【作者】 王跃春；

【导师】 张洹；

【作者基本信息】 暨南大学 ， 内科血液学， 2008， 博士

【摘要】 目的研究人胎儿来源骨髓间充质干细胞(mesenchymal stem cells derived from human fetalbone marrow,hfBM-MSCs)及肝脏间充质干细胞(mesenchymal stem cells derived fromhuman fetal liver,hfL-MSCs)的基本生物学特性;在体外诱导hfBM-MSCs及hfL-MSCs向胰岛β样细胞分化。方法取材12-20周流产胎儿。冲洗四肢长骨骨髓腔得到全骨髓细胞悬液;剪切、吹打胎肝组织获取肝细胞悬液,通过二步离心收集肝非实质细胞,再用羟乙基淀粉(hydroxyethylstarch,HES)沉淀红细胞,从而使有核细胞得到富集。接种、培养上述细胞,利用细胞差速贴壁生长的特性纯化MSCs。流式细胞仪检测hfBM-MSCs和hfL-MSCs的细胞周期和表面标志;RT-PCR及免疫(荧光)细胞化学染色检测AKP,hTERT,SSEA-4和Oct 4等胚胎干细胞特异性标志的在hfBM-MSCs和hfL-MSCs中的表达情况;用经典方法诱导hfBM-MSCs和hfL-MSCs向神经元、肌细胞(脂肪细胞、成骨细胞)和肝细胞所代表的外、中、内三个胚层细胞分化;对P8代及冻存半年后复苏的hfBM-MSCs和hfL-MSCs进行染色体分析;注射hfBM-MSCs和hfL-MSCs到裸鼠背部及肾被膜下以观察有无肿瘤形成;将hfBM-MSCs和hfL-MSCs与K562细胞共培养,观察其对肿瘤细胞生长的影响。采用各种方案诱导hfBM-MSCs和hfL-MSCs向胰岛β细胞分化,根据细胞在诱导前后的形态变化、胰岛相关基因及胰岛特异蛋白的表达情况(RT-PCR及免疫细胞化学染色),筛选、优化出最佳方案;对优化方案诱导的胰岛样细胞团(islet-like clusters,ILCs),进一步用透射和扫描电镜观察其表面及内部超微结构;双硫腙染色鉴定ILCs是否含有锌离子;化学发光法检测ILCs的胰岛素分泌量及胞浆内胰岛素含量;进行胰岛素释放实验以评价ILCs的功能;用Western blot鉴定ILCs分泌物的性质(是胰岛素,还是胰岛素原,或者兼而有之);最后,将ILCs移植到糖尿病小鼠左侧肾被膜下,每隔2-3d检测血糖变化;对血糖降至正常的小鼠,摘除其左肾以观察血糖是否反弹,最后取行异源移植小鼠的肾脏和胰腺进行免疫组化检测。结果从人胎儿骨髓及肝脏中分离、纯化得到MSCs;P3代hfBM-MSCs和hfL-MSCs均有90%以上处于G0/G1期,表达CD29、CD44和CD105,不表达CD15、CD34、CD45以及移植物抗宿主病(graft-versus-host disease,GVHD)相关的抗原如HLA-DR、CD40、CD80、CD86等;RT-PCR表明hfBM-MSCs和hfL-MSCs表达Oct 4,SSEA-4和hTERT等胚胎干细胞特异性标志,免疫细胞化学显示AKP,hTERT,SSEA-4和Oct 4等胚胎干细胞特异性蛋白呈阳性表达;在各种常规诱导条件下,hfBM-MSCs和hfL-MSCs可分化为类神经元、成脂细胞、成骨细胞、成肌细胞以及类肝细胞等;hfBM-MSCs和htL-MSCs在多次传代(P8代)及冻存半年后复苏仍保持正常核型;hfBM-MSCs和hfL-MSCs注射到裸鼠皮下及肾被膜下均未观察到肿瘤形成;hfBM-MSCs和hfL-MSCs与K562细胞共培养,可以抑制肿瘤细胞的生长。在向胰岛β细胞分化方面,诱导前hfBM-MSCs和hfL-MSCs呈梭形贴壁细胞,经最佳方案诱导后,细胞快速变成圆形或椭圆形,并聚集形成越来越多的ILCs(在25cm~2培养瓶的细胞生长面可见数百个ILCs);RT-PCR结果显示ILCs表达胰岛相关基因,如胰十二指肠同源异型看家基因盒基因-1(pancreatic duodenal homeobox-1,Pdx-1)、神经源素(neurogenin 3,ngn3)、胰岛1(Islet1,isl 1)、胰岛素(insulin)、胰高血糖素(glucagon)、葡萄糖转运子2(glucose transporter-2,glut2)等;免疫荧光染色结果表明ILCs强表达胰岛特异性蛋白,如胰岛素(Insulin)、C-肽(C-peptide)及胰高血糖素(Glucagon)等;ILCs经双硫腙染色后呈现猩红色阳性反应;扫描电镜显示未诱导MSCs表面无囊泡状突起,而ILCs表面分布有大小不等的囊泡样结构;透射电镜下可观察到ILCs的细胞表面有粗短的微绒毛,细胞内有较多大小不等、深浅不一的分泌颗粒;化学发光免疫分析法测得ILCs的培养液上清中免疫反应性胰岛素(immunoreactive insulin,IRI)大量分泌,在hfBM-MSCs和hfL-MSCs诱导的ILCs分别为(210±35.07)μU/mL和(106.7±28.21)μU/mL;胰岛素释放实验表明ILCs具有一定的糖反应性,刺激指数在hfBM-MSCs和hfL-MSCs诱导的ILCs分别为4.38±0.32和4.22±0.27;Western blot证实ILCs蛋白提取物中多为胰岛素原,提示体外诱导的ILCs不够成熟。移植ILCs到糖尿病小鼠的。肾被膜下,可观察到小鼠的血糖逐渐下降,而非移植组糖尿病小鼠则持续高血糖;摘除血糖降至正常的小鼠左肾,可见血糖出现反弹,小鼠很快死亡;该鼠肾被膜下移植物的免疫组织化学染色显示胰岛素阳性细胞,而其胰腺中胰岛萎缩,数量减少,胰岛素阳性细胞少见。结论hfBM-MSCs及hfL-MSCs具有类胚胎干细胞特性,体外诱导可向三个胚层来源的细胞分化,且免疫原性弱,无致瘤性,是组织工程和细胞治疗较为理想的种子细胞;hfBM-MSCs及hfL-MSCs在体外易于向胰岛β细胞的分化,但不够成熟,需移植糖尿病小鼠体内进一步发育以发挥调节血糖的作用。更多还原

【Abstract】 ObjectivesTo study the basic biological characteristics of the mesenchymal stem cells derived from human fetal bone marrow(hfBM-MSCs) and the mesenchymal stem cells derived from human fetal liver(hfL-MSCs),and to induce hfBM-MSCs and hfL-MSCs to differentiate into islet β-like cells in vitro.MethodsCells were isolated from the aborted fetuses of 12-20 week’s gestation.Rinsing the medullary cavity of the four limb bones to form the whole bone marrow suspension,cutting and pipetting the hepatic tissue to obtain the hepatocyte suspension,then collecting the non-parenchyma mesenchymal stem cell through two-step centrifugation,and enriching the karyota through removing the red blood cells by hydroxyethyl starch sedimentation.The cells in suspension were inoculated and cultured,and MSCs purification was done by adhesive screening method(because different kinds of cells have the different adherence to the wall of culture flask).The cell cycle and surface markers of hfL-MSCs and hfBM-MSCs were identified using flow cytometry.The expression of the embryonic stem cell-specific antigens such as AKP, SSEA-4,hTERT and Oct4 were detected with immunostaining or immunofluorescence staining at protein level,and the expression of Oct 4,SSEA-4 and hTERT were also tested by RT-PCR at RNA level.Inducing hfL-MSCs and hfBM-MSCs by routine inducing protocols to differentiate toward cells derived from three germ layers represented by nerve-like cells,muscle-like cells (adipocytes and osteoblastes) and hepatocytes respectively.Karyotype analysis of hfL-MSCs and hfBM-MSCs were made at passage 8 and after recovery from cryopreservation for 6 months. Grafting hfL-MSCs and hfBM-MSCs to the dorsum and renal subcapsule of nude mice to evaluate their oncogenicity.Culturing hfL-MSCs and hfBM-MSCs with K562 cells to observe the effect of hfL-MSCs and hfBM-MSCs on the growth of K562 cells.hfL-MSCs and hfBM-MSCs were induced to differentiate toward isletβcells under various inductive conditions,and the best protocols were finally selected and optimized basing on the results of morphological change,the expression of isletβcells related genes by RT-PCR and islet-specific proteins by immumofluorescent staining.Then,for the islet-like clusters(ILCs) induced by the optimized protocol,scanning and transmission electron microscope were used to show their exterior and interior ultramicroscopic structures.Dithizone(DTZ) staining was performed to identify the Zinc in ILCs.In addition,the quantity of insulin secretion and intracellular insulin were examined by chemiluminescence immunoassay.The test of glucose-simulated insulin release was made to evaluate the function of the ILCs.Western blot was conducted to identify the nature of secretion(insulin,proinsulin or both of them).Finally, the ILCs were implanted into the left renal subcapsular space of diabetic mice.Blood glucose levels were monitored every 2-3 days after implantation.Excising the left kidney of the xenotransplanted diabetic mice which glucose level had restored to normal to detect whether there is a rebound of blood glucose.Harvesting the pancreas tissue and the left kidney of the xenotransplanted diabetic mice for immunohistochemistry.ResultshfBM-MSCs and hfL-MSCs can be isolated and purified from human fetal bone marrow and liver.There are over 90%hfBM-MSCs and hfL-MSCs of passage 3 in G0/G1 phase. hfBM-MSCs and hfL-MSCs expressed adhesion molecules CD29 and CD44,but not antigens of hematopoietic CD15,CD34,CD45,and not antigens related to GVHD,such as HLA-DR,CD40, CD80 and CD86.hfBM-MSCs and hfL-MSCs expressed the embryonic stem cell-specific markers,such as Oct 4,SSEA-4 and hTERT at RNA level and were positive for Oct 4,SSEA-4, hTERT and AKP at protein level.Exposure of hfBM-MSCs and hfL-MSCs to routine inductive agents resulted in morphological changes towards nerve-like cells,adipocytes,osteoblasts, muscle-like cells and hepatocyte-like cells,and all these kinds of cells were identified by special staining or RT-PCR.hfBM-MSCs and hfL-MSCs showed normal karyotype at passage 8 and after cryopreservation for six months.hfL-MSCs and hfBM-MSCs were not cancerous after being grafted to nude mice.Co-culture of hfL-MSCs and hfBM-MSCs with K562 cells resulted in the growth inhibition of K562 cells.As for the induction of hfBM-MSCs and hfL-MSCs toward isletβcells,the untreated MSCs were spindle-shaped adherent cells,but during the course of differentiation induced by the optimized protocol,they changed quickly into round and oval shape and gathered more and more islet-like clusters(there were hundreds of clusters in the growth surface of a flask of T25). RT-PCR showed the ILCs expressed islet-related genes including Pdx-1,isl-1,ngn3,insulin and glucagon,and immumofluorescence demonstrated the ILCs were strongly positive for Insulin, C-peptide and Glucagon.The ILCs were stained crimson red by dithizone indicating the high zinc content in the cells.Scanning electron microscope showed there no vesicular prominency on the surface of untreated MSCs,but many vesicular structures of different size appeared on the surface of ILCs.Transmission electron microscope displayed the microvilli on the surface of ILCs and numerous secretory granules of variable size and color in the cytoplasm of ILCs. Chemiluminescence immunoassay demonstrated the ILCs were able to secrete a higher level of immunoreactive insulin into medium,the average level are(210±35.07)μU/mL for ILCs induced from hfBM-MSCs and(106.7±28.21)μU/mL for ILCs induced from hfL-MSCs, respectively.The test of glucose-simulated insulin release showed the ILCs could elevate the insulin secretion upon glucose challenge,and the stimulus index are 4.38±0.32 for ILCs induced from hfBM-MSCs and 4.22±0.27 for ILCs induced from hfL-MSCs,respectively.The results of Western blot showed that most of the protein extraction of ILCs was proinsulin suggesting the ILCs not enough mature.After the ILCs were implanted into the left renal subcapsular space of diabetic mice,the blood glucose levels decrease gradually,but control animals that did not receive implants exhibited persistent hyperglycemia.When the left kidneys that contain the implanted cells were removed,the hyperglycemia reappeared and the mice died rapidly.Immunohistochemistry staining showed the implanted cells under the kidney capsule were positive for insulin,and there were a few of atrophy islet in the pancreas of the xenotransplanted diabetic mice.ConclusionsThe hfBM-MSCs and hfL-MSCs possess the embryonic stem cell-like biological characteristics and have the ability to differentiate into the derivative cell types of the three embryonic germ layers,moreover,they have a lower immunogenic property and no tumorigenicity,so provide the ideal source for tissue engineering and cellular therapeutics.The hfBM-MSCs and hfL-MSCs can rapidly and easily be induced to differentiate into isletβ-like cells in vitro,but the induced ILCs are not enough mature.In order to exert the function of regulating the blood glucose level,it is necessary for the ILCs to be implanted into diabetic mice for further development in vivo.更多还原