Targeted silencing of heparanase gene by small interfering RNA inhibits invasiveness and metastasis of osteosarcoma cells

The effects of targeted silencing of heparanase gene by small interfering RNA(siRNA) on invasiveness and metastasis of osteosarcoma cells(MG63 cells) were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA(pGenesil-shRNA) was constructed successfully.MG63 cells were randomly allocated into 3 groups:blank group,empty vector(pGenesil) transfected group and expression vector(pGenesil-shRNA) transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%(P<0.01) and 75.3%(P<0.01) respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.     

干细胞移植治疗阿尔茨海默病的研究进展

干细胞移植可能足未来治疗阿尔茨海默病(AD)的可行性方法之一,许多动物实验都证实干细胞移植能改善记忆和认知功能;然而AD的病理改变,如B淀粉样蛋白(Aβ)沉积等对干细胞的存活和分化有不利影响.通过转染肾胰岛素残基溶酶于干细胞或使用苯羟基丙氨酸可减弱这些不利影响.增强干细胞表达神经营养因子不仅能减弱AD病理改变对干细胞的不良影响.更对脑内原有神经元起到保护作用.进一步研究如何减弱AD病理改变对干细胞的不良影响,充分发挥干细胞的治疗作用对将干细胞移植应用于临床具有重大意义. Abstract： Stem ceils transplantation is one of the potential therapeutic strategies for Alzheimer's disease (AD). Many animal models following stem cell transplantation showed improvement in memory and cognitive function. However, the pathological changes in AD, such as arnyloid beta deposits, negatively affect the survival and differentiaition of stem cells. Stem cells transfectcd with neprilysin or administration of phenserine could at-tenuate these adverse effects. Genetic modification of stem cell by over expression of neurotrophic factors could attenuate the adverse effects not only on stem cells but also on degenerative neurons. Further investigation on how to overcome the adverse effects of phathological factors in AD on stem cells and maximize the therapeutic effects of stem cells would support the hope for introduction of stem cells transplation into clinical application.     

Biocompatibility of KLD-12 peptide hydrogel as a scaffold in tissue engineering of intervertebral discs in rabbits

KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH2 was synthesized and its biocompatibility was assessed in animals. Rabbit MSCs were cultured in the hydrogel for 2 weeks. Live cells were counted by using Calcein-AM/P1 fluorescence staining. MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%, 0.03%, and 0.05%. Hemolysis test, skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues. Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape. Cell survival rate was 92.15% （mean： 92.15%±1.17%） at the 7th day and there was no difference in survival rate between day 7 and day 14. Cell proliferation test showed that the A value of the KLD-12 solutions was not significantly different from that of control groups （complete culture media） （P〉0.05） at the 24th and 48th h. The hemolysis rate of KLD-12 solution was 0.112%. Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0. The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes. It is concluded that KLD-12 peptide hydrogel bad a good biocompatibility with host rabbit and MSCs, and KLD-12 pep- tide hydrogel can provide an appropriate microenvironment for MSCs.     

美金刚胺治疗中重度阿尔茨海默病的Meta分析

目的 系统评价美金刚胺治疗中重度阿尔茨海默病(AD)的疗效与安全性. 方法检索中国期刊全文数据库(CJFD)、美国国立医学生物信息中心PubMed数据库(PubMed)、美国国立.卫生院临床试验数据库(Clinicaltrials.gov)、美金刚胺生产商麦氏(Merz)、灵北(Lundbeck)和森林实验室(Forest laboratories)的出版物以及森林实验室临床试验中心(Forest clinical trials registry)关于美金刚胺治疗中莺度AD的随机、双肓、安慰剂对照研究.对符合条件的研究结果用Revman 5.0软件进行Meta分析.以美金刚胺组相对于安慰剂组在总体功能、认知功能、日常生活能力和精神行为4个方面的加权均数差(WMD)为指标进行疗效评价,以两组在不良事件和严重不良事件两方面的相对危险度(RR)为指标进行安全性评价. 结果共纳入4项研究,1683名患者被随机分配,其中美金刚胺组849名,安慰剂组834名.Meta分析结果显示,美金刚胺组相对于安慰剂组在总体功能(CIBIC-PIus)、认知功能(SIB)、日常生活能力(ADCS-ADL19)和精神行为(NPI)4个方面的WMD分别为-0.29(P<0.00001)、2.79(P<0.00001)、1.03(P=0.002)、-2.85(P=0.002);美金刚胺组相对于安慰剂组在不良事件和严重不良事件的RR值分别为1.01(P=0.66)和0.98(P=0.90).除认知功能(P=0.05)外,4项研究在其他方面均未见明显统计异质性. 结论美金刚胺同安慰剂相比可以改善患者总体功能、认知功能、日常生活能力及精神行为症状,美金刚胺的安伞性和耐受性同安慰剂相比差异无统计学意义.     

雷帕霉素对不同肿瘤细胞Bax/Bcl-2和活性caspase-3表达的影响

目的:探讨雷帕霉素(rapamycin,RAPA)对不同肿瘤细胞Bax/Bcl-2和活性caspase-3表达的影响及其可能的机制.方法:采用RAPA处理肺腺癌A549细胞、人成神经细胞瘤SH-SY5Y细胞和人骨肉瘤MG63细胞后,MTT法检测细胞的相对增殖率,蛋白质印迹法检测细胞中Bcl-2、Bax和活性caspase-3的表达以及细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)和Akt活性的变化;RAPA联合ERK抑制剂U0126或PD98059以及联合Akt抑制剂wortmannin处理MG63和SH-SY5Y细胞后,分别用MTT法和蛋白质印迹法检测细胞的相对增殖率和Bcl-2、Bax、活性caspase-3的表达.结果:在A549细胞中,RAPA上调细胞Bax/Bcl-2和活性caspase-3的表达(P＜0.05),细胞的相对增殖率下降(P＜0.05).在MG63细胞中,RAPA通过ERK上调Bcl-2和Bax的表达,Bax/Bcl-2和活性caspase-3的表达以及细胞的相对增殖率无明显变化;ERK抑制剂U0126或PD98059逆转RAPA导致的Bcl-2上调,联合RAPA作用后细胞中Bax/Bcl-2和活性caspase-3的表达上调(P＜0.05),细胞的相对增殖率降低(P＜0.05).在SH-SY5Y细胞中,RAPA通过Akt上调Bcl-2的表达,Bax/Bcl-2降低(P＜0.05),细胞的相对增殖率上升(P＜0.05); Akt抑制剂wortmannin逆转RAPA导致的Bcl-2上调,联合RAPA作用后细胞中Bax/Bcl-2和活性caspase-3的表达上调(P＜0.05),细胞的相对增殖率降低(P＜0.05).结论:RAPA在不同肿瘤细胞中可能通过调节不同的激酶影响Bax/Bcl-2和活性caspase-3的表达.     

缺氧对骨肉瘤细胞MG63核干细胞因子表达调节及其机制的探讨

目的:探讨缺氧对骨肉瘤细胞MG63核干细胞因子(NS)表达的调节及其机制。方法:反转录聚合酶链式反应(RT-PCR)和蛋白质印迹法检测缺氧对NS转录和蛋白表达的影响;蛋白质印迹法检测在雷帕霉素抑制缺氧诱导因子-1α(HIF-1α)表达或激酶抑制剂预处理后NS在缺氧下的变化,检测NS调节的下游因子p53和活性Caspase-3蛋白表达的变化。结果:缺氧6、12和24 h后NS蛋白表达分别升高了1.26(P>0.05)、1.58(P<0.05)和1.92倍(P<0.01),NS的转录分别升高了1.33(P>0.05)、1.49(P<0.05)和1.54倍(P<0.05)。蛋白激酶B(Akt)抑制剂wortmannin逆转了缺氧对NS的上调,而细胞外信号调节激酶(ERK)抑制剂U0126、蛋白激酶A(PKA)抑制剂H89和雷帕霉素则没有逆转缺氧对NS的上调。Akt活性在缺氧6、12和24 h后分别升高了1.51(P>0.05)、1.56(P<0.05)和1.23倍(P>0.05)。尽管缺氧上调了NS的表达,但NS的下游因子p53与活性Caspase-3蛋白表达在缺氧后无明显变化。结论:缺氧通过增强Akt的活性上调了NS的表达。     

阿尔茨海默病与糖尿病之间关系的研究进展

阿尔茨海默病(AD)的发病与糖尿病之间关系被逐渐重视。AD患者同时存在脑内胰岛素缺乏与抵抗及外周高胰岛素血症,又被称为"3型糖尿病"。代谢综合征、胰岛素抵抗和糖尿病是发生AD的风险因素,AD发生后又常加重糖尿病发展,形成恶性循环。糖尿病患者的高胰岛素血症使脑内胰岛素降解酶被大量消耗,促进β淀粉样蛋白沉积;β淀粉样蛋白的沉积又加重脑内胰岛素缺乏与抵抗,形成恶性循环,促进AD进展。胰岛素及胰岛素增敏剂之一的过氧化物酶体增殖因子活化受体γ激动剂均被证实治疗AD有效,但可能只针对载脂蛋白E-ε4基因阴性的早期AD患者。同时,糖尿病患者因代谢障碍产生过多的糖化末端产物,导致氧化应激与炎症反应,也加重AD病情。干扰糖化末端产物作用的药物也是未来治疗AD的一个方向。     

Wnt/β-连环蛋白信号通路参与调节糖皮质激素诱导的阿尔茨海默病样病变

目的 探讨Wnt/β-连环蛋白(catenin)信号通路在糖皮质激素(glucocorticoids,GC)诱导的阿尔茨海默病样病变中的信号调节机制.方法 地塞米松(dexamethasone,DEX)分别处理转染人tau441的人胚肾293细胞(HEK293/tau)和野生型的人胚肾293细胞(HEK293/wt),采用细胞计数试剂盒( CCK-8)法检测DEX对细胞活力的影响,Western blot研究两组细胞tau蛋白磷酸化(p-T205,Tau-1)、β-catenin、p-β-catenin、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)及其上游激酶糖原合成激酶-3β(GSK-3β)、ps9-GSK-3β水平的变化,并加用GSK-3β的抑制剂氯化锂(LiCI),观察其对相关蛋白相应的逆转作用.结果 CCK-8细胞活力检测结果显示,1μmol/L DEX处理细胞48h,HEK293/wt组细胞活力(存活率)下降到95.5％±3.2％,HEK293/tau组下降到77.8％±4.4％,两者差异有统计学意义(t =6.60,P＜0.05);Western blot结果显示,1μmoL/L DEX处理两组细胞48 h,使得HEK293/tau细胞ps9-GSK3β、Tau -I、β-catenin、Bcl-2水平分别下降到对照组的47.8％±10.4％、53.9％±11.7％、50.9％±7.6％、48.4％±6.5％,差异均有统计学意义(t=7.01、3.86、7.09、7.30,均P＜0.05),而p-T205、p-β-catenin相对磷酸化水平分别增加到对照组的180.5％±22.2％、201.3％±27.6％,差异均有统计学意义(t=5.51、5.27,均P＜0.05);LiCI可以相应逆转上述改变.结论 在人tau存在的前提下,GC通过抑制Wnt/β-catenin信号转导通路,促进了HEK293/tau细胞的阿尔茨海默病样病变.