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1.
背景:多发性骨髓瘤骨病的发病机制目前尚未完全明确,骨髓间充质干细胞向成骨细胞分化障碍参与其中,而Notch1信号通路在间充质干细胞的增殖分化中起重要作用。 目的:探讨Notch1信号通路在多发性骨髓瘤骨病中的作用。 方法:分离培养多发性骨髓瘤患者和正常人骨髓间充质干细胞,Real-time PCR和Western blot检测成骨诱导分化前后Notch1和成骨基因Runx2的表达,以及Von Kossa染色鉴定钙质沉积程度。在多发性骨髓瘤患者间充质干细胞成骨诱导分化过程中,加入Notch1信号通路抑制剂DAPT和安慰剂,48 h后real-time PCR和western blot鉴定Notch1信号通路下游分子Hes1和成骨指标Runx2表达,2周后Von Kossa染色鉴定钙质沉积程度。 结果与结论:成骨诱导48 h后,间充质干细胞的Notch1表达减低,但是骨髓瘤患者间充质干细胞的降低幅度小于正常对照间充质干细胞;48 h后Runx2的表达在骨髓瘤患者间充质干细胞的表达明显弱于正常对照间充质干细胞;2周后,Von Kossa染色鉴定钙质沉积程度,骨髓瘤患者间充质干细胞明显弱于正常对照间充质干细胞;48 h后Hes1表达在DAPT组明显低于安慰剂组;而Runx2的表达在DAPT组明显高于安慰剂组。2周后 DAPT组钙质沉积明显强于安慰剂组。实验说明多发性骨髓瘤患者的间充质干细胞中,Notch1信号通路失活缺陷可能抑制其向成骨细胞分化。中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接:  相似文献   

2.
背景:脂肪来源的间充质干细胞是否具有和骨髓来源间充质干细胞类似的免疫调节作用? 目的:观察骨髓来源和脂肪来源间充质干细胞的免疫学特征。 方法:分离骨髓和脂肪来源的间充质干细胞,分别检测它们对T细胞周期、活化、抑制和增殖的作用情况。 结果与结论:骨髓来源和脂肪来源的间充质干细胞同样具有抑制T细胞增殖的能力,在有丝分裂原刺激和混合淋巴细胞反应的T细胞增殖中这种作用都是具有剂量依赖性的,在1︰2时有极强的抑制作用,但是在1︰100时这种作用基本消失,在共培养时骨髓来源和脂肪来源的间充质干细胞都可以使更多的T细胞被抑制在G0/G1期,同时也可以抑制T细胞的早期活化,但是上述作用脂肪来源的间充质干细胞均较骨髓来源间充质干细胞弱,且脂肪来源的间充质干细胞并不具有抑制T细胞凋亡的作用。  相似文献   

3.
目的 研究一氧化氮(nitric oxide,NO)在骨髓间充质干细胞(mesenchymal stem cells,MSCs)的免疫调节作用.方法 贴壁筛选法分离培养Lewis大鼠骨髓间充质干细胞.以刀豆蛋白A(concanavalin A,ConA)作为刺激因素,SD大鼠外周血分离的T淋巴细胞作为反应细胞,采用CCK-8法检测T淋巴细胞与MSCs共培养后其增殖能力的变化.然后采用RT-PCR、Western blot检测MSCs诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)mRNA与蛋白的表达;Griess法检测上清液亚硝酸盐含量;ELISA检测上清液Th1细胞因子IFN-γ和Th2细胞因子IL-4的水平.结果 MSCs明显抑制ConA诱导的T淋巴细胞增殖,抑制作用因细胞比例的不同而有所区别.当实验组MSC:T为1:10时最明显,增殖率为(33.83±2.10)%,而1:80组[(78.62±3.80)%]与不含MSCs的阳性对照组[(79.03±1.70)%]差异无统计学意义(P>0.05).实验组MSCs的iNOS mRNA与蛋白的表达以及上清液中亚硝酸盐含量均较阳性对照组明显上调,1:10~1:40的范围内呈明显递增趋势,但1:80组与1:40组差异无统计学意义(P>0.05).在加入iNOS特异性抑制剂2-甲基-2-巯基硫酸脲(S-methylisothiourea sulfate,SMT)后,各组T淋巴细胞增殖率基本恢复.实验组IFN-γ含量随MSCs所占比例的降低而增加,各组IL-4含最差异无统计学意义(P>0.05).结论 MSCs对同种异体外周血T淋巴细胞的增殖有免疫调节作用,机制可能与其分泌的可溶性因子NO影响了 Th1/Th2平衡有关.
Abstract:
Objective To observe the role of nitric oxide(NO) in the immune regulatory effect of bone marrow mesenchymal stem cells(MSCs). Methods Bone marrow MSCs were isolated from Lewis rats by adherence screening. Concanavalin A (ConA) was adopted as the stimulator and T-lymphocyte isolated from peripheral blood of SD rats as the reactive cells. The changes of the ability of T-lymphocyte proliferation, when co-cultured with MSCs, were measured by CCK-8 assay. The inducible nitric oxide synthase (iNOS) mRNA and protein expression on MSCs and were detected by RT-PCR, Western blot. The contents of nitrites and the levels of Th1 type cytokine IFN-γand Th2 type cytokine IL-4 were measured by Griess test and ELISA respectively, in the co-cultured supernatant. Results T-lymphocyte proliferation was inhibited by co-cultured MSCs, which was concentration-dependent. In this study, the inhibition was most obviously group[ (79.03 ± 1.70)% ] (P > 0.05 ). The I NOS mRNA expression, protein and nitrite levels were signifigroups, the proliferation rate of T-lymphocyte recovered. The content of IFN-γwas increased with the ratio decline of MSCs in the experimental group and IL-4 in each group has no significant difference( P > 0.05 ).Conclusion MSCs inhibited T-lymphocyte proliferation by influencing Th1/Th2 balance, and the secretion of soluble factor NO, which secreted by MSCs, may plays an important role in the immune regulation.  相似文献   

4.
间充质干细胞(MSC)已成功从动物体尤其骨髓中分离获得.干细胞,具有向多种组织和细胞如骨、脂肪、成软骨细胞等分化的潜能,这种潜能越来越引起人们关注,它可向受损的组织定向迁移以修复受损组织.体外资料研究表明,不管协同刺激信号存在与否,都具有低免疫原性.在体外MSC还具有免疫抑制活性,可抑制同种异体抗原或丝裂霉素刺激的T细胞扩增,阻止细胞毒性T细胞增殖,同时还具有调节炎性细胞的免疫活性使其向抗炎细胞分化的作用.因此,系统了解.MSC的生物学、免疫学特性将为人们深入阐明其免疫调节机制,并将其用于治疗自身免疫性病、组织修复及移植提供依据.  相似文献   

5.
目的比较脂肪源间充质干细胞(AMSCs)和骨髓来源间充质干细胞(BMSCs)的免疫调节能力的差异。方法用流式细胞仪分析AMSCs和BMSCs的表型。通过MSCs和淋巴细胞共培养体系,检测MSCs对T细胞增殖、周期、活化、凋亡以及Th细胞分化的影响。结果表型分析结果显示AMSCs和BMSCs的表型基本一致。AMSCs和BMSCs都能抑制T细胞的增殖和早期活化,并在调节辅助性T细胞亚群的分化上作用类似。但在MSC对活化后T细胞的凋亡影响方面,BMSCs能够抑制活化T细胞的凋亡,而AMSC没有这种作用。结论 AMSCs和BMSCs对T淋巴细胞的影响基本类似,其对活化T细胞凋亡影响的差异还有待进一步的机制研究。  相似文献   

6.
背景:近年来,骨髓微环境与多发性骨髓瘤细胞生长、存活及耐药性的关系成为科研人员研究热点,骨髓间充质干细胞条件培养液干预多发性骨髓瘤细胞株的实验鲜有报道。目的:探讨骨髓间充质干细胞条件培养液对多发性骨髓瘤细胞增殖的影响。方法:经Ficoll密度梯度离心法及贴壁纯化健康供者骨髓来源间充质干细胞,收集第3-5代骨髓间充质干细胞的上清培养基,超滤离心管制备浓缩骨髓间充质干细胞条件培养液,制备含10%人骨髓间充质干细胞条件培养液的1640完全培养基,诱导多发性骨髓瘤RPMI8226和U266细胞株培养48h,MTT法、EdU荧光染色检测细胞增殖能力;流式细胞仪和PI荧光染色检测细胞凋亡情况;Real-time PCR和Western blot检测细胞中BCL-2、BTK mRNA、蛋白表达水平。结果与结论:骨髓间充质干细胞条件培养液能促进多发性骨髓瘤细胞株的增殖(P <0.05),抑制多发性骨髓瘤细胞的凋亡(P <0.05),与BCL-2、BTK的mRNA和蛋白高表达密切相关(P <0.05)。结果表明,骨髓间充质干细胞条件培养液可促进多发性骨髓瘤细胞增殖。  相似文献   

7.
间充质干细胞具有独特的免疫负调节特性,能在不同层次作用于T淋巴细胞、B淋巴细胞、自然杀伤细胞(NK细胞)和树突状细胞,通过直接接触、分泌细胞因子转化生长因子(TGF-β)或调节这些细胞的代谢而发挥免疫调节作用,可望在干细胞移植后免疫重建及移植物抗宿主病的治疗中发挥重要作用。  相似文献   

8.
多发性骨髓瘤患者骨髓Flk1+间充质干细胞的成骨能力降低   总被引:1,自引:1,他引:1  
目的研究多发性骨髓瘤(MM)患者骨髓F lk1 间充质干细胞(F lk1 MSCs)的成骨特性,探讨其与多发性骨髓瘤骨病的关系。方法分离并鉴定MM患者骨髓源F lk1 MSCs;利用实时定量RT-PCR检测F lk1 MSCs成骨诱导前后成骨指标的表达,诱导2周后用Von-Kossa染色法测定钙盐沉积;同时测定F lk1 MSCs中转录共刺激因子(TAZ)的表达。结果MM患者骨髓F lk1 MSCs在体外经成骨诱导后,其成骨指标的表达较正常人明显降低,Von-Kossa染色也可见矿化基质沉积相应减低。同时F lk1 MSCs中TAZ的表达在MM中较正常人明显下调(P<0.05)。结论多发性骨髓瘤患者的骨髓F lk1 MSCs成骨能力较正常人减低,此因素可能参与了MM患者骨病的发病环节,而TAZ表达的减弱可能在此过程中发挥一定作用。  相似文献   

9.
背景:多发性骨髓瘤患者骨髓微环境中间充质干细胞有多种异常,但异常的间充质干细胞对骨髓瘤细胞的趋化功能影响目前仍不清楚。 目的:比较正常人与多发性骨髓瘤患者骨髓间充质干细胞在体外对骨髓瘤细胞株趋化迁移的影响。 方法:体外培养的正常人骨髓间充质干细胞或初诊骨髓瘤患者骨髓间充质干细胞,在有或无硼替佐米条件下,分别与骨髓瘤细胞株U266细胞直接共培养,比较U266细胞的Transwell迁移率、定量PCR检测mRNA的表达水平。同时还检测U266细胞对正常人骨髓间充质干细胞或骨髓瘤患者骨髓间充质干细胞培养液上清的Transwell迁移情况。 结果与结论:正常人骨髓间充质干细胞或骨髓瘤患者骨髓间充质干细胞与U266细胞直接共培养后,两组骨髓瘤细胞的趋化特性有区别,表现为骨髓瘤患者骨髓间充质干细胞组的U266细胞基础Transwell迁移率高、CCR1的表达水平高(P均 < 0.05)。硼替佐米处理U266细胞后并不能消除两组的的区别。然而,U266细胞对正常人骨髓间充质干细胞或骨髓瘤患者骨髓间充质干细胞培养液上清的Transwell迁移并没有明显区别。结果提示骨髓瘤患者骨髓间充质干细胞本身存在一些内在缺陷,在与骨髓瘤细胞株U266相互作用过程中,可以影响U266的体外趋化功能,并且这一影响主要是通过骨髓间充质干细胞与骨髓瘤细胞直接相互作用引起的。 中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程   相似文献   

10.
间充质干细胞在T细胞免疫反应中的调节作用   总被引:1,自引:1,他引:0  
间充质干细胞(MSCs)独特的免疫调节作用以T细胞为主,在混合淋巴细胞反应中,通过周期阻滞抑制T细胞增殖,但不引起T细胞凋亡增加、活化受抑。同时MSCs还能降低反应体系中的CD8+T细胞和Th1细胞,升高Th2细胞以抑制炎症反应,从而在T细胞介导的自身免疫性疾病中发挥治疗作用。  相似文献   

11.
目的 探讨骨髓间充质干细胞(mesenchymal stem cells, MSC)在体外对异体外周血B淋巴细胞的免疫调节作用.方法 用密度梯度离心法从骨髓中分离、培养MSC,从外周血中分离单个核细胞,L-亮氨酸甲酯去除单核细胞,以2-氨乙基硫脲溴化物(AET)处理绵羊红细胞(SRBC)的花环形成法,去除T淋巴细胞获得纯化的B淋巴细胞.用羊抗人IgM单克隆抗体(anti-IgM)刺激与或未与MSC或其培养上清共培养3d的B淋巴细胞,应用MTT法测8淋巴细胞的增殖,ELISA法测培养上清中免疫球蛋白IgG、lgM的产生,应用流式细胞术分别检测与MSC共培养24、48h后B淋巴细胞的凋亡.结果 MSC及其培养上清抑制由anti-IgM诱导的B淋巴细胞的增殖和分泌IgG、IgM,且随着MSC细胞数量及其培养上清浓度的增加,这种抑制越明显.流式细胞术结果显示,与MSC共培养不同时间的B淋巴细胞的凋亡变化无统计学意义,MSC抑制B淋巴细胞的增殖存在暂时性和可逆性.结论 MSC对异体外周血8淋巴细胞存在免疫调节作用,并且这种调控机制是复杂的,不仅与MSC细胞数量有关,还与细胞间的相互作用和MSC分泌的细胞因子有关.  相似文献   

12.
背景:多发性骨髓瘤患者的骨髓间充质干细胞具有多向分化、免疫调节和支持造血作用,但是这些功能是否受冻存的影响目前尚不清楚。 目的:探讨冻存对多发性骨髓瘤患者骨髓间充质干细胞生物学特性的影响。 方法:采用细胞贴壁法获取多发性骨髓瘤患者骨髓间充质干细胞,将传代后的细胞用IMDM细胞冻存液(含10%的二甲基亚砜和体积分数40%的胎牛血清)保存在-196 ℃液氮中。检测短期(1个月)和长期(12个月)冻存复苏后间充质干细胞的活性和增殖能力;将冻存后多发性骨髓瘤患者骨髓间充质干细胞作为滋养层,应用甲基纤维素半固体培养,检测其支持造血的能力;混合淋巴细胞反应检测冻存后多发性骨髓瘤患者骨髓间充质干细胞调控免疫能力。 结果与结论:经过短、长期冻存后多发性骨髓瘤患者骨髓间充质干细胞的细胞活性分别为(92.9±7.5)%和(86.7±9.2)%;短、长期冻存后细胞的增殖能力与冻存前间充质干细胞相似;冻存后多发性骨髓瘤患者骨髓间充质干细胞仍具有支持造血祖细胞生长的作用和抑制T淋巴细胞增殖的能力,与冻存前相比,没有明显差别。说明冻存可以降低多发性骨髓瘤患者骨髓间充质干细胞的细胞活性,但是并不影响间充质干细胞的增殖、支持造血和免疫调节的能力。  相似文献   

13.
Recent studies indicated that bone marrow mesenchymal stem cells (BM-MSCs) derived from multiple myeloma (MM) patients were different from those of normal subjects in a variety of aspects. However, it is largely unknown whether BM-MSCs derived from MM patients display any aberrant chemotactic migration. To this aim, we compared the chemotactic migration of BM-MSCs derived from MM patients with those from normal subjects. Our results showed that BM-MSCs derived from MM patients migrated more vigorously to myeloma cell line. Furthermore, proteasome inhibitor bortezomib was showed to suppress chemotactic migration of BM-MSCs whatever their origins. However, although the chemotactic migration of BM-MSCs derived from MM patients to myeloma cell line was more significantly suppressed by bortezomib treatment, migration to SDF-1 or FBS of BM-MSCs was less compromised. Both SDF-1 and TNF-α enhanced phosphorylation of iκ-Bα in BM-MSCs. Although bortezomib significantly inhibited the iκ-Bα phosphorylation by SDF-1, it had little effect on iκ-Bα phosphorylation by TNF-α. Collectively, our results suggested that aberrant chemotactic migration of BM-MSCs derived from MM patients and the possible migration-regulatory role of bortezomib treatment.  相似文献   

14.
目的:探讨大鼠骨髓间充质干细胞(Mesenchymal stemcells,MSCs)对脾脏T细胞的免疫调节作用。方法:从大鼠骨髓中分离培养间充质干细胞,通过瑞氏-姬姆萨染色观察细胞形态,并用流式细胞术(Flowcytometry,FCM)检测其细胞表面特征分子表达情况。MTT法测定经刀豆蛋白A(ConA)刺激后,不同数量MSCs对T细胞增殖能力的影响;ELISA法检测MSCs对T细胞分泌IFN-γ和IL-4水平的影响。FCM检测MSCs对脾脏T细胞凋亡水平的影响。MTT法测定MSCs对细胞毒性T细胞(Cytotoxic Tcells,CTL)杀伤活性的影响。结果:经不同数量MSCs作用后,脾脏T细胞增殖水平明显低于阳性对照组(P<0.01),而且MSCs比例越高,其抑制作用越强(P<0.01)。经MSCs作用后,T细胞分泌IFN-γ的水平明显降低,而分泌IL-4的水平明显升高,且这种作用随MSCs比例的增加而增强(P<0.01)。MSCs能够抑制在体外培养过程中T细胞的自发凋亡。与MSCs共培养后,T细胞对L1210细胞的杀伤活性和单独培养组相比明显下降(P<0.05)。结论:MSCs能够抑制T细胞增殖反应和CTL活性,该作用可能与MSCs改变T细胞分泌细胞因子水平有关,但与诱导T细胞凋亡无关。  相似文献   

15.
Oral leukoplakia is one of the common precancerous lesions in oral mucosa. To compare the biological characteristics and regenerative capacities of mesenchymal stem cells (MSCs) from oral leukoplakia (epithelial hyperplasia and dysplasia) and normal oral mucosa, MSCs were isolated by enzyme digestion. Then these cells were identified by the expression of MSC related markers, STRO-1, CD105 and CD90, with the absent for the hematopoietic stem cell marker CD34 by flow cytometric detection. The self-renewal ability of MSCs from oral leukoplakia was enhanced, while the multipotent differentiation was descended, compared with MSCs from normal oral mucosa. Fibrin gel was used as a carrier for MSCs transplanted into immunocompromised mice to detect their regenerative capacity. The regenerative capacities of MSCs from oral leukoplakia became impaired partly. Collagen IV (Col IV) and matrix metalloproteinases-9 (MMP-9) were selected to analyze the potential mechanism for the functional changes of MSCs from oral leukoplakia by immunochemical and western blot analysis. The expression of Col IV was decreased and that of MMP-9 was increased by MSCs with the progression of oral leukoplakia, especially in MSCs from epithelial dysplasia. The imbalance between regenerative and metabolic self-regulatory functions of MSCs from oral leukoplakia may be related to the progression of this premalignant disorder.  相似文献   

16.
间充质干细胞在自身免疫性疾病中的应用研究进展   总被引:1,自引:1,他引:0  
自身免疫性疾病(AID)的发病机制主要在于机体自身耐受的破坏,机体产生自身抗体和(或)自身反应性淋巴细胞,导致疾病的发生。目前临床上对AID的治疗主要是采取非特异性免疫抑制。虽然一定程度上可减轻症状,但不能根治疾病,而重症AID缺乏理想的治疗方法,预后差。因此,寻找有效的治疗方法仍然是目前临床亟待解决的问题。间充质干细胞(MSC)是一种非造血多能成体干细胞,具有多向分化以及促进组织修复等潜能,其免疫调控作用的发现是近年来干细胞研究领域的一项重要突破。最近,MSC移植治疗自身免疫性疾病的应用研究不断涌现,显示了MSC的免疫调节特性及其治疗AID的潜在能力,为进一步研究奠定了基础。  相似文献   

17.
The goal of the study was to investigate the expression of interleukin-17 (IL-17) and IL-17 receptor (IL-17R) in patients with myeloma bone diseases (MBD) and skeletal extramedullary disease (skeletal EMD). The levels of IL-17 were determined using ELISA. The expression of IL-17R on vascular endothelial cells of bone marrow (BM) and masses of skeletal EMD was detected using immunohistochemistry. The results showed an elevated IL-17 level in BM of BMD and skeletal EMD patients. The microvessel density (MVD) was significantly increased in the masses of skeletal EMD. IL-17R was almost exclusively expressed by endothelial cells, not by myeloma cells in the masses of skeletal EMD patients. We concluded that EMD masses showed increased angiogenesis mediated by IL-17 pathway and in part this may help in myeloma cell-growth under these conditions.  相似文献   

18.
Objective: To evaluate the therapeutic efficacy of uncultured bone marrow mononuclear cells (BMMCs) and bone mesenchymal stem cells in an osteoarthritis (OA) model of sheep. Methods: Induction of sheep OA was performed surgically through anterior cruciate ligament transection and medial meniscectomy. After 12 weeks, concentrated BMMCs obtained from autologous bone marrow harvested from anterior iliac crest or a single dose of 10 million autologous bone mesenchymal stem cells (BMSCs) suspended in phosphate-buffered saline (PBS) was delivered to the injured knee via direct intra-articular injection. Animals of the PBS group received vehicle alone. The contra-lateral joints were selected randomly as the control group. Knees of the four groups were compared macroscopically and histologically, and glycosaminoglycan (GAG) contents normalized to cartilage wet weight were measured at lesions of cartilage from medial condyle of the femur head. Gene expression levels of type II collagen (Col2A1), Aggrecan and matrix metalloproteinase-13 (MMP-13) in cartilage were measured based on RT-PCR and prostaglandin E2 (PGE2), Tumor Necrosis Factor-α (TNF-α) and Transforming Growth Factor beta (TGF-β) concentrations in synovial fluid were determined with ELISA assays at 8 weeks after injection. Results: At 8 weeks post cell transplantation, partial cartilage repair was observed in the cell therapy, but not the PBS group (P<0.05). The BMSCs group showed higher regeneration of cartilage and lower proteoglycan loss than the BMMCs group (P<0.05). Concentrated BMMCs injection led to a weaker treatment effect, but also inhibited PGE2, TNF-α and TGF-β levels in synovial fluid and promoted higher levels of Aggrecan and Col2A1 and downregulation of MMP-13 in sheep chondrocytes in a similar manner to BMSCs, compared with the PBS group. Conclusions: Bone marrow cells showed therapeutic efficacy in a sheep model of OA. Despite similar therapeutic potential, the easier and faster process of collection and isolation of BMMCs supports their utility as an effective alternative for OA treatment in the clinic.  相似文献   

19.
Both mesenchymal stem cells (MSCs) and dendritic cells (DCs) are engaged in the regulation of the immune response parallel to their numerous functions.The main objective of this study was to compare the effects of mesenchymal stem cells isolated from human adipose tissue or human bone marrow on the expression of specific cell surface markers as well as the secretion of some cytokines by monocyte-derived dendritic cells. The set of methods used includes cell cultures, magnetic beads isolation of cells, flow cytometry, ELISA and proteome profiler kit assays. The results obtained show that MSCs isolated from human adipose tissue are more potent immunomodulators of differentiation of human DCs in comparison to the bone marrow-derived MSCs. In both cases the percentages of CD14+ cells were increased in co-cultures of MSCs and DCs and at the same time down-regulated the expression of CD80, CD86 and CD83 as in all experiments the effect of adipose tissue MSCs was stronger. Similarly, the secretion of IL-10 by dendritic cells was up-regulated in co-cultures of MSCs and dendritic cells and the effect was stronger when adipose tissue-derived MSCs were used.Taken together all results presented reveal the higher potential of the adipose tissue-derived MSCs to inhibit the differentiation and expression of functionally important co-stimulatory molecules on the surface of monocyte-derived dendritic cells than the bone marrow-derived MSCs.  相似文献   

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