【摘要】：本文假設(shè)成體骨髓中存在一類處于非粘附狀態(tài)的、懸浮的、與造血細胞類似的多潛能骨髓基質(zhì)干細胞,它們雖具有貼壁潛能,但比貼壁的基質(zhì)干細胞更原始,稱為骨髓來源的非粘附基質(zhì)干細胞(non-adherent bone marrow-derived stromal cell,NA-BM-SC)。這類細胞能夠經(jīng)血液循環(huán)轉(zhuǎn)運到全身各器官,在特定的微環(huán)境下貼壁,增殖并分化為各器官的終末分化細胞,參與正常組織的再生及損傷組織的修復(fù)。 為了驗證這一假設(shè),從以下三個部分進行實驗研究: 第一部分采用反復(fù)轉(zhuǎn)移非貼壁骨髓細胞(non-adherent bone marrow cells,NA-BMC)體外培養(yǎng)法,取6周齡小鼠股骨和脛骨來源的骨髓總細胞(bone marrow cells,BMC)培養(yǎng)10-18天,每天轉(zhuǎn)移一次非貼壁骨髓細胞至新的培養(yǎng)皿,即“pour off”,至少4次。對一天后貼壁的細胞加入新鮮培養(yǎng)液繼續(xù)培養(yǎng)。實驗結(jié)果證實了不僅總BMC可以產(chǎn)生成纖維細胞克隆形成單位(Fibroblastoid Colony forming unit,CFU-F),非貼壁骨髓細胞也能產(chǎn)生CFU-F;地塞米松可以抑制CFU-F的形成效率,而1.25(OH)_2D_3則可以部分拮抗地塞米松的抑制作用。為了確定NA-BMC來源的基質(zhì)干細胞(NA-BM-SC)是否具有自我更新能力和多分化潛能,采用5-溴脫氧尿核苷(Bromodeoxyuridine,BrdU)摻入法、免疫細胞化學(xué)法和流式細胞儀法證實了NA-BM-SC不但具有自我更新能力,而且在體外不同條件下培養(yǎng),能分化為成骨細胞、軟骨細胞、脂肪細胞和神經(jīng)膠質(zhì)細胞。 第二部分采用維生素D受體(vitamin D receptor,VDR)基因敲除小鼠,經(jīng)致死劑量照射后,移植野生型小鼠的NA-BM-SC,通過VDR基因的示蹤作用,觀察NA-BM-SC是否具有造血重建和參與輻射損傷修復(fù)的作用。結(jié)果顯示,單純照射組的小鼠由于造血系統(tǒng)損傷、腸組織和多臟器壞死等原因,兩周內(nèi)全部死亡;然而經(jīng)過NA-BM-SC移植治療的照射移植組小鼠,在移植后30、60和90天的存活率分別達到75%、65%和60%。外周血細胞在照射移植后30天基本恢復(fù)到正常水平。通過PCR和免疫組織化學(xué)法檢測,VDR基因在照射移植組存活小鼠的多個器官表達,包括骨髓、脾臟和小腸。結(jié)果提示NA-BM-SC不僅可以重建小鼠的造血系統(tǒng),而且可
[Abstract]:In this paper, we hypothesize that there is a class of non-adherent, suspended, hematopoietic-like multipotent bone marrow stromal cells (NA-BM-SC) in adult bone marrow. Although they have adherent potential, they are more primitive than adherent stromal cells. These cells can be transported through blood circulation to all organs of the body, adhere to the wall in a specific microenvironment, proliferate and differentiate into terminal differentiation cells of various organs, and participate in the regeneration of normal tissues and repair of damaged tissues.
In order to test this hypothesis, the following three parts are studied:
In the first part, non-adherent bone marrow cells (NA-BMC) from femur and tibia of 6-week-old mice were cultured in vitro for 10-18 days. Non-adherent bone marrow cells were transferred to a new culture dish, i.e. pour off, at least four times a day. Adherent cells were further cultured in fresh medium. The results showed that not only total BMC could produce fibroblast cloning unit (CFU-F), but also non-adherent bone marrow cells could produce CFU-F. Dexamethasone could inhibit the formation of CFU-F and 1.25 (OH) _2D_3 could partially antagonize dexamethasone. To determine whether NA-BM-SC has the ability of self-renewal and multi-differentiation, 5-bromodeoxyuridine (BrdU) incorporation, immunocytochemistry and flow cytometry confirmed that NA-BM-SC not only has the ability of self-renewal, but also under different conditions in vitro. Under culture, they can differentiate into osteoblasts, chondrocytes, adipocytes and glial cells.
In the second part, we used vitamin D receptor (VDR) knockout mice. After lethal dose irradiation, NA-BM-SC was transplanted into wild type mice to observe whether NA-BM-SC could reconstruct hematopoiesis and participate in the repair of radiation injury. However, the survival rates of the irradiated mice treated with NA-BM-SC were 75%, 65% and 60% at 30, 60 and 90 days after transplantation, respectively. Peripheral blood cells returned to normal level at 30 days after transplantation. VDR gene was detected by PCR and immunohistochemistry. The expression of NA-BM-SC in multiple organs including bone marrow, spleen and small intestine of surviving mice in irradiated transplantation group suggested that NA-BM-SC could not only reconstruct the hematopoietic system of mice, but also reconstruct the hematopoietic system.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R329

【引證文獻】

相關(guān)期刊論文 前1條

1 萬建美;羅蔚鋒;李厚懷;包仕堯;劉春風(fēng);張志琳;李冰燕;;激光掃描共聚焦顯微鏡在骨髓基質(zhì)細胞移植中的應(yīng)用[J];激光雜志;2010年05期

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本文編號：2190500