發(fā)布時間：2018-03-05 20:40

  本文選題：骨髓　切入點：間充質(zhì)干細(xì)胞　出處：《泰山醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 骨髓間充質(zhì)干細(xì)胞(BMSCs)是一種存在骨髓中,來源于中胚層,且具有多向分化潛力的成體干細(xì)胞。在特定的誘導(dǎo)條件下可以向中胚層來源的各種細(xì)胞,如成骨細(xì)胞、脂肪細(xì)胞、肌細(xì)胞分化。近年來隨著對BMSCs的深入研究,發(fā)現(xiàn)BMSCs也具有跨胚層分化的潛能,BMSCs移植入活體的中樞神經(jīng)系統(tǒng)內(nèi)可以存活并可分化為神經(jīng)元細(xì)胞、神經(jīng)膠質(zhì)細(xì)胞,體外實驗已證實在一定的誘導(dǎo)條件下BMSCs向神經(jīng)元細(xì)胞、神經(jīng)膠質(zhì)細(xì)胞轉(zhuǎn)化,可不同程度的改善顱腦損傷、腦卒中等疾病的神經(jīng)功能的缺損狀態(tài)。因此,BMSCs是一種十分理想的移植治療神經(jīng)系統(tǒng)疾病的細(xì)胞,具有十分重要的臨床應(yīng)用價值與前景。本研究目的就是探討通過比較觀察使用①密度梯度離心提取+胎牛血清貼壁培養(yǎng)法②全骨髓胎牛血清貼壁培養(yǎng)法這兩種方法提取、培養(yǎng)SD大鼠的骨髓間充質(zhì)干細(xì)胞的細(xì)胞形態(tài)、生物學(xué)特性、細(xì)胞表型等生長增殖和生物活性各方面有無差異,從而評價兩種方法的優(yōu)勢與劣勢,以期待尋找一種更好的提取、培養(yǎng)骨髓間充質(zhì)干細(xì)胞的方法。 方法 在無菌的條件下用密度梯度離心法與全骨髓分別提取4-6周齡的SD大鼠的左、右下肢的BMSCs,繼而用含有10%胎牛血清的LG-DMEM(低糖DMEM)培養(yǎng)擴增BMSCs。用密度梯度離心提取出的原代BMSCs再經(jīng)胎牛血清貼壁培養(yǎng)法傳代后可得到較純化的可大量擴增的BMSCs細(xì)胞系(得到的細(xì)胞為A組);無菌條件下取全骨髓在胎牛血清中貼壁培養(yǎng)后經(jīng)換液、傳代也可得到大量的BMSCs(得到的細(xì)胞為B組)。流式細(xì)胞儀檢測密度梯度離心法與全骨髓兩種方法提取培養(yǎng)的細(xì)胞的CD29+,CD44-,CD45-的表達(dá),鑒定所培養(yǎng)的BMSCs,繪制BMSCs的生長曲線,并比較兩組BMSCs的細(xì)胞形態(tài)、凍存復(fù)蘇BMSCs后存活率和生長狀態(tài)等。 結(jié)果 1.A組與B組的BMSCs生物性狀皆較穩(wěn)定,初培養(yǎng)時細(xì)胞皆為圓形,傳代穩(wěn)定后細(xì)胞呈BMSCs典型的梭形,并以漩渦狀排列成長;有典型的特征性的貼壁生長; 2.經(jīng)流式細(xì)胞儀檢測A組與B組的BMSCs細(xì)胞表面標(biāo)記物:CD29+CD44-CD45-,結(jié)果A組CD29(+93.24%),CD44(-94.90%),CD45(-94.22%);B組CD29(+92.89%),CD44-(96.70%),CD45-(93.10%),均符合BMSCs的特征; 3.A組與B組BMSCs均貼壁生長,原代臺盼藍(lán)染色檢測細(xì)胞活性A組x為89.19%,貼壁法(B組) x為95.27%,并使用統(tǒng)計學(xué)軟件SPSS16.0進行統(tǒng)計學(xué)分析,差異存在統(tǒng)計學(xué)意義; 4.A組細(xì)胞生長特性:BMSCs貼壁生長,原代培養(yǎng)10天達(dá)到90%融合,傳代后的BMSCs增殖速度較原代(P0)培養(yǎng)增快,第三代(P3)的BMSCs已基本純化,BMSCs呈典型的梭形,旋渦狀排列生長,隨著傳代,細(xì)胞增殖分化能力減弱;B組細(xì)胞不同于A組在于:P0細(xì)胞為全骨髓細(xì)胞,換液,傳代過程是大幅度純化的主要 方法,第四代(P4)細(xì)胞鏡下觀察才可達(dá)到基本純化,但得到的細(xì)胞的活性卻強于A組。 結(jié)論 1.密度梯度離心提取和全骨髓胎牛血清貼壁培養(yǎng)這兩種方法提取、培養(yǎng)的細(xì)胞經(jīng)流式細(xì)胞儀進行表面標(biāo)志物鑒定符合BMSCs的特征;獲得的BMSCs純度較高,在顯微鏡下觀察兩組BMSCs的形態(tài)及生長狀態(tài)相似; 2.密度梯度離心提取間充質(zhì)干細(xì)胞細(xì)胞活力低于全骨髓法(存在統(tǒng)計學(xué)差異),這種差異可能與過程中的多次離心及洗滌對細(xì)胞造成丟失及損傷等原因有關(guān); 3.全骨髓法與密度梯度離心提取法相比操作簡單,花費較小,密度梯度離心提取法適于短時間內(nèi)要求得到純度較高的間充質(zhì)干細(xì)胞細(xì)胞。
[Abstract]:objective
Bone marrow mesenchymal stem cells (BMSCs) is a kind of bone marrow, derived from the mesoderm, and multilineage differentiation potential of adult stem cells. In certain conditions it can induce mesoderm from a variety of cells, such as osteoblasts, adipocytes and muscle cells. In recent years, with in-depth study of BMSCs the BMSCs also has the differentiation potential, BMSCs transplantation into the central nervous system in vivo can survive and can differentiate into neurons and glial cells, in vitro experiments have confirmed that under the condition of certain induction of BMSCs cells into neurons, glial cell transformation, can improve the brain injury in different degree, defect the state of stroke and other neurological diseases. Therefore, BMSCs is a kind of ideal transplantation for the treatment of diseases of the nervous system cells, has clinical application value and prospect is very important. The purpose of this study Is explored through comparative observation using the density gradient centrifugation extraction + fetal bovine serum adherent culture of whole bone marrow adherent culture method for fetal bovine serum of these two kinds of extraction methods, cultivation of SD rat bone marrow mesenchymal stem cells in cell morphology and biological characteristics, there are no differences in cell proliferation and phenotype. The biological activity, so as to evaluate the advantages and disadvantages of the two methods, in order to expect to find a better extraction of cultured bone marrow mesenchymal stem cells.
Method
Under sterile conditions by density gradient centrifugation and whole bone marrow were extracted from SD rats aged 4-6 weeks left and right lower limb BMSCs, then with LG-DMEM containing 10% fetal bovine serum (low glucose DMEM) culturing BMSCs. extracted by density gradient centrifugation after primary BMSCs fetal bovine serum adherent after passage culture method can get more purified BMSCs cells can be greatly amplified (the obtained cells were A group); under sterile conditions from whole bone marrow in fetal bovine serum in adherent culture after the change of liquid passage, can also get a lot of BMSCs (the B cell group) by flow cytometry. The detection method of density gradient centrifugation and two different extraction methods of whole bone marrow cells cultured in CD29+, CD44-, CD45- expression and identification of the cultured BMSCs, draw the growth curve of BMSCs, and compared between the two groups of BMSCs cells, cryopreservation and recovery after BMSCs survival rate and growth state.
Result
The BMSCs biological characteristics of 1.A group and B group were all stable. The cells in initial culture were all round. After passage, the cells were BMSCs typical spindle shape and arranged in a whirlpool pattern.
2., flow cytometry was used to detect the surface markers of BMSCs cells in group A and group B: CD29+CD44-CD45-, results A group CD29 (+93.24%), CD44 (-94.90%), CD45 (-94.22%), and -94.90% group, (96.70%), (93.10%) were all consistent with the characteristics of "Qi".
In group 3.A and group B, BMSCs was adhered to growth. Primary trypan blue staining was used to detect cell viability. A group X was 89.19%, adherence method (B group) x was 95.27%, and statistical software SPSS16.0 was used for statistical analysis, the difference was statistically significant.
The growth characteristics of 4.A group: BMSCs cells adherent growth, cultured for 10 days to reach the 90% fusion, the proliferation rate of BMSCs after passage than the primary (P0) develop faster, third generation (P3) BMSCs has been purified, BMSCs showed typical spindle shape, spiral arrangement growth, along with the passage, weakened the proliferation and differentiation of cells; A group is different from the B group cells: P0 cells for bone marrow cells was changed, the process is the main passage substantially purified
Methods the basic purification was achieved under the fourth generation (P4) cell microscope, but the activity of the obtained cells was stronger than that of the A group.
conclusion
1. density gradient centrifugation extraction and whole bone marrow adherent culture of fetal bovine serum from these two methods, the cultured cells by flow cytometry for surface markers with the characteristics of BMSCs; BMSCs get high purity, observe the morphology and growth state of two groups are similar to that of BMSCs under the microscope;
The cell viability of mesenchymal stem cells extracted by 2. density gradient centrifugation is lower than that of the whole bone marrow method.
3., compared with the density gradient centrifugation, the whole bone marrow extraction method is simpler and less expensive. The density gradient centrifugation method is suitable for obtaining high purity mesenchymal stem cells in a short time.

【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王婧,劉開彥;人骨髓間充質(zhì)干細(xì)胞體外分化為雪旺樣細(xì)胞的方法[J];北京大學(xué)學(xué)報(醫(yī)學(xué)版);2003年02期

2 鐘池,鐘春玖,羅玉敏,汪洋,秦震,沈馨亞;骨髓基質(zhì)細(xì)胞靜脈移植治療大鼠短暫性局灶性腦缺血[J];中華神經(jīng)醫(yī)學(xué)雜志;2004年02期

3 張勇,杜宏偉,鄒仲敏,郭朝華,周進明,王勁,范文輝,羅成基,程天民;人骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)及部分生物學(xué)特性的實驗研究[J];第三軍醫(yī)大學(xué)學(xué)報;2003年04期

4 路艷蒙,傅文玉,樸英杰;人骨髓間充質(zhì)干細(xì)胞的培養(yǎng)及性質(zhì)鑒定[J];第一軍醫(yī)大學(xué)學(xué)報;2001年08期

5 鄭有華;何滔;匡世軍;張志光;蘇凱;;人骨髓間充質(zhì)干細(xì)胞體外分離培養(yǎng)及其生物學(xué)特性[J];國際醫(yī)藥衛(wèi)生導(dǎo)報;2010年02期

6 王運濤;骨髓間充質(zhì)干細(xì)胞分離培養(yǎng)的研究進展[J];國外醫(yī)學(xué).生物醫(yī)學(xué)工程分冊;2002年04期

7 何少健,陳維平,鄺曉聰;成體大鼠的骨髓間充質(zhì)干細(xì)胞分離和體外培養(yǎng)的初步研究[J];廣西醫(yī)科大學(xué)學(xué)報;2002年03期

8 李秀森,郭子寬,楊靖清,劉曉丹,侯春梅,唐佩弦,毛寧;骨髓間充質(zhì)干細(xì)胞的生物學(xué)特性[J];解放軍醫(yī)學(xué)雜志;2000年05期

9 李佩英,路艷蒙,傅文玉,樸英杰;大鼠骨髓間質(zhì)干細(xì)胞的培養(yǎng)及表型特征[J];解剖學(xué)雜志;2002年05期

10 劉曉丹,郭子寬,李秀森,張雙喜,毛寧;人骨髓間充質(zhì)干細(xì)胞分離與培養(yǎng)方法的建立[J];軍事醫(yī)學(xué)科學(xué)院院刊;2000年04期

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