益气化瘀解毒方对MRP、GST-π和Topo Ⅱ基因在Sorafenib获得性耐药人肝癌QGY7702细胞表达的干预研究

目的探讨益气化瘀解毒方干预后对Sorafenib获得性耐药人肝癌QGY7702细胞(QGY7702/Sora)增殖及MRP、GST-π和Topo Ⅱ基因表达的影响。方法培养QGY7702/Sora细胞和QGY7702细胞,利用Cell Counting Kit-8(CCK-8)法检测Sorafenib对细胞的半数抑制率浓度(IC50值),计算耐药指数RI;观察益气化瘀解毒方对耐药细胞的增殖影响;采用荧光定量PCR检测药物干预前后2种细胞中MRP、GST-π和Topo Ⅱ基因表达水平。结果亲本细胞和耐药细胞Sorafenib的IC50值分别为(7.993±0.522)μmol/L和(19.651±1.216)μmol/L,RI约为2.5。益气化瘀解毒方可抑制耐药细胞的增殖活性。2种细胞的MRP、GST-π、Topo Ⅱ表达量无明显差异(P>0.05)。Sorafenib组可促进耐药细胞MRP 、GST-π基因的过表达(P<0.05),益气化瘀解毒方组可抑制GST-π基因的过表达(P<0.01),且联合Sorafenib可显著提高Topo Ⅱ基因的表达量(P<0.01)。结论 QGY7702/Sora细胞MRP、GST-π和Topo Ⅱ的表达水平与亲本细胞无显著差异。耐药细胞对Sorafenib敏感性降低与MRP、GST-π过表达相关,而益气化瘀解毒方拮抗Sorafenib耐药与抑制GST-π过表达相关。     

重组 GST-β-GT 融合蛋白的原核表达、纯化及活性鉴定

目的：构建噬菌体β‐GT蛋白的原核重组体系,诱导表达、纯化GST‐β‐GT 融合蛋白并对其进行酶活性测定。方法利用PCR技术从T4噬菌体中扩增β‐GT基因;经T‐A克隆,获得β‐GT基因片段,经酶切连接,构建原核表达载体pGEX‐6P‐1‐β‐GT ;经测序鉴定序列正确后,将所构建的载体转化进入大肠埃希菌BL21（DE3）中进行表达。利用IPTG诱导,经10％ SDS‐PAGE鉴定目的蛋白表达后,采用GST柱纯化目的蛋白;通过酶切和qPCR鉴定其活性。结果成功扩增了噬菌体β‐GT基因;重组质粒在大肠埃希菌BL21（DE3）中诱导表达出GST‐β‐GT融合蛋白;通过GST柱纯化后获得了GST‐β‐GT融合蛋白,纯度达95％;通过酶切和qPCR验证,此GST‐β‐GT融合蛋白具有T4‐β‐GT酶的活性。结论。原核表达GST‐β‐GT融合蛋白具有糖基转移酶活性。     

多耐药基因在乳腺癌组织中的表达及价值

目的：观察多药耐药基因（MDR1）及其编码的糖蛋白（P－gp）在乳腺癌组织中的表达及临床价值。方法：选取病历资料完整的乳腺癌根治术乳腺组织标本84份,使用S－P 法免疫组化法染色检测P－gp、谷光甘肽S转移酶（ GST－π）及热休克蛋白（ HSP70）、p53的表达情况,采用Kendall＇s taub相关性分析P－gp、p53、GST－π、及HSP70、p53之间协同表达关系。结果：P－gp 表达阳性率为50％（42／84）,HSP70表达阳性率为61．9％（52／84）,p53表达阳性率为52．4％（44／84）,GST－π表达阳性率为40．5％（34／84）;相关性分析显示,GST－π与P－gp无协同表达（r＝0．146,P＞0．05）,HSP70与p53有协同表达（r＝0．626,P＜0．05）,p53与P－gp有协同表达（r＝0．763,P＜0．05）;P－gp表达对对乳腺癌10年生存率有明显影响。 COX模型乳腺癌预后多因素分析表明,年龄因素、GST－π表达均不具有独立的预后意义;临床分期、组织学分级、P－gp 表达、p53表达、HSP70表达均具有独立的预后意义。结论：同时检测MDR1、HSP 7、p53和GST－π的表达,可为乳腺癌临床耐药性的判断提供更为全面的信息。     

奥替普拉在阻断细胞恶性转化时对某些基因表达的影响

奥替普拉（ｏｌｔｉｐｒａｚ）来源于十字花科（Ｃｒｕｃｉｆｅｒａｓｅ）蔬菜，全称５（２吡嗪基）４甲１，２２羟基３硫酮，文献报道它为一种防癌抑癌剂［１］。我们用致癌素香烟凝聚物（ｃｉｇａｒｅｔｅｓｍｏｋｉｎｇｃｏｎｄｅｎｓａｔｅＣＳＣ）诱发...     

肿瘤多药耐药蛋白MRP1、LRP和GST-π在胆囊和胆管癌组织中的表达

目的 探讨多药耐药(MDR)基因产物多药耐药相关蛋白(MRP1)、肺耐药蛋白(LRP)和谷胱甘肽S-转移酶-π(GST-π)在胆囊癌和胆管癌中的表达及其与耐药的关系.方法 采用免疫组化的方法(IHC)检测实验组18例胆囊癌、36例胆管癌及对照组13例胆囊胆管炎症组织中MRP1、LRP及GST-π的表达情况,采用x2检验和Spearman检验. 结果 MRP.、LRP和GST-π在胆囊癌组织中的表达阳性率分别72%(13/18)、78%(14/18)和61%(11/18),在胆管癌组织中的表达阳性率为86%(31/36)、75%(27/36)和69%(25/36),显著高于对照组的23%(3/13)、23%(3/13)和23%(3/13)(x2=4.5,P＜0.05).LRP在胆管癌年龄大于60岁组的表达阳性率93%(13/14)显著高于小于60岁组的64%(14/22)(X2=3.9,P＜0.05).上述指标与性别、年龄、病理分期、病理类型、分化程度和淋巴结转移均无明显相关(P>0.05);MRP,和GST-π在胆囊癌和胆管癌的表达具有相关性,Spearman系数=0.569(P＜0.05).结论 MRP.、LRP、GST-π在未经过化疗的胆囊癌和胆管癌组织中均有不同程度的高表达;胆囊癌和胆管癌的原发性多药耐药可能与MRP1、LRP、GST-π有关.     

耐药癫痫患者和难治性癫痫大鼠脑组织与外周血GST-π蛋白表达

目的考察耐药癫痫患者和难治性癫痫大鼠脑组织与外周血谷胱甘肽S-转移酶π(glutathione S-transferaseπ,GST-π)的表达。方法临床收集2010年1月至2014年3月在我院神经外科行手术治疗的耐药性癫痫患者32例,选择同期因脑血管畸形手术切除的10例正常脑组织为对照;动物试验:收集15只对照组和20只难治性癫痫大鼠组,难治性癫痫依据锂-匹罗卡品癫痫模型制备。检测人与大鼠外周血和脑的GST-π水平表达,并进行组间比较。结果耐药癫痫患者脑组织的颞叶20例,额叶6例,枕叶6例。GST-π在耐药癫痫患者和难治性癫痫大鼠脑的颞叶、额叶、枕叶中均有表达,存在于细胞胞浆,染成浅黄色至棕黄色颗粒,与对照组比,耐药癫痫患者和难治性癫痫大鼠脑和外周血GST-π表达明显增加(P0.05)。结论 GST-π可能参与耐药癫痫的发病,检测外周血GST-π为耐药性癫痫的治疗提供参考。     

Ligustrazine derivate DLJ14 reduces multidrug resistance of K562/A02 cells by modulating GSTπ activity

Multidrug resistance (MDR) of tumor cells is a major obstacle in chemotherapeutic cancer treatment. Over-expression of glutathione S-transferase π (GSTπ) is one of the mechanisms contributing to MDR. In this study, we investigated the reversal of MDR by DLJ14, a ligustrazine derivate, in adriamycin (Adr) resistant human myelogenous leukemia (K562/A02) cells by modulating the expression of GSTπ and the activity of GST-related enzymes. In the MTT test, DLJ14 showed a weak inhibition on proliferation of both K562/A02 and K562 cells, while verapamil at the same concentration showed a much stronger inhibition. The sensitivity of K562/A02 cells to cytotoxic killing by Adr was enhanced by incubation with DLJ14 as a result of the increased intracellular accumulation of Adr. The accumulation of Adr induced by DLJ14 may due to down regulation of GST-related enzyme activity. Western blot analysis and RT-PCR showed that DLJ14 was able to inhibit the protein expression and mRNA expression of GSTπ in K562/A02 cells. Moreover, DLJ14 increased the expression of cellular c-Jun NH₂-terminal kinase (JNK) in K562/A02 cells exposure to Adr. This is consistent with the inhibition of GSTπ. These results demonstrate that DLJ14 may be an attractive new agent for the chemosensitization of cancer cells.     

The potential of carcinoembryonic antigen, p53, Ki-67 and glutathion Stransferase-π as clinico-histopathological markers for colorectal cancer

Objective： Colorectal cancer is one of the major contributors to cancer death worldwide. Lack of reliable colorectal cancer markers has hampered the management of these cancer patients. Our main purpose was to study the correlation between histopathological variables of colorectal adenocarcinomas and identify histopathological markers that are of prognostic value in patients with colorectal cancer. Methods： In the present study, we examined the expression of carcinoembryonic antigen （CEA）, p53, Ki-67 and glutathion Stransferase （GST） -n by using immunohistochemical staining methods in 126 colorectal carcinoma patients and evaluated the lymph node metastasis status in these patients by histopathological examination. Results： The positive rates of CEA, p53, Ki-67 and GST-π expression in the colorectal cancer tissue specimens examined were 95.23%, 55.56%, 53.38% and 82.30%, respectively. Expression of p53 and Ki-67 was significantly correlated with the Dukes stages of the tumor, with higher levels of these proteins in Dukes＇ C and D tumors than those in Dukes＇ A and B tumors. Furthermore, the expression of p53, GST-π and Ki-67 correlated with prognosis of patients with colorectal cancer. Additionally, the expression of p53 in colorectal cancer was closely related to the expression of Ki-67 and the expression of GST-π was directly correlated with that of p53. Conclusion： The expression of CEA, p53, Ki-67 and GST-π was correlated with various clinical features of patients with colorectal cancer. The combined use of these histopathological markers appeared to be a promising tool in predicting the prognosis of patients with this type of cancer.     

GST-π在弥漫性大B细胞淋巴瘤中的表达及临床意义

目的：探讨谷胱甘肽S转移酶π（GST-π）在弥漫性大B细胞淋巴瘤（Diffuse large B cell lymphoma,DLBCL）组织中的表达状况及其与临床特征和疗效的关系.方法：采用免疫组织化学方法检测63例初治DLBCL患者肿瘤组织中GST-π的表达状况,分析该基因的表达状况与DLBCL临床特征、疗效等的关系.结果：GST-π在不同性别、年龄、B症状、结外病变间表达率差异无统计学意义（P＞0.05）,各临床分期GST-π表达率为Ⅰ期55.6％、Ⅱ期58.3％、Ⅲ期64.3％、Ⅳ期75.0％,各分期间比较差异无统计学意义（P＞0.05）;不同国际预后指数（IPI） GSTπ表达率为低危组61.1％、中低危组70.6％、高中危组70.6％、高危组63.7％,各组间比较差异无统计学意义（P＞0.05）;GST-π表达阳性组和阴性组,初治完全缓解（CR）率为28.6％ vs 66.7％,总有效（RR）率为47.6％ vs 90.5％,GS-Tπ表达阳性组CR率及RR率显著低于GST-π表达阴性组（P＜0.05）.结论：GST-π表达与性别、年龄、B症状、结外病变、临床分期、IPI无相关;近期疗效GST-π阴性组优于阳性组.     

Identification of postoperative adjuvant chemotherapy responders in non-small cell lung cancer by novel biomarker

Cisplatin-based (CDDP-based) adjuvant chemotherapy of non-small cell lung cancer (NSCLC) was reported to yield 5-15% improvement in 5-year survival compared to complete resection alone. The importance of information concerning preselection of good responders has become increasingly evident. The purpose of our study is the establishment of a preselection of good responders for CDDP-based adjuvant chemotherapy. We investigated protein expressions comparing intensity between parent strains (H69 and PC14 lung cancer cultured cells) and resistant strains against CDDP using 2-dimensional polyacrylamide gel electrophoresis (2-DE). Immunohistochemically, we evaluated the relationship between protein expression associated with CDDP-resistance and the clinical effects of platinum-based postoperative adjuvant chemotherapy using 126 surgically-resected NCLC materials. We detected 2 kinds of polypeptides that changed expression levels on 2-DE gels. The analyses of the amino acid sequence showed that these polypeptides were reticulocalbin (RCN) and glutathione-S-transferase-pi (GST-pi). The 2-DE analysis showed decreased expression in RCN and overexpression in GST-pi with the acquisition of CDDP-drug resistance. RCN-transfectant of H69 CDDP-resistant strain showed intermediate sensitivity between the parent strain and the CDDP-resistant strain. RCN-positive cases showed a statistically significant better disease-free survival only in the cases receiving postoperative platinum-based adjuvant chemotherapy after curative resection (p = 0.007). In addition, cases that were both RCN-positive and GST-pi-negative showed a statistically significantly better outcome (p = 0.0150). In the cases without postoperative adjuvant chemotherapy no relationship between the outcome and these expressions was seen. The evaluation of RCN and GST-pi might provide valuable information concerning postoperatively therapeutic strategy from the standpoint of individualized postoperative therapy.