点，目前该类药物不是根据组织放射敏感预测因子选择,疗效差别较大。前期研究证实S100A9、CyPA是肺腺癌中药放射增敏2个靶点。但这两个基因在非小细胞肺癌(NSCLC)放射增敏作用是协同还是相互独立，中药对这两个基因在放射增敏中调控机制如何,有待深入研究。本研究拟通过CRISPR/Cas9技术敲除相关基因（S100A9、CyPA、S100A9_CyPA）、逆转录病毒法转染相关基因，研究这些基因无表达与高表达时放射增敏比，以及观察放射敏感性不同NSCLC的S100A9和CyPA表达情况，确定S100A9与CyPA在放射增敏中相互作用关系;选用不同病理类型NSCLC，用扶正增效方对比观察CRISPR/Cas9技术敲出相关基因前后体内体外放射生物学实验，探讨其调控机制。此还为明确肺癌放射敏感性预测因子、促进靶向放射增敏药物与动物模型研发奠定理论基础.

研究背景：基因和分子靶向放射增敏药物是放射增敏剂研究热点，目前该类药物不是根据组织放射敏感预测因子选择,疗效差别较大。前期研究证实S100A9、CyPA是肺腺癌中药放射增敏2个靶点。但这两个基因在非小细胞肺癌(NSCLC)放射增敏作用是协同还是相互独立，中药对这两个基因在放射增敏中调控机制如何,有待深入研究。研究内容：①利用CRISPR/Cas9 基因敲除技术建立人肺腺癌H1975细胞Cypa基因敲除细胞系、S100A9基因敲除细胞系CyPA-S100A9基因敲除细胞系；②通过体内体外实验研究 S100A9、CyPA、S100A9_CyPA 无表达与放疗敏感性的关系；③利用逆转录病毒法稳转细胞构建H1975细胞 S100A9、CyPA、S100A9_CyPA 高表达模型；④通过体内体外实验研究 S100A9、CyPA、S100A9_CyPA 高表达与放疗敏感性的关系；⑤观察放射敏感性不同的肺癌细胞株放射后 S100A9 及 CyPA 表达变；⑥观察中药扶正增效方对肺腺癌(H1975) S100A9、CyPA、S100A9_CyPA基因敲除细胞系放射增敏的作用。研究结果：①成功构建肺腺癌细胞H1975 CyPA基因敲除、S100A9基因敲除、CyPA-S100A9基因敲除、CyPA过表达、S100A9过表达、CyPA-S100A9过表达细胞系；②CyPA和S100A9在肺腺癌放射敏感性中存在独立关系，敲除CyPA提高肺腺癌放射敏感性，但S100A9表达对肺腺癌放射敏感性无明显作用，CyPA可能作为NSCLC放射增敏的作用靶点；③CyPA表达与肺腺癌放射敏感性有关，可以依据CyPA的表达预测肺腺癌放射敏感性；④扶正增效方可增加放射敏感性，CyPA是扶正增效方起效靶点之一。