本文選題：骨髓間充質(zhì)干細(xì)胞 + 后腎　； 參考：《第三軍醫(yī)大學(xué)》2009年博士論文

【摘要】： 背景和目的: 慢性腎臟疾病(Chronic kidney disease,CKD)發(fā)病率不斷增加,在成人中的發(fā)生率已達(dá)10%,其主要特征是細(xì)胞外基質(zhì)(Extracellular matrix,ECM)過度沉積和纖維化,引起腎功能進(jìn)行性下降,發(fā)展的最終結(jié)局是終末期腎衰(end stage renal disease,ESRD),目前只能依賴血透、腹透和腎移植來治療。血透、腹透只能部分替代腎臟的濾過功能。腎移植供體短缺、抗排異藥物副作用大,這些治療方法均存在嚴(yán)重缺陷。尋找更完美的新治療方法和途徑是腎臟病工作者迫在眉睫的任務(wù)。 近來研究發(fā)現(xiàn)骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMSCs)具有高度可塑性(plasticity),在體的胚胎組織可誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向腎干細(xì)胞(renal stem cells)或腎臟固有細(xì)胞分化,異種胚胎組織也可誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞向腎干細(xì)胞或腎臟固有細(xì)胞分化,但在胚胎內(nèi)誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向腎干細(xì)胞分化無臨床應(yīng)用前景,因?yàn)殡y以從胚胎組織中分離純化由骨髓間充質(zhì)干細(xì)胞分化而來的腎干細(xì)胞。如果能在體外誘導(dǎo)骨髓間充質(zhì)干細(xì)胞分化為腎干細(xì)胞,則誘導(dǎo)的細(xì)胞可被輕而易舉地分離和純化,具有廣闊的臨床應(yīng)用前景。 大量資料表明,干細(xì)胞的分化發(fā)育及其發(fā)育方向,取決于其所處的微環(huán)境(microenvironment)。在后腎發(fā)育過程中,后腎間充質(zhì)細(xì)胞(metanephric mesenchyme cells,MMCs)即胚腎干細(xì)胞,具有自我更新及多向分化的能力,除了集合管起源于輸尿管芽(ureteric bud,UB)外,后腎間充質(zhì)細(xì)胞分化形成所有的腎單位和基質(zhì)細(xì)胞,在離開胚腎組織仍能發(fā)育成腎固有細(xì)胞,這提示胚腎組織具有干細(xì)胞微環(huán)境特征。 鑒于以上科學(xué)研究背景,本課題擬在體外,用Transwell小室共培養(yǎng)體系,將骨髓間充質(zhì)干細(xì)胞與后腎間充質(zhì)細(xì)胞和輸尿管芽分室共培養(yǎng),利用胚腎組織按內(nèi)在規(guī)律釋放的可溶性因子,誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向腎干細(xì)胞分化,觀察誘導(dǎo)后的干細(xì)胞在體外誘導(dǎo)輸尿管芽分枝的能力,為以后探討誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向腎干細(xì)胞分化所需的調(diào)控因子及建立單純用誘導(dǎo)因子在體外誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向腎干細(xì)胞分化的方法提供實(shí)驗(yàn)依據(jù),為腎臟組織工程研究以及將來采用腎干細(xì)胞在纖維化的腎組織形成有功能的新腎單位的實(shí)驗(yàn)研究提供充足的無免疫原性的腎臟種子細(xì)胞。 方法: 1.用密度梯度離心和貼壁培養(yǎng)相結(jié)合的方法分離培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞,倒置相差顯微鏡觀察細(xì)胞形態(tài)及生長特點(diǎn),四唑鹽比色法(Methylthio tetrazole, MTT)觀察細(xì)胞增殖及更新能力,透射電鏡觀察細(xì)胞內(nèi)部結(jié)構(gòu),流式細(xì)胞儀檢測細(xì)胞周期及細(xì)胞表面CD44、CD45、CD90分子的表達(dá),免疫細(xì)胞技術(shù)檢測細(xì)胞波形蛋白(Vimentin)、纖維連接蛋白(Fibronectin,FN)、角蛋白(Keratin)、E-鈣粘蛋白(E-cadherins)、CD34等的表達(dá),同時(shí)對(duì)培養(yǎng)細(xì)胞進(jìn)行成脂、成骨誘導(dǎo)分化,采用油紅(Oil red)O染色及骨鈣素(osteocalcim,OCN)觀察大鼠骨髓間充質(zhì)干細(xì)胞誘導(dǎo)分化情況。細(xì)胞鑒定后,將其接種于細(xì)胞外基質(zhì)膠中進(jìn)行三維立體(three-dimensional,3D)培養(yǎng),倒置相差顯微鏡觀察細(xì)胞在細(xì)胞外基質(zhì)膠中的形態(tài)變化,激光掃描共聚焦顯微鏡觀察細(xì)胞外基質(zhì)膠不同層面的細(xì)胞形態(tài)。 2.在解剖顯微鏡下分離孕13.5d(E13.5d)大鼠胚胎,獲取胚胎后腎并去除輸尿管芽,將胚胎后腎間充質(zhì)細(xì)胞以組織塊法進(jìn)行原代培養(yǎng),用倒置相差顯微鏡觀察細(xì)胞形態(tài)演變,MTT法觀察細(xì)胞增殖及更新能力,透射電鏡和掃描電鏡觀察細(xì)胞內(nèi)部結(jié)構(gòu)和表面結(jié)構(gòu),流式細(xì)胞儀檢測細(xì)胞周期及細(xì)胞表面CD44、CD45、CD90分子的表達(dá),免疫細(xì)胞技術(shù)檢測細(xì)胞Vimentin、Fibronectin、Keratin、E-cadherins及CD34的表達(dá),同時(shí)進(jìn)行細(xì)胞多向誘導(dǎo)分化(成脂肪細(xì)胞、成骨細(xì)胞)實(shí)驗(yàn),分別采用油紅O及OCN染色觀察誘導(dǎo)分化情況。細(xì)胞鑒定后,將其接種于細(xì)胞外基質(zhì)膠中進(jìn)行三維立體培養(yǎng),倒置相差顯微鏡觀察細(xì)胞在細(xì)胞外基質(zhì)膠中的形態(tài)變化,激光掃描共聚焦顯微鏡觀察細(xì)胞外基質(zhì)膠不同層面的細(xì)胞形態(tài)變化。 3.在解剖顯微鏡下分離孕13.5d(E13.5d)大鼠胚胎,獲取胚胎后腎,消化法得到完整的輸尿管芽,將其種植于Transwell小室上室中的細(xì)胞外基質(zhì)膠中,后腎間充質(zhì)細(xì)胞種植于Transwell小室下室中,二者進(jìn)行分室共培養(yǎng),倒置相差顯微鏡下觀察輸尿管芽的形態(tài),免疫細(xì)胞技術(shù)對(duì)其進(jìn)行鑒定,激光掃描共聚焦顯微鏡下觀察輸尿管芽的形態(tài)變化。 4.建立大鼠骨髓間充質(zhì)干細(xì)胞向腎干細(xì)胞分化的體外誘導(dǎo)共培養(yǎng)模型,并分為5組,A:骨髓間充質(zhì)干細(xì)胞+后腎間充質(zhì)細(xì)胞;B:骨髓間充質(zhì)干細(xì)胞+輸尿管芽;C:骨髓間充質(zhì)干細(xì)胞+后腎間充質(zhì)細(xì)胞+輸尿管芽;D:骨髓間充質(zhì)干細(xì)胞;E:空白對(duì)照組。用倒置相差顯微鏡觀察誘導(dǎo)后的大鼠骨髓間充質(zhì)干細(xì)胞形態(tài)變化,流式細(xì)胞儀檢測誘導(dǎo)后細(xì)胞周期及細(xì)胞表面CD44、CD45、CD90分子的表達(dá),免疫熒光化學(xué)法檢測誘導(dǎo)后細(xì)胞的細(xì)胞標(biāo)志物Vimentin、Fibronectin、Keratin、E-cadherins蛋白的表達(dá)。誘導(dǎo)后細(xì)胞與輸尿管芽共培養(yǎng),用激光掃描共聚焦顯微鏡觀察誘導(dǎo)后細(xì)胞對(duì)輸尿管芽的誘導(dǎo)能力。 結(jié)果: 1.大鼠原代BMSCs呈集落樣生長,細(xì)胞形態(tài)呈梭形,傳代后細(xì)胞生長迅速,形態(tài)比較均一,大部分細(xì)胞為成纖維樣或多角形,細(xì)胞增殖能力強(qiáng),具有自我更新能力;透射電鏡結(jié)果顯示細(xì)胞核較大,核呈橢圓形或不規(guī)則形,有核突,含有1-2個(gè)核仁,胞漿內(nèi)含有粗面內(nèi)質(zhì)網(wǎng)和線粒體,其他細(xì)胞器較少,胞漿內(nèi)可見分泌小泡,表現(xiàn)出未分化細(xì)胞的特征;細(xì)胞周期結(jié)果顯示,只有少數(shù)細(xì)胞進(jìn)入增殖活躍期,大部分細(xì)胞處于靜止期;流式細(xì)胞儀檢測細(xì)胞表面分子標(biāo)志物的結(jié)果顯示,細(xì)胞強(qiáng)表達(dá)CD44、CD90,不表達(dá)CD45;免疫細(xì)胞化學(xué)染色結(jié)果顯示BMSCs明顯表達(dá)Vimentin、Fibronectin,不表達(dá)Keratin、E-cadherin及CD34;BMSCs在特定誘導(dǎo)條件下可以向脂肪細(xì)胞、成骨細(xì)胞分化,加入成脂肪誘導(dǎo)培養(yǎng)基3w后,細(xì)胞內(nèi)可見多個(gè)折光性強(qiáng)的圓形脂肪滴,油紅O染色呈紅色;在加入成骨誘導(dǎo)培養(yǎng)基誘導(dǎo)后,可見圓形或卵圓形礦化結(jié)節(jié),OCN表達(dá)陽性。在本實(shí)驗(yàn)中,大鼠BMSCs在由80%Ⅰ型膠原與20%的微量生長因子基質(zhì)膠組成的細(xì)胞外基質(zhì)膠中生長狀態(tài)較好。 2.培養(yǎng)的大鼠MMSCs貼壁生長,梭形,成纖維細(xì)胞樣,單個(gè)核,核仁大,,形態(tài)均一,細(xì)胞增殖能力強(qiáng),具有自我更新能力;透射電鏡結(jié)果顯示,細(xì)胞為長梭形或不規(guī)則形狀,核不規(guī)則,核仁大,胞漿中滑面內(nèi)質(zhì)網(wǎng)發(fā)達(dá),可見分泌顆粒;掃描電鏡結(jié)果示,細(xì)胞呈梭形或多角形,核仁明顯,細(xì)胞連接緊密,表面有微絨毛突起,梭形細(xì)胞胞漿向兩極伸出長突起,多角形細(xì)胞突起呈樹枝狀,緊緊貼于蓋玻片上;細(xì)胞周期結(jié)果顯示,90%以上的細(xì)胞處于靜止期,只有少部分細(xì)胞處于增殖期;流式細(xì)胞儀檢測結(jié)果顯示,幾乎所有細(xì)胞都明顯表達(dá)CD44、CD90,幾乎所有細(xì)胞不表達(dá)CD45;免疫細(xì)胞化學(xué)染色法結(jié)果顯示,大鼠MMSCs明顯表達(dá)間充質(zhì)細(xì)胞標(biāo)志蛋白Vimentin、Fibronectin,不表達(dá)Keratin、E-cadherin及CD34;細(xì)胞在特定誘導(dǎo)條件下具有多向分化能力,可以向脂肪細(xì)胞、成骨細(xì)胞分化,誘導(dǎo)分化后的脂肪細(xì)胞油紅O染色呈特異性紅色,OCN表達(dá)呈陽性,并可形成鈣結(jié)節(jié)。倒置相差顯微鏡下觀察三維立體培養(yǎng)的大鼠MMSCs結(jié)果顯示,不同層面細(xì)胞都呈長梭形,培養(yǎng)2-3d可見細(xì)胞連接,細(xì)胞增殖能力強(qiáng);激光掃描共聚焦顯微鏡下觀察可見三維立體培養(yǎng)的細(xì)胞明顯表達(dá)Vimentin、Fibronectin,不表達(dá)Keratin及E-cadherin。三維立體培養(yǎng)體系中,大鼠MMSCs形狀立體感強(qiáng),生長狀態(tài)好。 3.倒置相差顯微鏡下觀察E13.5d大鼠的UB呈T形,接種于細(xì)胞外基質(zhì)中不斷分枝發(fā)芽。激光掃描共聚焦顯微鏡下,用FITC-DB標(biāo)記UB,結(jié)果顯示大鼠UB以二叉分枝方式進(jìn)行分枝、生長,UB頂端的細(xì)胞生長迅速,其頸部細(xì)胞較頂端(壺腹)部細(xì)胞的生長速度慢。隨著生長時(shí)間的延長,大鼠UB不斷以二叉分枝方式擴(kuò)展延伸,逐漸形成樹狀結(jié)構(gòu)。 4.倒置相差顯微鏡下觀察誘導(dǎo)后的大鼠BMSCs,結(jié)果顯示細(xì)胞生長密集,界限不清,呈梭形;流式細(xì)胞儀檢測誘導(dǎo)后的細(xì)胞周期結(jié)果顯示,90%以上的細(xì)胞處于靜止期,只有少部分細(xì)胞處于增殖期;流式細(xì)胞儀檢測誘導(dǎo)后的細(xì)胞表面標(biāo)記物,結(jié)果顯示幾乎所有細(xì)胞都明顯表達(dá)CD44、CD90,幾乎所有細(xì)胞不表達(dá)CD45;免疫熒光檢測誘導(dǎo)后的細(xì)胞結(jié)果顯示,大鼠后腎間充質(zhì)干細(xì)胞明顯表達(dá)間充質(zhì)細(xì)胞標(biāo)志蛋白Vimentin、Fibronectin,不表達(dá)上皮細(xì)胞標(biāo)志蛋白Keratin及E-cadherin;激光掃描共聚焦顯微鏡下觀察到C組誘導(dǎo)后的細(xì)胞對(duì)大鼠UB有誘導(dǎo)作用,C組的UB以二叉分枝的方式分枝發(fā)芽。 結(jié)論: 1.我們成功分離培養(yǎng)了大鼠BMSCs和MMSCs,這些細(xì)胞具有典型的間充質(zhì)細(xì)胞形態(tài)及干細(xì)胞表型,具有多向分化能力。 2.我們成功分離、培養(yǎng)、鑒定了大鼠UB。 3.我們建立了大鼠BMSCs在體外向腎干細(xì)胞分化模型,實(shí)現(xiàn)了BMSCs向腎干細(xì)胞的轉(zhuǎn)型。在體外誘導(dǎo)后的大鼠BMSCs能促進(jìn)大鼠UB分枝發(fā)芽。
[Abstract]:Background and Purpose :


Chronic kidney disease ( CKD ) is increasing and the incidence of chronic kidney disease ( CKD ) is up to 10 % . It is characterized by excessive deposition and fibrosis of extracellular matrix ( ECM ) , which causes progressive decline in renal function .


It has been found that bone marrow mesenchymal stem cells can induce differentiation of bone marrow mesenchymal stem cells into renal stem cells or kidney cells .


In the process of post - renal development , the differentiation of mesenchymal cells ( MMCs ) , i.e . , embryonic kidney stem cells , has the ability to self - renew and multi - directional differentiation .


In view of the above scientific research background , in vitro and in vitro , the bone marrow mesenchymal stem cells are co - cultured with the posterior renal mesenchymal stem cells and the ureter bud subchambers , and the differentiation of the bone marrow mesenchymal stem cells into the renal stem cells is induced by utilizing the soluble factors released by the embryo and kidney tissues in vitro , and the experimental research on the induction factor for inducing the differentiation of the bone marrow mesenchymal stem cells into the renal stem cells in vitro provides an experimental basis for further exploring the experimental research of inducing bone marrow mesenchymal stem cells to differentiate into renal stem cells in vitro , and provides sufficient non - immunogenic renal seed cells for experimental research on the renal tissue engineering research and the future use of renal stem cells to form a functional new kidney unit .


Method :


1 . The rat bone marrow mesenchymal stem cells were isolated and cultured by means of density gradient centrifugation and adherent culture . The cell morphology and growth characteristics were observed by reversed phase contrast microscope . The expression of CD44 , CD90 , E - cadherins , CD34 , etc . were observed .


2 . The rat embryo of 13.5 days ( E13.5 days ) was isolated under an anatomical microscope . After the embryo was obtained , the kidney was removed and the ureter bud was removed . The cell morphology and surface structure were observed by using an inverted phase contrast microscope . The cell cycle and the expression of CD44 , CD90 and E - cadherins and CD34 were observed by MTT assay . The morphological changes of the cells in extracellular matrix glue were observed by MTT assay . The morphological changes of the cells in extracellular matrix glue were observed by reversed phase contrast microscope .


3 . The rat embryo was isolated from 13.5 days ( E13.5 days ) under an anatomical microscope . After the embryo was obtained , the complete ureter bud was obtained by digestion method , and then it was planted in the extracellular matrix glue in the upper chamber of Transwell chamber . The posterior renal mesenchymal cells were cultured in the lower chamber of Transwell chamber . The morphology of the ureter bud was observed under the reversed phase contrast microscope . The morphological changes of the ureter bud were observed under the laser scanning confocal microscope .


4 . In vitro induction and co - culture of bone marrow mesenchymal stem cells into renal stem cells was established and divided into five groups : A : bone marrow mesenchymal stem cells + post - renal mesenchymal cells ; B : bone marrow mesenchymal stem cells + ureter buds ; C : bone marrow mesenchymal stem cells + post - renal mesenchymal stem cells + ureteral buds ; D : bone marrow mesenchymal stem cells ; E : blank control group .


Results :


The results of cell cycle showed that only a small number of cells entered the proliferative activity period , and the cells were in quiescent period . The results showed that only a small number of cells entered the proliferative activity period , and the cells were in the quiescent period . The results of the cell cycle showed that the cells were more likely to express vimentin , Fibronectin , and the cells were not expressed by Keratin , E - cadherin and CD34 .


The results showed that the cells were spindle - shaped or irregular . The results showed that the cells were spindle - shaped or irregular . The results showed that the cells were spindle - shaped or irregular . The results showed that the cells were spindle - shaped or polygonal . Fibronectin did not express Keratin and E - cadherin . In the three - dimensional three - dimensional culture system , the shape of MMSCs was strong and the growth state was good .


3 . The UB was T - shaped in E13.5 rats under reversed phase contrast microscope . UB was labeled with FITC - DB labeled UB . The results showed that the proliferation of UB at apical ( ampulla ) was slower than that of apical ( ampulla ) .


The results showed that almost all the cells expressed CD44 , CD90 and almost all the cells did not express CD90 . The results showed that almost all the cells expressed CD44 , CD90 , almost all the cells did not express CD90 . The results showed that almost all the cells expressed CD44 and CD90 . The results showed that almost all the cells did not express CD44 and CD90 . The results showed that almost all the cells did not express CD44 and CD90 .


Conclusion :


1 . We have successfully isolated the cultured and MMSCs , which have typical mesenchymal cell morphology and stem cell phenotype , and have multi - directional differentiation ability .


2 . We successfully isolated and cultured rat UB .


3 . We established a rat model of differentiation into renal stem cells in vitro , and the transformation of bone marrow cells into renal stem cells was achieved . In vitro , the rats were able to promote the sprouting of UB branches in rats .

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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