miR-19a在结肠癌中高表达以及通过靶定DLC1促进结肠癌细胞增殖

目的:探讨miR-19a在结肠癌组织中的表达对结肠癌细胞系SW620和SW480增殖的影响,以及其靶基因的确定和功能性研究.方法:实时荧光定量PCR检测miR-19a的表达.噻唑蓝(MTT)比色实验和克隆形成实验检测SW620和SW480细胞的增殖活性.生物信息学预测、荧光报告载体实验以及实时荧光定量PCR和western blot验证miR-19a下游靶基因.结果:miR-19a在结肠癌组织中表达增强(P＜0.01).miR-19a可以增强结肠癌细胞系SW620和SW480增殖能力.DLC1是miR-19a的候选靶基因,过表达miR-19a可以同时在mRNA和蛋白水平上抑制DLC1基因的表达(P＜0.01),反之抑制miR-19a的表达后DLC1的表达则升高(P＜0.01).过表达DLC1后,SW620和SW480细胞的活性和克隆形成能力减弱(P＜0.01).结论:DLC1是miR-19a的直接靶基因,miR-19a通过抑制DLC1的表达而促进结肠癌细胞的增殖.     

过表达miR-18a靶向ATM对结肠癌细胞增殖和迁移的影响

目的 研究miR-18a通过靶基因共济失调-毛细血管扩张突变基因 (ataxia telangiectasia mutated gene, ATM)调控结肠癌细胞的生物学特性。方法 通过软件预测miR-18a其中一个靶基因。设计合成miR-18a的模拟剂（mimics）和抑制剂（inhibitor）,通过转染不同组分上调或下调内源性miR-18a在结肠癌细胞株HCT116中的表达。采用实时荧光定量（qRT）-PCR、Western blot、 MTT法、克隆形成实验、Transwell等方法,检测过表达miR-18a调控ATM基因的表达对体外结肠癌细胞增殖及迁移能力的影响。结果 软件预测发现miR-18a的其中一个靶基因是ATM。通过瞬时转染,过表达内源性miR-18a,可降低HCT116细胞内靶基因ATM蛋白的表达水平。 转染miR-18a mimics后能够下调HCT116细胞的增殖活力,同时显著降低HCT116细胞的克隆形成能力、横向迁移能力及纵向侵袭能力,以上各项改变均与miR-18a过表达有关。结论 证实了miR-18a通过负向调控ATM的表达,抑制结肠癌细胞HCT116的增殖和迁移能力。     

miR-26a靶向HMGA1基因对结肠癌细胞生长、侵袭和迁移的影响

目的：探讨miR-26a靶向高迁移率族蛋白1（HMGA1）基因对结肠癌细胞生长、侵袭和迁移的影响,阐明HMGA1基因是否为miR-26a的靶基因。方法：将miR-NC （对照）、miR-26a mimics （模拟物）和miR-26a inhibitor （抑制物）转染人结肠癌SW480细胞作为miR-NC组、miR-26a mimics组和miR-26a inhibitor组。RT-PCR和Western blotting法分别检测各组SW480细胞中HMGA1 mRNA和蛋白表达水平。采用荧光素酶报告基因检测试剂盒检测各组SW480细胞中双荧光素酶活性,以此确定HMGA1是否为miR-26a的靶基因。采用CCK8实验检测各组细胞增殖活力,Transwell小室法检测各组细胞侵袭数和迁移数。结果：HMGA1基因是miR-26a的靶基因。与miR-NC组比较,转染24、48和72 h时miR-26a mimics组SW480细胞增殖活力明显降低（P<0.01）,而miR-26a inhibitor组细胞增殖活力明显升高（P<0.01）。与miR-NC组比较,各时间点miR-26a mimics组和miR-26a mimics+pcDNA3.1-HMGA1组细胞增殖活力、细胞侵袭数和细胞迁移数均降低（P<0.01）,而miR-NC+pcDNA3.1-HMGA1组细胞增殖活力、细胞侵袭数和细胞迁移数均升高（P<0.01）;与miR-26a mimics组比较,各时间点miR-26a mimics+pcDNA3.1-HMGA1组细胞增殖活力、细胞侵袭数和细胞迁移数均升高（P<0.01）;与miR-NC+pcDNA3.1-HMGA1组比较,各时间点miR-26a+pcDNA3.1-HMGA1组细胞增殖活力、细胞侵袭数和细胞迁移数均降低（P<0.01）。结论：HMGA1是miR-26a的靶基因。上调miR-26a表达可抑制人结肠癌SW480细胞增殖,下调miR-26a表达可促进人结肠癌SW480细胞增殖。     

miR-19a对溃疡性结肠炎的作用机制

目的探讨miR-19a对溃疡性结肠炎的作用机制。方法通过生物信息学分析预测miR-19a可能的靶基因,通过免疫组化及Western blotting技术检测靶基因在溃疡性结肠炎小鼠中表达的变化,并进一步通过绿色荧光蛋白报告载体实验对靶基因进行鉴定。结果生物信息学分析预测miR-19a可能的靶基因是TNF-α,免疫组化及Western blotting显示溃疡性结肠炎小鼠肠道组织TNF-α表达增加,miR-19a可抑制TNF-α-3’UTR-WT报告基因活性,而变异型TNF-α-3’UTR-mut报告基因活性不能被抑制。结论 miR-19a的靶基因为TNF-α,其结合位点为TNF-α3’UTR,miR-19a可能在肠道通过直接调控TNF-α而发挥作用。     

miR-185对肝癌细胞生长增殖及侵袭迁移的影响

目的 探讨miR-185对人肝癌细胞HepG2和SMMC-7721生长增殖、侵袭迁移能力的影响.方法 构建miR-185过表达载体、合成miR-185反义寡聚核苷酸(antisense oligonucleotide,ASO),经脂质体转染肝癌细胞HepG2和SMMC-7721.利用CCK-8和Transwell小室实验检测细胞的增殖、侵袭和迁移能力;生物信息学数据库预测结合基因芯片结果确定miR-185的候选靶基因NRl D1;利用实时荧光定量PCR技术和Western blot检测miR-185对细胞中NRlD1基因表达水平的影响;荧光报告载体实验验证miR-185对靶基因的调控作用.结果 miR-185在人肝癌细胞中的mRNA表达水平显著低于正常肝细胞(P＜0.01);过表达miR-185使人肝癌细胞的增殖、侵袭和迁移能力降低,下调肝癌细胞中NRl D1基因的mRNA和蛋白的表达水平(P＜0.01);荧光报告载体实验证明miR-185可以通过作用于NRlDl mRNA的3’端非翻译区(untranslated region,UTR)下调其表达水平(P＜0.05).结论 miR-185能抑制肝癌细胞的增殖、侵袭迁移能力,NRlD1是miR-185的直接靶基因.     

miR-181b通过靶向调控N-myc下游调节基因2影响骨肉瘤细胞的迁移和侵袭

目的探讨miR-181b对骨肉瘤细胞迁移和侵袭的影响。方法培养骨肉瘤细胞,实时荧光定量PCR检测miR-181b在骨肉 瘤细胞中的表达。抑制miR-181b的表达,Transwell检测对骨肉瘤迁移和侵袭的影响;生物信息学分析miR-181b靶基因并采用 荧光素酶报告基因分析miR-181b和靶基因在骨肉瘤中是否直接作用,同时采用Western Blotting检测靶基因在骨肉瘤细胞中的 表达以及Transwell检测靶基因对骨肉瘤迁移和侵袭的影响。结果实时荧光定量PCR结果表明miR-181b在骨肉瘤细胞中高 表达。抑制miR-181b 的表达能够抑制骨肉瘤细胞的迁移和侵袭;生物信息学分析表明N-myc 下游调节基因2（NDRG2）是 miR-181b直接作用的靶基因,荧光素酶报告基因分析验证了该结果;Western blotting检测表明NDRG2在骨肉瘤中低表达,而 抑制miR-181b 能够显著提高NDRG2 的表达。抑制miR-181b 的表达的同时抑制靶基因NDRG2 的表达,能够逆转抑制 miR-181b的表达对骨肉瘤细胞的迁移和侵袭的影响,从而显著提高骨肉瘤细胞的迁移和侵袭。结论miR-181b在骨肉瘤中过 表达,NDRG2为miR-181b直接调控靶基因,抑制miR-181b能够提高NDRG2的表达从而抑制骨肉瘤细胞的迁移侵袭。     

miR-593通过调控PLK1基因的表达抑制结肠癌细胞的增殖

目的探讨miR-593在结肠癌细胞增殖中的作用及分子机制。方法利用生物信息学分析筛选miR-593可能结合的靶基 因为PLK1;利用双荧光素酶报告基因实验验证miR-593 和PLK1 基因的结合;通过qRT-PCR、Western blotting 验证PLK1 为 miR-593直接作用的靶基因;通过CCK-8实验证明miR-593通过调控PLK1基因的表达抑制结肠癌细胞的增殖。结果运用三 种生物信息学软件对PLK1可能结合的miRNA进行了预测与分析,发现miR-593可能结合PLK1基因;双荧光素酶报告基因实 验证明miR-593 与PLK1 基因发生了结合;Western blotting 结果表明,PLK1 在miR-593 mimic 转染组表达量显著下降,而在 miR-593 inhibitor 组明显升高,相对于对照组均具有统计学差异（P<0.05）;qRT-PCR结果表明,PLK1 RNA水平在各组中表 达水平差异无统计学意义（P>0.05）;细胞增殖实验表明,miR-593与PLK1对结肠癌细胞增殖的影响为相反关系,且共转染miR-593 mimic与PLK1过表达质粒组对细胞增殖的影响介于单独转染miR-593 mimic组与PLK1过表达质粒组之间。结论miR-593通 过调控PLK1基因的表达抑制了结肠癌细胞的增殖,并发挥抑癌miRNA的作用。     

MiR-148a-3p靶基因的预测与验证

目的:预测和验证miR-148a-3p的靶基因.方法:利用生物信息学方法预测miR-148a-3p的靶基因;在神经胶质瘤细胞U87/U251中建立miR-148a过表达及抑制表达稳定细胞系.利用qPCR实验验证miR-148a-3p与靶基因的靶向关系;利用Western Blot实验检测靶蛋白表达水平.结果:生物信息学方法预测结果显示dynein light chain LC8-type 2(DYNLL2)为miR-148a-3p靶基因之一;qPCR实验结果显示DYNLL2在神经胶质细胞HA1800与神经胶质瘤细胞U87/U251中的表达有差异,在神经胶质瘤细胞miR-148a过表达及抑制表达的细胞中表达有差异;Western Blot实验结果显示DYNLL2的蛋白表达水平在神经胶质细胞HA1800与胶质瘤细胞U87/U251中有差异.结论:miR-148a-3p与DYNLL2有确切的靶向关系,DYNLL2在神经胶质细胞与胶质瘤细胞中均有表达,与正常胶质细胞相比,DYNLL2在神经胶质瘤细胞中表达下调,包括基因水平和蛋白水平,miR-148a-3p能够负向调控DYNLL2的表达.     

HIFU通过下调miR-181a进而促进ICAM-1表达增强小鼠抗肿瘤免疫

目的 探讨miR-181a在高强度聚焦超声(high-intensity focused ultrasound,HIFU)增强机体抗肿瘤免疫中的作用及其机制.方法 ①皮下构建供HIFU治疗的黑色素瘤移植瘤小鼠模型,采用简单随机法随机分为HIFU组与荷瘤组,每组30只,采用MTT方法检测各组脾淋巴细胞对B16细胞的杀伤活性,ELISA检测小鼠血清及共培养上清中TNF-α及IFN-γ的表达水平;②qRT-PCR检测HIFU组与荷瘤组脾淋巴细胞中的差异miRNAs;③用生物信息学方法预测差异miRNAs的潜在靶基因,通过RT-PCR及Western blot验证靶基因的表达;④构建荧光素酶报告质粒进一步验证差异miRNAs的靶基因,再将差异miRNAs的mimics转染脾淋巴细胞,RT-PCR及Western blot检测其靶基因表达的变化,以进一步确认差异miRNAs对靶基因表达的影响.结果 ①HIFU治疗能够抑制黑色素瘤生长,促进脾淋巴细胞分泌TNF-α和IFN-γ,增强脾淋巴细胞对肿瘤细胞的杀伤活性;②HIFU处理明显下调脾淋巴细胞中miR-181a的表达水平(P＜0.01);③生物信息学方法预测发现共刺激分子细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)可能是miR-181a的潜在靶基因,且HIFU处理明显促进脾淋巴细胞中ICAM-1的mRNA和蛋白质的表达(P＜0.01);④miR-181a mimics显著抑制pLUC-ICAM-1-3'UTR报告质粒荧光素酶的活性(P ＜0.01),并下调脾淋巴细胞中ICAM-1的mRNA(P＜0.05)和蛋白质的表达(P＜0.01).结论 HIFU通过抑制miR-181a对共刺激分子ICAM-1的负调控作用从而增强荷瘤小鼠的抗肿瘤免疫.     

胃癌SGC7901细胞长春新碱耐药性相关miRNA的初步分析

目的 探讨胃癌SGC7901细胞对长春新碱(VCR)化疗药物耐药相关的微小RNA(miRNA),并预测异常表达miRNA的靶基因.方法 体外培养胃癌SGC7901细胞和胃癌VCR耐药细胞SGC7901/VCR;提取总RNA并质检,采用miRNA 8.0微阵列芯片检测分析miRNA表达谱;荧光定量RT-PCR(qRT-PCR)验证差异表达的miRNA,生物信息学分析软件预测可能调控的靶基因;采用Western blot方法检测靶基因的蛋白表达水平.结果 芯片检测发现,SGC7901和SGC7901/ VCR细胞中63个异常表达的miRNA;其中miR-1267、miR-221、miR-223、miR-200c、miR-99a表达显著性上调;miR-122、miR-1246、miR-302a、miR-195、miR-124表达显著性下调.在胃癌细胞和胃癌组织中验证结果初步显示,miRNA-122和miR-302a可能分别调控靶基因IGF-IR和WDR62.结论 获得的差异表达miRNA,以及预测的靶基因IGF-IR和WDR62可能在胃癌化疗药物VCR耐药中起关键性作用.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

Acupuncture Treatment of Vomiting

CASE HISTORY A female patient, 46 years old, head of the foreign affairs department of a certain university in Beijing, paid her first visit on October 9, 2006, with the chief complaint of vomiting for one month. She got vomiting after meals in early September. Before that, she had discomfortable sensation in the stomach due to angry with others, but she didn't pay much attention. Later, it developed into vomiting after eating. After the vomiting, the discomfort would be relieved, but with slight hypodynamia. She was once diagnosed as having 'neurogenic vomiting'. Having taken some western and Chinese drugs, the above symptoms were a little bit improved, but she would have nausea upon eating and with regurgitation. Because of the fear for vomiting, she did not dare to have food intake, with body weight reduction of 6 kilos in one month.     

Qiu Baoguo''''s Experience in Treating Consumptive Syndromes after Radio-Chemotherapies

Radiotherapy and chemotherapy are the important modern medical therapies for malignant tumors,yet they can also bring about serious local and systemic toxic side reactions so to decrease the patient;'s life quality,manifested by a series of consumptive symptoms.Having engaged in the combined work of Chinese and western medicine for nearly 50 years,the research fellow Qiu Baoguo in Henan Provincial Academy of TCM has developed his unique views on the TCM study of consumptive syndromes.The author of this essay had once the fortune tO follow Dr.Qiu in clinic,and specially would like to introduce in the following Dr.Qiu's experience in treating consumptive syndromes after radio-chemotherapies for patients with malignant tumor.     

Insomnia Due to Deficiency of Both the Heart and Spleen Treated by Acupuncture-Moxibustion and Chinese Tuina

OBJECTIVE: To observe therapeutic effects of the comprehensive therapy of acupuncture-moxibustion and Chinese Tuina for treatment of insomnia due to deficiency of both the heart and spleen. METHODS: 92 cases were divided randomly into the treatment group (treated by acupuncture-moxibustion and Chinese Tuina) and the control group (treated by acupuncture-moxibustion). RESULTS: The therapeutic effect of the treatment group was obviously superior to that of the control group (the CHI2 test showed P < 0.01). CONCLUSIONS: The comprehensive therapy of acupuncture-moxibustion and Chinese Tuina can give marked therapeutic effects for treatment of insomnia due to deficiency of both the heart and spleen.     

Analysis of CK-19,PDX-1,Nestin,Ngn3 in different donor islet purity in rats

Objective: To investigate if there are the CK-19, PDX-1, Nestin, Ngn3 positive cells in the donor islets of different purity in rats. Methods: Thirty male adult SD rats were randomly divided into 3 groups. Islets were isolated using digestion by ductal injection of collagenase. Group Ⅰ (n=10): Separating cell preparations were not purified, Group Ⅱ(n=10): Islet sediment was purified with 25% Ficoll400 ,Group Ⅲ (n=10): Islet sediment was purified with 25% and 11% Ficoll-400. The levels of protein of CK-19, PDX-1, Nestin and Ngn3 were detected by immunohistochemistry and the mRNA of CK-19, PDX-1, Nestin, Ngn3 was amplified by RT-PCR. Results: After two different purification methods applied, three islet preparations of different purities were obtained. The difference of islet purity was significant among various groups (P＜0.05). Compared with group Ⅱ and group Ⅲ,the protein and mRNA of CK-19, PDX-1, Nestin,Ngn3 were both higher in group Ⅰ; group Ⅲ was poorly expressed. Conclusions: The three different islet purity donor islet have different CK-19, PDX-1, Nestin, Ngn3 positive cells within them, indicating that there are some islet stem cells in the purified donor islet.     

Dr.Zhang Ren''''s Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.     

Luo Lingjie''''s Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.