RNAi沉默肺腺癌A549细胞系PD-L1基因表达对其增殖和凋亡的影响

目的:沉默肺癌细胞系A549的程序性死亡配体1(programmed death ligand 1,PD-L1)基因,观察其对A549细胞增殖和凋亡的影响。方法:构建PD-L1 shRNA重组质粒p GPU6/PD-L1,并应用脂质体转染法将其转染入A549细胞。RT-PCR法检测PD-L1基因的表达;Western blotting法检测PD-L1蛋白的表达;MTT法检测p GPU6/PD-L1对A549细胞增殖的影响;AnnexinⅤ-FITC/PI双染法检测p GPU6/PD-L1对A549细胞凋亡的影响。结果:成功将p GPU6/PD-L1转染入A549细胞;RT-PCR结果显示p GPU6/PD-L1使A549细胞PD-L1基因表达水平降低;Western blotting结果显示p GPU6/PD-L1转染A549细胞使PDL1蛋白表达减少;MTT结果显示p GPU6/PD-L1能够抑制A549细胞增殖;AnnexinⅤ-FITC/PI双染法检测结果显示p GPU6/PD-L1能够诱导A549细胞凋亡。结论:p GPU6/PD-L1能够下调肺癌细胞A549 PD-L1的表达,抑制细胞增殖,并诱导细胞凋亡。     

Modulatory effects of adiponectin on the polarization of tumor‐associated macrophages

The plasticity of macrophages with selective functional phenotypes partially arises in respective to their microenvironment. Tumor‐associated macrophages (TAMs) may promote disease progression with tumor specific manner. Here we report that in pediatric malignant soft‐tissue tumors, the presence of TAMs and expression of adiponectin (APN) are heterogeneous. Both APN and TAMs had high expression in rhabdomyosarcoma, especially in the malignant subtype, alveolar rhabdomyosarcoma. To investigate the mode of action of APN on TAM activation, a murine MN/MCA1 sarcoma model was used. The Results revealed that exogenous APN had no effect on MN/MCA1 proliferation but tumor size was markedly reduced in apn^?/? mice versus WT controls. The accumulation of TAMs in apn^?/? mice was also reduced which correlated to downregulated serum levels of MCP‐1. Likewise, TAMs in apn^?/? mice exhibited a M1‐like phenotype, characterized by increase in MHC II^high population and M1 phenotypic markers, such as iNOS gene and serum TNF‐α accompanied by a decrease in M2 markers, namely YM1 gene and serum IL‐10. In addition, APN deficiency increased the number of CD4⁺ T cells, CD8⁺ T cells and NK cells in tumors and reduced tumor metastasis. The altered phenotype of TAMs in apn^?/? mice was associated with a marked decrease in phospho‐p38 and treatment with a p38 MAPK inhibitor significantly reduced tumor size and increased MHC II expression on TAMs in WT mice, implying p38 MAPK signaling pathway may contribute to APN‐mediated TAM polarization. Collectively, our findings suggest that APN may have a potential role in regulating soft tissue sarcoma growth.     

紫外线诱导体外培养的LOVO、HT-29细胞的凋亡

目的研究紫外线照射体外培养的大肠癌ＬＯＶＯ和ＨＴ┐２９细胞后，诱导这两株大肠癌细胞系发生凋亡的作用。方法紫外线照射两株大肠癌细胞系后２４～３６ｈ，通过琼脂糖凝胶电泳、ＨＥ染色和流式细胞仪检测两株细胞发生凋亡的情况。结果两株大肠癌细胞在形态学上出现典型的细胞核固缩、碎裂，琼脂糖凝胶电泳显现特征性的“梯状”带，流式细胞仪分别在２４和３６ｈ出现亚二倍体峰。结论紫外线具有杀灭癌细胞的作用，其机制与诱导细胞凋亡有关；其方法简单、易行，可作为研究细胞凋亡的较好的体外模型。     

Frequent hypermethylation of RASSF1A and TSLC1, and high viral load of Epstein-Barr Virus DNA in nasopharyngeal carcinoma and matched tumor-adjacent tissues

We examined the promoter hypermethylation of tumor-suppressor genes RASSF1A and TSLC1, quantitated EBV DNA load in nasopharyngeal carcinoma (NPC) tissues (T tissues), and matched tumor-adjacent tissues outside 0.5 cm (P tissues) and outside 1.0 cm (Z tissues) to evaluate the role of promoter hypermethylation of RASSF1A and TSLC1 as well as viral load in the pathogenesis of NPC. Methylation-specific polymerase chain reaction (PCR) for RASSF1A and TSLC1 and quantitative real-time PCR analysis of EBV DNA were performed on matched T, P, and Z tissues (n = 28) as well as chronic nasopharyngitis tissues (n = 8). Hypermethylated RASSF1A was frequently detected in the T (82%) and P tissues (75%), but less frequently in Z tissues (46%). he average quantities of EBV DNA (copies/microg DNA) in matched T, P, and Z tissues were 673,000, 90,000, and 7000. The differences of promoter hypermethylation of RASSF1A and EBV viral load among T, P, and Z tissues were statistically significant, with more frequent methylation and higher viral load detected when tissues examined were nearer to the NPC tissues. Our results suggest that aberrant hypermethylation of RASSF1A and high EBV load might be important events in NPC pathogenesis, and they may be useful molecular diagnostic markers for this cancer.     

NEDD9在食管鳞状细胞癌中表达的意义

目的:探讨NEDD9在食管鳞癌中表达的意义及与肿瘤发生发展和转移的关系。方法:采用免疫组化方法及Real-time PCR技术检测NEDD9在40例食管鳞癌癌组织、癌旁正常组织中的表达情况,并分析NEDD9的表达与肿瘤临床病理特征之间的关系。结果:食管癌组织与癌旁正常组织中NEDD9蛋白阳性表达率分别为77.5%、25.0%,mRNA表达CT值分别为1.704±0.360、0.843±0.526,食管癌组织中NEDD9蛋白及mRNA水平均明显高于癌旁组织及正常食管组织。NEDD9的表达与患者年龄、性别无关(P>0.05),与肿瘤组织分化程度、TNM分期及有无淋巴结转移有关(P<0.05)。结论:NEDD9在食管癌组织中表达明显高于正常癌旁食管组织,可能促进食管癌的侵袭及转移,可作为食管癌治疗的潜在靶点。     

扶桑花抗生育成分对早孕小鼠黄体影响的定量研究

本文处用图像分析仪 ,以核浆比和数密度为指标 ,研究了不同浓度的扶桑花提取物—HR 1对早孕小鼠黄本组织学的影响。结果表明 :(1)小鼠黄体细胞的核浆比和数密度 ,随给药剂量 (0、 4、 10、 10 0、10 0 0mg/kg/d)的增加而增加 ,其中 10 0和 10 0 0mg/kg/d两组的黄体细胞核浆比和数密度明显高于对照组(p <0 .0 5) ;(2 )黄体组织学的定性观察显示 ,给药各组的黄体细胞明显退化 ,细胞缩小 ,细胞界限不清。这些结果提示 ,扶桑花提取物的抗早孕作用与妊娠黄体受损有关。     

携带靶向沉默LSD1基因表达shRNA慢病毒载体的构建与鉴定

目的:构建慢病毒干扰LSD1表达的载体并鉴定。方法:设计并合成3对针对LSD1基因的shRNA链,退火形成双链,与酶切的质粒连接,转化DH5a菌株,提取质粒,进行酶切鉴定和测序分析。脂质体法转染人胃癌MKN-28细胞,通过荧光倒置显微镜判定转染效率,并通过real time-PCR法检测细胞中LSD1基因的表达水平。结果:经酶切鉴定筛选出的重组质粒测序结果与目的序列相同,重组质粒构建成功;转染胃癌MKN-28细胞后,LSD1基因在mRNA水平的表达降低。结论:成功构建了靶向LSD1基因的shRNA慢病毒载体,并筛选出1种对LSD1基因有显著抑制作用的shRNA。     

Second primary neoplasms after 19281 endocrine gland tumours: aetiological links?

The nationwide Swedish Family-Cancer Database of 9.6 million individuals was used to analyse the development of second neoplasia after 6909 thyroid and 12697 other endocrine tumours. Tumour cases were retrieved from the Swedish Cancer Registry from 1958 to 1996. The risk of a second endocrine tumour was markedly increased compared with first endocrine tumour; e.g. the standardised incidence ratios (SIRs) were well over 10 for adrenal tumours after thyroid cancer, and vice versa. Familial risks were higher for the development of second compared with first neoplasms, and the SIRs for men were usually higher than those for women. Many increases between different endocrine glands can probably be ascribed to known cancer syndromes. Even cancers at some other sites were increased after the development of primary endocrine tumours. Notably, small intestinal carcinoids were increased after thyroid and other endocrine tumours, and brain menigiomas were increased after parathyroid and pituitary adenomas. These novel associations suggest shared risk factors for these sites. However, many endocrine tumours are benign and the diagnosis of the first tumour may increase the likelihood of a second diagnosis.     

急性髓系白血病17种基因异常的检测

目的:探讨应用多重巢式RT-PCR、荧光定量PCR、PCR-SSCP银染技术检测初诊急性髓系白血病(acute myeloid leukemia,AML)中17种基因异常表达及在各亚型的分布情况,为个体化治疗提供依据.方法:采用多重巢式RT-PCR检测融合基因,PCR检测FLT3-ITD,荧光定量PCR检测NPM1的突变类型(A、B、D、I和R)及C-kit/D816V,PCR-SSCP银染技术检测CEBPA.对140例初诊AML患者(APL除外)的骨髓进行17种基因异常分析,并与骨髓染色体检测结果进行对比.结果:在140例初诊AML患者中检出基因异常占69例(49.3％),其中包括FLT3-ITD、NPM1、C-kit/D816V、CEBPA、HOX11、CBFβ/MYH11、AML-ETO、MLL/AF6、MLL/AF10、dupMLL、EVI1.同时对140例初治AML患者采用G显带技术行染色体核型分析,128例获得可供分析的染色体核型,其中57例(44.5％)检出染色体结构和数目异常.PCR在急性髓系白血病基因检测中较染色体核型分析具有更高的检出率.结论:PCR协同染色体核型分析检测初诊AML患者的基因异常,能提高临床诊断率、指导疾病危险度分组,为判断预后及监测微小残留病提供更好的理论依据.     

miR-145调控肝癌腹水模型Th9细胞升高的机制研究

目的:探讨miR-145调控肝癌腹水模型Th9细胞升高的作用机制。方法:构建小鼠H22肝癌腹水模型。造模两周后处死小鼠并分离出脾脏组织,流式细胞术分析肝癌腹水组（MA组）与正常对照组（Control 组）小鼠脾脏Th9细胞表达水平。ELISA法检测脾脏IL-9表达水平。RT-PCR法检测脾脏miR-145表达水平。Western Blot法检测脾脏中PI3K/Akt/mTOR/P70S6K/HIF-1α相关蛋白表达水平。分选小鼠脾脏CD4+T细胞,随机分为miR-145 mimics组和NC组,分别应用miR-145-5P mimics、阴性对照寡核苷酸（negative control,NC）进行转染,RT-PCR检测各组miR-145、HIF-1α mRNA及IL-9 mRNA表达水平。结果:与Control 组相比,MA组Th9细胞及其细胞因子IL-9表达均升高（P<0.05）,miR-145表达降低（P<0.05）,p-PI3K、p-Akt、mTOR、p-mTOR、p-P70S6K、HIF-1α蛋白均升高（P<0.05）。与NC组相比,miR-145 mimics组miR-145显著升高,HIF-1α mRNA及IL-9 mRNA表达下降。结论:肝癌腹水中Th9细胞升高可能与miR-145下降导致的PI3K/Akt/mTOR/P70S6K/HIF-1α通路激活有关。