本文關鍵詞：人AB型血清培養(yǎng)體系體外培養(yǎng)擴增人臍血源基質細胞的實驗研究　出處：《第三軍醫(yī)大學》2009年碩士論文　論文類型：學位論文

  更多相關文章： 造血微環(huán)境 人臍血源基質細胞 人AB型血清 體外培養(yǎng) 細胞擴增

【摘要】： 造血微環(huán)境(Hematopoietic inductive microenvironment,HIM)是造血干細胞產生、增殖和分化發(fā)育的場所,基質細胞是造血微環(huán)境的重要組成成分,不僅與造血干細胞的自我更新、增殖、分化和歸巢現(xiàn)象關系密切,而且對血液系統(tǒng)疾病的發(fā)生、進展和預后產生重要影響。人臍血源基質細胞(human stromal cells derived from umbilical cord blood, hUCBDSCs)可促進骨髓造血微環(huán)境損傷的修復,具有取材方便、細胞增殖活性高和免疫原性弱等優(yōu)點。已有研究表明,從人臍血標本中分離的基質細胞通過直接接觸促進造血干細胞自我更新、增殖和分化的作用.基質細胞可以分泌多種促進造血的細胞因子如TPO、GM-CSF和SCF等,可增強造血干細胞的骨髓遷移和發(fā)育,并可以分泌細胞外基質成分(extracellular matrix ,ECM)參與造血微環(huán)境的組成。然而,臍帶血來源的基質細胞存在數(shù)量有限,且其體外擴增培養(yǎng)必須嚴格依賴于特定的胎牛血清等缺點,這阻礙了人臍血源基質細胞的臨床應用。理想的體外培養(yǎng)體系應避免使用動物血清成分帶來臨床應用上的風險。本研究嘗試以人AB型血清培養(yǎng)體系體外培養(yǎng)人臍血基質細胞,獲得了較好的效果,建立了一種穩(wěn)定的體外培養(yǎng)擴增人臍血基質細胞方法,為人臍血基質細胞的臨床應用奠定基礎。主要的實驗結果如下: 1.人臍血基質細胞生長狀態(tài)的動態(tài)觀察:在培養(yǎng)48～96 h后,可見少數(shù)細胞已貼壁。貼壁細胞呈不規(guī)則形并可見胞漿突起形成,大約1周后貼壁細胞開始出現(xiàn)明顯的分裂增殖,并可見CFU-F集落形成,繼續(xù)培養(yǎng)至CFU-F集落融合形成單層細胞。經HE染色觀察,人臍血基質細胞呈梭形、圓形或不規(guī)則形,核為圓形或橢圓形,CFU-F集落內細胞排列呈放射狀和渦流狀。集落間細胞相互連接、交錯成片。 2.15%人AB型血清和bFGF細胞因子濃度對人臍血基質細胞CFU-F的影響:15%人AB型血清能有效支持臍血基質細胞在體外的擴增培養(yǎng)。在0～10μg/L范圍內bFGF的濃度越高,形成的集落數(shù)也越多,但當其濃度大于10μg/L時,對人臍血基質細胞形成集落數(shù)的刺激作用無明顯增加。 3.臍血基質細胞表面標志的檢測:應用免疫組化染色,檢測了Vimentin、CD34、TdT、CD45和CK等在臍血基質細胞中的表達。生長狀態(tài)良好的第1-2次傳代細胞用于進行免疫組化檢測,結果表明人臍血基質細胞的表型為Vimentin(+)、CD34(+)、TdT(-)、CD45(-)和CK(-)。 主要結論:成功建立一種人AB型血清培養(yǎng)體系體外培養(yǎng)擴增人臍血源基質細胞的方法,為下一步的治療性研究,如體內促進放、化療后骨髓造血功能損傷的修復和臨床臍血基質細胞治療技術的開展,提供了前期的條件準備。
[Abstract]:The hematopoietic microenvironment (Hematopoietic inductive microenvironment, HIM) is a hematopoietic stem cell, proliferation and differentiation and development of the place, stromal cells are important components of hematopoietic microenvironment, and hematopoietic stem cell self-renewal, proliferation, differentiation and homing related phenomenon, and on the blood system diseases have an important impact on progress and prognosis. Human umbilical cord blood stromal cells (human stromal cells derived from umbilical cord blood, hUCBDSCs) can promote the repair of bone marrow hematopoietic micro environment damage, it is a convenient, high cell proliferation activity and weak immunogenicity and other advantages. Studies have shown that stromal cells isolated from human umbilical cord blood samples by direct contact to promote hematopoietic stem cell self-renewal, proliferation and differentiation. Stromal cells can secrete a variety of cells such as TPO promote hematopoietic factor, GM-CSF and SCF, can be increased Strong bone marrow hematopoietic stem cell migration and development, and can secrete extracellular matrix components (extracellular, matrix, ECM) components involved in hematopoietic microenvironment. However, stromal cells derived from umbilical cord blood has a limited number, and the in vitro culture must be strictly dependent on fetal bovine serum specific shortcomings, which hinders the clinical application human umbilical cord blood stromal cells cultured in vitro. The ideal system should avoid the use of animal serum components risks in clinical practice. This study attempts to use human AB type serum cultured human umbilical cord blood stromal cell system in vitro, to obtain good results, establish a stable in vitro culture method of human umbilical cord blood stromal cells, which the basis for the clinical application of human umbilical cord blood stromal cells. The main results are as follows:
Dynamic observation of umbilical cord blood stromal cell growth state in 1.: after 48~96 h culture, showing a few cells adherent. Adherent cells were irregular in shape and the formation of cytoplasmic protrusions, marked the beginning of the proliferation of adherent cells after about 1 weeks, and visible CFU-F colony formation, continue to develop CFU-F colony formation of monolayer cells. Fusion by HE staining, human umbilical cord blood stromal cells were spindle shaped, round or irregular shape, nucleus was round or oval, CFU-F colony cells arranged radially and swirling. Inter colony cells are connected to each other and goes into the film.
Effect of serum bFGF and cytokine concentrations of 2.15% AB of human umbilical cord blood stromal cell CFU-F: 15% human AB type serum can effectively support umbilical cord blood stromal cells in vitro culture. In 0 ~ 10 g/L concentration range of bFGF is high, the colony counts more, but when the concentration is greater than 10 g/L, on human umbilical cord blood stromal cell colony formation stimulation number were not significantly increased.
3. detection of umbilical cord blood stromal cell surface markers: immunohistochemical staining, detection of Vimentin, CD34, TdT, CD45 and CK in umbilical cord blood stromal cells. The expression of 1-2 cell growth in good condition for immunohistochemical detection results showed that the phenotype of human umbilical cord blood stromal cells (Vimentin +), CD34 (+), TdT (-), CD45 (-) and CK (-).
The main conclusion: we have successfully established a human AB type serum culture system in vitro amplification of human umbilical cord blood stromal cells, on the treatment of the next step, such as promoting the body, repair of bone marrow hematopoietic function injury and clinical treatment of umbilical cord blood stromal cells after chemotherapy development, provide the conditions of preparation.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

【參考文獻】

相關期刊論文 前10條

1 馮樂平;梁瑜禎;喬偉;;臍帶血來源基質細胞培養(yǎng)及造血微環(huán)境的建立[J];吉林大學學報(醫(yī)學版);2008年03期

2 張曦,王蘋,陳幸華,劉林,彭賢貴,孔佩艷,劉紅;TNF-α對人臍血基質細胞體外擴增作用的觀察[J];重慶醫(yī)學;2003年10期

3 陳幸華;;造血功能障礙的修復與重建[J];重慶醫(yī)學;2007年17期

4 張曦;陳幸華;司英健;劉耀;高蕾;高力;張誠;彭賢貴;王慶余;;人臍血源基質細胞移植促進裸鼠造血損傷修復的實驗研究[J];重慶醫(yī)學;2007年17期

5 羅成基,陳幸華,鄒仲敏,郭朝華,周燕虹;造血基質細胞在造血重建中的作用[J];基礎醫(yī)學與臨床;2000年06期

6 羅成基;造血微環(huán)境基質細胞的研究概況及前景[J];解放軍醫(yī)學雜志;1999年01期

7 高蕾,陳幸華,張曦,彭賢貴,劉耀,孔佩艷,劉林,劉紅,張怡;人臍血基質細胞對臍血CD34~+細胞體外擴增的作用[J];醫(yī)學研究生學報;2005年06期

8 趙杰,李紅燕,郝紅櫻,姚程,易永林;人骨髓基質細胞體外擴增方法的研究[J];武警醫(yī)學;1999年03期

9 蔡瓊霞;熊華鋒;胡葵葵;張繼平;李潔;李蜀光;賀文風;;臍血間充質干細胞分離培養(yǎng)的理論研究與應用[J];中國組織工程研究與臨床康復;2007年28期

10 張曦,王蘋,陳幸華,劉林,彭賢貴,孔佩艷,劉紅,張怡,王慶余;人臍血貼壁細胞生物學特點的觀察[J];中國實驗血液學雜志;2005年01期

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