脐血间充质干细胞静脉移植治疗新生大鼠缺氧缺血性脑损伤的时效性研究

目的探讨脐血间充质干细胞(MSCs)静脉移植治疗新生鼠缺氧缺血性脑损伤(HIBD)的可行性及其时效性。方法脐血MSCs移植使用前以4′,6-二脒基-2-苯吲哚盐酸(DAPI)体外标记。实验选用7日龄SD大鼠38只制备HIBD模型,死亡3只,余35只共分3组:空白对照组(n=11);移植1组(n=12),在HIBD后第2天经鼠尾静脉注入脐血MSCs;移植2组(n=12),在HIBD后1周开始移植。两组均于移植后第2天以及HIBD后2周分别随机将鼠处死、取脑,用于脑组织病理形态学观察,并取海马回区相同部位的缺血脑组织切片,荧光显微镜下观察DAPI阳性细胞数。结果移植2组,1周后缺血脑组织细胞外间隙缩小,细胞数明显增加,脑组织水肿已明显减轻,在大鼠病灶侧脑内,可见大量的DAPI阳性细胞向病灶区及周围迁移和扩散,没有明显的界限。而移植1组于移植后病灶侧脑内很少见到DAPI阳性脐血MSCs分布,其脑组织水肿程度及细胞外间隙的改善和细胞数目的增加也不明显。结论脐血MSCs移植治疗新生大鼠HIBD,能有效透过血脑屏障,在病灶脑组织周围迁移、扩散、整合;移植时间选择HIBD后1周时有良好疗效。移植治疗过程中未见植入反应和其他不良反应。     

BMP2诱导骨骼肌成骨的研究进展

20世纪90年代以来,组织工程技术不断发展,在组织工程化骨领域取得了较大进展,然而,迄今为止,组织工程化骨修复缺损,离临床的要求还有较大距离。目前认为,主要问题是种子细胞在一定传代后发生衰老而不能继续增殖。骨组织工程种子细胞多集中在骨髓基质干细胞的研究上,实际上机体骨髓中所含的基质干细胞数量非常有限^[1],     

源于骨髓间充质干细胞的神经元存活与c-myc

目的：探讨源于骨髓问充质干细胞的神经元的存活与原癌基因蛋白c-myc的关系。方法：从成年大鼠骨髓分离、扩增rMSCs至6代。给予不同的诱导条件,采用免疫组化方法观察神经分化过程中神经特异抗原和c-myc蛋白的表达,并计算分化率。结果：在不同的分化诱导条件下,MSCs呈现典型的神经元形态。加入MNM组分化率高于对照组。分化后4～36h,可检测到c-myc蛋白的持续表达。结论：c-myc在MSCs向神经元分化过程中有重要作用。     

Biomaterials reinforced MSCs transplantation for spinal cord injury repair

Due to the complex pathophysiological mechanism, spinal cord injury (SCI) has become one of the most intractable central nervous system (CNS) diseases to therapy. Stem cell transplantation, mesenchymal stem cells (MSCs) particularly, appeals to more and more attention along with the encouraging therapeutic results for the functional regeneration of SCI. However, traditional cell transplantation strategies have some limitations, including the unsatisfying survival rate of MSCs and their random di...     

Therapeutic potential of bone marrow-derived mesenchymal stem cells on experimental liver fibrosis

OBJECTIVE: To study the effect of mesenchymal stem cells (MSC) on experimental liver fibrosis in rats. DESIGN AND METHOD: MSC were derived from bone marrow obtained from femoral and tibial bones of male albino rats. MSC were separated, grown, and propagated in culture for 4 weeks and were characterized morphologically and by detection of CD29 by RT-PCR. They were then infused into the tail vein of female rats that received CCl4 injection to induce liver fibrosis. Rats were divided into 4 groups: control, CCl4, CCl4 plus MSC, and MSC. Liver tissue was examined histopathologically and liver functions (ALT and serum albumin) were estimated for all groups. Y-chromosome gene (sry) was assessed by PCR in liver tissue of the female rats to confirm uptake of the male stem cells. Hydroxyproline content in liver tissue was assessed by chemical methods and expression of the collagen gene (type I) was detected as a marker for liver fibrosis. Results of the present study showed that MSC have a significant antifibrotic effect as evidenced by the significant decrease in liver collagen gene expression as well as the decrease in hydroxyproline content in the CCl4/MSC group (p<0.001) compared to the CCl4 group. The Y-chromosome gene (sry) was detected by RT-PCR in the CCl4/MSC group, but was not detected in control group and other groups. The CD29 gene was expressed in MSC culture, and this confirmed the efficiency of isolation and propagation of MSC in culture. With regard to liver function, there was also a significant improvement and elevation of serum albumin in the CCl4/MSC group compared to the CCl4 group (p<0.05). As regard to the liver enzyme ALT, there was a decrease of its level in the CCl4/MSC group compared to the CCl4 group. However, this was statistically nonsignificant (p>0.05). In conclusion, MSC have a potential therapeutic effect against the fibrotic process through their effect in minimizing collagen deposition in addition to their capacity to differentiate into hepatocytes.     

丹酚酸B体外干预骨髓间充质干细胞分化过程中cTnT的表达

[目的]观察丹酚酸B(SalB)体外干预骨髓间充质干细胞(MSCs)后细胞形态及心肌特异性蛋白肌钙蛋白T(cTnT)的表达。[方法]培养、纯化及鉴定MSCs,用SalB、5-氮胞苷组(5-aza)、SalB联合5-aza(SalB 5-aza)分别定向诱导分化,观察此过程中MSCs的形态学变化并采用免疫细胞化学法鉴定诱导后MSCs心肌特异性肌钙蛋白T(cTnT)的表达。[结果]诱导后的MSCs体积增大,增殖减慢,并出现肌管样结构;免疫细胞化学结果显示诱导后MSCs表达心肌特异性蛋白cTnT。[结论]SalB在体外定向诱导MSCs分化为心肌细胞的过程中发挥了一定的作用。     

骨髓间充质干细胞移植在心肌病大鼠体内存活、分化的实验研究

目的:研究骨髓间充质干细胞（MSCs）移植在心肌病大鼠体内存活、分化的情况。方法:将雌性W istar大鼠30只经腹腔注射阿霉素6次（总剂量15mg/kg）,建立心肌病的动物模型,在此模型的左室前壁采用微静脉注射法植入MSCs。8周后处死大鼠,取出心脏标本,做冰冻切片进行HE染色,观察植入的MSCs在病损心肌组织中的变化及免疫荧光检查观察植入MSCs心肌肌球蛋白重链（MHC）及心肌特有的连接蛋白（CX43）表达情况。结果:HE染色检查显示,植入的MSCs存活并有新生血管形成;免疫荧光检查显示植入的MSCs,表达MHC及CX43,但较宿主表达弱。结论:MSCs移植在心肌病大鼠体内心肌微环境中能存活,并向心肌细胞分化。     

Changes of mesenchymal stromal cells mobilization and bone turnover in an experimental bone fracture model in ovariectomized mice

Objective: The aim of this study was to characterize the mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs) mobilization, and bone turnover in osteoporotic fracture healing in ovariectomized mice. Methods: In total, 112 female C57/BL mice were divided into two groups. The first group was sham-operated (SO), and the other group was ovariectomized (OVX). After three weeks, the right femora of the mice were fractured under anesthesia and internally fixed with steel pin. Peripheral blood and bone marrow were was collected for flow cytometry analysis, at 0 hours (h), 12 h, 24 h, 72 h and 168 h after fracture. MSCs and EPCs levels were assessed using cell surface antigens in different combinations (CD44+ CD34-CD45-, and CD34+ KDR+CD45-) by flow cytometry. At 0, 14, 28 and 42 days after fracture, sera were assayed for circulating levels of procollagen type I-N-terminal propeptide (P1NP) and C-terminal telopeptide of type I-collagen (CTX) by ELISA. Femurs were harvested at 2 weeks and 6 weeks after fracture for X-ray radiography, micro-computed tomography (micro-CT) and histology. Results: Our results showed that bone marrow and peripheral blood MSCs numbers of the OVX mice were significantly lower than the SO mice, at 12 h, 24 h and 72 h after fracture. In addition, circulating P1NP and CTX levels of the OVX mice were significantly higher than the SO mice, at 2 and 4 weeks. Conclusion: Results of the present study revealed disorders of bone marrow MSCs mobilization and bone turnover may partially account for the delay of osteoporotic fracture healing.     

Biosynthesis of collagen I,II, RUNX2 and lubricin at different time points of chondrogenic differentiation in a 3D in vitro model of human mesenchymal stem cells derived from adipose tissue

The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called ‘cell pellets’. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.     

MSCs-肝细胞共培养上清对体外培养L02细胞的影响

目的以体外培养L02细胞为实验对象,了解骨髓间充质干细胞(MSCs)与肝细胞共培养上清的生物活性。方法采用两步酶分离法、全骨髓贴壁筛选法分别分离肝细胞、MSCs,按比例直接共培养,收集不同时段的共培养上清,用于人肝细胞系-L02细胞的培养。分别以单独培养的肝细胞上清、MSCs上清为对照,观察共培养上清对L02细胞活力和总蛋白合成的影响;同时观察其对L02细胞损伤模型的AST、LDH及细胞凋亡的影响。结果肝细胞上清、MSCs上清以及共培养上清对L02细胞的活力和增殖均有显著促进作用,表现为L02增殖活跃,总蛋白合成量增加;加入上述细胞上清的酒精损伤L02细胞AST、LDH释放和细胞凋亡显著低于未加细胞上清的对照组,其中共培养上清效果最为显著(P<0.05)。结论共培养上清对L02有明显的刺激增殖和损伤保护作用。