CD34+细胞在干细胞旁分泌下向内皮细胞分化的研究

Objective To study whether the paracrine action of umbilical cord-derived mesenchymal stem cells (UC-MSC) can induce differentiation of human umbilical cord blood-derived CD34+ cells into endothelial cells in vitro. Methods The 20 fresh umbilical cord blood samples were collected with volume of (103. 80 ± 19. 77) ml. CD34+ cells were isolated from the mononuclear cells by magnetic activated cell sorting system (MACS) , and mesenchymal stem cells (MSCs) were isolated from umbilical cord by collagenase and trypsin digestion. Three groups were set up: CD34+ cells pure culture group, cytokineinduced group and two stem cells co-culture group with noncontact. Results The average purity of enriched CD34+ cells as assessed by FACS was (95. 02 ± 3. 81) %. Freshly isolated CD34+ cells were small and round which suspended in culture medium. Attached cord-like structure cells of CD34+ cells appeared after 7 days coculture with noncontacted MSC, and when the CD34+ cells grew into large number, they formed colonies. These cells expressed endothelial specific markers, including CD144 (54. 40 ±4. 13)% ,vWF (47. 53 ± 3. 96) % , CD31 (65. 43 ± 5. 61) % ( P ＜ 0. 05, as compared with CD34+ cells pure culture group) , partially expressed CD34 and, the leukocyte common antigen CD45 was negatively expressed.Conclusion Human umbilical cord blood-derived CD34+ cells could be induced into endothelial cells under the paracrine action of umbilical cord-derive mesenchymal stem cells which has the same effect of cytokines.     

CD34+细胞在干细胞旁分泌下向内皮细胞分化的研究

目的 探讨人脐血CD34+细胞在脐带间充质干细胞(UC-MSCs)旁分泌作用下向内皮细胞诱导分化的可行性.方法 收集20份脐血,体积(103.80±19.77)ml.免疫磁珠(MACS)分选CD34+细胞;取脐带用消化贴壁法获得UC-MSC.流式鉴定干细胞表型.实验分单纯培养组、诱导组、共培养组.结果 流式鉴定CD34+细胞纯度(95.02±3.81)%.培养14 d流式检测共培养组表达CD31、CD144、VWF分别为(65.43±5.61)%、(54.40±4.13)%、(47.53±3.96)%(与单纯培养组比较P＜0.05),部分表达CD34,阴性表达CD45,这与诱导组及成熟脐静脉内皮细胞表达率一致.结论 UC-MSCs旁分泌作用与外源性细胞因子都具有促分化作用,均能使脐血CD34+细胞向内皮细胞分化.

Abstract：

Objective To study whether the paracrine action of umbilical cord-derived mesenchymal stem cells (UC-MSC) can induce differentiation of human umbilical cord blood-derived CD34+ cells into endothelial cells in vitro. Methods The 20 fresh umbilical cord blood samples were collected with volume of (103. 80 ± 19. 77) ml. CD34+ cells were isolated from the mononuclear cells by magnetic activated cell sorting system (MACS) , and mesenchymal stem cells (MSCs) were isolated from umbilical cord by collagenase and trypsin digestion. Three groups were set up: CD34+ cells pure culture group, cytokineinduced group and two stem cells co-culture group with noncontact. Results The average purity of enriched CD34+ cells as assessed by FACS was (95. 02 ± 3. 81) %. Freshly isolated CD34+ cells were small and round which suspended in culture medium. Attached cord-like structure cells of CD34+ cells appeared after 7 days coculture with noncontacted MSC, and when the CD34+ cells grew into large number, they formed colonies. These cells expressed endothelial specific markers, including CD144 (54. 40 ±4. 13)% ,vWF (47. 53 ± 3. 96) % , CD31 (65. 43 ± 5. 61) % ( P ＜ 0. 05, as compared with CD34+ cells pure culture group) , partially expressed CD34 and, the leukocyte common antigen CD45 was negatively expressed.Conclusion Human umbilical cord blood-derived CD34+ cells could be induced into endothelial cells under the paracrine action of umbilical cord-derive mesenchymal stem cells which has the same effect of cytokines.     

人脐带间充质干细胞分泌物对肝细胞增殖和凋亡的影响

Objective To investigate the effect of human umbilical cord mesenchymal stem cell paracrine substance on proliferation and apoptosis of liver cells in vitro. Methods Mesenchymal stem cells (MSC)were separated from human umbilical cord with type Ⅳ collagenase and trypsogen digestion method and cultured in vitro. The human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) which contain paracrine substance of human umbilical cord mesenchymal stem cells (HUCMSC) was prepared. Hepatocytes were isolated from SD rats by low concentration collagenase perfusion procedure. There were three groups in the experiment, control group, 2% MSC-CM group and 8% MSC-CM group. The proliferation of normal hepatocytes were assayed with MTT method. We detected the urea and albumin level in culture supernatant to assay the hepatocyte function under different concentration MSC-CM. Hepatocytes were induced for apoptosis by Actinomycin D and tumor necrosis factor alpha (TNF-α),and the apoptosis effect of different concentration MSC-CM was assayed with LIVE/DEAD Viability/Cytotoxicity Kit. Results The MTT assay showed that the absorbance of 2% MSC-CM group was significantly increased (P＜0. 01), and the urea and albumin levels of 2 % MSC-CM group were also significantly increased when compared with control group(P＜0. 01).LIVE/DEAD Viability/Cytotoxicity Kit revealed that hepatocyte survival rate of 2 % MSC-CM group was increased when compared with control group(P＜0. 05), there were no significant differences in above-mentioned experiments when 8% MSC-CM group compared with control group. Conclusion The low concentration MSC-CM could stimulate normal hepatocyte proliferation, inhibit impaired hepatocyte apoptosis and improve hepatocyte function.     

人脐带间充质干细胞分泌物对肝细胞增殖和凋亡的影响

Objective To investigate the effect of human umbilical cord mesenchymal stem cell paracrine substance on proliferation and apoptosis of liver cells in vitro. Methods Mesenchymal stem cells (MSC)were separated from human umbilical cord with type Ⅳ collagenase and trypsogen digestion method and cultured in vitro. The human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) which contain paracrine substance of human umbilical cord mesenchymal stem cells (HUCMSC) was prepared. Hepatocytes were isolated from SD rats by low concentration collagenase perfusion procedure. There were three groups in the experiment, control group, 2% MSC-CM group and 8% MSC-CM group. The proliferation of normal hepatocytes were assayed with MTT method. We detected the urea and albumin level in culture supernatant to assay the hepatocyte function under different concentration MSC-CM. Hepatocytes were induced for apoptosis by Actinomycin D and tumor necrosis factor alpha (TNF-α),and the apoptosis effect of different concentration MSC-CM was assayed with LIVE/DEAD Viability/Cytotoxicity Kit. Results The MTT assay showed that the absorbance of 2% MSC-CM group was significantly increased (P＜0. 01), and the urea and albumin levels of 2 % MSC-CM group were also significantly increased when compared with control group(P＜0. 01).LIVE/DEAD Viability/Cytotoxicity Kit revealed that hepatocyte survival rate of 2 % MSC-CM group was increased when compared with control group(P＜0. 05), there were no significant differences in above-mentioned experiments when 8% MSC-CM group compared with control group. Conclusion The low concentration MSC-CM could stimulate normal hepatocyte proliferation, inhibit impaired hepatocyte apoptosis and improve hepatocyte function.     

人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究

Objective To explore the efficient way to purify and to enrich umbilical cord mesenchymal stem cells which have abilities to differentiate into chondrocytes. Methods Umbilical cord was acquired from new born infants and chopped into 1 mm2 cubics, then they were pretreated with colleganase and trypsin so as to get the mixed cell suspension. CD45(-),CD90(+) cells were acquired from the mixed cell suspension via FCM sorting and were cultured in the medium as umbilical cord mesenchymal stem PO cells. The PO cells were detached and passaged by multiplex collagenase NB4 and the positive rate of CD90 and CD45 of the P1-P3 cells were observeed via FCM ; the P3 cells were induced to differentiated into chondrocytes. Results FCM results showed that the positive rate of CD90 on P1, P2, F3 was (80.86 ± 7.85)%,(95.86± 3.28)%,(97.15± 1.43)%, while the positive rate of CD45 on P1, P2, P3 cells was (2.53 ±0.28)% and (0.97 ± 0.48)% and (0.05 ± 0.01)% respectively; P3 cells had differenciated into chondrocytes. Conclusion FCM presorting and Multiplex collagenase detachment procedures can swiftly acquire highly purified human umbilical cord mesenchymal stem cells which have the ability to differentiate into chondroeytes.     

人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究

Objective To explore the efficient way to purify and to enrich umbilical cord mesenchymal stem cells which have abilities to differentiate into chondrocytes. Methods Umbilical cord was acquired from new born infants and chopped into 1 mm2 cubics, then they were pretreated with colleganase and trypsin so as to get the mixed cell suspension. CD45(-),CD90(+) cells were acquired from the mixed cell suspension via FCM sorting and were cultured in the medium as umbilical cord mesenchymal stem PO cells. The PO cells were detached and passaged by multiplex collagenase NB4 and the positive rate of CD90 and CD45 of the P1-P3 cells were observeed via FCM ; the P3 cells were induced to differentiated into chondrocytes. Results FCM results showed that the positive rate of CD90 on P1, P2, F3 was (80.86 ± 7.85)%,(95.86± 3.28)%,(97.15± 1.43)%, while the positive rate of CD45 on P1, P2, P3 cells was (2.53 ±0.28)% and (0.97 ± 0.48)% and (0.05 ± 0.01)% respectively; P3 cells had differenciated into chondrocytes. Conclusion FCM presorting and Multiplex collagenase detachment procedures can swiftly acquire highly purified human umbilical cord mesenchymal stem cells which have the ability to differentiate into chondroeytes.     

人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究

Objective To explore the efficient way to purify and to enrich umbilical cord mesenchymal stem cells which have abilities to differentiate into chondrocytes. Methods Umbilical cord was acquired from new born infants and chopped into 1 mm2 cubics, then they were pretreated with colleganase and trypsin so as to get the mixed cell suspension. CD45(-),CD90(+) cells were acquired from the mixed cell suspension via FCM sorting and were cultured in the medium as umbilical cord mesenchymal stem PO cells. The PO cells were detached and passaged by multiplex collagenase NB4 and the positive rate of CD90 and CD45 of the P1-P3 cells were observeed via FCM ; the P3 cells were induced to differentiated into chondrocytes. Results FCM results showed that the positive rate of CD90 on P1, P2, F3 was (80.86 ± 7.85)%,(95.86± 3.28)%,(97.15± 1.43)%, while the positive rate of CD45 on P1, P2, P3 cells was (2.53 ±0.28)% and (0.97 ± 0.48)% and (0.05 ± 0.01)% respectively; P3 cells had differenciated into chondrocytes. Conclusion FCM presorting and Multiplex collagenase detachment procedures can swiftly acquire highly purified human umbilical cord mesenchymal stem cells which have the ability to differentiate into chondroeytes.     

人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究

Objective To explore the efficient way to purify and to enrich umbilical cord mesenchymal stem cells which have abilities to differentiate into chondrocytes. Methods Umbilical cord was acquired from new born infants and chopped into 1 mm2 cubics, then they were pretreated with colleganase and trypsin so as to get the mixed cell suspension. CD45(-),CD90(+) cells were acquired from the mixed cell suspension via FCM sorting and were cultured in the medium as umbilical cord mesenchymal stem PO cells. The PO cells were detached and passaged by multiplex collagenase NB4 and the positive rate of CD90 and CD45 of the P1-P3 cells were observeed via FCM ; the P3 cells were induced to differentiated into chondrocytes. Results FCM results showed that the positive rate of CD90 on P1, P2, F3 was (80.86 ± 7.85)%,(95.86± 3.28)%,(97.15± 1.43)%, while the positive rate of CD45 on P1, P2, P3 cells was (2.53 ±0.28)% and (0.97 ± 0.48)% and (0.05 ± 0.01)% respectively; P3 cells had differenciated into chondrocytes. Conclusion FCM presorting and Multiplex collagenase detachment procedures can swiftly acquire highly purified human umbilical cord mesenchymal stem cells which have the ability to differentiate into chondroeytes.     

人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究

Objective To explore the efficient way to purify and to enrich umbilical cord mesenchymal stem cells which have abilities to differentiate into chondrocytes. Methods Umbilical cord was acquired from new born infants and chopped into 1 mm2 cubics, then they were pretreated with colleganase and trypsin so as to get the mixed cell suspension. CD45(-),CD90(+) cells were acquired from the mixed cell suspension via FCM sorting and were cultured in the medium as umbilical cord mesenchymal stem PO cells. The PO cells were detached and passaged by multiplex collagenase NB4 and the positive rate of CD90 and CD45 of the P1-P3 cells were observeed via FCM ; the P3 cells were induced to differentiated into chondrocytes. Results FCM results showed that the positive rate of CD90 on P1, P2, F3 was (80.86 ± 7.85)%,(95.86± 3.28)%,(97.15± 1.43)%, while the positive rate of CD45 on P1, P2, P3 cells was (2.53 ±0.28)% and (0.97 ± 0.48)% and (0.05 ± 0.01)% respectively; P3 cells had differenciated into chondrocytes. Conclusion FCM presorting and Multiplex collagenase detachment procedures can swiftly acquire highly purified human umbilical cord mesenchymal stem cells which have the ability to differentiate into chondroeytes.     

人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究

Objective To explore the efficient way to purify and to enrich umbilical cord mesenchymal stem cells which have abilities to differentiate into chondrocytes. Methods Umbilical cord was acquired from new born infants and chopped into 1 mm2 cubics, then they were pretreated with colleganase and trypsin so as to get the mixed cell suspension. CD45(-),CD90(+) cells were acquired from the mixed cell suspension via FCM sorting and were cultured in the medium as umbilical cord mesenchymal stem PO cells. The PO cells were detached and passaged by multiplex collagenase NB4 and the positive rate of CD90 and CD45 of the P1-P3 cells were observeed via FCM ; the P3 cells were induced to differentiated into chondrocytes. Results FCM results showed that the positive rate of CD90 on P1, P2, F3 was (80.86 ± 7.85)%,(95.86± 3.28)%,(97.15± 1.43)%, while the positive rate of CD45 on P1, P2, P3 cells was (2.53 ±0.28)% and (0.97 ± 0.48)% and (0.05 ± 0.01)% respectively; P3 cells had differenciated into chondrocytes. Conclusion FCM presorting and Multiplex collagenase detachment procedures can swiftly acquire highly purified human umbilical cord mesenchymal stem cells which have the ability to differentiate into chondroeytes.     

人脐血来源内皮祖细胞的纯化鉴定及定向分化的研究

目的 检测CD133^＋血管内皮祖细胞（EPC）的细胞表面标志物，鉴定其生物学特性。方法 采用免疫磁珠法分离人脐血EPC，用EGM-2MV培养基[含表皮生长因子（EGF）、血管内皮生长因子（VEGF）、成纤维细胞生长因子（FGF）2等具有诱导分化作用的细胞因子]进行体外培养。应用流式细胞术、免疫组织化学等方法检测CD133^＋细胞的比例、原代EPC的生长曲线及生长特性。透射电镜查找Weibel-Palade小体。同时设计EPC裸鼠成瘤实验，观察瘤体生长及血管增生情况，以鉴定EPC的生物学特性。结果 CD133^＋细胞在免疫磁珠分离前后的比例各为0．91％及85．52％。EPC原代培养时细胞形态正常、密度均匀，前3d为相对抑制期，此后快速增殖，10d后达80％～90％融合。EPC生长曲线显示，接种后5d内细胞数量无明显变化，从第7天开始明显增加，第17天达高峰。光学显微镜下可见，原代EPC贴壁后呈梭形，接种后7d数量明显增加，呈克隆状生长。透射电镜观察到，细胞膜伸出许多伪足丝，基底膜呈多层；细胞质内可见一种短棒状小体，含平行管状的内部结构，即Weibel-Palade小体，确定其为EPC。裸鼠成瘤实验可见EPC组肿瘤瘕体及血管数量大于或多于对照组；抗人血管性假血友病因子（vWF）免疫荧光染色显示，大量EPC参与肿瘤血管生成。结论本实验分离培养的CD133^＋细胞具有分化为成熟血管内皮细胞的能力，即为EPC。裸鼠成瘤实验初步证明EPC参与血管重建，具有促进血管新生、加速缺血组织血管化的作用。     

Transplantation of human CD34+ stem cells from umbilical cord blood to rats with thioacetamide-induced liver cirrhosis

BACKGROUND: Liver fibrosis results from accumulation of extracellular matrix components and is associated with many chronic hepatic diseases. There is to date no specific therapy for this disease, and patients receive treatment for its associated complications. Specific progenitor cells, known as oval cells, are present in the liver. As oval cells express markers such as CD34, they are thought to arise from a hematopoietic precursor. The aim of this work was to investigate whether transplantation of hematopoietic CD34(+) stem cells could improve hepatic fibrosis by their differentiation into hepatocytes. METHODS: CD34(+) stem cells from human umbilical cord blood were purified, transduced with a lentiviral vector containing the green fluorescent protein (GFP) gene and injected via portal vein into rats with liver cirrhosis induced by the 4-month administration of thioacetamide. Rats were killed 15 and 60 days post-transplantation. RESULTS: Up to 37% and 22% fluorescent cells were observed in the blood of control and cirrhotic rats, respectively, at 15 days post-transplantation. At 60 days post-transplantation, however, fluorescent cells were completely absent from the blood. Fluorescence was not detected in liver sections at either 15 or 60 days post-transplantation. Polymerase chain-reaction study to detect the GFP gene ruled out silencing of the transgene. CONCLUSIONS: These results suggest that the transplanted cells did not engraft in the liver and were eliminated from the rats.     

Immunosuppression and xenotransplantation of cells for cardiac repair

The death of highly vulnerable cardiomyocytes during ischemia leads to cardiac dysfunction, including heart failure. Due to limited proliferation of adult mammalian cardiomyocytes, the dead myocardium is replaced by noncontractile fibrotic tissue. Introducing exogenous cells to participate in the regeneration of infarcted myocardium has thus been proposed as a novel therapeutic approach. In view of the availability of various xenogeneic cells and fewer ethical and political concerns that surround human embryonic stem cells and fetal cardiomyocytes, cellular xenotransplantation may be a potential alternative approach for cardiac repair in humans. However, one of the most daunting challenges of xenotransplantation is immunorejection. This article summarizes the progress in cellular xenotransplantation for cardiac repair in experimental settings and the current understanding of possible immune responses following the engraftment of xenogeneic cells. The public attitude towards xenotransplantation is reportedly more favorable to receiving cells or tissues than a whole organ, but many scientific obstacles need to be overcome before the utilization of xenogeneic cells for cardiac repair in patients with heart disease becomes applicable to clinical practice.     

探讨CD34+CD44+细胞输入量对无关脐血移植后造血恢复的影响

目的　研究用无关脐血移植治疗儿童急性白血病时 ,CD3 4 CD44 细胞输入量对中性粒细胞和血小板恢复时间的影响。方法　用流式细胞术分析复苏后的CD3 4 CD44 细胞数 ,并对 18例儿童急性白血病患者在无关脐血移植后的中性粒细胞和血小板恢复时间等进行测定。结果　 18例患者中 ,CD3 4 CD44 细胞输入量为 (5.90～ 2 97.0 2 )× 10 4 /kg(中位数 54.11× 10 4 /kg)。移植后中性粒细胞 >0 .5× 10 9/L的时间为 11～ 2 3d(中位数为 17.5d) ,血小板 >2 0× 10 9/L的时间为 12～ 118d(中位数 41d)。中性粒细胞恢复时间与CD3 4 CD44 细胞输入量存在相关性 ,γ值为 -0 .657,P <0 .0 1;血小板恢复时间与CD3 4 CD44 细胞输入量也有相关性 ,γ值为 -0 .490 ,P <0 .0 5。结论　CD3 4 CD44 细胞输入量与无关脐血移植后的造血重建有关     

犬骨髓CD34＋细胞种植人工血管置换后短期内皮化的实验研究

目的：探讨骨髓CD34＋细胞种植于常用膨体聚四氟乙烯（expanded polytera fluoroethylene，ePTFE）人工血管和涤纶人工血管后二者的内皮化程度。方法：选杂种犬8条，依支架覆膜不同分为ePTFE组和涤纶组，每组实验犬2条，对照犬2条。实验犬采自体骨髓，提取CD34＋细胞种植覆膜支架，对照犬采用单纯自体血预凝覆膜支架。将人工血管植入犬的下腔静脉和腹主动脉。在术后第10、30天观察植入的人工血管通畅情况，采用免疫组织化学方法鉴定内膜细胞来源，在光镜和电镜下观察人工血管新生内膜表面内皮化情况。结果：术后第10天实验组与对照组差异无统计学意义；术后第30天腔面新生内膜内皮细胞自人工血管吻合口向中间逐渐减少（P〈0．05）；而对照组内膜表面第10、30天均无内皮细胞覆盖。结论：经纯化的CD34＋细胞种植于ePTFE和涤纶人工血管，均获得理想的内皮化。     

Xenoreactivity and engraftment of human mesenchymal stem cells transplanted into infarcted rat myocardium

OBJECTIVE: It is thought that adult human mesenchymal stem cells do not induce immunoreactivity even to xenografts. We wanted to study whether adult human mesenchymal stem cells survive and engraft in experimentally induced ischemic rat myocardium. METHODS: Bone marrow-derived adult human mesenchymal stem cells (2.5 x 10(6)) were injected into the myocardium of 4 Sprague-Dawley rats. One week after injection, peripheral blood rat lymphocytes were added to adult human mesenchymal stem cells in a mixed lymphocyte reaction. Furthermore, an infarction was created by left anterior descending artery ligation of 8 Sprague-Dawley rats, 4 of which were immunosuppressed with tacrolimus (0.1 mg/kg/d) and 4 RNU athymic rats. One week after left anterior descending artery ligation, 2.5 to 3.5 x 10(6) adult human mesenchymal stem cells were injected around the infarcted area. The adult human mesenchymal stem cells were identified with fluorescence in situ hybridization technique and myocardial antigens by immunohistochemistry. The immune response was studied by hematoxylin and eosin staining and by antibodies directed toward macrophages. RESULTS: Significant rat lymphocyte proliferation was observed when adult human mesenchymal stem cells were added to peripheral blood from Sprague-Dawley rats previously exposed to adult human mesenchymal stem cells. No reactivity was seen in lymphocytes from untreated Sprague-Dawley rats and athymic rats. Adult human mesenchymal stem cells could only be identified in the myocardium of athymic rats. Further, in normal Sprague-Dawley rats, there was a significant myocardial infiltration of round cells, mostly macrophages, in the area of injection of adult human mesenchymal stem cells. In RNU rats, this reaction was less intense. CONCLUSION: Adult human mesenchymal stem cells did not induce xenoreactivity in vitro in previously unexposed immunocompetent Sprague-Dawley rats. However, although mesenchymal stem cells are transplantable across allogeneic barriers, transplant rejection can occur in a xenogenic model. When transplanted into an immunoincompetent host, adult human mesenchymal stem cells showed persistent engraftment.     

胚胎干细胞在转化生长因子-β1诱导下表达vWF、CD34的研究

目的　探讨胚胎干 (ES)细胞在转化生长因子 β1(TGF β1)诱导条件下的分化表现 ,为组织工程种子细胞来源提供诱导方法。方法　小鼠ES细胞 ,以 1× 10 - 9mol L维甲酸 (RA)和 3μg LTGF β1进行诱导分化。应用逆转录 聚合酶链反应 (RT PCR)及免疫组织化学验证细胞性质。结果　证实ES细胞经RA和TGF β1的诱导 ,3～ 4d出现圆形前体细胞 ,6d形成管状血管样结构。分化细胞可表达内皮细胞的标志vWF、CD34。结论　ES细胞在特定的条件下可分化为血管内皮细胞 ,有可能为构建组织工程化血管及其他人工血管的内皮化提供种子细胞来源。