COA: siRNA靶向PPARγ基因重组腺病毒穿梭载体对激素诱导兔骨髓间充质干细胞成脂分化的抑制作用

背景：激素是诱发股骨头坏死（osteonecrosis of the femoral head,ONFH)的重要原因之一,可导致骨细胞内脂肪沉积而变性坏死从而抑制成骨分化及骨的形成。过氧化物酶体增殖子活化受体-γ(peroxisome proliferator-activated receptor-γ,PPARγ)是一种成脂转录因子,ONFH的发生与PPARγ在骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）的高表达有密切关系。BMSCs具有分化为成骨细胞与脂肪细胞的潜能,BMSCs内PPARγ高表达,则BMSCs分化为脂肪细胞。因此,通过RNA干扰（RNA interference,RNAi）调控PPARγ的表达可抑制BMSCs的脂肪分化,保持MSCs的成骨分化特性。 方法：取新西兰大白兔骨髓,制备兔BMSCs,并随机分6组。S1、S2、S3：干扰1、2、3组,Con：感染无关siRNA组,M：模型组,N：正常组。10-7mol/L地塞米松诱导和siRNA病毒载体感染细胞,倒置显微镜观察BMSCs的形态及生长情况,在第1d、3d、5d、7d时,检测各组BMSCs内PPARγ、osteocalcin和Runx2 mRNA及蛋白的表达。14d时,检测细胞内甘油三酯( triglyceride,TG )含量、细胞培养液中骨钙素(osteocalcin,OC)分泌量和碱性磷酸酶(alkaline phosphatase,ALP)活性。21d,苏丹Ⅲ染色BMSCs,并在显微镜下作脂肪细胞计数。 结果：M组和Con组中PPARγ mRNA及其蛋白表达、TG含量和脂肪细胞计数均明显高于N组(P <0.05),而Osteocalcin和Runx2 mRNA及其蛋白表达、培养液中OC含量和细胞内ALP活性明显低于N组(P <0.05)。S1、S2、S3组中PPARγ mRNA及其蛋白表达显著低于M组和Con组(P <0.05),接近N组 (P>0.05),其中S2组的干扰作用最强。 结论：siRNA靶向PPARγ基因腺病毒穿梭载体能够有效抑制激素诱导的BMSCs成脂分化,维持BMSCs成骨分化。     

shRNA干扰PPARγ基因表达预防兔激素性股骨头坏死的研究

目的 研究激素性股骨头坏死(steroid-induced avascular necrosis of the femoral head,SANFH)的发病机制,观察靶向过氧化物酶体增殖激活受体γ(PPARγ)基因shRNA腺病毒载体预防兔SANFH的作用效果.方法 健康新西兰兔48只,随机分为4组,每组12只.单纯激素组(M组)仅肌注地塞米松;实验组(S组)在肌注地塞米松同时,加用靶向PPARγ基因shRNA腺病毒载体感染兔活体股骨头内骨髓基质干细胞(bone marrow stromal cells,BMSCs);无关序列组(E组)在肌注地塞米松同时,加用搭载无关序列的shRNA重组腺病毒感染兔活体股骨头内BMSCs;对照组(N组)肌注等量生理盐水.测定4组兔血清甘油三酯(TG)、胆固醇(CHO)含量.观察股骨头组织病理学改变.应用RT-PCR 技术检测PPARγ mRNA和Runx2 mRNA表达;应用免疫组化技术检测PPARγ和Runx2蛋白表达情况.结果 M、E、S组血清中TG和CHO含量增高,与N组比较差异均有统计学意义(P<0.05).M组和E组可见明显骨坏死,脂肪细胞增生肥大,骨小梁变细和稀疏,大量空骨陷凹,S组和N组未见骨坏死及上述改变.S组中PPARγ mRNA和蛋白均呈低表达,Runx2 mRNA及蛋白呈高表达,与M组和E组比较,差异有统计学意义(P<0.05);与N组比较,差异无统计学意义(P>0.05).结论 靶向PPARγ基因shRNA腺病毒载体可有效阻断BMSCs内PPARγ mRNA表达,抑制其成脂分化;增强Runx2 mRNA表达,促进其成骨分化,预防SANFH的发生,为通过基因靶向治疗SANFH提供新思路.     

人绒毛膜促性腺激素对3T3-L1脂肪细胞过氧化物酶增殖体激活受体γ及脂联素mRNA表达的影响

目的:探讨人绒毛膜促性腺激素(HCG)对3T3-L1脂肪细胞过氧化物酶增殖体激活受体γ(PPARγ)及脂联素mRNA表达的影响.方法:分别用50、500和5000 U/L HCG干预未分化脂肪细胞2、4和6 d,未用HCG干预为空白对照组,PT-PCR测定PPARγ mRNA的表达;用诱导分化液(1 μmol/L地塞米松+0.5 mmol/L 3-异丁基-1-甲基黄嘌呤+10 ng/L胰岛素)诱导3T3-L1脂肪细胞分化为成熟脂肪细胞,油红O染色鉴定后用5000 U/L HCG干预成熟脂肪细胞6、12和24 h,未用HCG干预为空白对照组,用RT-PCR测定PPARγ及脂联素mRAN的表达情况.结果:不同剂量HCG干预未分化3T3-L1脂肪细胞,与空白对照组相比,不同剂量干预组未分化脂肪细胞PPARγ mRNA的表达量均增加,差异有统计学意义(F=19.823、21.352和23.519,P均<0.001);不同剂量HCG干预组组间比较,5000 U/L剂量干预组PPARγ的表达量较50和500 U/L剂量组增加,差异均有统计学意义(P均<0.05):50和500 U/L剂量组比较,PPARγ mRNA的表达量差异无统计学意义(P>0.05).5000 U/L HCG干预已分化3T3-L1脂肪细胞后,空白对照组、干预6、12和24 h组相比,脂肪细胞PPARγ mRNA的表达差异有统计学意义(F=26.907,P<0.001),脂联素mRNA的表达差异无统计学意义(F=0.066,P=0.976).结论:HCG可能促进313-L1脂肪细胞的分化,但对脂联素的分泌无影响.     

靶向PPARγ基因RNA干扰对激素抑制家兔骨髓基质细胞成骨分化的影响

目的:观察靶向过氧化物酶体增殖子活化受体-γ(PPARγ)基因RNA干扰阻断激素作用保持骨髓基质细胞(MSCs)成骨分化的作用.方法:选用家兔第2代MSCs,随机分为7组.对照组(C):无特殊处理;模型组(M):给予10-7 mol/L地塞米松;感染空载体组(O):给予空载体腺病毒1 × 105 U和地塞米松;感染无关siRNA组(N):给予无关序列siRNA腺病毒1×105 U和地塞米松;干扰1、2、3组(I1、I2、I3):分别给予靶向PPARγ siRNA 1、2、3腺病毒1 × 105 U和地塞米松.7d后采用RT-PCR技术检测各组细胞中PPARγ mRNA和核心结合因子 al(Cbfal) mRNA表达.14 d后测定各组培养液骨钙素含量和细胞内碱性磷酸酶(ALP)活性.结果:M组、O组和N组PPARγ mRNA呈高表达,cbfal mRNA呈低表达,骨钙素含量和ALP活性均下降,与C组比较差异均有统计学意义(P均<0.05).I1组、I2组、I3组PPARγ,mRNA呈低表达,Cbfal mRNA呈高表达,骨钙素含量和ALP活性均接近C组,与C组比较差异均无统计学意义(P均>0.05).结论:靶向PPARγ基因RNA干扰能够阻断激素作用,下调PPARγ基因表达,上调Cbfal基因表达,保持细胞的正常成骨分化.     

Effect of EphB6 on osteogenic differentiation of bone marrow-derived mesenchymal stem cells

目的 探讨EphB6在小鼠骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSC)成骨分化中的作用及其可能机制.方法 将BMSC通过双相接种法接种于脱钙骨基质,分为4组:成骨诱导5、14 d(A、B组),普通培养5、14 d(C、D组),茜素红染色检测钙结节,定量检测各组碱性磷酸酶活性,Runx2、Osterix和EphB6的mRNA表达,采用游离态配体ephrinB1-Fc激活EphB6并重复检测上述指标.结果 ALP活性、Runx2和Osterix在A、B组增高,且B组检测到最高的ALP活性[(7.32±2.02)金氏单位/100 ml]、Runx2( 3.442 6±0.2585)倍和Osterix(2.4823±0.329 2)倍.EphB6的表达水平在A、B组降低,且在B组最低(0.754 3±0.023 5)倍.以ephrinB1-Fc激活EphB6后,茜素红染色显示钙结节明显减少,ALP活性显著下降(P＜0.05),在高浓度配体作用后ALP活性[(12.20±1.30)金氏单位/100 ml]较低浓度配体[(15.40±1.03)金氏单位/100ml]有进一步下降(P＝0.008).配体作用后,BMSC成骨分化中Runx2和Osterix 的表达显著下降.结论 EphB6通过调控Runx2和Osterix抑制 BMSC成骨分化.     

EphB6在小鼠骨髓间充质干细胞成骨分化中的初步研究

目的探讨EphB6在小鼠骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSC)成骨分化中的作用及其可能机制。方法将BMSC通过双相接种法接种于脱钙骨基质,分为4组:成骨诱导5、14 d(A、B组),普通培养5、14 d(C、D组),茜素红染色检测钙结节,定量检测各组碱性磷酸酶活性,Runx2、Osterix和EphB6的mRNA表达,采用游离态配体ephrinB1-Fc激活EphB6并重复检测上述指标。结果 ALP活性、Runx2和Osterix在A、B组增高,且B组检测到最高的ALP活性[(7.32±2.02)金氏单位/100 ml]、Runx2(3.442 6±0.258 5)倍和Osterix(2.482 3±0.329 2)倍。EphB6的表达水平在A、B组降低,且在B组最低(0.754 3±0.023 5)倍。以ephrinB1-Fc激活EphB6后,茜素红染色显示钙结节明显减少,ALP活性显著下降(P<0.05),在高浓度配体作用后ALP活性[(12.20±1.30)金氏单位/100 ml]较低浓度配体[(15.40±1.03)金氏单位/100 ml]有进一步下降(P=0.008)。配体作用后,BMSC成骨分化中Runx2和Osterix的表达显著下降。结论 EphB6通过调控Runx2和Osterix抑制BMSC成骨分化。     

淫羊藿含药血清对骨质疏松大鼠骨髓基质干细胞成脂分化PPARγ mRNA表达的影响

目的探讨淫羊藿含药血清对绝经后骨质疏松症大鼠骨髓基质干细胞(MSCs)成脂分化PPARγ mRNA表达的干预作用。方法 15只SPF级SD雌性大鼠,无菌条件下分离培养骨髓间基质干细胞,传至3代。将所获的细胞随机分为模型组、成脂诱导组、西药组、成脂诱导西药组、中药组和成脂诱导中药高、中、低剂量组。使用小牛血清,淫羊藿高、中、低剂量含药血清以及尼尔雌醇含药血清配合成脂诱导剂分别对MSCs进行干预,观察各组干预后7 d PPARγ mRNA表达情况。结果与模型组比较,西药组、成脂诱导西药组、中药高剂量组PPARγ mRNA表达均有下降,差异显著(P<0.05),中药低剂量组明显降低(P<0.01);与成脂诱导组比较,成脂诱导中药低剂量组PPARγmRNA表达明显降低(P<0.01),成脂诱导中药高剂量组表达降低具有显著性差异(P<0.05);成脂诱导中药各组中,以低剂量组抑制PPARγ mRNA表达最为明显,中药高剂量组其次。结论通过7 d的干预,各组均有PPARγmRNA表达,其中以模型组表达最为明显;在抑制PPARγ mRNA表达方面以中药低、高剂量组为最优。     

葡萄糖、胰岛素对大鼠骨髓间充质干细胞向成骨细胞分化的影响

目的 观察不同浓度葡萄糖及胰岛素(INs)对骨髓间充质干细胞(BMSC)向成骨细胞(OB)分化能力的影响,探讨葡萄糖及INs对骨代谢的作用机制.方法 采用体外细胞培养技术自大鼠股骨和胫骨中分离BMSC进行纯化,培养,然后在成骨培养基中诱导BMSC分化为OB,并用不同浓度的葡萄糖(5.6、25、50 mmol/L)及加或不加INs(0.6 μg/ml)干预诱导过程,光镜下观察茜素红染色分化后的OB钙结节的细胞比例.Realtime PCR测定OB的标记物碱性磷酸酶(ALP)、骨钙素(BGP)及转录因子Runx2 mRNA表达.结果 随着糖浓度升高,茜素红染色红色钙结节数目明显减少,PCR结果显示ALP,BGP及Runx2 mRNA表达也明显降低,差异有统计学意义(P<0.05).提示随着糖浓度增加成骨分化逐渐减少,在同等糖浓度下,加入INs干预组,茜素红染色阳性细胞计数明显增加,PCR结果ALP,BGP及Runx2 mRNA表达明显升高(P<0.01),提示INs可提高OB分化.结论 葡萄糖呈量效性地抑制BMSC向OB分化,这可能为糖尿病性骨质疏松形成的重要机制之一.INs可促进BMSC向OB分化,并可改善高糖对BMSC向OB分化的抑制作用.     

胰岛素对稳定表达人脂联素3T3-L1细胞P PARγ2m RNA表达的影响

目的 探讨胰岛素对稳定表达人脂联素3T3-L1细胞PPARγ2mRNA表达的影响.方法 重组脂联素真核表达质粒(pcDNA3.1+-hADPN)脂质体法稳定转染3T3-L1细胞.用胰岛素(500ng/mL)处理未转染、转染空载载体、转染pcDNA3.1+-hADPN的3T3-L1细胞,RT-pCR检测PPARγ2 mRNA的表达量.结果 ①稳定转染了pcDNA3.1+-hADPN的未分化和已分化3T3-L1细胞,PPARγ2表达量较未转染组明显增加(P＜0.01).②胰岛素可明显抑制PPARγ2 mRNA的表达(P＜0.05).③稳定转染pcDNA3.1+-hADPN可改善胰岛素抑制作用(P＜0.05).结论 稳定转染pcDNA3.1+-hADPN可明显增加未分化和已分化的3T3-L1细胞PPARγ 2 mRNA表达.胰岛素抑制PPARγ 2 mRNA表达,转染PcDNA3.1+-hADPN可改善胰岛素抑制作用.     

靶向PPARγ基因RNA干扰对乙醇诱导家兔骨髓基质细胞成脂分化的影响

目的:观察靶向过氧化物酶体增殖子活化受体-γ(PPARγ)基因RNA干扰阻断乙醇诱导骨髓基质细胞的成脂分化效应.方法:选用家兔第2代骨髓基质细胞,随机分为7组.对照组(N):无特殊处理;模型组(M):给予0.09 mol/L乙醇;感染空载体组(CO):给予空载体腺病毒1×105U和乙醇;感染无关siRNA组(C1):给予无关序列siRNA腺病毒1×105U和乙醇;干扰1、2、3组(S1、S2、S3):分别给予靶向PPAR-γ siRNA 1、2、3 腺病毒1×105U和乙醇.7d后采用RT-PCR技术检测MSCs内PPARγmRNA表达,14 d后测定MSCs内甘油三酯含量,21d后MSCs苏丹Ⅲ染色,计数脂肪细胞.结果:M组、C0组、C1组PPARγ mRNA呈高表达,脂肪细胞数增多,细胞甘油三酯含量升高.与N组比较差异均有统计学意义(P均<0.05);S1组、S2组、S3组PPARγ mRNA呈低表达,脂肪细胞数少,细胞内甘油=三酯含量稍高,与M组、CO组、C1组比较差异均有统计学意义(P均<0.05),与N组比较差异无统计学意义(P>0.05).结论:靶向PPARγ,基因RNA干扰能够有效地阻断乙醇诱导的骨髓基质细胞中PPARγmRNA高表达和成脂分化.     

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…