miR-30a-5p在人骨髓间充质干细胞向成骨细胞分化过程中的生物学功能及验证

目的：探讨并验证重组miRNA-30a-5p体外转染对人骨髓间充质干细胞（MSCs）向成骨细胞分化的生物学作用。方法：人工重组合成从人骨髓MSCs定向成骨分化差异性表达miRNA基因芯片结果中筛选成骨性表达量显著增高的miRNA-30a-5p。从人体骨髓中分离培养MSCs,成骨诱导分化过程中于体外转染重组miRNA-30a-5p,并通过茜素红S法染色检测钙盐沉积、碱性磷酸酶（ALP）钙钴法染色及ALP比色法定量检测成骨细胞ALP活性,对比观察研究重组miRNA-30a-5p在人骨髓MSCs定向成骨诱导分化过程中的生物学功能。结果：成功分离培养人骨髓MSCs并诱导其成骨定向分化。经体外向MSCs转染miRNA-30a-5p并诱导其成骨定向分化,转染效率为（37.32±2.43）%。分别于诱导培养第14、21天行ALP染色观察、ALP活性测定、茜素红钙盐结节染色检测各组细胞成骨活性,结果显示miRNA-30a-5p mimics转染组MSCs成骨分化特性显著性增强（P〈0.05）。结论：miRNA-30a-5p在MSCs定向成骨分化的过程中具有一定的促进增强作用,被转染的MSCs向成骨细胞定向分化表达有所增加,为进一步阐明人骨髓MSCs定向成骨分化的分子生化机制,细胞移植修复治疗骨缺损奠定了理论基础。     

兔自体骨髓间充质干细胞移植修复关节软骨缺损的研究

目的:研究将体外培养的兔骨髓间充质干细胞(MSCs)定向诱导分化为成骨细胞的方法, 探讨其作为组织工程化骨的种子细胞的可行性.方法:采用Percoll分离液,从兔的骨髓组织中分离骨髓间充质干细胞,用低糖型的DMEM( Dulbecco's modified Eagle's medium)培养液中添加成骨诱导剂地塞米松、β-甘油磷酸钠及抗坏血酸进行培养.诱导2周后钙-钴法染色检测碱性磷酸酶(ALP)表达, 诱导4周后VonKos-sa染色检测钙结节形成.结果:第3代兔MSCs呈典型的成骨细胞形态,诱导2周ALP染色阳性率达80%,诱导4周后VonKossa染色可见钙结节形成.结论:经过分离纯化后的兔骨髓基质细胞在体外培养条件下可以分化为成骨细胞.     

培养的人骨髓间充质干细胞向成骨细胞、脂肪细胞分化

目的探讨体外培养的人骨髓间充质干细胞(MSCs)向成骨细胞、脂肪细胞分化的方法,以及钙结节形成、Ⅰ型胶原表达等情况. 方法分离人骨髓间充质干细胞,经过原代培养和传代培养,分别加入成骨诱导、脂肪诱导培养体系,经过倒置显微镜观测与HE染色了解诱导后细胞形态变化,并通过von Kossa染色、Ⅰ型胶原免疫组化染色、苏丹黑染色与油红O法染色鉴定细胞的性质.另取2代细胞体外培养,加入成骨诱导分化试剂,以碱性磷酸酶(ALP)为检测指标计数细胞阳性率,比较成骨诱导1、2、3、4周时细胞碱性磷酸酶的变化.结果体外培养的MSCs在合适的条件下能够向成骨细胞、脂肪细胞分化.细胞经成骨诱导2～3周时细胞碱性磷酸酶阳性率最高,达到85%.结论 MSCs体外培养在一定条件下向成骨细胞、软骨细胞与脂肪细胞分化,体外培养时形成钙结节,并表达Ⅰ型胶原.培养2～3周后细胞经成骨诱导分化细胞ALP阳性率较高.     

人骨髓间质干细胞体外培养及诱导成骨的鉴定

目的 优化人骨髓间质干细胞(human bone marrow mesenchymal stem cells,hBMSCs)体外分离培养方法,对hBMSCs体外培养及诱导成骨能力进行鉴定.方法 密度梯度离心法分离hBMSCs,流式细胞分析法(FACS)对细胞表面抗原、细胞活力、细胞周期进行检测.以含地塞米松(1×10-7 mmol/L)、β-甘油磷酸钠(10 mmol/L)、抗坏血酸(35.22 mg/L)的DMEM培养液对hBMSCs进行诱导分化,相差显微镜和透视电镜观察细胞形态,Gomori钙钴法进行碱性磷酸酶染色(ALP),免疫组化检测Ⅰ型胶原,茜素红法进行钙结节染色.结果 hBMSCs可在体外分离扩增,表达CD44、CD105和CD106,不表达CD34和CD45,细胞活力为92.3%,G0-G1细胞占94.2%.经成骨培养液诱导后细胞形态由长梭形向多边形转变,ALP染色、Ⅰ型胶原染色及钙结节染色均为强阳性.结论 hBMSCs可在体外长期、稳定培养,具有向成骨细胞分化的潜能.     

富血小板血浆对骨髓间充质干细胞向成骨细胞定向分化的影响

目的 观察富血小板血浆（platelet-rich plasma,PRP）对兔源骨髓间充质于细胞（bone mesenchymal stem cells,BMSCs）定向分化为成骨细胞的作用及机制. 方法 离心法分离兔骨髓后,体外培养BMSCs,体积分数5％和10％的PRP与BMSCs共培养,观察BMSCs诱导分化过程中茜素红染色阳性率,应用酶标仪检测碱性磷酸酶（alkaliphosphatase,ALP）活性,应用ELISA法检测BMSCs诱导分化过程中骨钙素（osteocalcin,OCN）及结缔组织生长因子（connective tissue growth factor,CTGF）的含量,应用real time-PCR检测成骨相关基因CTGF、ALP、成骨细胞核心结合因子α1（core-binding factor alpha 1,Cbf-α1）、兔Ⅰ型胶原（Col-Ⅰ）和OCN的表达. 结果 BMSCs诱导分化后细胞茜素红染色阳性率、ALP活性、OCN及CTGF含量升高,CT-GF、ALP、Cbf-α1、Col-Ⅰ和OCN的mRNA表达增加. 结论 PRP可促进BMSCs向成骨细胞分化,可能与PRP促进BMSCs中CTGF、ALP、Cbf-α1、Col-Ⅰ和OCN表达有关.     

骨髓间充质干细胞建立的成骨细胞培养模型的研究

目的：为骨折骨缺损治疗的基础实验研究建立理想的细胞模型,观察兔骨髓间充质干细胞（BMSCs）在体外培养状态下的生长情况,对其向成骨细胞分化及分化能力进行研究.方法：采用全骨髓密度梯度离心法联合差速贴壁法从兔股骨骨髓中提取、分离、纯化BMSCs,取第3代BMSCs细胞分为两组进行培养,A组：为用含有诱导剂的低糖DMEM培养基的诱导培养组;B组：为常规培养组.倒置显微镜及HE染色观察记录各代次细胞形态等特征;CD44,CD90检测细胞表形,通过碱性磷酸酶（ALP）染色及标准化钙结节计数,鉴定细胞成骨活性.结果：兔骨髓间充质干细胞生长状态良好,培养7 d后各组细胞形态趋于一致,多呈长梭形,培养2 wk后对两组细胞进行ALP染色,可见A组细胞质内ALP染色阳性反应明显,B组染色显色弱.21 d时A组细胞茜素红染色后钙化结节数量多且较大,B组有少量钙结节形成,但较小.A组标准化钙结节计数显著高于B组差异具有显著统计学意义（P〈0.05）.结论：通过密度梯度离心法联合差速贴壁法所获得的BMSCs在常规培养或诱导培养时均可表现出成骨潜能,但诱导培养组的BMSCs的ALP活性更强,成骨能力更确切,可为骨组织工程远期实验研究提供理想的细胞模型.     

体外培养大鼠颌骨成骨细胞成骨及成血管相关因子表达检测

目的 探讨体外培养的大鼠颌骨成骨细胞成骨及成血管相关因子的表达情况。 方法 体外分离培养大鼠颌骨成骨细胞,采用碱性磷酸酶染色(ALP)与矿化结节茜素红染色对细胞进行鉴定。应用实时荧光定量聚合酶链反应及酶联免疫吸附法检测细胞成骨与成血管相关细胞因子的表达情况。 结果 体外培养的大鼠颌骨成骨细胞ALP及矿化结节茜素红染色均为阳性。颌骨成骨细胞在高表达成骨相关基因、Runt相关转录因子2、Ⅰ型胶原和骨钙素的同时,在蛋白及基因水平均高表达成血管相关细胞因子血管内皮生长因子、血管生成素1和成纤维生长因子2。 结论 颌骨成骨细胞在成骨分化的同时可通过分泌成血管相关细胞因子参与血管化调控。     

兔骨髓间充质干细胞的体外分离、培养及诱导分化

目的：研究兔骨髓间充质干细胞（Rabbit bone marrow mesenchymal stem cells,rBMSCs）的体外分离培养,观察其生长特性并探讨其定向诱导分化为成骨细胞和成脂肪细胞的途径,为种子细胞的选择提供一定的实验基础。方法：乌拉坦麻醉下耳缘静脉空气栓塞处死兔子,无菌条件下取出兔股骨,用percoll液密度梯度离心法+全骨髓贴壁培养法相结合进行体外培养、传代,倒置显微镜下观察细胞形态。取第3代细胞向成骨细胞和成脂肪细胞诱导,14 d后成骨细胞诱导组检测碱性磷酸酶,成脂肪细胞诱导组进行油红O染色。结果：percoll密度梯度离心法＋全骨髓贴壁培养法可成功获得rBMSCs,具有活跃的增殖能力。第3代细胞的生物学特征基本一致,并能定向诱导分化为成骨细胞及成脂细胞。成骨细胞诱导组碱性磷酸酶检测表达呈强阳性,成脂肪细胞诱导组油红O染色见胞浆内出现大量红染脂滴。结论：percoll密度梯度离心法+全骨髓贴壁培养法可成功分离和培养rBMSCs,其生长稳定,增殖力强,能定向诱导分化为成骨细胞及成脂细胞,可见rBMSCs是组织工程的优良种子细胞。     

17β-雌二醇对大鼠骨髓间充质干细胞成骨分化的作用

目的: 探讨不同剂量17β-雌二醇(17β-E2)对大鼠骨髓间充质干细胞(MSCs)成骨分化和骨钙素(BGP)基因表达的影响,为应用17β-E2调控MSCs向成骨细胞分化提供依据。方法: 应用密度梯度离心法与贴壁筛选法分离出MSCs,连续传代3次,进行成骨细胞的诱导分化。MSCs分为对照组[单纯成骨细胞培养基(OBM)培养]和不同剂量17β-E2组(在成骨诱导分化液中分别添加0.1、1.0、10.0和100.0 pmol·L^-1 17β-E2)。各组细胞在第7 天利用放射免疫法测定碱性磷酸酶(ALP)活性、Von Kossa染色检测钙盐沉积;第14天应用酶联免疫吸附法测定Ⅰ型胶原(ColⅠ)水平;第21天应用实时荧光定量PCR检测BGP mRNA表达水平,采用茜素红染色及在酶标仪上560 nm波长下测定各组细胞的吸光度(A)值,定量分析成骨矿化能力。结果: 应用密度梯度离心法与贴壁筛选法成功分离出MSCs,细胞呈成纤维细胞样,椭圆形核,核仁可见。传代培养的MSCs生长旺盛,保持原代细胞的形态特征。与对照组比较,不同剂量17β-E2组细胞ALP活性和ColⅠ水平增加(P<0.05或P<0.01),骨基质的钙化能力增强(P<0.05或P<0.01),并呈剂量依赖性;100.0 pmol·L^-1 17β-E2组 BGP mRNA表达水平升高(P<0.01)。结论: 密度梯度与贴壁筛选方法分离所得细胞为大鼠MSCs,17β-E2可增强MSCs成骨分化能力,上调BGP mRNA表达,并呈剂量依赖关系。     

兔骨髓间充质干细胞的体外培养及生物学活性观察

目的建立兔骨髓间充质干细胞(MSCs)体外分离、培养、扩增的方法，并进行生物学活性观察。方法穿刺抽取新西兰大耳白兔骨髓5ml，通过密度梯度离心法获取MSCs，体外贴壁培养、扩增、传代，倒置显微镜下观察原代及各代细胞形态、数量生长情况，描绘生长曲线。第2代细胞用矿化液连续培养后，进行ALP及矿化结节染色。结果体外成功建立兔骨髓间充质干细胞分离扩增传代的方法，能够连续传至8～9代；矿化液培养的细胞ALP及矿化结节染色阳性。结论兔骨髓间充质干细胞能在体外成功培养，具有向成骨细胞诱导分化的特性，可以作为骨组织工程的种子细胞。     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

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…     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

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.     

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.     

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.