骨髓基质干细胞-藻酸钙复合物构建骨组织的实验研究

目的研究犬骨髓基质干细胞(BMSCs)体外成骨活性和在犬体内异位成骨能力,探讨其作为骨组织工程种子细胞的可能性. 方法抽取成年犬骨髓,梯度离心法获取单个核细胞,条件培养液体外培养、诱导、扩增后,进行组织化学、免疫细胞化学染色,透射电镜下观察其成骨活性.将培养的第3代细胞与藻酸钙所形成的复合物植于犬皮下,3月后行组织学检测. 结果体外培养的BMSCs Von Kossa染色可见钙结节,AKP染色及I型胶原、Osf2/Cbfα1、骨钙素免疫细胞化学染色先后呈现阳性;透射电镜下见基质钙化;组织学检测提示细胞-藻酸钙复合物体内培养3月后形成骨样组织. 结论BMSCs在大量扩增的同时,在一定条件下能向成骨细胞分化,细胞与藻酸钙复合物能在具免疫力的哺乳动物皮下形成骨样组织.BMSCs可作为骨组织工程较理想的种子细胞.     

Bio-Oss~/PRF复合支架结合骨髓基质细胞构建组织工程骨实验研究

[目的]探讨Bio-Oss/PRF复合物作为骨组织工程支架材料的可行性.[方法]取实验犬全血,利用乙醇沉淀法制备富血小板纤维蛋白(PRF);取实验犬髂骨骨髓进行全骨髓培养,收集扩增的第3代骨髓基质细胞(BMSCs)与骨形态发生蛋白-2(BMP-2)接种于Bio-Oss/PRF复合支架,植入裸鼠背部皮下6个月后进行组织学检测.[结果]Bio-Oss/PRF/BMSCs/BMP-2复合物在体内均无炎症反应,标本体积维持率均达到90%以上,新骨形成率为16.8%±6.4%,异种骨吸收率为6.93%±0.69%.[结论]Bio-Oss/PRF复合物是具有良好成骨活性的骨组织工程支架材料.     

兔骨髓基质干细胞在琼脂糖和藻酸钙凝胶中体外培养的观察

目的 观察兔骨髓基质干细胞(BMSCs)在琼脂糖(Agarose)和藻酸钙(Calcium alginate)二种凝胶中体外培养的生长分化状况，以探索一种理想的骨软骨组织工程载体。方法 将传代培养至21d经诱导的兔BMSCs分别接种至1．5％琼脂糖和1．2％藻酸钙凝胶中立体培养，通过细胞计数、HE染色和钙结节检测来观察BMSCs的生长分化。结果在培养第16d琼脂糖凝胶表面有钙结节形成，藻酸钙凝胶中无钙结节形成；在琼脂糖凝胶立体培养中，细胞数量变化不明显，但向成软骨样细胞转化，在藻酸钙凝胶中，部分向成软骨样细胞转化，部分细胞坏死。结论细胞外基质(ECM)可明显影响BMSCs分化，琼脂糖作为BMSCs的载体可尝试用于动物体内的骨软骨修复，而藻酸钙凝胶作为细胞载体尚待进一步研究。     

应用GFP标记技术示踪裸鼠体内组织工程化骨的形成

目的 应用绿色荧光蛋白(GFP)标记技术，观察组织工程化骨体内形成过程中种子细胞的变化与转归。方法 用GFP重组逆转录病毒载体(GFP-RV)转染犬骨髓基质于细胞(BMSCs)，将其接种于β-磷酸三钙(β-TCP)，形成细胞—材料复合物，移植于裸鼠皮下。术后8周，HE染色观察组织结构，碱性磷酸酶(AKP)染色和骨钙素(OCN)免疫组化检测功能蛋白，激光共聚焦显微镜下对GFP进行示踪观察。结果 8周后，大体可见组织工程化新骨形成，组织学示新生骨小梁围绕材料孔隙生成，AKP染色和OCN免疫组化结果阳性，并可见新生组织内有呈绿色的GFP标记细胞，β-TCP部分降解。结论 组织工程化骨组织学结构与松质骨小梁类似。新生组织表达GFP，证实组织工程化骨组织的形成来源于供体细胞。     

犬牙周膜细胞与骨髓基质细胞组织再生能力比较的实验研究

目的 比较犬骨髓基质细胞(BMSC)和牙周膜细胞(PDLC)体内成骨及成韧带样组织的能力,为牙周组织工程种子细胞的选择提供实验依据. 方法 将体外培养Beagle犬BMSC及PDLC与脱细胞真皮基质(ADM)膜复合后植入裸鼠皮下,以单纯ADM为对照组,分别于术后4周和8周进行标本组织学及免疫组织化学染色分析,比较骨保护素(OPG)和胶原Ⅻ的表达. 结果 复合物植入4周和8周组织学观察显示,BMSC形成骨样组织多于PDLC,面形成韧带样组织少于PDLC.免疫组织化学染色显示,BMSC组的OPG表达量高于PDLC组,但胶原Ⅻ的表达量低于PDLC组. 结论 BMSC体内成骨能力高于PDLC组,但成韧带样组织能力低于PDLC组.     

骨髓基质细胞体外培养的生物学特性和成骨能力初步鉴定

目的体外分离培养狗的骨髓基质细胞（BMSCs），诱导其向成骨细胞分化，初步鉴定其成骨潜能和生物学特性。方法抽取毕格犬髂骨骨髓体外分离培养获得BMSCs，DMEM、新生牛血清培养基进行原代培养，部分传代细胞以含10mmol／L地塞米松、50μg／ml抗坏血酸和10mmol／L β-甘油磷酸钠的矿化培养液诱导其向成骨细胞增殖分化。倒置相差显微镜进行细胞形态学和细胞生长增殖观察，VonKossa法染色检测体外矿化结节形成，细胞碱性磷酸酶染色检测BMSCs的成骨活性。结果体外分离培养的BMSCs经条件培养基诱导后表现出明显的成骨活性，体外矿化（骨样）结节的Von Kossa染色阳性；传代BMSCs碱性磷酸酶染色阳性。结论体外分离培养的BMSCs中含有骨源性前体细胞．传代细胞具有较强的成骨潜能。     

应用GFP标记技术示踪裸鼠体内组织工程化骨的形成

目的应用绿色荧光蛋白(GFP)标记技术,观察组织工程化骨体内形成过程中种子细胞的变化与转归.方法用GFP重组逆转录病毒载体(GFP-RV)转染犬骨髓基质干细胞(BMSCs),将其接种于β-磷酸三钙(β-TCP),形成细胞-材料复合物,移植于裸鼠皮下.术后8周,HE染色观察组织结构,碱性磷酸酶(AKP)染色和骨钙素(OCN)免疫组化检测功能蛋白,激光共聚焦显微镜下对GFP进行示踪观察.结果 8周后,大体可见组织工程化新骨形成,组织学示新生骨小梁围绕材料孔隙生成,AKP染色和OCN免疫组化结果阳性,并可见新生组织内有呈绿色的GFP标记细胞,β-TCP部分降解.结论组织工程化骨组织学结构与松质骨小梁类似.新生组织表达GFP,证实组织工程化骨组织的形成来源于供体细胞.     

组织工程技术修复犬牙槽骨缺损的实验研究

目的　研究以犬骨髓基质细胞 (bonemarrowstromalcells ,BMSC)为种子细胞 ,利用组织工程技术修复水平型牙槽骨缺损的可行性。方法　抽取成年犬骨髓 ,用梯度离心法获取单个核细胞 ,经条件培养液体外诱导培养后 ,进行组织化学、免疫细胞化学、扫描电镜检测 ,并将培养的第 3代细胞与藻酸钙形成复合物。在犬双侧下颌第 3、4前磨牙和第 1磨牙颊侧制备冠根向深 5mm的水平型牙槽骨缺损 ,用以下方法进行治疗 :(1)细胞 藻酸钙复合物修复组 ;(2 )藻酸钙对照组 ;(3)空白对照组。4、12、2 4周后经组织学检查骨缺损修复情况。并比较 12周时实验组与对照组的修复效果。结果在体外经诱导培养的BMSC可表达AKP、cbfa1、Ⅰ型胶原等 ,表现出成骨细胞活性 ;4、12、2 4周细胞 藻酸钙复合物修复部位显示逐渐有骨形成 ;第 12周时 ,实验组牙槽嵴高度可增高 2 4mm± 0 9mm ,达到原来的 4 8 5 9% ,空白对照组增高 0 8mm± 0 8mm ,达到原来的 15 76 % ,材料对照组增高 1 0mm± 0 9mm ,达到原来的 19 74 % ,实验组的牙槽嵴增高度和修复率与两对照组均有显著性差异(P <0 0 1)。结论　能以BMSC为种子细胞 ,以藻酸钙为支架材料 ,利用组织工程技术部分修复水平型牙槽骨缺损。     

全骨髓贴壁法分离培养rBMSCs及成骨诱导探讨

[摘要]　目的 通过研究在体外培养大鼠骨髓基质干细胞(rBMSCs)及其成骨生物学特性,探讨构建骨组织工程种子细胞的方法。方法 通过全骨髓贴壁培养法分离大鼠骨髓基质干细胞,应用含地塞米松、维生素C、β2甘油磷酸钠的诱导培养液定向诱导传代细胞向成骨细胞分化,并检测碱性磷酸酶活性及钙结节茜素红染色反应,进行成骨细胞鉴定。结果 全骨髓贴壁培养下,骨髓基质干细胞增殖能力强,生长迅速,呈成纤维细胞样生长。诱导条件下细胞的碱性磷酸酶活性明显增高,并且钙结节茜素红染色反应阳性。结论 采用全骨髓贴壁培养法,骨髓基质干细胞分离培养及体外扩增更为简便迅速,诱导条件下成骨能力肯定,可为骨组织工程构建提供大量种子细胞。     

非诱导条件下犬骨髓基质细胞的生物学特性

目的 研究体外培养骨髓基质细胞(BMSC)的生长特点和非诱导条件下的成骨特性。方法 采用犬来源BMSC体外扩增培养，观察非诱导条件下BMSC生长变化和成骨分化。结果 形态学观察表明，BMSC贴壁细胞呈集落生长，有成纤维细胞样外观，未加入成骨诱导剂，细胞形态发生变化，钙沉积出现，碱性磷酸酶(ALP)表达。3代内扩增的BMSC有成骨活性，但原代细胞成骨活性优于传代后细胞。结论 体外培养BMSC能大量扩增，具有自然向成骨细胞分化的能力，是骨组织工程理想的种子细胞。本实验所培养的BMSC具有骨祖细胞特性。     

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.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

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.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.