本文選題：人 + 胎兒��； 參考：《暨南大學》2008年博士論文

【摘要】： 目的 研究人胎兒來源骨髓間充質(zhì)干細胞(mesenchymal stem cells derived from human fetalbone marrow,hfBM-MSCs)及肝臟間充質(zhì)干細胞(mesenchymal stem cells derived fromhuman fetal liver,hfL-MSCs)的基本生物學特性;在體外誘導hfBM-MSCs及hfL-MSCs向胰島β樣細胞分化。 方法 取材12-20周流產(chǎn)胎兒。沖洗四肢長骨骨髓腔得到全骨髓細胞懸液;剪切、吹打胎肝組織獲取肝細胞懸液,通過二步離心收集肝非實質(zhì)細胞,再用羥乙基淀粉(hydroxyethylstarch,HES)沉淀紅細胞,從而使有核細胞得到富集。接種、培養(yǎng)上述細胞,利用細胞差速貼壁生長的特性純化MSCs。 流式細胞儀檢測hfBM-MSCs和hfL-MSCs的細胞周期和表面標志;RT-PCR及免疫(熒光)細胞化學染色檢測AKP,hTERT,SSEA-4和Oct 4等胚胎干細胞特異性標志的在hfBM-MSCs和hfL-MSCs中的表達情況;用經(jīng)典方法誘導hfBM-MSCs和hfL-MSCs向神經(jīng)元、肌細胞(脂肪細胞、成骨細胞)和肝細胞所代表的外、中、內(nèi)三個胚層細胞分化;對P8代及凍存半年后復蘇的hfBM-MSCs和hfL-MSCs進行染色體分析;注射hfBM-MSCs和hfL-MSCs到裸鼠背部及腎被膜下以觀察有無腫瘤形成;將hfBM-MSCs和hfL-MSCs與K562細胞共培養(yǎng),觀察其對腫瘤細胞生長的影響。 采用各種方案誘導hfBM-MSCs和hfL-MSCs向胰島β細胞分化,根據(jù)細胞在誘導前后的形態(tài)變化、胰島相關基因及胰島特異蛋白的表達情況(RT-PCR及免疫細胞化學染色),篩選、優(yōu)化出最佳方案;對優(yōu)化方案誘導的胰島樣細胞團(islet-like clusters,ILCs),進一步用透射和掃描電鏡觀察其表面及內(nèi)部超微結構;雙硫腙染色鑒定ILCs是否含有鋅離子;化學發(fā)光法檢測ILCs的胰島素分泌量及胞漿內(nèi)胰島素含量;進行胰島素釋放實驗以評價ILCs的功能;用Western blot鑒定ILCs分泌物的性質(zhì)(是胰島素,還是胰島素原,或者兼而有之);最后,將ILCs移植到糖尿病小鼠左側腎被膜下,每隔2-3d檢測血糖變化;對血糖降至正常的小鼠,摘除其左腎以觀察血糖是否反彈,最后取行異源移植小鼠的腎臟和胰腺進行免疫組化檢測。 結果 從人胎兒骨髓及肝臟中分離、純化得到MSCs;P3代hfBM-MSCs和hfL-MSCs均有90%以上處于G0/G1期,表達CD29、CD44和CD105,不表達CD15、CD34、CD45以及移植物抗宿主病(graft-versus-host disease,GVHD)相關的抗原如HLA-DR、CD40、CD80、CD86等;RT-PCR表明hfBM-MSCs和hfL-MSCs表達Oct 4,SSEA-4和hTERT等胚胎干細胞特異性標志,免疫細胞化學顯示AKP,hTERT,SSEA-4和Oct 4等胚胎干細胞特異性蛋白呈陽性表達;在各種常規(guī)誘導條件下,hfBM-MSCs和hfL-MSCs可分化為類神經(jīng)元、成脂細胞、成骨細胞、成肌細胞以及類肝細胞等;hfBM-MSCs和htL-MSCs在多次傳代(P8代)及凍存半年后復蘇仍保持正常核型;hfBM-MSCs和hfL-MSCs注射到裸鼠皮下及腎被膜下均未觀察到腫瘤形成;hfBM-MSCs和hfL-MSCs與K562細胞共培養(yǎng),可以抑制腫瘤細胞的生長。 在向胰島β細胞分化方面,誘導前hfBM-MSCs和hfL-MSCs呈梭形貼壁細胞,經(jīng)最佳方案誘導后,細胞快速變成圓形或橢圓形,并聚集形成越來越多的ILCs(在25cm~2培養(yǎng)瓶的細胞生長面可見數(shù)百個ILCs);RT-PCR結果顯示ILCs表達胰島相關基因,如胰十二指腸同源異型看家基因盒基因-1(pancreatic duodenal homeobox-1,Pdx-1)、神經(jīng)源素(neurogenin 3,ngn3)、胰島1(Islet1,isl 1)、胰島素(insulin)、胰高血糖素(glucagon)、葡萄糖轉(zhuǎn)運子2(glucose transporter-2,glut2)等;免疫熒光染色結果表明ILCs強表達胰島特異性蛋白,如胰島素(Insulin)、C-肽(C-peptide)及胰高血糖素(Glucagon)等;ILCs經(jīng)雙硫腙染色后呈現(xiàn)猩紅色陽性反應;掃描電鏡顯示未誘導MSCs表面無囊泡狀突起,而ILCs表面分布有大小不等的囊泡樣結構;透射電鏡下可觀察到ILCs的細胞表面有粗短的微絨毛,細胞內(nèi)有較多大小不等、深淺不一的分泌顆粒;化學發(fā)光免疫分析法測得ILCs的培養(yǎng)液上清中免疫反應性胰島素(immunoreactive insulin,IRI)大量分泌,在hfBM-MSCs和hfL-MSCs誘導的ILCs分別為(210±35.07)μU/mL和(106.7±28.21)μU/mL;胰島素釋放實驗表明ILCs具有一定的糖反應性,刺激指數(shù)在hfBM-MSCs和hfL-MSCs誘導的ILCs分別為4.38±0.32和4.22±0.27;Western blot證實ILCs蛋白提取物中多為胰島素原,提示體外誘導的ILCs不夠成熟。 移植ILCs到糖尿病小鼠的。腎被膜下,可觀察到小鼠的血糖逐漸下降,而非移植組糖尿病小鼠則持續(xù)高血糖;摘除血糖降至正常的小鼠左腎,可見血糖出現(xiàn)反彈,小鼠很快死亡;該鼠腎被膜下移植物的免疫組織化學染色顯示胰島素陽性細胞,而其胰腺中胰島萎縮,數(shù)量減少,胰島素陽性細胞少見。 結論 hfBM-MSCs及hfL-MSCs具有類胚胎干細胞特性,體外誘導可向三個胚層來源的細胞分化,且免疫原性弱,無致瘤性,是組織工程和細胞治療較為理想的種子細胞;hfBM-MSCs及hfL-MSCs在體外易于向胰島β細胞的分化,但不夠成熟,需移植糖尿病小鼠體內(nèi)進一步發(fā)育以發(fā)揮調(diào)節(jié)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.

【學位授予單位】：暨南大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R587.1;R329

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