神经干细胞在缺血缺氧性脑病中的治疗前景

过去认为神经元受损伤后难以再生.近年发现神经干细胞(neuralstemcells,NSC)主要存在于胚胎和成熟个体的中枢神经系统(CNS)中,具有增殖和分化的潜能.NSC成为神经学科的热点课题,是神经发育和疾病研究的重要平台,作为新生神经细胞的“种子”,它为治疗缺血缺氧性脑病提供了新策略,尤其是中枢神经细胞的治疗性再生和基因治疗.对NSC的发育、组织学特点、增殖分化的调控及治疗前景进行了阐述.     

VEGF与缺血缺氧性脑损伤

缺血缺氧可造成全身多个系统受损,尤其是中枢神经系统,缺血缺氧对脑损害最严重,血管内皮生长因子(VEGF)的血管生成作用、神经保护作用、神经再生作用能在一定程度上改善缺血缺氧性脑损伤,是一种潜在的缺血缺氧保护剂,本文就近年来VEGF在缺血缺氧性脑损伤方面的相关研究综述如下.     

针灸治疗幼鼠缺血缺氧性脑病的实验研究

目的:观察针灸治疗幼鼠缺血缺氧性脑病(HIE)的临床效果.方法:利用"针刺通经健脑"法选与幼鼠相对应的穴位,观察针刺治疗前、后脑部血流量、脑组织含水量、一氧化氮(NO)、丙二醛(MDA)含量.结果:HIE组经针刺治疗,头部血流量增加了1.3倍;脑组织含水量于第二天下降了0 64%,统计学比较有显著差异(P＜0.01);NO及MDA含量分别下降了21.21%及38.09%(P＜0.01).结论:幼鼠HIE经针刺治疗可促进脑部血液循环,减弱脂质过氧化及NO对神经细胞的损伤作用.     

少突胶质前体细胞移植对新生儿缺氧缺血性脑病炎性反应的影响

目的研究少突胶质细胞-Ⅱ型-星形胶质祖细胞(oligodendrocyte-type II astrocyte progenitor,O2A)移植对新生儿缺氧缺血性脑病(hypoxic-ischemic encephalopathy,HIE)炎性反应的影响,探讨O2A细胞移植对HIE的修复机制。方法采用C57小鼠制成HIE模型。①对照组:即假手术组,尾静脉注入生理盐水2 mL;②HIE非移植组:于成模后48h行尾静脉注入生理盐水2 mL,7、14、28 d脱颈处死动物,甲醛灌注后分离全脑组织、甘油梯度脱水后置冰冻切片机上切20μm脑片,行F4/80及BrdU免疫荧光染色;③HIE移植组:将提取、分离并PKH-26荧光染色的O2A细胞于HIE成模后48 h行尾静脉注入,7、14、28 d脱颈处死动物,甲醛灌注后分离全脑组织、甘油梯度脱水后置冰冻切片机上切20μm脑片,行F4/80,BrdU及DAPI免疫荧光染色。结果 O2A细胞移植后7、14、28 d在HIE小鼠脑内均未见PKH-26阳性的O2A细胞存留;而移植组侧脑室膜下区的内源性神经干细胞较对照组明显增殖旺盛。HIE损伤后在海马及丘脑内可见较多F4/80阳性的小胶质细胞,以第14天为著;O2A细胞移植后上述F4/80阳性的小胶质细胞明显减少。结论细胞移植激活和促进HIE小鼠脑内源性神经干细胞增殖;O2A细胞移植对HIE脑组织的炎性损伤具有抑制作用。提示O2A细胞移植具有促进HIE病变修复、阻止HIE的病情进展的作用。     

自体骨髓单个核细胞移植治疗缺血性下肢血管病变患者254例

目的探讨自体骨髓单个核细胞移植在下肢缺血性疾病治疗中的应用并评价其疗效。方法回顾性总结分析解放军第四六三医院细胞治疗中心2003年11月至2008年8月住院的具有完整随访资料的下肢缺血性疾病患者254例,经动员后采集及分离自体骨髓,行下肢自体骨髓单个核细胞移植术。随访时间为1年。术后随访指标：3、6、12个月后复查皮温、深感觉、经皮氧分压和踝肱比,随访疼痛、冷感和跛行距离,观察溃疡和坏疽情况。采用F检验分析差异的统计学意义。结果完成随访254例患者。（1）患者的疼痛缓解率为61．8％（157例／254例）,冷感缓解率缓解率74．0％（188例／254例）,跛行好转率40．2％（102例／254例）,溃疡好转率59．0％（36例／61例）,坏疽截肢6例,脱落愈合5例,无变化8例,扩大7例;（2）患者下肢皮温由32．89℃±2．19℃上升至35．52℃±2．26℃（t=13．32,P=0．000）,深感觉缓解由（26．20±15．78）mV下降至（20．34±10．86）mV（t=4．901,P=0．000）,经皮氧分压由（26．46±18．49）mmHg上升至（34．14±14．99）mmHg（t=5．157,P=0．000）,踝肱比由0．62±0．36上升至0．84±0．24（t=8．104,P=0．000）。结论骨髓单个核细胞移植治疗下肢缺血性血管病有效,是一种简单的、有效的治疗下肢动脉缺血性疾病的方法。     

纯化自体外周血CD34^＋细胞移植治疗肢体重度缺血的中期疗效

目的探讨纯化CD34^＋细胞移植治疗肢体重度缺血的中期疗效。方法自2009年5月至2011年12月录入30例肢体重度缺血患者,男性29例,女性1例,平均年龄43 ± 12岁（23 - 75岁）,均不具备外科血管重建条件,共治疗31条下肢和4条上肢。G-CSF（5-10μg/kg）动员,第5天采集外周血单个核细胞,分选获得纯化CD34^＋细胞,肢体肌肉局部注射。根据移植细胞数分为3组：低剂量组（105/kg）、中剂量组（5 × 105/kg）和高剂量组（106/kg）。结果随访16 - 48个月,1个月和2个月时Wong-Baker FACES疼痛评分由术前7 ± 2降到3 ± 3（P〈 0.001）和1 ± 2（P〈 0.001）。术后3个月和6个月：最长无痛步行时间由术前的（5 ± 3）min分别延长至（12 ± 6）min（P〈 0.001）和（19 ± 5）min（P〈 0.001）;踝肱指数由术前的0.44 ± 0.20提高至0.62 ± 0.18（P= 0.04）和0.66 ± 0.14（P〈 0.001）;经皮氧分压由术前（26 ± 11）mmHg分别上升至（42 ± 11）mmHg（P 〈 0.001）和（56 ± 12）mmHg（P〈 0.001）。16例溃疡患者愈合11例。Kaplan Meier生存分析法计算16个月保肢率为84﹪（95﹪可信区间为0.63 - 0.94）。3个不同剂量组之间的比较,治疗效果差异无统计学意义,除了1个月时WFPRSC检测结果显示：高剂量组明显优于低、中剂量组。结论纯化自体外周血CD34^＋细胞移植治疗肢体重度缺血性疾病安全、可行,能够较为持久地缓解缺血,挽救肢体,改善患者生活质量。     

新生儿缺血缺氧性脑病发病机理及其治疗方法

新生儿缺血缺氧性脑病 (HIE)主要是指新生儿在围产期窒息导致的缺血缺氧性损害 ,临床上出现一系列脑病的表现。 HIE至今仍是威胁新生儿生命和健康的严重疾病。近年来 ,国内外对此病的研究较多 ,现就其发病机理及其治疗方法综述如下。1　发病机理　　新生儿缺血缺氧性脑病的发病原因很多 ,目前认为主要是围产期窒息 ,其发病机理大多认为与氧自由基、Ca2 内流及再灌注有关。1 .1　 HIE与氧自由基　正常生物体内有少量自由基产生 ,并经清除系统不断清除 ,从而维持动态平衡 ,使机体免遭损害。当机体自由基浓度高于正常水平时 ,自由基清除系…     

缺血再灌注对大鼠肾脏足细胞podocin的表达影响及意义

目的:观察SD大鼠缺血再灌注肾损伤足细胞表面相关蛋白(podocin)表达的变化,探讨podocin分子在足细胞损伤中的作用.方法:60只雄性SD大鼠随机分为假手术组(S组)30只和模型组(M组)30只,各组又分为lh、3h、6h、12h、24h、48h六个亚组,每个亚组各5只.采用Westemblot蛋白印迹法检测肾皮质足细胞podocin蛋白表达.并观察各组血肌酐、尿素氮的变化.结果:与S组相比,肾缺血再灌注损伤后,M组尿素氮于6h开始升高,且48h达高峰(P＜0.05);血肌酐48 h才升高(P＜0.05).Podocin 蛋白在3h表达开始下降,以后随缺血再灌注时间的延长podocin蛋白的表达逐渐下降,且48h组下降最明显(P＜0.01).结论:(1)缺血再灌注肾损伤时大鼠肾功明显发生改变,尿素氮早期开始升高,血肌酐晚期升高明显,说明造模成功.(2)大鼠肾脏足细胞podocin的蛋白表达随着缺血再灌注时间的延长而改变,早期下降不明显,3h组开始逐渐下降,48h组下降最明显.此结果表明podocin蛋白在肾脏缺血再灌注肾损伤表达减少.我们可以推想在缺血再灌注肾损伤时,podocin随缺血时间延长而表达逐渐减少的同时,也破坏了与足细胞相关蛋白之间的联系,这种关系的破坏最终导致足细胞受损.通过对足细胞表面蛋白podocin的研究,对将来肾脏足细胞损伤的防治提供新的思路.     

缺氧缺血诱导对原代新生大鼠前脑混合细胞髓鞘形成和细胞骨架的影响

目的:该研究探讨了缺氧缺血诱导对新生大鼠前脑混合细胞髓鞘形成和细胞骨架的影响。方法:在原代培养的新生大鼠前脑混合细胞中,应用免疫组化染色髓鞘碱性蛋白(MBP)和劳克坚牢蓝(LFB)染色检测髓鞘和轴突的发育情况;Western印迹分析NF155蛋白的表达情况;免疫荧光分析参与细胞骨架调节的ERM-Rho GTP酶家族相关蛋白表达。结果:缺氧缺血对混合细胞的进一步分化和成熟起到抑制作用,MBP染色阳性率降低57%,髓鞘和轴突染色阳性率降低74%;NF155蛋白表达降低51%;Rho GTP酶家族成员Rac1、Cdc42分别降低81%和75%。结论:缺氧缺血使细胞突起的形成和骨架重组受到阻碍,继而影响髓鞘的发育和成熟,这一过程与ERM-Rho GTP酶细胞骨架通路有关。     

延迟5分钟剖宫产造全脑缺氧缺血新生大鼠模型

目的建立围产期全脑缺氧缺血性损伤的新生大鼠模型。方法 SD雌性大鼠妊娠21 d时,颈椎脱臼法处死,用止血钳夹闭双侧子宫角血管5 min后,剖宫产取出新生大鼠,交由代乳鼠喂养。结果造模组雌性大鼠9只,共娩出新生大鼠91只,出生3 d内死亡7只,死亡率7.7%。新生大鼠出生第2天进行翻身实验,第14天进行悬吊实验和斜坡实验,造模组和其余各组均有显著性差异。新生大鼠出生后21 d,取脑组织切片行HE染色,显示大脑皮层典型的缺氧缺血性损伤,与正常组相比,可见神经细胞明显的病理形态学改变。结论采用延迟5min剖宫产和代乳鼠喂养的方法,操作简便,并结合行为学测试筛选行为异常者,可建立稳定可靠、可供长期实验使用的围产期全脑缺氧缺血性损伤的新生大鼠模型。     

Secretome of Peripheral Blood Mononuclear Cells Enhances Wound Healing

Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SEC^PBMC). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SEC^PBMC containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SEC^PBMC on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SEC^PBMC containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SEC^PBMC induced proliferation and tube-formation in a matrigel-assay. In addition, SEC^PBMC treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers.     

Isolation of Peripheral Blood Mononuclear Cells Using Glycerol Density Gradient

Isolation of peripheral blood mononuclear cells (PBMCs) is fraught with challenges including, but not limited to, the cost of limited gradients available for the isolation of PBMCs. Glycerol gradient (1.077 g/ml) was used to isolate PBMCs from adult peripheral blood. The differentiation potential of the isolated cells was assessed by culturing the cells in MEM at 37 °C in 5 % CO₂. The results demonstrated that the isolated cells could differentiate into committed linages of the erythroid progeny. LDH assay revealed that glycerol was not cytotoxic to the cells. The use of glycerol density as an alternative could be significant in cell culture experiments.     

Interindividual Variation in the Proteome of Human Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMCs) are main actors in inflammatory processes and linked to many diseases, including rheumatoid arthritis, atherosclerosis, asthma, HIV and cancer. Moreover, they seem an interesting ‘surrogate tissue’ that can be used in biomarker discovery. In order to get a good experimental design for quantitative expression studies, the knowledge of the interindividual variation is an essential part. Therefore, PBMCs were isolated from 24 healthy volunteers (15 males, 9 females, ages 63–86) with no clinical signs of inflammation. The extracted proteins were separated using the two dimensional difference in gel electrophoresis technology (2D-DIGE), and the gel images were processed with the DeCyder 2D software. Protein spots present in at least 22 out of 24 healthy volunteers were selected for further statistical analysis. Determination of the coefficient of variation (CV) of the normalized spot volume values of these proteins, reveals that the total variation of the PBMC proteome varies between 12,99% to 148,45%, with a mean value of 28%. A supplemental look at the causes of technical variation showed that the isolation of PBMCs from whole blood is the factor which influences the experimental variance the most. This isolation should be handled with extra care and an additional washing step would be beneficial. Knowing the extent of variation, we show that at least 10 independent samples per group are needed to obtain statistical powerful data. This study demonstrates the importance of considering variance of a human population for a good experimental design for future protein profiling or biomarker studies.     

Influence of Genes Suppressing Interferon Effects in Peripheral Blood Mononuclear Cells during Triple Antiviral Therapy for Chronic Hepatitis C

The levels of expression of interferon-stimulated genes (ISGs) in liver are associated with response to treatment with pegylated interferon (PEG-IFN) plus ribavirin (RBV). However, associations between the responses of ISGs to IFN-based therapy and treatment efficacy or interleukin-28B (IL28B) genotype have not yet been determined. Therefore, we investigated the early responses of ISGs and interferon-lambdas (IFN-λs) in peripheral blood mononuclear cells (PBMCs) during PEG-IFN/RBV plus NS3/4 protease inhibitor (PI) therapy. We prospectively enrolled 50 chronic hepatitis C patients with HCV genotype 1, and collected PBMCs at baseline, 8 and 24 h after the initial administration of PEG-IFN/RBV/PI. Levels of mRNAs for selected ISGs and IFN-λs were evaluated by real-time PCR. All 31 patients with a favorable IL28B genotype and 13 of 19 with an unfavorable genotype achieved sustained virological responses (SVR). Levels of mRNA for A20, SOCS1, and SOCS3, known to suppress antiviral activity by interfering with the IFN signaling pathway, as well as IRF1 were significantly higher at 8 h in patients with an unfavorable IL28B genotype than in those with a favorable one (P = 0.007, 0.026, 0.0004, 0.0006, respectively), especially in the non-SVR group. Particularly, the fold-change of IRF1 at 8 h relative to baseline was significantly higher in non-SVR than in SVR cases with an unfavorable IL28B genotype (P = 0.035). In conclusion, levels of several mRNAs of genes suppressing antiviral activity in PBMCs during PEG-IFN/RBV/PI differed according to IL28B genotypes, paralleling treatment efficacy.     

Circulating Angiogenic Cells can be Derived from Cryopreserved Peripheral Blood Mononuclear Cells

Background

Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs).

Methods

PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw) or 28 (late thaw) days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential.

Results

The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34⁺VEGFR2⁺CD133⁺ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle.

Conclusion

Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34⁺VEGFR2⁺CD133⁺ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.     

Intracardiac Injection of Dental Pulp Stem Cells After Neonatal Hypoxia-Ischemia Prevents Cognitive Deficits in Rats

Neonatal hypoxia-ischemia (HI) is associated to cognitive and motor impairments and until the moment there is no proven treatment. The underlying neuroprotective mechanisms of stem cells are partially understood and include decrease in excitotoxicity, apoptosis and inflammation suppression. This study was conducted in order to test the effects of intracardiac transplantation of human dental pulp stem cells (hDPSCs) for treating HI damage. Seven-day-old Wistar rats were divided into four groups: sham-saline, sham-hDPSCs, HI-saline, and HI-hDPSCs. Motor and cognitive tasks were performed from postnatal day 30. HI-induced cognitive deficits in the novel-object recognition test and in spatial reference memory impairment which were prevented by hDPSCs. No motor impairments were observed in HI animals. Immunofluorescence analysis showed human-positive nuclei in hDPSC-treated animals closely associated with anti-GFAP staining in the lesion scar tissue, suggesting that these cells were able to migrate to the injury site and could be providing support to CNS cells. Our study evidence novel evidence that hDPSC can contribute to the recovery following hypoxia-ischemia and highlight the need of further investigation in order to better understand the exact mechanisms underlying its neuroprotective effects.     

Inhibition of Varicella-Zoster Virus Glycoprotein Expression by Peripheral Blood Mononuclear Cells

The effect of peripheral blood mononuclear cells (PBMC) on expression of varicella-zoster virus (VZV) glycoproteins (Gps) was analyzed by flow cytometry. PBMC from VZV seropositive and seronegative donors and supernatant of PBMC co-cultured with VZV-infected human embryonic fibroblasts reduced VZV Gp expression. Neutralization of supernatant fluid with mixture of anti-interferons (IFN)-α, -β, -γ, and tumor necrosis factor (TNF)-α partially reduced inhibitory activity of supernatant on VZV Gp expression. Deletion of natural killer (NK) cells and adherent cells from PBMC reduced inhibitory activity of PBMC on VZV Gp expression. These results suggest that IFN-α, -β, -γ, TNF-α and other soluble factors released from NK cells and monocytes by co-cultivation with VZV-infected fibroblasts inhibit VZV Gp expression.     

轮状病毒感染患者外周血单个核细胞的转录组学分析

轮状病毒(Rotavirus,RV)是引起急性肠胃炎的主要病原体,分析RV感染患者的人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)中差异表达基因(Differentially expressed genes,DEGs)有利于探讨人PBMC在清除RV中的作用。为此,本研究采集2019年2月-2019年6月长春儿童医院中RV感染患者和健康儿童血液,分离PBMC,通过转录组测序(RNA sequencing,RNA-seq)技术比较RV感染患者与健康儿童之间的RNA表达图谱,借助基因本体论(Gene Ontology,GO)数据库功能富集分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)、Reactome通路富集分析DEGs,使用实时荧光定量PCR(Real-time quantitative PCR,qPCR)技术进行验证。结果显示,与健康对照组相比,RV感染轻症患者PBMC中有1619个DEGs;重症患者PBMC中有2816个DEGs,主要与干扰素(Interferon,IFN)反应、中性粒细胞、溶酶体、核小体、染色质等相关。qPCR验证轻症患者干扰素刺激基因(IFN-stimulated genes,ISGs)15表达上调,白介素(Interleukin,IL)1β表达下调;重症患者IL15、ISG15表达上调,IL1β表达下调,与转录组结果相一致。本研究提示,RV感染可能激活人I型和II型IFNs反应抵御病毒感染,但也会抑制溶酶体相关基因,对细胞自噬过程产生影响。