小鼠骨生成诱导因子基因逆转录病毒载体的构建及其在293T细胞中的表达

目的构建小鼠骨生成诱导因子（OIF）基因重组逆转录病毒载体,鉴定其在293T细胞中的表达。方法 PCR法扩增OIF-3FLAG基因,测序后克隆入逆转录病毒载体pMSCV PIG（Puro-IRES-GFP）的相应位点,构建重组逆转录病毒表达载体pMSCV PIG-OIF-3FLAG,并对重组体进行测序鉴定。应用脂质体分别将PMSCV PIG-OIF-3FLAG和VSVG、GAG-POL辅助病毒包装载体共转染至293T包装细胞,48h后荧光显微镜下观察绿色荧光蛋白（GFP）表达;收集包装细胞上清液,进一步用含有包装病毒的上清液再感染293T细胞,48 h后加入3μg/mL嘌呤霉素（puromycin）,连续7d,筛选病毒感染的稳定表达pMSCVPIG-OIF-3FLAG的293T细胞株,分别采用Real-time PCR技术和Western blotting方法检测293T细胞OIF mRNA及其蛋白表达。结果构建OIF基因逆转录病毒表达载体PMSCV PIG-OIF-3FLAG,经酶切及测序鉴定证实目的基因插入位点和读码框架正确;病毒感染293T细胞,经嘌呤霉素筛选稳定表达小鼠OIF的293T细胞株;Real-time PCR和Western blotting鉴定证实OIF mRNA和蛋白在该细胞株中呈高表达。结论成功构建小鼠OIF基因的逆转录病毒载体,并获得稳定高表达OIF基因的293T细胞株。     

小鼠骨生成诱导因子基因逆转录病毒载体的构建及其在293T细胞中的表达

目的 构建小鼠骨生成诱导因子(OIF)基因重组逆转录病毒载体,鉴定其在293T细胞中的表达。方法 PCR法扩增OIF-3FLAG基因,测序后克隆入逆转录病毒载体pMSCV PIG（Puro-IRES-GFP）的相应位点,构建重组逆转录病毒表达载体pMSCV PIG-OIF-3FLAG,并对重组体进行测序鉴定。应用脂质体分别将PMSCV PIG-OIF-3FLAG和VSVG、GAG-POL辅助病毒包装载体共转染至293T包装细胞,48 h后荧光显微镜下观察绿色荧光蛋白（GFP）表达;收集包装细胞上清液,进一步用含有包装病毒的上清液再感染293T细胞,48 h后加入3 μg/mL嘌呤霉素（puromycin）,连续7 d,筛选病毒感染的稳定表达pMSCV PIG-OIF-3FLAG 的293T细胞株,分别采用Real-time PCR技术和Western blotting方法检测293T细胞OIF mRNA及其蛋白表达。结果 构建OIF基因逆转录病毒表达载体PMSCV PIG-OIF-3FLAG,经酶切及测序鉴定证实目的基因插入位点和读码框架正确;病毒感染293T细胞,经嘌呤霉素筛选稳定表达小鼠OIF的293T细胞株;Real-time PCR和Western blotting鉴定证实OIF mRNA和蛋白在该细胞株中呈高表达。结论 成功构建小鼠OIF基因的逆转录病毒载体,并获得稳定高表达OIF基因的293T细胞株。     

NOC2L基因重组慢病毒表达载体的构建及其在293T细胞中的表达

目的构建NOC2L基因重组慢病毒并使其在293T细胞中表达。方法通过设计引物及PCR扩增获得NOC2L全基因片段,将pCDH-GFP载体分别用XbaⅠ和BamHⅠ双酶切制备线性化载体;NOC2L全基因片段和线性化载体经切胶回收后,通过同源重组反应将NOC2L全基因片段连接到线性化的pCDH-GFP载体上,构建pCDH-NOC2L-GFP慢病毒表达载体;通过菌落PCR、质粒双酶切、测序等方法对构建的NOC2L基因重组慢病毒表达载体进行鉴定,利用构建的pCDH-NOC2L-GFP包装NOC2L慢病毒并使用该慢病毒感染293T细胞。结果菌落PCR、质粒双酶切和测序结果显示成功将NOC2L基因连接到pCDH-GFP载体上,使用构建成功的pCDH-NOC2L-GFP转染293T细胞,能够成功转染及包装NOC2L基因重组慢病毒,同时包装好的NOC2L基因重组慢病毒能够成功感染293T细胞并过表达293T细胞中的NOC2L基因。结论采用本研究方法能成功构建NOC2L基因重组慢病毒表达载体。     

表达HIF1α与EGFP融合蛋白的重组逆转录病毒载体构建及鉴定

目的构建表达人缺氧诱导因子-1α(HIF-1α)与EGFP融合蛋白的重组逆转录病毒载体,并观察其对NIH3T3细胞感染效率.方法采用基因工程技术,将HIF-1α基因片段克隆至逆转录病毒载体pLEGFP-N1上,鉴定后用脂质体法转染PT67细胞包装、扩增,最后用重组逆转录病毒感染NIH3T3细胞.其中采用PCR方法对重组病毒进行鉴定,利用绿色荧光蛋白作为报告基因,对病毒滴度和感染效率进行监测.结果酶切、测序及PCR结果与HIF-1α基因重组逆转录病毒载体的预期结果一致,病毒滴度达1.2×106 pfu/ml,并对NIH3T3细胞有强感染能力.结论应用基因工程技术,成功构建了表达HIF-1α与EGFP融合蛋白的重组逆转录病毒载体,为应用于治疗性血管生成创造了条件.     

人硫氧还蛋白基因克隆及其重组逆转录病毒载体的构建和鉴定

目的 克隆人硫氧还蛋白（hTRX）基因,并构建含有该目的 基因的重组逆转录病毒载体。方法 利用RT-PCR法,以PHA活化的人外周血单个核细胞总RNA为模板,扩增hTRX编码蛋白的cDNA基因,并亚克隆至逆转录病毒载体pSIV-1中进行PCR、双酶切和测序鉴定。结果 将所得的序列与GenBank（BC003377）报道的序列比较,其酶活性中心（Trp-Cys-Gly-Pro-Cys）与已知序列一致,第132、136、170、264位碱基与已知序列不同,其中第136、170位相应密码子编码的氨基酸发生了变化（Phe→Leu,Ile→Thr）,成功获得了pSIV-1-hTRX重组逆转录病毒载体。结论 hTRX基因的克隆及其重组逆转录病毒载体pSIV-1-hTRX的构建,为进一步探讨hTRX的生物学活性和应用奠定了基础。     

HBX基因逆转录病毒载体的构建及在LO_2细胞内的稳定表达

目的 构建乙型肝炎病毒X基因(HBX)逆转录病毒载体,并使其在人正常肝细胞LO2内的进行稳定表达.方法 PCR扩增HBX基因,克隆到逆转录病毒载体质粒pSEB-Flag中,构建表达HBX的重组逆转录病毒载体质粒pSEB-Flag-HBX,通过通过PCR、酶切、测序验证HBX基因后;体外将重组质粒pSEB-Flag-HBX与包装质粒pAmpho共同转染人胚胎肾上皮细胞系293T细胞,包装获得携带HBX基因的重组逆转录病毒,并感染靶细胞人正常肝细胞LO2,稻瘟菌素(Blasticidin)筛选,获得成功转入HBX的LO2细胞.通过RT-PCR和Western blot进一步验证HBX基因及HBx蛋白在LO2细胞内的表达.结果 (1)成功获得携带HBX基因的阳性重组质粒pSEB-Flag-HBX;(2)HBX基因被逆转录病毒成功地导入靶细胞LO2细胞,并实现稳定表达,RT-PCR检测到HBX mRNA在靶细胞中的表达,Western blot检测到HBx蛋白在靶细胞LO2-HBx细胞中的表达.结论 成功构建了携带HBX基因的重组逆转录病毒载体,感染人正常肝细胞LO2后能够稳定表达HBx蛋白,为进一步探讨HBx在肝癌发生发展中的作用提供了较为理想的实验模型.     

人DC-SIGN基因克隆及其转基因细胞的构建

目的克隆人DC-SIGN基因,并构建含有该目的基因的重组逆转录病毒载体,获得稳定表达DC-SIGN分子的L929基因转染细胞。方法利用RT-PCR的方法从人外周血单核细胞来源的树突状细胞（DC）总RNA中克隆出人DC-SIGN基因,通过双酶切（EcoRI,BamHI）装入逆转录病毒载体pGEZ-Term中,脂质体法共转染包装细胞293T,用含有完整病毒颗粒的293T细胞的培养上清感染L929细胞,72h后,加入Zeocin进行筛选,挑选出能稳定表达DC-SIGN蛋白的L929细胞株。结果构建了用于表达的含DC-SIGN基因的重组逆转录病毒载体,经转染包装细胞293T后,获得具有感染能力的重组DC-SIGN逆转录病毒和转染L929细胞,继而经RT-PCR、流式细胞仪表型检测,筛选出了稳定表达人DC-SIGN蛋白的L929转基因细胞。结论构建了含人DC-SIGN基因重组逆转录病毒载体和稳定表达人DC-SIGN蛋白的细胞株,为该基因功能的后续研究和单克隆抗体的研制奠定了基础。     

逆转录病毒Pmscv/Hyg介导的RNA干扰表达载体的构建与鉴定

目的构建逆转录病毒Pmscv/Hyg介导的RNA干扰表达载体,并在HEK293细胞株中观察其基因沉默效果。方法PCR方法扩增人U6启动子,下游引入核酸内切酶位点BamHI、SalI,反向插入质粒Pmscv/Hyg的3′端LTR上游。扩增EGFP基因,插入载体Pmscv/Hyg的多克隆位点。合成干扰P53基因表达的寡核苷酸序列,退火复性后插入U6启动子下游BamHI、SalI酶切位点间。重组质粒经酶切、测序鉴定正确后转染病毒包装细胞PT67,产生具有一次感染能力的病毒颗粒。病毒上清感染靶细胞HEK293,经潮霉素筛选,RT-PCR和western blot检测靶基因P53表达情况。结果质粒酶切及DNA测序表明成功构建重组病毒载体,并在PT67细胞中包装成具有一次感染能力的逆转录病毒。经Hygromycin筛选,HEK293细胞中P53蛋白和mRNA表达显著下调。结论成功构建以Pmscv/Hyg为基础的RNA干扰表达载体,重组载体包装出的逆转录病毒可有效介导基因沉默。     

Math1基因重组慢病毒的构建及其在293T细胞中的表达

目的 构建携带Math1基因的重组慢病毒载体,检测其滴度,检测其在293T细胞中的表达.方法 PCR扩增Math1基因,将其连入慢病毒栽体pLenti-GFP中;在感受态细胞DH5α中培养扩增,并行Math1基因的测序鉴定;将重组的慢病毒四质粒共转染293T细胞,收获并浓缩病毒;感染293T细胞和提取细胞DNA后用实时定量PCR法检测病毒滴度.用逆转录PCR和westem blot法检测Mathl基因在感染病毒的293T细胞中的表达.结果 构建的慢病毒载体pLenti-Math1-GFP经测序分析证实基因序列正确.四质粒共转染293T细胞后,荧光显微镜下可见大量绿色荧光.包装后慢病毒测定滴度约为3X10"Tu/L.逆转录PCR和Western blot法均能检测Math1基因在感染病毒的293T细胞中的表达.结论 成功构建携带Math1基因的重组慢病毒,并能在293T细胞中表达.     

Delta1-IRES-EGFP双顺反子逆转录病毒表达载体的构建及鉴定

目的构建并鉴定人Delta1和增强型绿色荧光蛋白(EGFP)基因双顺反子逆转录病毒表达载体.方法利用基因重组技术将人Delta1基因构建到含IRES-EGFP逆转录病毒载体中;电穿孔法将重组质粒pDelta1-IRES-EGFP分别转入单、双嗜性包装细胞GP E86和PA317,嘌呤霉素(puromycin)筛选获得阳性表达克隆;"乒乓球"法提高病毒滴度;重组病毒感染NIH3T3细胞,流式细胞仪(FACS)及RT-PCR法分别检测细胞荧光和Delta1 mRNA表达变化;C2C12共培养实验检测蛋白的功能.结果酶切、PCR鉴定及DNA测序证实逆转录病毒表达载体pDelta1-IRES-EGFP构建正确;经体外包装,获得最高滴度9.7×105 CFU/ml的重组病毒液;重组病毒液感染NIH3T3细胞,FACS检测部分细胞荧光强度显著提高;RT-PCR证实细胞内有外源基因Delta1表达;C2C12共培养实验证实外源蛋白Delta1可抑制C2C12细胞的分化.结论逆转录病毒基因转移系统安全,效率较高,可获得外源基因长期、稳定的表达.     

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…     

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.     

猪肺磷脂注射液联合经鼻持续气道正压通气对呼吸衰竭早产儿的临床疗效及肌酸激酶同工酶活性的影响

目的 探讨猪肺磷脂注射液联合经鼻持续气道正压通气(NCPAP)对呼吸衰竭早产儿的临床疗效及肌酸激酶同工酶活性(CK-MB)的影响.方法 选取呼吸衰竭早产儿80例,分为观察组和对照组各40例.对照组采用NCPAP给氧治疗,观察组给予NCPAP给氧联合猪肺磷脂气管内给药.观察两组患儿治疗前及治疗12h、24 h后PaO2、PaCO2、血氧饱和度(SaO2)、pH的变化情况,检测治疗前及治疗5d后血清CK-MB水平;评估两组患儿的临床治疗效果.结果 两组患儿PaO2、PaCO2、SaO2、pH比较,差异均有统计学意义(P＜0.05),其中观察组治疗后的PaO2、SaO2、pH均高于对照组,PaCO2则低于对照组.两组的PaO2、SaO2、pH均随观察时间延长而升高(P＜0.05),PaCO2均随观察时间的延长而降低(P＜0.05).观察组治疗有效率为87.5％,显著高于对照组的70.0％ (P ＜0.05).治疗5d后两组患儿血清CK-MB水平均较前降低(P＜0.05),且观察组明显低于对照组(P＜0.05).结论 猪肺磷脂注射液气管内给药联合NCPAP可以显著降低呼吸衰竭早产儿CK-MB的含量,提高治疗有效率,起到很好的呼吸循环支持作用.     

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.