SD大鼠超排卵及胚胎冷冻方法比较

目的优化超排卵效果及建立大鼠胚胎冷冻方法。方法腹腔注射三种不同剂量PMSG—HCG（15IU／只、20IU／只、25IU／只）超排5周龄SD大鼠，当日与正常雄鼠1：1合笼交配，比较三种激素剂量的超排卵效果。应用输卵管吹卵法收集2-细胞胚胎，采用小鼠胚胎冷冻方法对大鼠胚胎进行冷冻，通过胚胎复苏率比较不同冷冻时间（方法Ⅰ：5min，方法Ⅱ：1min）的冷冻效果。结果采用三种剂量进行超排分别获得13±12．7、31±14．2、41±25．4枚胚胎，其中激素剂量25IU／只超排效果最好；方法Ⅱ在操作简便性上明显优于方法Ⅰ，但冷冻效果无显著性差异（平均复苏率88．3％与85．0％）。采用方法Ⅱ冻存大鼠胚胎总计152枚，复苏后形态正常胚胎共计139枚（91．44％），培养20min后，对存活胚胎共计105枚（75．53％）进行胚胎移植，产仔39只（37．14％）。结论成功优化大鼠超排及建立大鼠胚胎冷冻方法。     

潮霉素抗性转基因BDF1小鼠模型的建立

目的建立具有潮霉素（hygromycin）抗性转基因BDF1小鼠，用于制备携有hygromycin抗性筛选标志ES阳性细胞克隆的饲养层。方法通过显微注射的方法，将含有潮霉素B磷酸转移酶基因片段（5.1kb）导入BDF1受精卵雄原核中，共注射169枚受精卵，然后将129枚受精卵细胞植入同期受孕的受体母鼠输卵管内。结果共产生37只转基因小鼠，经PCR和Southern检测获得9只阳性小鼠，对一只子代鼠进行RT-PCR检测证明hyg基因已经在肾、肌肉、脾内表达。结论成功的建立具有潮霉素抗性的BDF1转基因鼠，该模型动物可以为基因敲除研究提供良好的基础条件。     

Dicer 1转基因小鼠模型的建立

目的 建立Dicer1转基因小鼠模型.方法 构建pcDNA3.I-Dicer1转基因构件,经酶切、纯化后通过显微注射方法导人BDF1小鼠受精卵原核并移植到同期受孕的ICR受体母鼠输卵管内.出生后仔鼠用PCR和Southern方法检测鼠尾DNA鉴定基因型,通过免疫组化检测Dicer1基冈表达.结果 显微注射172枚卵,移植119枚卵于3只受体输卵管中,2只怀孕,共产仔15只,经PCR检测获得6只阳性鼠,Southern榆测6只均为阳性.对Southern检测阳性转基因小鼠子代进行RT-PCR检测和免疫组化分析证明Dicer1基因在肝脏、肾脏、肺内均有表达.对腹腔肿胀的转基因阳性1号鼠解剖发现肝脏、脾脏明显增大,胚胎发育异常.结论 成功建立Dicer1基因表达的转基因小鼠模型,该模型为进一步研究DICER1基因功能及miRNA的表达及功能等奠定基础.     

角质细胞特异性表达Cre重组酶转基因小鼠的研制

本研究采用转基因技术中最常用的受精卵原核显微注射法，制备角质细胞特异性表达Cre重组酶转基因小鼠。我们将经过超排处理的供体雌鼠拉颈处死，从其输卵管壶腹部取出卵丘细胞团，用透明质酸酶消化、清洗，得到形态饱满、极体和双原核明显的受精卵培养。将构建好的包括角质细胞特异性角质素5（K5）启动子、Cre重组酶基因和人生长激素（hGH）polyA的转基因载体pK5-Cre-hGH制备成一定浓度的注射片段，通过显微注射的方法将4．2kb的转基因片段引入小鼠受精卵的雄原核。共注射了720枚受精卵，移植至29只假孕母鼠的输卵管中发育，获得子代小鼠48只，经PCR鉴定有12只小鼠在基因组上整合有Cre基因，整合率为25％。此结果通过Southern杂交得到进一步证实。     

大鼠显微注射胚胎采集方法的比较

目的优化大鼠显微注射前胚胎采集的方法。方法选用最佳超排5周龄SD大鼠，腹腔注射PMSG-HCG，分别用撕开输卵管壶腹部和输卵管伞端冲洗两种方法采集胚胎，比较获得卵数，受精率，注射存活率等的差异。结果冲洗输卵管伞端采集胚胎的方法所得到的受精率、存活率都较高，与撕开输卵管壶腹部方法比较差异显著（P〈0．01），胚胎总数无显著性差异（P〉0．05）。结论冲洗输卵管伞端采集胚胎的方法为大鼠显微注射前胚胎采集比较好的方法。     

TNNI2突变转基因小鼠模型的建立

目的建立TNNI2突变转基因小鼠模型。方法构建pEGFP-tnni2转基因构件，TNNI2基因的第175个氨基酸缺失，通过原核显微注射法。把线性化、纯化后的外源基因pEGFP-tnni2注射入BDF1小鼠受精卵中。胚胎移植给同期发情的假孕受体母鼠，获得子代小鼠。用PCR和Southern方法检测子代鼠尾DNA鉴定基因型。通过RT-PCR方法检测tnni2基因表达。结果移植注射胚胎115枚给4只假孕小鼠共出生了23只后代鼠。经PCR和Southern方法检测得到4只阳性小鼠。对其子代进行RT-PCR检测，tnni2基因在肌肉、心脏内表达。结论通过显微注射法使外源基因pEGFP-tnni2（TNNI2基因的第175个氨基酸缺失）在小鼠基因组中得到整合，建立了转pEGFP-tnni2的转基因小鼠模型。     

fat-1转基因小鼠的构建与鉴定

目的构建和鉴定携带有外源fat-1基因的转基因小鼠。方法将fat-1基因的cDNA与动物表达载体pEF—neo连接，构建pEF-fat-1重组质粒，酶切、测序鉴定正确后，以显微注射法把线性化重组质粒注射到小鼠受精卵的雄原核中，并将受精卵移植到受体鼠的输卵管中产出转基因小鼠，通过PCR、Southern blot杂交等方法确立阳性整合有目的基因的G0代小鼠。结果成功构建了pEF-fat-1重组质粒，将其显微注射到小鼠受精卵中，得到G0代小鼠，PCR、Southern blot杂交确立了4只整合有fat-1基因的首建鼠。结论fat-1基因可整合到小鼠体内，得到的转基因小鼠为研究fat-1基因的生物学功能提供了动物模型。     

四环素调控SV40Tag转基因小鼠模型的建立

目的构建四环素调控的SV40T转基因小鼠模型。方法同时显微注射外源基因p205-rtTA-C3和pTRE-Tag至FVB小鼠原核,注射受精卵移植到同期发情的假孕受体出生个体,经PCR和Southern检测获得阳性转基因小鼠。结果经PCR结合Southern检测得到rtTA和Tag双阳性转基因小鼠一只,rtTA单阳性两只和Tag单阳性一只。结论通过饮水给与四环素的双阳性小鼠可在卵巢中检测到Tag mRNA的表达。     

EB病毒早期基因BHRF1转基因模型鼠的建立

目的构建稳定表达EB病毒早期基因BHRF1蛋白的重组载体,通过显微注射法建立BHRF1转基因鼠模型。方法自EBV阳性细胞系B95-8提取总RNA,应用RT-PCR扩增BHRF1基因,然后与pcDNA3.1(+)连接,构建pcDNA3.1-BHRF1重组载体。重组载体用NruⅠ和DraⅢ双酶切,电泳后纯化回收长约2000 bp的目的片段溶于显微注射缓冲液。从超排小鼠取健康受精卵,通过显微注射技术,将纯化的CMV+BHRF1+polyA DNA片段注射入小鼠受精卵,短暂培养后选择健康受精卵移入假孕母鼠输卵管内。采用PCR检测子鼠鼠尾组织DNA,鉴定筛选BHRF1阳性首建鼠。结果经PCR、限制性内切酶鉴定及测序证实pcDNA3.1-BHRF1重组载体构建成功,DraⅢ和NruⅠ双酶切获得长约2 000 bp的CMV+BHRF1+polyA DNA片段。自18只见栓超排小鼠获取健康受精卵480枚,分别注入纯化的目的片段,经短暂培养后390枚受精卵仍健康,移入14只假孕母鼠输卵管内,11只受孕,产下74只子鼠。PCR检测子鼠鼠尾DNA获得16只阳性首建鼠,阳性率为21.6%(16/74)。结论本实验首次建立了BHRF1转基因鼠模型,为深入研究BHRF1的生物学特性及其在EBV相关肿瘤发生发展中的作用提供了理想的动物模型。     

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.     

Survey and practice of reporting quality of randomized controlled clinical trials on traditional Chinese medicine

Evidence obtained from randomized controlled trials （RCTs） has been generally accepted as the gold standard in the evaluation of clinical effectiveness. Readers need to understand the trial design, implementation, results, analysis and interpretation, so as to fully Jnderstand the results of RCTs. Thus, the investigators of RCTs have to report these items in a complete, accurate and clear manner. Since 1998, we have conducted several evaluations on the reporting quality of RCTs published in Chinese journals on traditional Chinese medicine （TCM） and results have shown that there is an urgent need for higher quality RCTs on TCM.     

TCM Treatment of Ankylosing Spondylitis by Tonifying the Kidney and Strengthening the Governor Vessel

Ankylosing spondylitis is a chronic and progressive disorder with inflammation mainly involving the central axis joints. It mainly affects the cervical spine and the lumbosacral area, with the pathogenesis closely related to the kidney and the Governor Vessel (GV). TCM holds that the syndrome is deficiency in origin and excess in superficiality, which is due to insufficiency of the kidney, deficiency of GV, and blocking of the channels with the invasion of exogenous evil, leading to poor circulation of qi and blood and malnutrition of the bones, muscles and joints. The TCM method of tonifying the kidney and strengthening GV to regulate circulation of qi and blood and check the arthralgia pain should be adopted, with the Kidney-Tonifying and GV Strengthening Decoction (益肾强督汤) prescribed.     

IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

CHEMOTHERAPY playsa greatrolein the treat- ment of malignanttumors,especiallyingynecolo- gicalones.But inanticancerchemotherapy,leuko-cytopeniaisfrequentlytheprimarydose-limitingsideeffect factor.Moreover,cancersarefrequentlychemoresistantbe-causeof overexpressionof P-glycoprotein(P-gp), which isencodedby multidrugresistancegene (MDR1 ) and detectableinup to50% ofhuman cancersand renderscellsresistancetoanticancerdrugs.The safetyand potentialtherapeuticbenefitof mdr1 gene transferredto h…