小凹蛋白-1在大鼠骨髓间质干细胞分化为神经元样细胞中的作用

目的 应用RNA干扰(RNA interference)技术,抑制沉默小凹蛋白-1(caveolin-1)基因的表达,观察小凹蛋白-1在骨髓间充质干细胞(MSCs)分化为神经元样细胞的作用.方法 构建大鼠caveolin-1 siRNA(small interfering RNA),然后传染大鼠MSCs,诱导分化并在倒置显微镜下进行形态学观察,用MTT比色法检测细胞存活率,采用免疫细胞化学染色法检测Nestin、 NSE(神经元标记物) 、GFAP(神经胶质细胞标记物)的表达,设诱导未传染组和传染对照组作为对照研究.结果 用caveolin-1 siRNA传染MSCs后,caveolin-1基因表达消失,传染后24 h和3 d MSCs细胞存活率下降,与诱导未传染组和传染对照组比较差异有统计学意义(P<0.01),传染caveolin-1 siRNA的MSCs向神经元样细胞分化效率显著提高,诱导24 h后NSE阳性细胞率为(75.5±2.5)%,6 d后达(81.6±3.5)%,且未见GFAP的表达,与诱导未传染组和传染对照组比较差异有统计学意义(P<0.01).结论 caveolin-1 siRNA抑制caveolin-1基因的表达,可提高MSCs向神经元样细胞的分化效率. Abstract： Objective To investigate the role of caveolin-1 in bone marrow mesenchymal stem cells(MSCs) differentiating into neuron-like cells by transfecting MSCs with small interfering RNA(siRNA). Methods Construct caveolin-1 siRNA and then transfect into MSCs cultured in vitro. Cell growth was observed by an inverted phase contrast microscope. The survival ratio of MSCs was determined by MTT before transfection,24 hour, 3 days after transfection,respectively. MSCs was induced to differentiate into neuron-like cells by-ME. The neural cell specific marker Nestin,NSE,GFAP were determined by immunocytochemistry stain technique. Results Expresion of caveolin-1 vanished by tranfection of caveolin-1 siRNA,survival ratio of MSCs by transfection decreased and there was significant differences between no-transfection and transfection-controlling group after 24 hours and 3 days of transfection(P<0.01).The differentiation efficiency of MSCs transfected by caveolin-1 siRNA into neuron-like cells and the expression of NSE were increased significantly than no-transfection and transfection-controlling group, The percentage of positive NSE was(75.5±2.5)% after 24 hours and(81.6±3.5)% after 6 days, there was no expression of GFAP. There was significant differences between no-transfection and transfection-controlling group(P<0.01). Conclusions The differentiation efficiency of MSCs increased by suppressing caveolin-1 expression.     

Inactivation of the Rho-ROCK signaling pathway to promote neurologic recovery after spinal cord injuries in rats

Background After injury,axonal regeneration of the adult central nervous system （CNS） is inhibited by myelin-derived growth-suppressing proteins.These axonal growth inhibitory proteins are mediated via activation of Rho,a small GTP-binding protein.The activated form of Rho,which is bound to GTP,is the direct activator of Rho kinase （ROCK） through serial downstream effector proteins to inhibit axonal regeneration.The objective of this study was to observe the therapeutic effect of inactivation of the Rho-ROCK signaling pathway to promote neurologic recovery after spinal cord injuries in rats.Methods One hundred and twenty adult female Sprague-Dawley rats were randomly divided into three groups.Laminectomies alone were conducted in 40 rats in the sham group.Laminectomies and spinal cord transections were performed in 40 rats in the control group （treated with normal saline administered intraperitoneally）.Laminectomies and spinal cord transections were performed in 40 rats in the fasudil-treated group （treated with fasudil administered intraperitoneally）.Neurologic recovery was evaluated before surgery and 3 days,and 1,2,3,and 4 weeks after surgery using the Basso-Beattie-Bresnahan （BBB） scale of hind limb movement.At the same time,the expression of RhoA mRNA was determined with RT-PCR.Histopathologic examinations and immunofiuorescence staining of NF were performed 1 month after surgery.Results Compared with the control group,the BBB scores of the fasudil-treated group were significantly increased and the expression of RhoA mRNA was significantly decreased.In the fasudil-treated group,a large number of NF-positive regenerating fibers was observed; some fibers crossed the slit of the lesion.Conclusion Inactivation of the Rho-ROCK signaling pathway promotes CNS axonal regeneration and neurologic recovery after spinal cord injuries in rats.     

Inhibition of rho kinase attenuates high flow induced pulmonary hypertension in rats

Background The RhoA/Rho kinase pathway may participate in the pathogenesis of hypoxia and monocrotaline induced pulmonary hypertension. This study tested whether RhoA/Rho kinase pathway is involved in the pathogenesis of high flow induced pulmonary hypertension in rats. Methods Male Wistar rats （4 weeks） were randomly divided into 4 shunt groups, 4 treated groups and 4 control groups. Shunt and treated groups underwent left common carotid artery/external jugular vein shunt operation. Control groups underwent sham operation. Treated groups received fasudil treatment and the others received same dose of saline. At weeks 1, 2, 4 and 8 of the study, nght ventricular systolic pressure was measured and blood gases were analysed to calculate Qp/Qs. The weight ratio of right ventricle to left ventricle plus septum and the mean percentage of medial wall thickness in moderate sized pulmonary arteries were obtained. RhoA activity in pulmonary arteries was detected using Rho activity assay reagent. Rho kinase activity was quantified by the extent of MYPT1 phosphorylation with Western blot. Proliferating cells were evaluated using proliferating cell nuclear antigen immunohistological staining, Results Carotid artery/jugular vein shunt resulted in high pulmonary blood flow, both an acute and a chronic elevation of right ventricular systolic pressure, significant medial wall thickening characterized by smooth muscle cells proliferation, nght ventricular hypertrophy and increased activation of RhoA and Rho kinase. Fasudil treatment lowered pulmonary artery systolic pressure, suppressed pulmonary artery smooth muscle cells proliferation, attenuated pulmonary artery medial wall thickening and inhibited right ventricular hypertrophy together with significant suppression of Rho kinase activity but not Rho activity. Conclusions Activated RhoNRho kinase pathway is associated with both the acute pulmonary vasoconstriction and the chronic pulmonary artery remodelling of high flow induced pulmonary hypertension. Fasudil treatment could improve pulmonary hypertension by inhibiting Rho kinase activity.     

A Rho-kinase inhibitor,fasudil, attenuates progressive glomerulosclerosis induced by daunorubicin in rats

Accumulating evidence suggests that the small G protein Rho and its downstream effec-tor Rho kinase may play important roles in kidney biology. The present study examined the effects of a Rho-kinase inhibitor, fasudil, on daunorubicin-induced progressive glomerulosclerosis and explored the underlying mechanism by which fasudil ameliorates glomerulosclerosis. Thirty-six male SD rats were randomly allocated into sham-operation group （sham group, n=12）, unilateral nephrectomy （UNX）＋daunorubicin （DRB） group （model group, n=12）, UNX＋DRB＋Fasudil group （treatment group, n=12）. Two to four weeks after the establishment of the animal model, 6 rats in each group were taken randomly for the detection of 24-h urine protein excretion. Kidney sections were exam-ined by HE and PAS staining, immunohistochemistry and transmission electric microscopy （TEM）. The expression of Rho-kinase mRNA and P27 mRNA in kidney were detected by RT-PCR. It was found that the 24-h urine protein excretion in model group was increased significantly as compared with sham group （P〈0.01）. But this increase was significantly suppressed by fasudil （P〈0.05）. At 4 week, the foot process effacement in podocytes, mesangial proliferation and ECM accumulation were observed in model group, presenting as focal segmental glomerulosclerosis. But in the treatment group, the fasudil alleviated glomerular injury, with proliferating cell nuclear antigen （PCNA）-positive cell infiltration ameliorated and the expression of P27 increased. The expression of Rho-kinase mRNA was significantly enhanced in model group and was suppressed in treatment group. Moreover, fasudil up-regulated the mRNA expression of P27. Our study demonstrated that the glomerulosclerosis was substantially ameliorated by inhibiting the expression of Rho-kinase. It is suggested that Rho-kinase pathway is involved in the renal injury and the inhibition of Rho-kinase may constitute a therapeutic strategy for the treatment of renal injury.     

Adipose tissue-derived stromal cells express neuronal phenotypes

Background Adipose tissue-derived stromal cell (ADSCs) can be greatly expanded in vitro, and induced to differentiate into multiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic, and adipogenic cells. This study was designed to investigate the possibility of ADSCs differentiating into neurons. Methods Adipose tissue from rats was digested with collagenase, and adherent stromal cells were cultured. A medium containing a low concentration of fetal bovine serum was adopted to induce the cells to differentiate. ADSCs were identified by immunocytochemistry, and semi-quantitative RT-PCR was applied to detect mRNA expression of neurofilament 1 (NF1), nestin, and neuron-specific enolase (NSE). Results Nestin-positive cells were found occasionally among ADSCs. ADSCs were found to express NSE mRNA and nestin mRNA, but not NF1 mRNA. ADSCs could differentiate into neuron-like cells in a medium composed of a low concentration of fetal bovine serum, and these differentiated cells displayed complicated neuron-like morphologies. Conclusions The data support the hypothesis that adipose tissue contains stem cells capable of differentiating into neurons. These stem cells can overcome their mesenchymal commitment, and may represent an alternative autologous stem cell source for CNS cell transplantation.     

Protective effects of trimetazidine on bone marrow mesenchymal stem cells viability in an ex vivo model of hypoxia and in vivo model of locally myocardial ischemia

Bone marrow mesenchymal stem cells (MSCs) have shown potential for cardiac repair following myocardial injury,but this approach is limited by their poor viability after transplantation.The present study was to investigate whether trimetazidine (TMZ) could improve survival of MSCs in an ex vitro model of hypoxia,as well as survival,differentiation,and subsequent activities of transplanted MSCs in rat hearts with acute myocardial infarction (AMI).MSCs at passage 3 were examined for their viability and apoptosis under a transmission electron microscope,and by using flow cytometry following culture in serumfree medium and exposure to hypoxia (5% CO2,95% N2) for 12 h with or without TMZ.Thirty Wistar rats were divided into 3 groups (n=10 each group),including groupⅠ(AMI control),groupⅡ (MSCs transplantation alone),and group Ⅲ (TMZ+MSCs).Rat MSCs (4×107) were injected into peri-infarct myocardium (MSCs group and TMZ+MSCs group) 30 min after coronary artery ligation.The rats in TMZ+MSCs group were additionally fed on TMZ (2.08 mg?kg-1?day-1) from day 3 before AMI to day 28 after AMI.Cardiac structure and function were assessed by echocardiography at 28th day after transplantation.Blood samples were collected before the start of TMZ therapy (baseline),and 24 and 48 h after AMI,and inflammatory cytokines (CRP,TNF-α) were measured.Then the sur-vival and differentiation of transplanted cells in vivo were detected by immunofluorescent staining.The cellular apoptosis in the peri-infarct region was detected by using TUNEL assay.Furthermore,apoptosis-related proteins (Bcl-2,Bax) within the post-infarcted myocardium were detected by using Western blotting.In hypoxic culture,the TMZ-treated MSCs displayed a two-fold decrease in apoptosis under serumfree medium and hypoxia environment.In vivo,cardiac infarct size was significantly reduced,and cardiac function significantly improved in MSCs and TMZ+MSCs groups as compared with those in the AMI control group.Combined treatment of TMZ with MSCs implantation demonstrated further decreased MSCs apoptosis,further increased MSCs viability,further decreased infarct size,and further improved cardiac function as compared with MSCs alone.The baseline levels of inflammatory cyto-kines (CRP,TNF-α) had no significant difference among the groups.In contrast,all parameters at 24 h were lower in TMZ+MSCs group than those in MSCs group.Furthermore,Western blotting indicated that the expression of antiapoptotic protein Bcl-2 was upregulated,while the proapoptotic protein Bax was down-regulated in the TMZ+MSCs group,compared with that in the MSCs group.It is suggested that implantation of MSCs combined with TMZ treatment is superior to MSCs monotherapy for MSCs viability and cardiac function recovery.     

盐酸法舒地尔体外诱导大鼠骨髓间充质干细胞向神经元样细胞分化的可行性

目的:探讨Rho/Rho激酶(ROCK)抑制剂盐酸法舒地尔体外诱导大鼠骨髓间充质干细胞(MSCs)向神经元样细胞分化的可行性.方法:全骨髓培养法分离培养大鼠骨髓MSCs,用200μmol/L盐酸法舒地尔诱导分化.倒置显微镜观察诱导不同时间细胞的形态学变化;AO-EB染色法鉴定诱导后细胞存活率;免疫荧光法鉴定诱导后神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF200)与胶质纤维酸性蛋白(GFAP)的表达,判断其分化情况.结果:盐酸法舒地尔诱导30、60、90、120及180 min后细胞存活率分别为(96.7±2.2)%、(95.3±1.9)%、(93.8±1.8)%、(92.5±2.1)%及(90.1±1.3)%,其中以盐酸法舒地尔诱导120 min后,形态变化最为理想.盐酸法舒地尔诱导60、90、120及180min后,NSE阳性表达率分别为(66.5±1.9)%、(88.1±3.2)%、(93.6±1.9)%和(93.5±5.4)%,NF200阳性表达翠分别为(70.1±2.9)%、(89.5 ±1.3)%、(98.1±1.6)%和(98.3±1.9)%,而GFAP阳性表达率均小于5%.结论:盐酸法舒地尔可在体外快速、高效地诱导大鼠MSCs向神经元样细胞分化.     

An Improved Method for Directional Differentiation and Efficient Production of Neurons from Embryonic Stem Cells in vitro

To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4 protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium(ACM) in Vitro. The totipotency of ES cells was identified by observation of cells‘ morphology and formations of teratoma in immunocompromised mice. The cells‘ differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes,including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells‘ ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells‘ ratio in the ceils induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5 % among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRAand ACM.     

Distribution and differentiation of mesenchymal stem cells in tumor tissue

Background Tumor has an ability to become enriched in mesenchymal stem cells （MSCs） and of guiding MSCs to migrate to tumor tissue. But there are lack of relevant reports on the distribution and differentiation of MSCs in tumor tissue and the effect on tumor growth after MSCs engrafted in tumor tissue. In this study, we observed the distribution of bone marrow MSCs in tumor tissue and the possibility of MSCs differentiating into myofibroblast under the induction of local tumor microenvironment.
Methods Twenty-four New Zealand rabbits were randomly classified into the control group and the test group. MSCs were isolated and cultured for each animal, vx-2 tumor tissue was transplanted under the bladder mucosa of each animal One week after the transplantation, the self F2 passage MSCs marked by 4＇,6-diamidino-2-phenylindole were transplanted into tumor tissue in the test group while only Dulbecco＇s modified Eagle＇s medium-low glucose was infused into the control group. Ultrasonography was performed for each animal 1, 2, 3 and 4 week（s） after the vx-2 tumor mass was transplanted. The maximum bladder tumor diameter of each animal was recorded and the mean value of each group was calculated. One animal from each group was sacrificed in the third week and the remaining animals in the fourth week to observe the tumor development. Another animal treated the same as the test group was sacrificed to observe the distribution of MSCs in tumor tissue one week after self MSCs transplantation. Immunofluorescence was used to trace MSCs in tumor tissue. The double labeling immunofluorescence for α-smooth muscle actin （α-SMA） and vimentin was performed to identify whether the MSCs can differentiate into myofibroblast.
Results The ultrasonography showed no tumor mass one week after the vx-2 tumor mass transplantation. The mean maximum tumor diameter of the control group and test group was （0.70±0.14） cm and （0.78±0.14） cm, respectively, and there was no significant difference （t=1.308, P=0.204）. The tumor growth rate of the test group increased gradually in the third and fourth weeks, and the difference of the mean maximum tumor diameter between the two groups also increased gradually and was statistically significant （P 〈0.05）. MSCs distributed uniformly in tumor tissue one week after transplantation while most were distributed in the tumor stroma three weeks after transplantation. The double labeling immunofluorescence showed that the expression of α-SMA as well as Vimentin increased significantly three weeks after mesenchymal stem cells engrafted into tumor, indicating that MSCs had differentiated into myofibroblasts under the induction of the tumor microenvironment.
Conclusion MSCs can accelerate the tumor development and can differentiate into myofibroblast under the induction of tumor microenvironment.     

Over-expression of VEGF in marrow stromal cells promotes angiogenesis in rats with cerebral infarction via the synergistic effects of VEGF and Ang-2

This study explored whether the transplantation of modified marrow stromal cells (MSCs) has angiogenic effects in a left middle cerebral artery occlusion infarction/reperfusion (MCAO I/R) rat model and preliminarily examined the mechanism of angiogenesis following cerebral infarction.MSCs were isolated by using a direct adherent method and cultured.Vascular endothelial growth factor (VEGF) was transfected into MSCs by employing the liposome transfection.The transfection efficiency was measured by the optical density method.The protein expression of VEGF gene before and after transfection was measured by Western blotting.SD rat model of transient occlusion of the left middle cerebral artery was established by using an approach of intra-luminal occlusion.Tetrazolium (TTC) and HE staining were performed to observe the cerebral infarction.ELISAs were used to measure the levels of VEGF in the rat cerebral tissues.The expression patterns of angiopoietin-2 (Ang-2) and CD34 in cells surrounding the area of infarction were immunohistochemistrically oserved.Ang-2 protein expression in the tissue surrounding the area of infarction was measured by Western blotting.VEGF expression in the MSCs increased after transfection at a rate of approximately 28%±3.4%.ELISA showed that the expression of VEGF in the cerebral tissue was significantly increased after induction of infarction,peaking on the 4th day and decreasing to the levels of the sham surgery group (normal) within 7 to 10 days.The VEGF level was significantly higher at each time point in the VEGF-MSC and MSC groups compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group than in the MSC group and stayed relatively high until the 10th day.The immunohistochemical results showed that 10 days after the infarction,the number of Ang-2 and CD34-expressing cells in the area surrounding the infarction was significantly higher in the VEGF-MSC group and the MSC group compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group th     

人骨髓间充质干细胞向血管内皮细胞诱导的研究

目的 研究人骨髓间充质干细胞(HBMCs)的体外培养和向血管内皮细胞的诱导分化.方法 利用密度梯度离心法将HBMCs从人骨髓中分离出来,体外扩增.将第三代的细胞以含VEGF,bFGF的培养基定向诱导,使其向内皮细胞方向分化.利用免疫细胞化学和流式细胞学检测诱导后的细胞表型.结果 原代未经诱导的骨髓干细胞,培养3周后细胞形态呈梭形,诱导后第7天的细胞呈椭圆形或不规则形,第14天细胞大致呈铺路石样改变.免疫细胞化学显示CD31、CD34、vWF因子呈阳性,流式细胞仪测定CD31、vWF因子阳性率分别为87.5%、82.6%,双阳性率为71.2%.结论 骨髓间充质干细胞在内皮细胞生长因子和碱性成纤维细胞因子的诱导下向血管内皮细胞方向分化. Abstract： Objective To study the culture methods of human bone marrow mesenchymal stem cells(MSCs) and differentiation into vascular endothelial cells in vitro. Methods Bone marrow mesenchymal stem cells were isolated and cultivated by density gradient centrifugation method. Induce the third generation to vascular endothelial cells with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Immunophenotypes of cells were detected by flow cytometry techniques and immunocyte chemistry after the transfection. Results After three weeks of primary culture of MSCs, the cell showed on fusiform shape. After 7 days induction, the cell showed on ellipse and irregular shape. After two weeks transfection, the cell exhibited a Cobblestone-like morphology. The cell expressed CD34,CD31,vWF after transfection by immunocyte chemistry. The positive rates of CD31 vWF were 87.5% and 82.6%, and the double positive rate was 71.2%. Conclusions Bone marrow mesenchymal stem cells can differentiate into vascular endothelial cells by treating with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).     

骨髓干细胞和伞状支撑骨移植治疗股骨头坏死的动物实验研究报告

目的 探讨骨髓干细胞移植和伞状支撑骨移植治疗股骨头坏死的效果.方法 取6只健康山羊,采用液氮冷冻法制作单侧股骨头坏死动物模型,8周后在坏死侧行骨髓干细胞移植和伞状支撑骨移植术.分别于术后3、6个月做放射学检查,观察股骨头内骨质变化.结果 实验动物一般状况良好.液氮冷冻法8周后达成股骨头坏死模型,模型侧后肢出现跛行.骨髓干细胞移植和伞状支撑骨移植术后3个月X线显示,股骨头外形恢复,股骨头内囊性低密度区消失,可见骨柱影,股骨头内骨质愈合状况良好,原塌陷已修复,股骨头无再塌陷.6个月时,治疗侧股骨头外形正常,骨密度基本均匀,骨柱影已模糊,植骨已融合,股骨头无再塌陷.正常对照侧股骨头无异常变化.结论 骨髓干细胞移植和伞状支撑骨移植术可以有效治疗骨坏死,防治股骨头塌陷,效果良好. Abstract： Objective To study the usefulness to treat the osteonecrosis of the femoral head (ONFH) with bone marrow mesenchymal stem cell transplantation and umbrella strut bone grafting. Methods Six goats were established of osteonecrosis of the femoral head models on one side by method of liquid nitrogen frozen, and then they were taken into bone marrow mesenchymal stem cell transplantation and umbrella strut bone grafting after 8 weeks. The radiological examination was made at 3 and 6 months after the bong grafting, and observed changes of bone union in the femoral head. Results The general state of the experimental animal was fine after the operation. The model of ONFH was reached on 8 weeks after liquid nitrogen frozen, and limping. After 3 months of bone marrow mesenchymal stem cell transplantation and umbrella strut bone grafting, X-ray film showed that low bone density disappeared, the shape of the femoral heads, grafting strut bone and initial bone union were fine in the head, and no repeated collapse of the head. The X-ray film showed that the shape of the femoral heads was normal, grafting strut bone was union in the head, and no repeated collapse of the head after 6 months. There was normal on the control side. Conclusions The ONFH can be treated effectively by bone marrow mesenchymal stem cell transplantation and umbrella strut bone grafting, and the collapse of the femoral head is prevented.     

法舒地尔对急性冠状动脉综合征患者血管内皮功能的影响

目的 研究法舒地尔对急性冠状动脉综合征(以下简称急性冠脉综合征)患者血管内皮功能的影响.方法 随机选择急性冠脉综合征患者80例,将入选患者按随机单盲法分为法舒地尔治疗组40例,常规治疗组40例,另外随机选择40例正常人做对照组.分别测定两组治疗前及治疗后4周血浆内皮素、一氧化氮含量.结果 急性冠脉综合征患者血浆一氧化氮水平明显下降,与对照组比较差异有统计学意义(P<0.01);血浆内皮素水平明显升高,与对照组比较差异有统计学意义(P<0.01);急性冠脉综合征组和常规治疗组两组治疗前血浆内皮素、一氧化氮水平比较差异无统计学意义(P>0.05).急性冠脉综合征组治疗后一氧化氮水平较前明显升高, 血浆内皮素水平较前明显降低,与治疗前比较差异有统计学意义(P<0.01),与常规治疗组治疗后比较差异亦有统计学意义(P<0.05).结论 法舒地尔对急性冠脉综合征患者具有保护血管内皮功能的作用. Abstract： Objective To explore the effect of fasudil on vascular endothelial function in patients with acute coronary syndrome.Methods Randomly selected patients with acute coronary syndrome in 80 cases, all patients were selected randomly divided into single-blind fasudil group of 40 patients, 40 patients with conventional treatment group, and randomly selected 40 cases of normal people to the control group. The two groups were measured before and after treatment for 2 weeks ET, nitric oxide.Results Acute coronary syndrome patients with decreased levels of nitric oxide, there was a significant difference compared with the control group (P<0.01).Plasma ET levels were significantly increased compared with the control group,and there was a significant difference (P<0.01).ET, nitric oxide levels were not significantly different between the two groups before treatment (P>0.05). The level of nitric oxide significantly increased, plasma ET levels significantly decreased than that before treatment and the difference was significant (P<0.01), and compared with the conventional treatment group, the difference was also significant (P<0.05).Conclusions Fasudil on patients with acute coronary syndrome has the protection of vascular endothelial function.     

骨髓间充质干细胞移植对肺气肿大鼠血浆TNF-α的作用

目的 研究移植骨髓间充质干细胞(MSCs)对肺气肿大鼠血浆肿瘤坏死因子-α(TNF-α)水平的影响.方法 将30只Wistar大鼠随机分为对照组、肺气肿组、肺气肿+MSCs治疗组,每组30只.在气管内注入脂多糖(LPS)并烟雾暴露,建立肺气肿大鼠模型,然后将培养的MSCs经尾静脉注入到肺气肿大鼠体内.观察移植后大鼠肺组织的变化,酶联免疫吸附测定(ELISA)法检测血浆TNF-α含量.结果 肺气肿组、肺气肿+MSCs移植组均出现肺气肿样病理改变,但后者较前者明显减轻.与肺气肿组比较,肺气肿+MSCs组TNF-α含量显著降低.结论 MSCs移植对肺气肿大鼠有保护作用. Abstract： Objective To investigate the effect of bone marrow mesenchymal stem cell (MSCs) transplantation on plasma TNF-α levels in rats of pulmonary emphysema. Methods Thirty wistar rats were randomized into three groupes:normal control group, pulmonary emphysema group, pulmonary emphysema +MSCs transplantation groupe. The pulmonary emphysema model of rats were established by intratracheal instillation of lipopolysaccharide twice and daily exposure to smog. Bone marrow MSCs were infused through tail vein. Morphologic changes of the lung tissues were observed. The level of plasma TNF-α were dectected using enzyme-linked immunosorbent assay. Results Destruction of alveolar walls was observed in rat lungs from pulmonary emphysema group and pulmonary emphysema +MSCs transplantation group. MSCs transplantation group significantly ameliorated the emphysematous changes. Significant differences in the levels of TNF-α between pulmonary emphysema group and pulmonary emphysema +MSCs transplantation group were observed. Conclusions MSCs therapy shows protective effects against pulmonary emphysema.     

Pdx-1真核表达载体构建及其在大鼠骨髓间充质干细胞中的表达

目的 构建含有胰腺-十二指肠同源盒-1(Pdx-1)的真核表达载体,并研究其在大鼠骨髓间充质干细胞(MSCs)中的表达情况.方法 克隆Pdx-1基因,构建含Pdx-1的真核表达载体pEGFP-Pdx-1及pcDNA3.1-Pdx-1,转染MSCs,倒置荧光显微镜及逆转录聚合酶链反应(RT-PCR)检测MSCs中Pdx-1的表达.结果 测序结果 表明克隆的Pdx-1基因序列正确,构建的含Pdx-1的真核表达载体能有效的转染MSCs,并检测到该基因mRNA的表达.结论 完成Pdx-1基因克隆,成功构建含有Pdx-1基因真核表达载体,并在转化株中检测到该基因的表达,为糖尿病的细胞替代及基因治疗提供基础. Abstract： Objective To construct eukaryotic expression vector containing the pancreatic-duodenum homeobox-1(Pdx-1)and to study the expression in rat bone marrow-derived mesenchymal stem cells (MSCs). Methods Cloned Pdx-1 gene and constructed containing Pdx-1 eukaryotic expression vector pEGFP-Pdx-1 and pcDNA3.1-Pdx-1, transfected MSCs, detected the Pdx-1 expression in MSCs by invert fluorescence microscope and RT-PCR.Results Sequencing results showed that the cloned Pdx-1 gene sequence was correct, the eukaryotic expression vector containing Pdx-1 could effectively transfected MSCs and detected the gene expression at mRNA level.Conclusions The completion of Pdx-1 gene is cloned successfully and constructed containing Pdx-1 gene eukaryotic expression vector, detected the expression of the gene in the stable transfected MSCs,making preparations for the cell replacement and gene therapy of diabetes.     

布托啡诺复合小剂量氯氨酮用于剖宫产术后镇痛

目的 观察术后硬膜外持续泵入布托啡诺复合小剂量氯胺酮用于剖宫产术后镇痛的效果和对初乳及不良反应的影响. 方法选择40例Ⅰ～Ⅱ级择期或急症孕足月行剖宫产手术后的产妇,年龄23～35岁,随机分为两组.A组为观察组(n=20),用布托啡诺0.1 mg/kg+氯胺酮1 mg/kg+生理盐水至 100 ml持续泵入;B组为对照组(n=20),用0.15%罗哌卡因+0.004%芬太尼+0.1%地塞米松+生理盐水总量100 ml持续泵入.观察产妇术后各时间段的视觉模拟镇痛评分(VAS)、镇静评分、泌乳时间,并对术后产妇的总体感觉满意度进行评分.结果 A、B两组在泵药后4、12、24、48 h的VAS评分、镇静评分差异无统计学意义(P>0.05).A组产妇的总体感觉满意度高于B组(P<0.05),两组的初乳时间差异无统计学意义(P>0.05).结论 布托啡诺复合小剂量氯胺酮硬膜外持续泵入用于剖宫产术后镇痛可以提供满意的镇痛效果,而且也有利于术后产妇的恢复,并且不影响泌乳. Abstract： Objective To observe the analgesia effects of buprenorphine hydrochloride combined with low ketamine that continued to pump into epidural space,which were used to treat postoperative analgesia of cesarean section and to study their on colostrums and side reactions. Methods Forty patients were at age of 23-35 years and term pregnancy, who were 1-2 grade or emergency.These cases were randomly divided into two groups: group A(study group,n=20) and group B(control group,n=20),group A were treated with buprenorphine hydrochloride(0.1 mg/kg)+ ketamine(1 mg/kg)+NS(coming to 100 ml),which continued to pump into epidural space. Group B were treated with ropivacaine(0.15%)+febtanyl citrate(0.004%)+dexame thasone(0.1%)(coming to 100 ml), which continued to pump into epidural space.We observe postoperative parturients mimic analgesia VAS of visual sense,sedation VAS,lactigenous time and the total degree of satisfaction. Results There were no significant difference between group A and group B about mimic analgesia VAS of visual sense(P>0.05) and lactigenous time(P>0.05),the total degree of satisfaction in group A was higher than that in group B (P<0.05). Conclusions Buprenorphine hydrochloride combined with low ketamine continued to pump into epidural space,which are used to treat postoperative analgesia of cesarean section, show satisfactory analgesia effect, and it is propitious to recovery of posteroprative parturients and did not influence lactation.     

利维爱联合盐酸米多君治疗绝经后女性尿失禁的临床研究

目的 探讨利维爱联合盐酸米多君治疗绝经后女性尿失禁的有效性和可行性.方法 采用随机、双盲、安慰剂平行对照的方法对120例绝经期女性尿失禁患者进行利维爱联合盐酸米多君与安慰剂对照组的对比研究.试验组60例,利维爱2.5 mg/次,1次/d,同时加用盐酸米多君2.5 mg/次,3次/d,疗程3个月;对照组60例,给予安慰剂.均分别在给药4周和3个月时观察疗效.结果 120例均未失访,治疗组症状改善率达56.1%,恶化率达26.7%;对照组改善率达22.6%,恶化率达33.8%.治疗组症状改善率明显高于对照组,两组比较差异有统计学意义(uc≈59.9658,P＜0.01),但治疗时间的长短对疗效无明显影响(χ2≈0.6265,P＞0.05).结论 利维爱联合盐酸米多君可以改善绝经后女性尿失禁的症状,是治疗绝经后妇女尿失禁的一种有效方法. Abstract： Objective To investigate the efficacy and feasibility of liviral plus midodrine in postmenopause women with incontinence. Methods A randomized,double-blind,parallel,placebo-controlled study was carried out.One hundred and twenty postmenopause women with incontinence were involved in. Sixty cases in study group received liviral(2.5 mg/d) and midodrine (2.5 mg,thrice daily)for three months.Sixty cases in control group received placebo in the same way. Results One hundred and twenty cases completed the treatment. Incontinence improved in 22.6% of the women assigned to placebo compared with 56.1% assigned to liviral and midorine, while 33.8% of the placebo group worsened compared with 26.7% of the liviral and midorine group (P＜0.01). This difference was evident by 4 weeks of treatment,but there was no obvious greater improvement for three months of treatment (P＞0.05). Conclusions Liviral combined with midodrine hydrocholoride therapy is associated with improving urinary incontinence in older postmenopausal women with incontinence. We believe this therapy will be effective for the treatment of incontinence in postmenopause women.     

原因不明的血小板下降在白血病异基因干细胞移植术后早期复发中的预测作用

目的 探讨白血病患者异基因造血干细胞移植(Allo-HSCT)术后原因不明的血小板(PLT)下降与白血病复发之间的关系.方法 51例白血病患者于2006年4月至2009年10月在我科接受了Allo-HSCT术,其中5例患者在术后2年内出现了复发,我们回顾性分析了其复发前外周血PLT计数、骨髓形态学及植入状态的动态变化.结果 5例移植后复发患者移植早期中性粒细胞和PLT造血重建平均时间分别为15.8、25.8 d.当PLT计数降至50×109/L左右时,骨髓巨核细胞进一步减少,骨髓幼稚细胞比率虽较PLT开始下降时有所增多,但均未达到复发的标准,STR检查所有患者从FDC转变为进展性混合型嵌合体(PMC);5例患者在此后的3个月内先后发生形态学复发,复发时骨髓巨核细胞几乎消失,STR检查除1例患者仍为PMC外,其余4例患者均转变为植入失败;统计学分析发现Allo-HSCT术后PLT进行性下降可较出现PMC提前平均75 d、形态学复发提前平均164 d.结论 白血病患者Allo-HSCT术后出现原因不明的血小板进行性下降这一现象,虽然不能用来作为白血病复发的诊断,但对其早期复发有一定的预测作用. Abstract： Objective To explore the relationship between unexplained decline in platelets (PLT) and relapse in leukemia patients after allo-geneic haematopoietic stem cell transplantation(Allo-HSCT). Methods Fifty-one patients with leukemia received Allo-HSCT in our department between April 2006 to October 2009 and 5 of them relapsed within 2 years after transplantation.We retrospectively analyzed the dynamic alteration about the platelets in peripheral blood, bone marrow morphology and the state of implantation in the 5 relapsed patients. Results The average reconstitution time for granulocytes and PLTs was respectively 15.8 days and 25.8 days in the 5 relapsed patients. When PLTs decreased to about 50×109/L, megakaryocytes in bone marrow in all the five patients further reduced. The radio of blast cells in bone marrow increased compared with that when PLT started to decline, but failed to meet the criteria for relapse. STR for all five patients had transformed from FDC to progressive mixed chimerism (PMC). The five patients occurred morphological relapse in the subsequent three months. When the relapse happened, megakaryocytes in bone marrow almost disappeared, STR in 4 patients had transformed to graft failure(GF),except one case was still in PMC. Statistical analysis showed that the progressive decline in PLTs could be an average of 75 days in advance than the appearance of PMC, and an average of 164 days than morphological relapse in the five relapsed patients. Conclusions Although the phenomenon that unexplained progressive decline in PLTs in leukemia patients after Allo-HSCT can not be used as a diagnosis for leukemia relapse, it may be a predict for the early relapse.     

胎盘、脐血及外周血造血干细胞免疫原性初步比较

目的 初步比较胎盘、脐血和外周血来源的造血干细胞的免疫原性.方法 应用单向混合淋巴细胞培养的方法将胎盘、脐血和外周血三种来源单个核细胞的增殖情况进行比较.结果 单向混合淋巴细胞培养中胎盘、脐血和外周血来源的细胞的增殖比较差异有统计学意义(P＜0.01).结论 胎盘较脐血和外周血来源的造血干细胞的免疫原性低,更适合用于异基因造血干细胞移植. Abstract： Objective To compare phenotypic characteristics and immunog- genicity of hematopoietic stem cell(HSC)from placenta(PT), from cord blood(CB)and from peripheral blood(PB).Methods Comparing cell proliferation of PT, CB and PB using mixed - lymphocyte reactions(MLR).Results There was a difference in proliferation of PT, CB and PB(P ＜ 0.01).Conclusions Immunogenicity of HSC from PT are lower than that what from CB and PB.HSC of PT are more suitful for transplant of allogen hematopoietic stem cell.