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人胎儿来源骨髓和肝脏MSCs的生物学特性及其向胰岛β样细胞分化的研究

发布时间:2018-05-05 14:12

  本文选题: + 胎儿 ; 参考:《暨南大学》2008年博士论文


【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。
[Abstract]:objective
The basic biological characteristics of human fetal bone marrow mesenchymal stem cells (mesenchymal stem cells derived from human fetalbone marrow, hfBM-MSCs) and liver mesenchymal stem cells (mesenchymal stem cells) were studied and differentiated into islet beta like cells in vitro.
Method
Fetuses 12-20 weeks aborted fetus. Rinse the bone marrow cavity of the long bone to get the whole bone marrow cell suspension; cut, blow the fetal liver tissue to obtain the liver cell suspension, collect the liver non parenchymal cells by two steps, and then precipitate the red blood cells with hydroxyethyl starch (hydroxyethylstarch, HES), so that the nucleated cells are enriched. Characteristic purification of cell differential adherent growth MSCs.
Cell cycle and surface markers of hfBM-MSCs and hfL-MSCs were detected by flow cytometry; RT-PCR and immunofluorescent cytochemical staining were used to detect the expression of specific markers of AKP, hTERT, SSEA-4 and Oct 4 in hfBM-MSCs and hfL-MSCs; hfBM-MSCs and hfL-MSCs to neurons, muscle cells (adipocytes) were induced by classical methods. Osteoblasts and hepatocytes were divided into three outer, middle, and internal cells, and the P8 generation and the hfBM-MSCs and hfL-MSCs of the resuscitation after half a year were analyzed; hfBM-MSCs and hfL-MSCs were injected under the dorsal and renal membrane of nude mice to observe the formation of tumor; and hfBM-MSCs and hfL-MSCs were co cultured with K562 cells to observe the tumor. The effect of cell growth.
Various schemes were used to induce the differentiation of hfBM-MSCs and hfL-MSCs to islet beta cells. According to the morphological changes of the cells before and after induction, the expression of islet related genes and islet specific proteins (RT-PCR and immunocytochemical staining), the optimal scheme was optimized. The optimized scheme induced islet like cell mass (islet-like clusters, ILCs) was optimized. The surface and the internal ultrastructure were observed by transmission and scanning electron microscopy. Dithizone staining was used to identify whether ILCs contained zinc ions; chemiluminescence assay was used to detect the insulin secretion and intracellular insulin content of ILCs; the insulin release test was used to evaluate the function of ILCs; Western blot was used to identify the properties of ILCs secretions (insulin, In the end, ILCs was transplanted to the left kidney of diabetic mice to detect the changes of blood sugar every 2-3D; to the mice that had been reduced to normal blood sugar, the left kidney was removed to observe whether the blood sugar rebounded. Finally, the kidney and pancreas of the allograft mice were detected by immunohistochemistry.
Result
MSCs was purified from the bone marrow and liver of human fetus, and more than 90% of the P3 generation hfBM-MSCs and hfL-MSCs were in G0/G1 stage, expressed CD29, CD44 and CD105, and did not express CD15, CD34, CD45, and graft versus host disease. 4, SSEA-4 and hTERT and other embryonic stem cell specific markers, immunocytochemistry showed that the specific protein of AKP, hTERT, SSEA-4 and Oct 4 was positive; hfBM-MSCs and hfL-MSCs could be differentiated into neurons, adipocytes, osteoblasts, myoblasts and liver like cells, and hfBM-MSCs and htL under a variety of conventional induction conditions. -MSCs remained normal karyotype after multiple passages (P8 generation) and frozen storage for half a year. No tumor formation was observed under the subcutaneous and renal capsule of nude mice by hfBM-MSCs and hfL-MSCs; the growth of tumor cells could be inhibited by co culture of hfBM-MSCs and hfL-MSCs with K562 cells.
In the differentiation of islet beta cells, hfBM-MSCs and hfL-MSCs were spindle shaped parietal cells before induction. After optimal induction, the cells quickly became round or elliptical and gathered to form more and more ILCs (hundreds of ILCs) were found in the cell growth surface of 25cm~2 culture bottles. RT-PCR results showed that ILCs expressed islet related genes, such as pancreas twelve. -1 (pancreatic duodenal homeobox-1, Pdx-1), neurogenic hormone (neurogenin 3, Ngn3), islet 1 (Islet1, ISL 1), insulin (insulin), glucagon (glucagon), glucose transporter 2 (glucose), etc.); immunofluorescence staining results showed that the islet specific proteins were strongly expressed, such as Insulin (Insulin), C- peptide (C-peptide) and glucagon (Glucagon), ILCs was dyed by Dithizone and showed scarlet positive reaction; scanning electron microscopy showed that there was no vesicular protuberance on the surface of MSCs, and the surface of ILCs had a vesicular structure with different sizes, and the cell surface of ILCs could be observed to have short microvilli on the surface of ILCs. There were many different sizes and different secretory granules in the cells. The chemiluminescence immunoassay was used to measure the secretion of immunoreactive insulin (IRI) in the supernatant of ILCs culture. The ILCs induced by hfBM-MSCs and hfL-MSCs was (210 + 35.07) mu U/mL and (106.7 + 28.21) micron respectively, and the insulin release experiment showed ILC. S has a certain sugar reactivity, and the stimulation index of ILCs induced by hfBM-MSCs and hfL-MSCs is 4.38 + 0.32 and 4.22 + 0.27, respectively. Western blot confirms that the ILCs protein extract is mostly proinsulin, suggesting that the induced ILCs in vitro is not mature enough.
ILCs was transplanted into the diabetic mice. The blood sugar of the mice decreased gradually under the renal capsule, while the non transplanted diabetic mice continued to be hyperglycemic; the blood sugar dropped to the normal mouse left kidney, the blood sugar rebounded and the mice died quickly; the immuno histochemical staining of the transplanted kidney in the rat showed the insulin positive cells. Pancreatic islets atrophy and decrease, and insulin positive cells are rare.
conclusion
HfBM-MSCs and hfL-MSCs have the characteristics of embryonic stem cells, which can differentiate into three germ cells in vitro, and have weak immunogenicity and no tumorigenicity. It is an ideal seed cell for tissue engineering and cell therapy. HfBM-MSCs and hfL-MSCs are easy to differentiate into islet beta cells in vitro, but they are not mature enough to transplant diabetic mice. It is further developed to play the role of regulating blood sugar.

【学位授予单位】:暨南大学
【学位级别】:博士
【学位授予年份】:2008
【分类号】:R587.1;R329

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