本文關(guān)鍵詞：大鼠骨髓間充質(zhì)干細(xì)胞的生物學(xué)特性及其對急性化學(xué)性肝損傷的修復(fù)作用　出處：《天津醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 慢病毒 增強(qiáng)綠色熒光蛋白 轉(zhuǎn)染 示蹤 肝損傷 干細(xì)胞移植

【摘要】： 一、大鼠骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)、鑒定及EGFP標(biāo)記 目的:本研究旨在體外分離培養(yǎng)和純化大鼠骨髓間充質(zhì)干細(xì)胞(Bone marrowmesenchymal stem cells,BMSCs),并初步鑒定BMSCs的生物學(xué)特性,為利用骨髓間充質(zhì)干細(xì)胞作細(xì)胞治療提供實驗依據(jù);探討不同濃度慢病毒載體對體外培養(yǎng)的BMSCs的轉(zhuǎn)染效率,為進(jìn)一步觀察體內(nèi)向肝細(xì)胞分化奠定基礎(chǔ)。 方法:在無菌條件下取四周齡100～120g健康雄性Wistar大鼠雙后肢的骨髓細(xì)胞,采用密度梯度離心法與貼壁培養(yǎng)相結(jié)合的方法,體外分離BMSCs。采用24小時后貼壁的細(xì)胞用含10%～15%胎牛血清(Fetal calf serum,FCS)的DMEM培養(yǎng)基(Dulbecco's Modified Eagle Media,DMEM)進(jìn)行培養(yǎng),以后每隔3天換液一次。待細(xì)胞80%～90%融合后,消化傳代,使之逐漸純化。1.BMSCs的觀察與鑒定:(1)倒置顯微鏡下觀察細(xì)胞形態(tài);(2)透射電鏡下觀察細(xì)胞超微結(jié)構(gòu);(3) MTT法測定細(xì)胞生長曲線;(4)流式細(xì)胞儀檢測CD29,CD34,CD45,CD90的表達(dá)。2.BMSCs的增強(qiáng)綠色熒光蛋白(enhanced green fluorescentprotein,EGFP)轉(zhuǎn)染:將分離的BMSCs分為空白對照組和實驗組,實驗組按照不同感染復(fù)數(shù)(multiplicity of infection,MOI)(1、10、30、50、70、100)加入攜帶EGFP的慢病毒液,空白組加入磷酸鹽緩沖液(phosphate-buffered saline,PBS)。轉(zhuǎn)染后即刻到14天持續(xù)于熒光顯微鏡藍(lán)光激發(fā)下觀察綠色熒光蛋白表達(dá)情況及細(xì)胞形態(tài)變化,確定最佳MOI。 結(jié)果: 1.采用Percoll密度梯度離心與貼壁培養(yǎng)相結(jié)合的方法所培養(yǎng)的BMSCs呈長梭形,胞核圓形或橢圓形,位于中央。流式細(xì)胞檢測結(jié)果為:CD29+/CD34-/CD45-/CD90+; 2.從傳代細(xì)胞生長曲線可見:第1～2天為細(xì)胞生長潛伏期,第3～7天為對數(shù)生長期,8天以后進(jìn)入平臺期。 3.透射電鏡結(jié)果顯示:BMSCs有兩種不同的形態(tài)結(jié)構(gòu),一種是處于未分化或分化較低狀態(tài)的小幼稚性細(xì)胞;另一種是處于相對活躍期的成熟大細(xì)胞。 4.熒光顯微鏡下觀察骨髓間充質(zhì)干細(xì)胞轉(zhuǎn)染后24小時后熒光開始發(fā)出,5～7天綠色熒光表達(dá)穩(wěn)定,表達(dá)效率最高能達(dá)到(79.85±4.92)%(MOI=30,第7天);轉(zhuǎn)染效率隨MOI的增加而增加,MOI=30,50,70,100時的轉(zhuǎn)染效率顯著高于MOI=1,10時的轉(zhuǎn)染效率(P＜0.05);熒光顯微鏡藍(lán)光下觀察細(xì)胞形態(tài)與普通顯微鏡下觀察空白組細(xì)胞形態(tài)一致。 結(jié)論:本論文通過體外分離、純化和鑒定BMSCs,建立了一套較合理的間充質(zhì)干細(xì)胞體外培養(yǎng)的技術(shù)方案,成功分離出BMSCs,并對BMSCs的生物學(xué)特性有進(jìn)一步的了解。含EGFP基因的慢病毒載體系統(tǒng)能夠成功轉(zhuǎn)染BMSCs,并且能高效的表達(dá)EGFP,且不會對細(xì)胞的生物學(xué)特性產(chǎn)生明顯的影響,為進(jìn)一步觀察BMSCs在體內(nèi)向肝細(xì)胞分化奠定基礎(chǔ)。 二、EGFP標(biāo)記的大鼠BMSCs同種異體移植對急性化學(xué)性肝損傷的修復(fù)作用 目的:觀察穩(wěn)定表達(dá)EGFP的BMSCs在肝損傷環(huán)境下的定居及對肝損傷的修復(fù)作用,了解EGFP標(biāo)記的BMSCs在肝損傷模型內(nèi)的變化,為同種異體移植BMSCs為基礎(chǔ)的肝損傷治療提供理論依據(jù)。 方法:健康雄性Wistar大鼠30只,體重150～180克,飼養(yǎng)一周后隨機(jī)分為六組,每組5只,肝損傷組采用2%四氯化碳(carbon tetrachloride,CCL_4)連續(xù)灌胃七天誘導(dǎo)急性肝損傷模型,于造模第7天行細(xì)胞移植:A組(正常對照組):正常大鼠,不行細(xì)胞移植,尾靜脈內(nèi)注入等量PBS;B組(正常+未標(biāo)記組):正常大鼠,向尾靜脈內(nèi)移植未標(biāo)記的BMSCs;C組(正常+標(biāo)記組):正常大鼠,向尾靜脈內(nèi)移植EGFP標(biāo)記的BMSCs;D組(肝損傷對照組):肝損傷大鼠,不行細(xì)胞移植,尾靜脈內(nèi)注入PBS;E組(肝損傷+未標(biāo)記組):肝損傷大鼠,向尾靜脈內(nèi)移植未標(biāo)記的BMSCs;F組(肝損傷+標(biāo)記組):肝損傷大鼠,尾靜脈內(nèi)移植標(biāo)記的BMSCs。移植后1周處死全部受試動物,血清檢測肝功能、HE染色行組織學(xué)觀察,熒光顯微鏡下確認(rèn)EGFP陽性細(xì)胞在肝損傷大鼠的定位及對大鼠肝損傷的修復(fù)作用。 結(jié)果: 1.采用CCL_4制作大鼠急性化學(xué)性肝損傷模型1周后,肝損傷組(D、E、F)血清丙氨酸氨基轉(zhuǎn)移酶(alanine aminotransferase,ALT)、天門冬氨酸氨基轉(zhuǎn)移酶(aspartate aminotransferase,AST)、總膽紅素(total bilirubin,TBIL)等指標(biāo)較正常喂養(yǎng)大鼠(A、B、C)相應(yīng)指標(biāo)升高,差異有統(tǒng)計學(xué)意義(P＜0.05)。肝損傷大鼠肝內(nèi)出現(xiàn)彌漫性分布的點狀壞死、橋接壞死,肝血竇充血明顯,彌漫炎性細(xì)胞浸潤。 2.同種異體移植BMSCs1周后E、F組大鼠血清ALT、AST、TBIL等指標(biāo)較移植前有所恢復(fù),與未移植BMSCs的肝損傷組(D組)的相應(yīng)指標(biāo)比較,差異有統(tǒng)計學(xué)意義(P＜0.05)。BMSCs移植組大鼠組織中壞死、充血、炎性細(xì)胞浸潤情況較未移植組減輕。無論是在正常組大鼠還是在肝損傷大鼠組,EGFP標(biāo)記的BMSCs移植與未標(biāo)記的BMSCs移植后,大鼠血清學(xué)、肝臟組織病理學(xué)情況類似。F組大鼠肝臟冰凍切片在熒光顯微鏡下可以在肝組織觀察到EGFP陽性的細(xì)胞。 結(jié)論: 1.CCL_4灌胃可以獲得比較可靠的肝損傷動物模型。 2.BMSCs移植后可修復(fù)急性化學(xué)性肝損傷所致的肝功能損害。 3.EGFP標(biāo)記不影響正常大鼠的肝功能,也不影響B(tài)MSCs對急性化學(xué)性損傷的修復(fù)作用。
[Abstract]:Isolation, culture, identification and EGFP markers of rat bone marrow mesenchymal stem cells
Objective: the purpose of this study is to isolate and purify rat bone marrow mesenchymal stem cells (Bone marrowmesenchymal stem cells, BMSCs), the biological characteristics and preliminary identification of BMSCs, to provide the experimental basis for the use of bone marrow mesenchymal stem cells for cell therapy; to investigate the transfection efficiency of different concentrations of lentiviral vector of BMSCs in vitro that is to lay a foundation for further study of liver cell differentiation in vivo.
Methods: under sterile conditions, take 100 ~ 4 weeks old healthy male 120g Wistar bipedal rat bone marrow cells by density gradient centrifugation combined with adherent culture, in vitro by BMSCs. after 24 hours of adherent cells with 10% ~ 15% fetal bovine serum (Fetal calf, serum, FCS) DMEM medium (Dulbecco's Modified Eagle Media, DMEM) were cultured, after every 3 days. After the cell was changed every 80% to 90% after the fusion of digestive passage, so that gradually purified observation and identification of.1.BMSCs: (1) the cells were observed under inverted microscope; (2) the ultrastructure was observed by transmission electron microscope; (3) the cell growth curve was determined by MTT; (4) the detection of CD29, flow cytometry, CD34, CD45, enhanced green fluorescent protein expression of.2.BMSCs CD90 (enhanced green fluorescentprotein, EGFP) transfection: the isolated BMSCs were divided into control group and experimental group, experimental Group in accordance with different multiplicities of infection (multiplicity of, infection, MOI) (1,10,30,50,70100) with lentivirus carrying EGFP liquid, adding phosphate buffer (phosphate-buffered saline, PBS group). After transfection for 14 days immediately to fluorescent microscopy under blue light to observe the expression of green fluorescent protein and cell morphological changes, determine the best MOI.
Result:
1., the BMSCs cultured by Percoll density gradient centrifugation and adherent culture was spindle shaped, and the nucleus was round or oval. It was located in the center. The result of flow cytometry was CD29+/CD34-/CD45-/CD90+.
2. from the cell growth curve, the incubation period of cell growth was seen from first to 2 days, the logarithmic growth period was third to 7 days, and the platform stage was entered after 8 days.
3. transmission electron microscopy showed that BMSCs has two different morphological structures, one is immature cells in undifferentiated or differentiated state, and the other is mature cells in relatively active stage.
4. observation under fluorescence microscopy of bone marrow mesenchymal stem cells 24 hours after transfection, fluorescence began to produce 5 ~ 7 days, the expression of green fluorescence expression stability, efficiency can reach a maximum (79.85 + 4.92)% (MOI=30, seventh days); transfection efficiency increased with the increase of MOI, MOI=30,50,70100, transfection efficiency was significantly higher than that of MOI=1,10 the transfection efficiency (P < 0.05); control group observation of cell morphology and cell morphology consistent with ordinary microscope fluorescence microscope under blue light.
Conclusion: the in vitro isolation, purification and identification of BMSCs, to establish a set of reasonable technical scheme of mesenchymal stem cells in vitro, BMSCs successfully isolated, and the biological characteristics of BMSCs have further understanding. Lentiviral vector containing EGFP gene can be successfully transfected into BMSCs, and the expression of EGFP efficient, and does not obviously affect on cell biological characteristics, lay the foundation for further study on BMSCs differentiation to hepatocytes in vivo.
Two, EGFP labelled rat BMSCs allograft for the repair of acute chemical liver injury
Objective: To observe the colonization and BMSCs repair of BMSCs with stable expression of EGFP in the liver injury environment, and to understand the change of EGFP labeled BMSCs in the liver injury model, so as to provide a theoretical basis for the treatment of BMSCs based liver injury.
鏂規(guī)硶:鍋ュ悍闆勬，

本文編號：1382746