骨髓间充质干细胞诱导的胰岛样细胞治疗大鼠胰腺损伤

目的 探讨骨髓间充质干细胞(MSCs)诱导后促进胰腺损伤修复的方法.方法 应用贴壁法分离、纯化大鼠MSCs,经bFGF、EGF、HGF、2%B27、activeA、β巯基乙醇、尼克酰胺诱导12～14 d,RT-PCR鉴定后,以DAPI标记治疗胰腺损伤模型大鼠.治疗1 w后激光共聚焦显微镜观察胰岛样细胞的迁移、定位,RT-PCR鉴定参与胰腺损伤修复的细胞来源.结果 MSCs经诱导12～14 d,分化为可表达Ins1、Ins2的胰岛样细胞,经DAPI标记后治疗胰腺损伤模型大鼠1 w,使炎性细胞浸润明显、细胞结构不清、胰岛结构崩解的损伤区的胰腺组织结构开始恢复,胰岛再建;RT-PCR检测Sry进一步证实损伤区胰腺组织内存在输注的胰岛样细胞.结论 MSCs可诱导分化成胰岛样细胞,该细胞参与损伤胰腺组织的修复.     

犬骨髓间充质干细胞体外诱导分化为胰岛样细胞的研究

目的 研究犬骨髓间充质干细胞(MSC)体外诱导分化为胰岛样细胞的可能性.方法 分离纯化犬骨髓MSC,予EGF、bFGF和β细胞调节素、尼克酰胺、B27添加剂诱导.双硫腙染色、 Western blot、免疫荧光染色、胰岛素分泌量测定和葡萄糖刺激胰岛素释放试验鉴定诱导前后的细胞功能.结果 未诱导细胞呈长梭形贴壁生长,诱导第2周细胞逐渐变圆,聚集成团,双硫腙染色呈棕红色;Western blot检测诱导后细胞表达PDX-1;免疫荧光染色显示诱导后细胞表达胰岛素、胰升血糖素、生长抑素; ELISA结果表明诱导后细胞可分泌胰岛素,体外葡萄糖刺激胰岛素释放试验阳性.结论 犬骨髓MSC在体外能被诱导分化为胰岛样细胞.     

骨髓间充质干细胞转化为胰岛样细胞的研究进展

骨髓间充质干细胞是存在于骨髓中的一群具有自我更新和多向分化潜能的细胞。新近研究表明，骨髓间充质干细胞在体内微环境及体外细胞因子的作用下不仅能够分化为胰岛β样细胞，而且具有一定的β细胞分泌功能，能够降低血糖，克服了胰岛移植治疗糖尿病所面临的供体细胞缺乏和免疫排斥反应问题，从而为临床糖尿病细胞治疗开辟了一条新的途径。     

链脲佐菌素所致糖尿病大鼠骨髓中存在胰岛样细胞

本研究证实糖尿病大鼠骨髓中存在表达胰岛素、C肽、胰高血糖素、生长抑素和胰淀粉样多肽的细胞簇，并检测到胰岛发育和功能相关基因的表达，这些胰岛样细胞可能是骨髓中的成体干细胞转分化而来。     

2型糖尿病患者血糖波动对胰岛β细胞功能的影响

回顾性分析2017年3月至2018年4月71例2型糖尿病患者的临床资料。结果通过Pearson相关性分析发现,C肽释放指数、0min、120min胰岛细胞分泌功能指数、餐后2hC肽、空腹C肽与血糖标准差呈负相关性(r<0,P<0.05)。结论2型糖尿病患者血糖波动与其胰岛β细胞功能存在相关性,即患者血糖控制越差,其胰岛β细胞功能越不理想。     

体外诱导大鼠骨髓间质干细胞分化为胰岛样细胞的研究

目的探索大鼠骨髓间质干细胞(MSCs)体外诱导分化为胰岛素分泌细胞的方法,为解决胰岛移植物来源匮乏问题提供新的思路。方法采用横向分化技术,将成年大鼠MSCs诱导成胰岛素分泌细胞;间接免疫荧光法鉴定诱导前后的细胞巢蛋白、胰岛素、胰高血糖素、生长抑素的表达;RTPCR法检测诱导前后的细胞胰岛素1、Pdx1、Pax6mRNA的表达;24h累积分泌量测定和胰岛素刺激实验评价诱导前后细胞的功能。结果诱导5h,巢蛋白阳性细胞为(44.6±7.3)%,诱导24h增至(61.8±8.4)%。此后,巢蛋白阳性细胞数开始下降,诱导第14天后,巢蛋白表达基本消失。而且,诱导后细胞可表达胰岛素、胰高血糖素、生长抑素等蛋白;表达胰岛素1及其多种转录因子基因mRNA;胰岛素刺激实验反应敏感,而诱导前MSCs不具备上述特点。结论体外大鼠MSCs可诱导成为胰岛素分泌细胞,为胰岛移植开辟新的研究思路。     

骨髓间充质干细胞诱导为胰岛样细胞后自体移植治疗糖尿病猕猴

目的 观察胰腺提取物诱导骨髓间充质干细胞(BMSC)为胰岛样细胞(IC)的变化及其自体移植对糖尿病猕猴的影响.方法 分离猕猴BMSC,纯化、扩增、鉴定后分别用Ⅰ液[含20 ng/ml碱性成纤维细胞生长因子(b FGF)、20 ng/ml干细胞生长因子(SCF)、2%B27和2%新生牛血清的DMEM/F12]和Ⅱ液(Ⅰ液加10%胰腺提取物和20 mmol/L尼克酰胺)诱导培养,镜检法观察细胞形态的变化,免疫细胞化学法鉴定相关蛋白的表达.将IC自体移植到链脲佐菌素(STZ)所致糖尿病猕猴胰腺,观察移植对血糖、胰岛素和C肽分泌的影响.结果 分离到CD105阳性的BMSC,经体外诱导后,由长梭形变为神经元样细胞(nestin染色阳性),随后细胞逐渐聚集呈丛状排列的IC(胰岛素和C肽染色阳性).自体移植IC后,糖尿病猕猴血糖浓度逐渐降低,而胰岛素与C肽浓度则逐渐升高,3个月后分别与移植前的浓度相比差异均具有统计学意义(P＜0.01),但仍未恢复到注射STZ前的正常水平.结论 BMSC可以在体外被含有10%胰腺提取物的条件培养基诱导为IC,自体移植IC到糖尿病猕猴后可以纠正血糖、胰岛素和C肽的分泌.     

大鼠胰岛与睾丸细胞共移植诱导同种胰岛移植免疫豁免

目的　探究大鼠胰岛与睾丸细胞共移植后睾丸细胞Fas配体表达能否对胰岛移植物提供免疫豁免作用。方法　将同种大鼠胰岛及睾丸细胞移植于糖尿病受体,观察移植物存活情况,并体外检测睾丸Sertoli细胞对活化淋巴细胞的抑制作用以及移植物内淋巴细胞凋亡情况。结果　单纯移植胰岛组移植胰岛平均存活期为(6±1)天。睾丸细胞和胰岛细胞共移植,当睾丸细胞数量为5×10     

胰升血糖素样肽-1改善波动性高糖诱导大鼠胰岛细胞增殖功能机制的研究

目的 研究胰升血糖素样肽-1 （GLP-1）对波动性高糖诱导大鼠胰岛细胞增殖功能的影响及机制. 方法 将获取的原代胰岛细胞分为5组,即正常葡萄糖（5.5 mmol/L）组、恒定高糖（30.0 mmol/L）组、波动高糖（每24 h轮换培养于5.5、30.0 mmol/L）组、恒定高糖＋GLP-1 （100 nmol/L）组和波动高糖＋GLP-1组.干预7d后检测细胞增殖活性、活性氧簇（ROS）、细胞周期及周期蛋白cyclinD1、p21、p27、Skp2的表达. 结果 GLP-1干预可降低波动性高糖诱导的细胞内ROS水平[（194.40±19.20）vs（406.78±18.40）,P＜0.05],上调促细胞周期cyclinD1、Skp2表达,下调周期抑制蛋白p21、p27表达,使停滞在G0/G1期的细胞比例减少,改善细胞增殖活性[（1.38±0.09）vs（0.44±0.10）,P＜0.05]. 结论 GLP-1可通过降低氧化应激水平及对不同细胞周期蛋白表达的调节改善波动性高糖抑制的胰岛细胞增殖活性.     

骨质疏松大鼠骨髓白细胞介素6受体亚单位gp80和gp130基因的表达

以竞争性RT-PCR研究骨质疏松模型大鼠骨髓白细胞介素6受体亚单位gp80和gpl30基因的表达。结果表明这些骨质疏松大鼠的gp80和gp130 mRNA基因表达增加，该变化可能为雌激素丢失所致骨质疏忪的形成机制之。     

Reversal of hyperglycemia in diabetic rats by portal vein transplantation of islet-like cells generated from bone marrow mesenchymal stem cells

AIM： To study the capacity of bone marrow mesenchymal stem cells （BM-MSCs） trans-differentiating into islet-like cells and to observe the effect of portal vein transplantation of islet-like cells in the treatment of streptozotocin-induced diabetic rat.
METHODS： BM-MSCs were isolated from SD rats and induced to differentiate into islet-like cells under defined conditions. Differentiation was evaluated with electron microscopy, RT-PCR, immunofluorescence and flow cytometry. Insulin release after glucose challenge was tested with ELISA. Then allogeneic islet-like cells were transplanted into diabetic rats via portal vein. Blood glucose levels were monitored and islet hormones were detected in the liver and pancreas of the recipient by immunohistochemistry.
RESULTS： BM-MSCs were spheroid adherent monolayers with high CD90, CD29 and very low CD45 expression. Typical islet-like cells clusters were formed after induction. Electron microscopy revealed that secretory granules were densely packed within the cytoplasm of the differentiated cells. The spheroid cells expressed islet related genes and hormones. The insulin-positive cells accounted for 19.8% and mean fluorescence intensity increased by 2.6 fold after induction. The cells secreted a small amount of insulin that was increased 1.5 fold after glucose challenge. After transplantation, islet-like cells could locate in the liver expressing islet hormones and lower the glucose levels of diabetic rats during d 6 to d 20.
CONCLUSION： Rat BM-MSCs could be transdifferentiated into islet-like cells in vitro. Portal vein transplantation of islet-like cells could alleviate the hyperglycemia of diabetic rats.     

骨髓干细胞在大鼠肝再生环境中的分化

研究大鼠骨髓干细胞在部分肝切除后肝再生环境中的分化。从部分肝切除模型大鼠的胫骨中提取骨髓细胞,应用流式细胞仪富集骨髓干细胞,以PKH26-GL体外标记后通过门静脉进行自体移植,2周后行白蛋白和角蛋白8免疫组化检查。结果肝板肝细胞间PKH26-GL标记骨髓干细胞表达白蛋白、角蛋白8。提示骨髓干细胞在部分肝切除后肝再生环境中能分化为肝细胞,骨髓干细胞可能参与部分肝切除后的肝再生过程。     

Pancreatic islet-like clusters from bone marrow mesenchymal stem cells of human first-trimester abortus can cure streptozocin-induced mouse diabetes

Bone marrow mesenchymal stem cells (BMSCs) have been reported to possess low immunogenicity and cause immunosuppression of recipients when allografted. They can differentiate into insulin-producing cells and may be a valuable source for islet formation. However, the extremely low differentiating rate of adult BMSCs toward insulin-producing cells and the insufficient insulin secretion of the differentiated BMSCs in vitro prevent their clinical use in diabetes treatment. Little is known about the potential of cell replacement therapy with human BMSCs. Previously, we isolated and identified human first-trimester fetal BMSCs (hfBMSCs). Under a novel four-step induction procedure established in this study, the hfBMSCs effectively differentiated into functional pancreatic islet-like cell clusters that contained 62 ± 14% insulin-producing cells, expressed a broad gene profile related to pancreatic islet β-cell development, and released high levels of insulin (2.245 ± 0.222 pmol/100 clusters per 30 min) and C-peptide (2.200 ± 0.468 pmol/100 clusters per 30 min) in response to 25 mmol/L glucose stimulus in vitro. The pancreatic islet-like cell clusters normalized the blood glucose level of diabetic model mice for at least 9 weeks when xenografted; blood glucose levels in these mice rose abnormally again when the grafts were removed. Examination of the grafts indicated that the transplanted cells survived in recipients and produced human insulin and C-peptide in situ. These results demonstrate that hfBMSCs derived from a human first-trimester abortus can differentiate into pancreatic islet-like cell clusters following an established four-step induction. The insulin-producing clusters present advantages in cell replacement therapy of type 1 diabetic model mice.     

Rapid reduction of methemoglobin in rat bone marrow erythroid cells

Methemoglobin reduction was shown to proceed much more rapidly in erythroid cells from rat bone marrow than in rat erythrocytes. Methemoglobin reduction in suspensions of intact, nitrite-treated bone marrow cells does not depend on the presence of glucose in the incubation mixture, even after the cells have been stored in substrate-free medium. 2-Deoxyglucose and iodoacetate prevent the reduction from proceeding to completion. The results suggest that, relative to erythrocytes, immature erythroid cells more efficiently catalyze methemoglobin reduction and more effectively store metabolites which provide electrons for this reaction.     

Purification and characterization of rat bone marrow endothelial cells

A method is described to obtain endothelial cells from rat bone marrow with high purity and viability. Marrow cell suspensions were prepared by collagenase and subjected to discontinuous gradient centrifugation on Percoll (densities 1.04 and 1.06). Endothelial cells were concentrated in the middle layer as demonstrated by electron microscopy and flow cytometry as well as fluorescent microscopy after staining for factor VIII antigen. Cells of this layer were then subjected to centrifugal elutriation and highly purified endothelial cell preparations were obtained with flow rates of 15-20 ml/min. By fluorescent microscopy, 49%-51% of these cells were factor VIII positive. Identification by means of electron microscopy indicated a much higher purity of endothelium ranging from 63% to 90% with a yield in the range of 10(6) cells and a viability exceeding 90%. Some technical considerations in the development of this method are discussed. This method permits in vitro experiments on relatively high purity, high viability preparations of marrow endothelium.     

骨髓干细胞在大鼠肝纤维化形成环境中的分化

目的 研究骨髓干细胞在肝纤维化形成环境中向肝细胞定向分化。 方法 采用四氯化碳皮下注射法诱导大鼠肝纤维化,应用流式细胞仪分选富集Thy+CD3-CD45RA-的骨髓干细胞,采用红色荧光染料PKH26-GL对其标记后进行自体移植,6周后通过免疫组织化学方法检测大鼠肝组织白蛋白、ck 8、α-平滑肌肌动蛋白表达。 结果 PKH 26-GL标记的细胞在纤维化肝脏中表达白蛋白和ck8,占肝细胞总数的(0.17±0.02)％;未见表达α-平滑肌肌动蛋白。 结论 骨髓干细胞在肝纤维化形成环境中可以向肝细胞定向分化,不向肌成纤维样细胞分化。     

Renin-angiotensin system expression in rat bone marrow haematopoietic and stromal cells

The existence of a bone marrow renin-angiotensin system (RAS) is evidenced by the association of renin, angiotensin converting enzyme (ACE), and angiotensin (Ang) II and its AT(1) and AT(2) receptors with both normal and disturbed haematopoiesis. The expression of RAS components by rat unfractionated bone marrow cells (BMC), haematopoietic-lineage BMC and cultured marrow stromal cells (MSC) was investigated to determine which specific cell types may contribute to a local bone marrow RAS. The mRNAs for angiotensinogen, renin, ACE, and AT(1a) and AT(2) receptors were present in BMC and in cultured MSC; ACE2 mRNA was detected only in BMC. Two-colour flow fluorocytometry analysis showed immunodetectable angiotensinogen, ACE, AT(1) and AT(2) receptors, and Ang II, as well as binding of Ang II to AT(1) and AT(2) receptors, in CD4(+), CD11b/c(+), CD45R(+) and CD90(+) BMC and cultured MSC; renin was found in all cell types with the exception of CD4(+) BMC. Furthermore, Ang II was detected by radioimmunoassay in MSC homogenates as well as conditioned culture medium. The presence of Ang II receptors in both haematopoietic-lineage BMC and MSC, and the de novo synthesis of Ang II by MSC suggest a potential autocrine-paracrine mechanism for local RAS-mediated regulation of haematopoiesis.