【作者】 王旭；

【导师】 范平生；

【作者基本信息】 山东大学 ， 肿瘤学（专业学位）， 2021， 博士

【摘要】 研究背景:胃癌(gastric carcinoma)是消化系统最为常见的恶性肿瘤之一,其起源于胃腔内的粘膜上皮组织。据2021年资料数字统计,去年全球范围内新确诊胃癌的患者高达108万例,占比5.6%,位居第5位,因罹患胃癌而死亡的患者累计有76万例,高居恶性肿瘤死亡排行榜第4位。据2020年中国癌症年报统计,我国胃癌新发病几率和死亡人数均高居第三位。早期胃癌患者因无明显的症状通常被忽略,其具有隐蔽性且无特异性,可时隐时现,也可长期存在,等出现不适症状时就诊已确诊进展期,这也和由我国医学卫生资源的分配不平衡且癌症普查率较低有着密切关系,此时罹患胃癌的患者遗憾地错过了最佳的手术根治时机,临床上针对进展期胃癌患者则采用化学治疗、放射治疗、生物靶向精准治疗等综合治疗。同时由于胃癌对放疗的不敏感,而且放疗产生较多的副作用限制了放疗的使用,内科多线综合化疗已然成为治疗中晚期胃癌的主要手段之一,希望能以此延长患者的生命周期,提高患者的生活质量。对于早期胃癌患者,即使有机会行根治性手术,大约有80%的早期胃癌患者在手术根治治疗后的3年内出现局灶复发或远处脏器侵袭转移,其5年生存率低于30%。而中晚期胃癌的首选治疗方案是内科综合化疗,国内外多项研究表明:肿瘤多药耐药(multi drug resistance,MDR)是造成肿瘤内科化疗无效的关键因素。肿瘤多药耐药是指肿瘤细胞在一种抗癌药物治疗后所产生的耐药性,后期该肿瘤细胞对其它未曾接触过的、作用机制及分子结构完全不同的其它抗癌药物也同时产生交叉耐药的一种现象。肿瘤多药耐药分为天然耐药和获得性耐药。多因素共同导致的胃癌细胞对抗癌药物产生耐药性是当下治疗中晚期胃癌患者十分突出的问题。因此,如何克服肿瘤细胞的多药耐药,更进一步提高抗癌药物的疗效已然成为目前治疗肿瘤亟待解决的问题,进一步深入的研究胃癌细胞耐药所产生的分子机制、寻求能运用于临床的药物及方法,是研究胃癌治疗的重要契点。随着多种用于临床治疗的手段不断被发明和发展,特别是针对肿瘤治疗的方式也日益丰富,除手术、化疗、放疗外,新型的介入治疗学使肿瘤患者在整个治疗过程中受益。靶动脉灌注化疗是联合穿刺、插管,在X线引导下将导管精准地选择性插入瘤灶的供血靶动脉内,通过DSA对病灶的部位、数量、形态等进行分析、诊断后,将高浓度的抗癌药物通过导管精准地直接灌注于瘤体,而除瘤体外其它组织器官药物浓度低,极大地降低全身副反应。从19世纪80年代开始,有研究者开始尝试使用通过靶动脉精准局部灌注化疗药物以此来治疗食道恶性肿瘤,研究发现通过靶动脉灌注化疗药物的治疗方法能够显著提高抗癌治疗疗效,且副反应明显小于传统化疗。维拉帕米(Verapamil,VER)是一类Ca2+通道阻滞剂,是治疗心脏病的常见药物,同时维拉帕米也是早期发现的可以通过改变细胞内药物浓度从而降低肿瘤耐药性的药物。既往研究认为VER主要是通过抑制P-糖蛋白(P-glycoprotein,P-gp)的表达及活性来实现抗肿瘤多药耐药的,然而,近来多数研究发现VER还可通过其他非经典途径来增加其抗耐药性。体外实验表明,VER对多数肿瘤细胞具有抗耐药性,且有效作用浓度为6.0～10.0 μmol/L,但静脉用VER的安全浓度为1.0～2.0 μmoL/L,超出此范围则会导致心率下降、血压下降、房室传导阻滞等严重的不良反应,导致VER作为肿瘤耐药逆转剂不能在临床治疗晚期恶性肿瘤中广泛应用。本课题组通过前期犬实验证实,当VER在组织浓度是外周静脉血浓度的50-100倍时,VER能成功逆转肝恶性肿瘤的多药耐药,且实验犬未发生不良的心血管反应。本研究采用靶动脉灌注VER联合静脉应用抗癌药物治疗中晚期胃癌患者,结果表明能显著提高治疗疗效,但仍有近30%的患者治疗效果不理想。因此,寻找VER逆转肿瘤细胞耐药的关键基因,以期增强其疗效成为亟待解决的问题。有学者提出寻找凋亡相关因子,并验证其为肿瘤细胞多药耐药的新靶点。研究表明,抗凋亡基因Bcl-2在肿瘤耐药形成过程中表达量明显增高,导致肿瘤细胞的多药耐药。这些基因相互作用调控细胞的凋亡程序的同时,也在细胞耐药中也有一定影响。葡萄糖神经酰胺合成酶(glucosylcemmide synthase,GCS)是神经酰胺代谢合成葡糖脑苷脂的关键酶,其活性增高可导致肿瘤细胞获得性多药耐药的产生。本团队前期研究表明VER能够逆转部分晚期恶性肿瘤细胞的多药耐药。本研究通过筛选出有效预测VER逆转化疗耐药能力的个体差异的分子标志物,将为TACE+VER联合使用提供临床疗效预测手段和理论依据,对于完善VER个体化治疗有着重要意义。本课题组前期研究通过实时荧光定量PCR及Western blotting实验表明化疗联合VER通过抑制Bcl-2表达及活性从而促进恶性肿瘤细胞的凋亡、增强细胞对化疗敏感性,但其中的具体作用机制有待进一步深入研究,旨在寻找与VER逆转恶性肿瘤细胞耐药的关键基因,为靶动脉灌注VER治疗胃恶性肿瘤提供理论依据。第一章胃癌细胞hMDR1/P-gp表达水平与维拉帕米逆转化疗耐药能力相关性研究目的:探讨在胃癌细胞株中,hMDR1/P-gp水平与VER抗耐药能力差异的相关性。方法:1.实时荧光定量PCR和Western blot方法分别检测hMDR1/P-gp在3株胃癌细胞株(差分化人胃癌细胞株AGS、低分化人胃癌细胞株BGC-823和中分化人胃癌细胞株SGC-7901)中表达水平;2.CCK-8法分别检测不同胃癌细胞株对抗癌药物(5-Fu、ADM、DDP)的敏感性;3.CCK-8法分别检测胃癌细胞株对VER联合上述3种抗癌药物的药物敏感性;4.VER逆转胃癌化疗耐药能力的判断。结果:1.hMDR1/P-gp在基因和蛋白层面的表达水平在不同分化程度的胃癌细胞株中的表达水平差异明显,与SGC-7901相比,AGS中的表达量最高;2.BGC-823和AGS对顺铂耐药性大于SGC-7901;SGC-7901和AGS对阿霉素耐药性显著大于BGC-823,SGC-7901和AGS对5-氟尿嘧啶耐药性大于BGC-823;3.用固定浓度的VER(4.91μg/mL)联合抗癌药物分别处理胃癌细胞,均不同程度降低了胃癌细胞对3种抗癌药物的IC50值,提示VER提高了抗癌药物的敏感性;4.在SGC-7901细胞中,VER逆转ADM化疗耐药能力最强(Relative IC50=6.77),与BGC-823细胞相比有显著性差异(Relative IC50=1.66)。结论:hMDR1/P-gp水平与VER逆转胃癌细胞对ADM化疗耐药能力之间无相关一致性。第二章胃癌耐药细胞株的构建并验证其耐药效率目的:建立人胃癌SGC-7901/5-Fu耐药细胞株。方法:应用间歇作用体外诱导法构建胃癌耐5-Fu细胞株SGC-7901/5-Fu,反复短期暴露并逐步增加5-Fu的浓度,每月做相关检测,CCK-8法对药物的敏感性进行检测,并在耐药细胞株成功构建后第3天、第15天、后每间隔15天对其稳定性进行检测。结果:经历6个月建成人胃癌5-氟尿嘧啶耐药细胞株SGC-7901/5-Fu,并与ADM抗癌药存在交叉耐药。结论:SGC-7901/5-Fu细胞株具有耐药性,且耐药性稳定。第三章介导维拉帕米逆转胃癌细胞化疗耐药能力差异性基因的筛选、验证及机制研究目的:筛选出介导VER逆转胃癌细胞化疗耐药能力的差异基因并验证,初步探讨其作用机制。方法:1.通过生物信息学分析及文献资料筛选出可能的差异基因;2.实时荧光定量PCR/Western blot法检测候选基因/蛋白在胃癌细胞中的表达;3.GCS基因表达沉默或过表达后,CCK-8法检测VER逆转胃癌耐药细胞对ADM化疗耐药能力的变化;4.GCS基因表达沉默及过表达后,Annexin V-PI双染法检测VER促胃癌细胞凋亡能力的变化。结果:1.我们筛选出8个候选基因,分别为hMDR1、LRP、GST-π、GCS、TOPO Ⅱ、PrPc、MGr1-Ag、CIAPIN1;2.实时荧光定量PCR验证表明经过VER处理后,LRP、GCS、TOPOⅡ的表达存在差异,其中GCS差异最为明显(p<0.01);3.western blot验证经VER处理后GCS蛋白表达存在差异,结合实时荧光定量PCR结果,本研究着重从GCS出发,初步探讨其参与调控VER逆转耐药的可能机制;4.在SGC-7901和SGC-7901/5-Fu、SGC-7901/ADM细胞株中,过表达GCS基因(pEGFP-GCS),VER逆转胃癌细胞ADM化疗耐药能力明显减弱,表达沉默GCS基因(siR-GCS),VER逆转胃癌细胞ADM化疗耐药能力明显增强;5.过表达GCS基因(pEGFP-GCS)后,VER促进ADM胃癌细胞凋亡能力明显减弱,表达沉默GCS基因(siR-GCS)后,VER促进ADM胃癌细胞凋亡能力较前增强;6.western blot法检测沉默GCS基因(siR-GCS)后,VER促进ADM胃癌细胞凋亡蛋白Bcl-2及ERK表达量明显减弱。结论:GCS介导了 VER逆转胃癌细胞ADM化疗耐药能力差异,VER可能通过改变GCS基因表达水平,可明显改变其逆转ADM化疗耐药及促进细胞凋亡能力。第四章 临床研究P-gp、GCS蛋白在胃癌组织样本中的表达目的:临床研究P-gp、GCS蛋白在胃癌两组极端病例(治疗明显好转组/治疗疾病进展组)组织样本中的表达。方法:1.选取2014.1-2017.12入住我院就诊中晚期胃癌患者11例,均经胃镜技术提取病变标本,病理证实为胃癌,每位患者每月介入1次共介入2-4次,评估患者治疗疗效,分为两组:维拉帕米抗耐药治疗有效组(治疗明显好转病例,CR)7例,维拉帕米抗耐药治疗无效组(治疗疾病进展病例,PD)5例;2.免疫组化法检测P-gp、GCS蛋白在两组胃恶性肿瘤组织样本中的表达。结果:1.P-gp蛋白主要表达于癌细胞胞浆/胞膜中,有效组的P-gp蛋白平均光密度值与无效组比较无明显差异;2.GCS蛋白主要表达于癌细胞胞核/胞浆中,在癌组织中,有效组的GCS平均光密度值明显低于无效组(p<0.01)。结论:1.维拉帕米抗耐药治疗有效组P-gp蛋白表达水平与无效组比较无明显差异;2.维拉帕米抗耐药治疗有效组GCS蛋白表达水平明显低于抗耐药治疗无效组。更多还原

【Abstract】 Background:Gastric carcinoma is one of the most common digestive malignant tumors in the world,originating from gastric mucosal epithelium.According to the statistics of 2021,there are nearly 1.08 million new gastric cancer cases in the world,accounting for 5.6%of all new gastric cancer cases.There are about 760,000 cases of gastric cancer death every year,making it the fourth leading cause of cancer death worldwide.According to the statistics in 2020,the new incidence rate and mortality rate of gastric cancer in China are the third highest.Early gastric cancer patients often have no obvious clinical symptoms or atypical symptoms,with concealment,no specificity,can be hidden,can also exist for a long time.This is also closely related to the imbalance in the distribution of medical and health resources in China and the low cancer screening rate.At this time,patients with gastric cancer regrettably missed the best opportunity for radical surgery.At this time,they lost the best opportunity for surgical treatment,most advanced gastric cancer patients are treated by surgery combined with perioperative chemotherapy,radiotherapy and biological targeted therapy.At the same time,because of the insensitivity of gastric cancer to radiation and the side effects of radiotherapy,the use of radiotherapy is limited.Therefore,medical chemotherapy has become one of the main treatment for advanced gastric cancer,in order to prolong the survival period and improve patient quality of life.For patients with early gastric cancer,even if they have the opportunity to undergo radical surgery,at least 80%of patients will have local recurrence or distant metastasis within 2 to 3 years after surgery,and the 5-year survival rate is less than 30%.The preferred treatment for advanced gastric cancer is internal medicine chemotherapy.Studies showed that multiple drug resistance(MDR)is the main cause of chemotherapy failure.Tumor multi drug resistance refers to the phenomenon that after a drug acts on tumor cells and produces drug resistance,tumor cells also produce cross resistance to other chemotherapy drugs with different molecular structures and mechanisms.The multi drug resistance of tumor cells can be divided into natural resistance and acquired resistance.The drug resistance of tumor involves many mechanisms,such as the decrease of intracellular drug concentration,the change of drug target molecules,the metabolic detoxification,the imbalance of DNA damage repair function and so on.The resistance of gastric cancer cells to chemotherapeutic drugs caused by various reasons is an extremely difficult problem in multidisciplinary treatment of gastric cancer,which seriously restricts the survival of cancer patients.Therefore,overcoming multi drug resistance of tumor cells and improving the efficacy of chemotherapeutic drugs have become an urgent problem for cancer treatment.Further in-depth study of the molecular mechanism of drug resistance of gastric cancer cells and search for drugs and methods that can be used in clinical practice are important points for the study of gastric cancer treatment.With the continuous invention and development of a variety of methods for clinical treatment methods,especially for the treatment of cancer is also increasingly abundant.In addition to simple surgery,chemotherapy,radiotherapy,new interventional therapy makes tumor patients venefit in the entire treatment.Target artery infusion chemotherapy is the application of puncture,intubation technology,under the guidance of X-ray fluoroscopy angiography,the catheter will be accurately and selectively inserted into the related blood supply target artery of the tumor focus,through DSA to analyze and diagnose the location,quantity and shape of the focus,and then the high concentration of chemotherapy drugs will be accurately and directly infused into the focus through the catheter,which has strong lethality and high curative effect The distribution of drugs in unrelated tissues and organs is less,and the systemic side effects are very low.Since the 1880s,foreign doctors have tried to use target artery infusion drugs to treat esophageal cancer,and found that they can significantly improve the lethal effect of chemotherapy drugs on tumor tissues,and the toxic and side effects are significantly less than that of systemic meridional chemotherapy.Verapamil(VER)is a widely used clinical calcium channel blocker in heart disease,which has the effect of reversing MDR in tumor cells.It has been thought that anti-tumor drug resistance is mainly achieved through suppression of P-glycoprotein(P-gp)pathway.However,recently,a growing number of researchers have found that it increases the cells’ susceptibility to drugs through nonclassical pathways.Experimental studies show that VER can significantly improve the sensitivity of various tumor cells to chemical drugs.In-vitro study reported that 6.0-10.0 μmol/L VER concentration,higher than the safe vein concentration(1.0-2.0μmoL/L),can effectively reverse tumor drug resistance.Exceeding the safe vein concentration of VER causes sinus bradycardia,atrioventricular block,and other serious toxic and side effects,thus restricting its wide application in clinics as a reversal agent of tumor multi drug resistance.Previous animal experiments have shown that the concentration of verapamil in tissue is 50-100 times higher than that in peripheral blood,which can successfully reverse multi-drug resistance of tumors without causing adverse cardiovascular reactions.In our study,the target artery perfusion with verapamil combined with chemotherapy can significantly improved the efficacy of advanced gastric cancer,with an overall effective rate of up to 75%,but there were still some poor efficacy.Therefore,it is urgent to find the key genes of VER to reverse the drug resistance of tumor cells and enhance its efficacy.Most cytotoxic agents kill tumor cells by inducing apoptosis.The tolerance of tumor cells to apoptosis is one of the important mechanisms of MDR.Apoptosis related factors become the targets of drug resistance and can mediate MDR together with other pathways.Autophagy has dual effects on the drug resistance of tumor cells.On the one hand,apoptosis tolerance is an important mechanism of tumor drug resistance.Autophagy can promote tumor drug resistance by antagonizing apoptosis induced by chemotherapy drugs;on the other hand,autophagy may be a way of death of apoptosis tolerant tumor cells.Studies have shown that autophagy and apoptosis are closely related.Firstly,there is a cross between the two signaling pathways.For example,the interaction between Bcl-2 family members and autophagy protein Beclinl through BH3 domain prevents Beclinl from participating in the assembly of pre autophagosome and the binding of Bcl-2 and apoptosis protein,thus promoting apoptosis and inhibiting autophagy.In addition,ATG5 translocated from cytoplasm to mitochondria after calpain cleavage,and combined with Bcl-x to promote the release of cytochromec and promote apoptosis.Glucosylcemmide Synthase(GCS)is the key enzyme in the glycosylation of ceramide,and the increase of its activity can lead to the generation of tumor acquired multi-drug resistance.GCS can catalytic uridine diphosphate glucose sugar base combined with ceramide,make the ceramide into no cytotoxicity of glucose amide(GlcCer),GlcCer is decorated a series of glycosyl transferase,synthetic higher forms of sheath glycolipids,the sheath of sugar fat cells not only maintain the normal structure and function,is also involved in cell proliferation,differentiation,and can be against the apoptosis induced by drugs.Previous studies of our group showed that verapamil reversed the multi-drug resistance of some tumor cells.Several studies have suggested that verapamil competitiveness combines with P-gp to achieve reverse effects by acting as a competitive substrate for the drug resistant cell efflux pump and by increasing intracellular chemotherapeutic drug accumulation.In this study,molecular markers that can effectively predict individual differences in VER’s ability to reverse chemotherapy resistance are selected to provide a clinical efficacy prediction method and theoretical basis for the combined use of TACE+VER,which is of great significance for the improvement of verapamil individualized treatment.In our previous study,real-time PCR and Western blotting showed that the expression of Bcl-2 in verapamil combined with doxorubicin group was low.The specific mechanism of verapamil combined with chemotherapeutic drugs to enhance the sensitivity of gastric cancer cells to chemotherapeutic drugs by inhibiting Bcl-2 is not clear,so we continue to further study to find P-gp In addition to protein related genes,more genes related to the reversal of drug resistance of verapamil were identified,which provided a theoretical basis for the treatment of gastric cancer with verapamil combined with chemotherapy drugs via target artery infusion.Chapter 1.The correlation between the expression level of P-gp and the reversal of VER’s resistance to chemotherapy in gastric carcinoma cellsObjective:To investigate the correlation between hMDR1/P-gp level and the difference of VER resistance in gastric cancer cell lines.Methods:1.The expression levels of hMDR1/P-gp in gastric carcinoma cell lines(SGC-7901,BGC-823 and AGS)were detected by real-time quantitative PCR and western blot;2.The drug sensitivity of gastric carcinoma cell lines to three chemotherapeutic drugs(5-FU,ADM and DDP)was detected by CCK-8;3.The drug sensitivity of gastric carcinoma cell lines to verapamil combined with three chemotherapeutic drugs(5-FU,ADM and DDP)was detected by CCK-8;4.Evaluation of the ability of verapamil to reverse chemotherapy drug resistance in gastric carcinoma cells.Result:1.The expression level of P-gp was significantly different in GC cell lines.Compared with SGC-7901,the expression level of AGS was the highest.2.The IC50 values of DDP in BGC-823 and AGS are significantly higher than those in SGC-7901.The IC50 value of ADM and 5-Fu in BGC-823 is lower than those in the rest of the two cell lines.3.After VER is added,the IC50 values of DDP,ADM,and 5-Fu in the three cell lines(SGC-7901,BGC-823 and AGS)declined to different extents,indicating that VER can increase the sensitivity of the three aforementioned chemotherapeutics to different extents.4.The resistance of SGC-7901 to VER reversal ADM,which has significant difference with BGC-823(Relative IC50=1.66),is the strongest(Relative IC50=6.77).Conclusion:No simple correlation between the expression level of hMDR1/P-gp and VER resistance is found.Chapter 2.Construct gastric cancer drug-resistant cell line and verify its drug-resistant efficiencyObjective:To construct a 5-Fu resistant gastric carcinoma cell line.Methods:Drug concentration gradient increasing method combined with intermittent induction was used to establish the 5-Fu resistant sub-line of gastric carcinoma cell line SGC-7901/5-Fu.Relevant tests were performed monthly,and drug sensitivity was determined by CCK-8 method.Result:After 6 months,SGC-7901/5-Fu cell line was established,and it had cross resistance to ADM,DDP and other anticancer drugs.Conclusion:The SGC-7901/5-Fu cell line has the phenotypic characteristics of drug resistance,and drug resistance can be stable.Chapter 3.Screening,confirmation and mechanism study of differential genes mediated by VER in the reversal of chemotherapy resistance of gastric carcinoma cellsObjective:To screen the differential gene and confirm the differential resistance of VER to the chemotherapeutic drug resistance of gastric carcinoma cells,and to explore the mechanism of its action.Methods:1.Screening of possible differential genes by bioinformatics analysis and literature;2.Real-time quantitative PCR test of expressions of candidate genes in gastric carcinoma cells;3.Western blot test of expression of candidate proteins in gastric carcinoma cells;4.Changes in VER reversal of chemotherapy resistance following GCS upregulation or downregulation.5.Annexin V-FITC/PI double-staining test of gastric carcinoma cell apoptosis.Result:1.We found a total of 8 genes,namely,hMDR1,LRP,GST-π,GCS,TOPO Ⅱ,PrPc,MGrl-Ag and CIAPIN1.2.The qRT-PCR showed that the expression of LRP,GCS and TOPO II was different after VER treatment.The GCS gene was the most significant difference(p<0.01).So this research focuses on the possible mechanism of VER resistance reversal from GCS.3.Western blot verified the differences expression of GCS protein after treatment with VER.Combined with qRT-PCR results,we focus on the possible mechanism of VER resistance reversal from GCS.4.In the SGC-7901 and SGC-7901/5-Fu cells,overexpression of the GCS gene(pEGFP-GCS),VER reversal of ADM chemotherapy resistance was significantly enhanced.While downregulation of GCS gene(siR-GCS),VER reversal of ADM chemotherapy resistance significantly reduced.5.VER promotes the apoptosis of gastric carcinoma cells obviously in the presence of ADM which overexpression of GCS genes.Silencing of GCS gene(siR-GCS)expression,the cell apoptosis rates were reduced by VER.6.After overexpression of GCS gene(pEGFP-GCS),the expression levels of Bcl-2 and ERK were significantly reduced by VER.Conclusion:GCS mediated the difference in VER’s ability to reverse ADM chemotherapy resistance in gastric cancer cells,and VER may significantly alter its ability to reverse ADM chemotherapy resistance and promote cell apoptosis by changing the expression level of GCS gene.Chapter 4.Clinical study on the expression of P-gp and GCS in tissue samples of gastric cancer casesObjective:The expression of P-gp and GCS genes in the tissue samples of extreme cases of gastric carcinoma(treatment group/disease progression group).Methods:1.A total of 11 cases of gastric carcinoma in middle and terminal stages were selected in our hospitals.Lesion samples were extracted using endoscopy,and all cases were diagnosed as gastric carcinoma.Each patient received one intervention every month or a total of two to four interventions.The therapeutic effects of these interventions to the patients were evaluated into two groups,namely,7 cases of VER effective group(high cure rate,CR)and 5 cases of VER ineffective group(progression of disease,PD).2.P-gp,GCS protein expression test in cancer tissue samples of GC patents by immunohistochemical method.Result:1.The P-gp protein was mainly expressed in the cytoplasm of cancer cells.In the cancer tissue and adjacent tissues,the average P-gp protein density of the effective group and the ineffective group(p>0.05).2.The GCS protein is mainly expressed in the nucleus and cytoplasm of cancer cells.In cancer tissues,the IOD/area of P15 of the VER effective group is significantly higher than that of the VER ineffective group(p<0.01)Conclusion:1.There was no significant difference in the P-gp protein expression between the effective group and the ineffective group in verapamil resistance treatment.2.The expression level of GCS protein in verapamil resistance treatment effective group was significantly lower than that in verapamil resistance treatment ineffective group.更多还原