發(fā)布時間：2018-09-03 10:38

【摘要】：目的:對Sprague-Dwley(SD)大鼠骨髓間充質(zhì)干細(xì)胞(Bone Marrow derived Mesenchymal Stem Cells,BMSCs)進(jìn)行體外抽提,并進(jìn)行分離、培養(yǎng)、純化,對生物學(xué)特性進(jìn)行鑒定。用綠色熒光蛋白(Green Fluorenscent Protein,GFP)標(biāo)記慢病毒載體進(jìn)行體內(nèi)示蹤。觀察BMSCs移植治療大鼠橫紋肌溶解致急性腎損傷(Rhabdomyolysis-induced AKI,RIAKI)的可行性,評價其對RIAKI大鼠腎臟形態(tài)結(jié)構(gòu)、功能的影響,并從細(xì)胞水平觀察BMSCs治療RIAKI的效果。觀察BMSCs移植后RIAKI大鼠Th17細(xì)胞及Treg細(xì)胞變化及炎癥介質(zhì)水平變化,探討B(tài)MSCs治療橫紋肌溶解致急性腎損傷的機制。方法:1、無菌條件選取健康雄性SD大鼠雙后肢的骨髓細(xì)胞,采用密度梯度離心法體外分離、培養(yǎng)BMSCs并傳代,保存P2~P6代BMSCs,治療時用P3代BMSCs。流式細(xì)胞儀檢測細(xì)胞表面標(biāo)志分子及生物學(xué)性質(zhì),包括生長曲線及細(xì)胞周期分析。細(xì)胞化學(xué)法鑒定BMSCs的多向分化潛能。BMSCs慢病毒載體介導(dǎo)GFP標(biāo)記和擴增,以感染復(fù)數(shù)(multiplicity of infection,MOI)(5、10、25、50)進(jìn)行感染,得到最佳感染效率和細(xì)胞活性。2、BMSCs移植治療RIAKI大鼠效果:⑴成年健康雄性SD大鼠禁水24h后,雙后肢肌內(nèi)注射50%甘油(生理鹽水稀釋)10ml/Kg制作RIAKI大鼠模型。⑵將SD大鼠隨機分為4組:正常對照組(Normal組);假手術(shù)組(NS組);模型組(RIAKI組);治療組(BMSCs組)。分別觀察各組大鼠一般情況;血清生化指標(biāo):尿素氮(BUN)、肌酐(Scr)、肌酸激酶(CK);形態(tài)學(xué)與病理學(xué)改變:雙后肢肌肉(HE染色);腎(HE、PAS染色);腎臟細(xì)胞凋亡(TUNEL凋亡分析,Western Blot檢測腎組織caspase3,caspase 9,Bcl-2,cytochrome C蛋白表達(dá))。3、BMSCs移植治療大鼠RIAKI作用機制:⑴細(xì)胞水平:流式細(xì)胞技術(shù)檢測各組大鼠脾臟組織中Treg細(xì)胞(CD4+CD25+Foxp3+)在CD4+T細(xì)胞中的比例及Th17細(xì)胞(CD4+、IL-17+代表Th17)比例。⑵基因水平:RT-PCR檢測各組大鼠脾臟及腎組織Th17細(xì)胞特異性轉(zhuǎn)錄因子RORγtm RNA及Treg細(xì)胞特異性轉(zhuǎn)錄因子Foxp3m RNA水平⑶特異性細(xì)胞因子水平:ELISA檢測各組大鼠血清及腎組織中Treg細(xì)胞釋放的特異性細(xì)胞因子:TGF-β、IL-10(抗炎);Th17細(xì)胞釋放IL-17(促炎);相關(guān)炎性細(xì)胞因子TNF-α、IL-6的水平。結(jié)果:1、采用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,胞核位于中央,呈圓形或橢圓形。流式細(xì)胞檢測結(jié)果為:BMSCs均表達(dá)CD44,CD90,陽性率分別為89.9%,91.3%;而CD45,呈陰性,陽性率分別為2.8%。從傳代細(xì)胞生長曲線可見:第1~2d為細(xì)胞生長潛伏期,第3~7d為對數(shù)生長期,7d后進(jìn)入平臺期。感染復(fù)數(shù)(multiplicity of infection,MOI)=25時高表達(dá)綠色熒光蛋白,在熒光顯微鏡下綠色熒光明顯可見,細(xì)胞生長速度快,BMSCs-GFP90%,本實驗最佳MOI確定為25。2、生化指標(biāo)變化:RIAKI組大鼠后肢甘油注射后血BUN、Scr和CK顯著升高,第6h、24h、48h、72h、96h及1W(周)后與干預(yù)前及Normal組比較差異顯著(P0.01),其中血BUN、SCr于第72h達(dá)峰值,CK于第24h達(dá)峰值,上述生化指標(biāo)升高水平及變化趨勢符合橫紋肌溶解并急性腎損傷的實驗室診斷標(biāo)準(zhǔn)。BMSCs組血BUN、Scr水平于第24h、48h、72h、96h及1W較RIAKI組相同時間點降低,差異顯著,有統(tǒng)計學(xué)意義(P0.01)。BMSCs組第24h、48h、72h、96h肌肉組織損傷指標(biāo)血CK值較RIAKI組相同時間點降低,差異顯著,有統(tǒng)計學(xué)意義(P0.01)。NS組與Normal組兩組間各指標(biāo)比較無統(tǒng)計學(xué)意義(P0.05)。光鏡下可見RIAKI組及BMSCs組大鼠雙后肢局部肌肉組織呈肌溶解性病理改變,BMSCs組大鼠24h、48h、72h橫紋肌溶解損傷程度較RIAKI組明顯減輕,BMSCs組大鼠24h、48h、72h腎組織損傷程度較RIAKI組減輕,表現(xiàn)為腎小管內(nèi)管型減少,上皮細(xì)胞壞死程度減輕。TUNEL法檢測細(xì)胞凋亡情況,熒光顯微鏡觀察BMSCs組較RIAKI組第24h、48h、72h、1w大鼠腎臟凋亡細(xì)胞減少,凋亡指數(shù)(AI)降低(P0.01)。WB檢測結(jié)果顯示Normal組及NS組caspase 3、caspase 9、cytochrome C蛋白極低水平表達(dá),Bcl-2蛋白表達(dá)水平較高。BMSCs組第24h、48h、72h、1w各時間點caspase 3、caspase 9、cytochrome C蛋白含量與均低于RIAKI組表達(dá)水平(P0.05),BMSCs組各時間點Bcl-2蛋白表達(dá)水平均高于RIAKI組(P0.05)。3、流式技術(shù)檢測大鼠脾臟Treg細(xì)胞比例變化,結(jié)果顯示BMSCs組與RIAKI組比較第24h、48h、72h Treg細(xì)胞比例升高,Th17細(xì)胞比例降低,差異顯著,有統(tǒng)計學(xué)意義(P0.05)。RT-PCR檢測RORγt m RNA和Foxp3 m RNA的表達(dá)發(fā)現(xiàn)BMSCs組與RIAKI組比較脾臟及腎組織RORγt m RNA水平下調(diào),Foxp3m RNA水平上調(diào),差異顯著,有統(tǒng)計學(xué)意義(P0.05)。ELISA檢測血清及腎組織細(xì)胞因子IL-17、IL-10、TGF-β、IL-6和TNF-α的含量發(fā)現(xiàn):BMSCs組與RIAKI組比較IL-17水平下,L-10水平升高,TGF-β水平升高,IL-6、TNF-α水平降低,差異顯著,有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:1、通過密度梯度離心法聯(lián)合BMSCs貼壁篩選法可獲得足量純化的BMSCs,GFP慢病毒載體成功轉(zhuǎn)染BMSCs,并高效的表達(dá)GFP,不影響B(tài)MSCs生長、分化等生物學(xué)特性及功能,可作為移植BMSCs細(xì)胞標(biāo)記示蹤的手段。2、雙后肢肌肉注射50%甘油溶液可成功誘導(dǎo)了RIAKI大鼠模型,BMSCs治療降低RIAKI大鼠的血BUN、SCr和CK水平,在一定程度上改善了急性損傷腎臟的結(jié)構(gòu)與功能,證實了BMSCs對RIAKI治療效果;進(jìn)一步從細(xì)胞水平觀察發(fā)現(xiàn)BMSCs治療可減少RIAKI大鼠腎臟凋亡細(xì)胞數(shù)量,降低凋亡指數(shù)。BMSCs移植上調(diào)RIAKI大鼠腎組織抗凋亡蛋白Bcl-2的表達(dá),抑制促凋亡因子cyt C釋放,下調(diào)凋亡效應(yīng)因子caspase-9、caspase-3表達(dá),BMSCs抑制腎臟細(xì)胞凋亡可能是通過使細(xì)胞凋亡通路中線粒體途徑受抑,從而發(fā)揮腎保護(hù)作用。未發(fā)現(xiàn)BMSCs在腎臟的定植,推測BMSCs可能通過“非分化修復(fù)”的旁分泌及免疫調(diào)節(jié)等減輕RIAKI。3、在第二部分實驗基礎(chǔ)上進(jìn)一步研究BMSCs改善RIAKI的“非分化修復(fù)”機制,結(jié)果證實RIAKI大鼠機體存在Thl7/Treg細(xì)胞免疫失衡及其特異性、炎性細(xì)胞因子水變化,機體存在促炎-抗炎反應(yīng)失衡。BMSCs治療可上調(diào)Treg細(xì)胞比例,在基因水平下調(diào)Th17細(xì)胞特異RORγtm RNA表達(dá),促進(jìn)Treg細(xì)胞特異性轉(zhuǎn)錄因子Foxp3m RNA上調(diào);BMSCs抑制Thl7細(xì)胞特異性促炎性細(xì)胞因子IL-17的表達(dá),促進(jìn)Treg細(xì)胞因子抗炎性細(xì)胞因子IL-10和TGF-β的表達(dá),同時抑制炎性效應(yīng)因子IL-6、TNF-α表達(dá),從而驗證了BMSCs可以調(diào)節(jié)RIAKI大鼠體內(nèi)Thl7/Treg細(xì)胞免疫失衡、抑制炎癥反應(yīng),這可能是BMSCs減輕橫紋肌溶解致急性腎損傷程度,發(fā)揮腎保護(hù)作用的重要機制。
[Abstract]:AIM: To isolate, culture, purify and identify the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) from Sprague-Dwley (SD) rats in vitro. The lentiviral vectors were labeled with green fluorescent protein (GFP). The feasibility of treating acute renal injury (RIAKI) induced by rhabdomyolysis in rats was evaluated. The effects of BMSCs on renal morphology, structure and function in RIAKI rats were evaluated. The effects of BMSCs on RIAKI were observed at cellular level. The changes of Th17 cells and Treg cells and inflammatory mediators in RIAKI rats after BMSCs transplantation were observed. Methods: 1. Bone marrow cells from both hind limbs of healthy male SD rats were isolated by density gradient centrifugation in vitro. BMSCs were cultured and passaged. P2-P6 generation BMSCs were preserved. P3 generation BMSCs were used for treatment. Flow cytometry was used to detect cell surface markers and biological properties, including biological properties. Long curve and cell cycle analysis. Multidifferentiation potential of BMSCs was identified by cytochemical method. GFP marking and amplification mediated by BMSCs lentiviral vector were carried out. The optimal infection efficiency and cell viability were obtained by multiple infection (MOI) (5,10,25,50). 2. Effect of BMSCs transplantation on RIAKI rats: _Adult healthy male SD was large. Rats were randomly divided into 4 groups: normal control group (Normal group); sham operation group (NS group); model group (RIAKI group); treatment group (BMSCs group). The general conditions of rats in each group were observed; serum biochemical indexes: BUN, Scr, and so on. Creatine kinase (CK); morphological and pathological changes: muscle of both hind limbs (HE staining); kidney (HE, PAS staining); renal cell apoptosis (TUNEL apoptosis analysis, Western Blot detection of caspase 3, caspase 9, Bcl-2, cytochrome C protein expression) 3, BMSCs transplantation treatment of RIAKI mechanism: _Cell level: flow cytometry detection of rats in each group The proportion of Treg cells (CD4+CD25+Foxp3+) in CD4+T cells and the proportion of Th17 cells (CD4+, IL-17+ representing Th17) in spleen tissue were measured. _Gene level: RT-PCR was used to detect the levels of RORgamtmRNA and Foxp3m RNA in spleen and kidney tissue of rats. The levels of specific cytokines (TGF-beta, IL-10, anti-inflammation), IL-17, TNF-alpha and IL-6) released by Treg cells in serum and kidney of rats were detected. The positive rate of BMSCs was 89.9% and 91.3% respectively, while that of CD45 was negative with a positive rate of 2.8%. From the growth curve of passaged cells, it was found that the cell growth incubation period was 1-2 days, the logarithmic growth period was 3-7 days, and the plateau period was 7 days later. High expression of green fluorescent protein was observed under fluorescence microscope. The growth rate of BMSCs-GFP was 90%. The optimum MOI was 25.2. The biochemical indexes of RIAKI group increased significantly after glycerol injection in hind limbs. The BUN, Scr and CK of RIAKI group increased significantly after 6 h, 24 h, 48 h, 72 h, 96 h and 1 W (week) compared with that before intervention and Normal group. Blood BUN, SCr reached the peak at 72h, CK reached the peak at 24h. The above biochemical indexes were in accordance with the laboratory diagnostic criteria of rhabdomyolysis and acute renal injury. The levels of BUN, Scr in BMSCs group were lower at 24h, 48h, 72h, 96h and 1W than RIAKI group at the same time point, the difference was significant (P 0.01). Compared with RIAKI group at the same time point, the level of serum CK in MSCs group at 24h, 48h, 72h and 96h decreased significantly (P 0.01). There was no significant difference between NS group and Normal group (P 0.05). The degree of rhabdomyolysis injury in rats at 24h, 48h, 72h was significantly less than that in RIAKI group. The degree of renal tissue injury in BMSCs group was less than that in RIAKI group at 24h, 48h, 72h. The renal tubular type was reduced and the degree of epithelial cell necrosis was reduced. The expression of caspase 3, caspase 9 and cytochrome C protein was very low and the expression of Bcl-2 protein was high in Normal group and NS group. The expression of caspase 3, caspase 9 and cytochrome C protein in BMSCs group was lower than that in RIAKI group at 24, 48, 72, and 1 W time points (P 0.05). The expression of Bcl-2 protein in BMSCs group was higher than that in RIAKI group (P 0.05). The percentage of Treg cells in spleen was detected by flow cytometry. The percentage of Treg cells in BMSCs group was higher than that in RIAKI group at 24h, 48h, 72h, and the percentage of Th17 cells was lower than that in RIAKI group (P 0.05). Compared with RIAKI group, the levels of ROR-gamma t RNA in spleen and kidney were down-regulated and Foxp3m RNA was up-regulated in BMSCs group (P 0.05). The levels of IL-17, IL-10, TGF-beta, IL-6 and TNF-alpha in serum and kidney tissue were detected by ELISA. The results showed that the levels of L-10 and TGF-beta were up-regulated in BMSCs group compared with RIAKI group. Conclusion: 1. Full purified BMSCs can be obtained by density gradient centrifugation combined with BMSCs adherence screening method. GFP lentiviral vector can successfully transfect BMSCs and efficiently express GFP, which does not affect the biological characteristics and functions of BMSCs, such as growth and differentiation, and can be used as fine BMSCs transplantation. 2. Intramuscular injection of 50% glycerol solution into both hind limbs could induce RIAKI rat model successfully. BMSCs treatment reduced the levels of BUN, SCr and CK in RIAKI rats, and improved the structure and function of acute injured kidney to a certain extent, which confirmed the therapeutic effect of BMSCs on RIAKI. BMSCs transplantation can up-regulate the expression of anti-apoptotic protein Bcl-2, inhibit the release of pro-apoptotic factor cyt C, and down-regulate the expression of apoptotic effector caspase-9 and Caspase-3 in RIAKI rats kidney. BMSCs may inhibit the apoptosis of renal cells through the mitochondrial pathway of apoptosis pathway. No BMSCs were found to colonize the kidneys. It is speculated that BMSCs may alleviate RIAKI.3 by "undifferentiated repair" paracrine and immunoregulation. On the basis of the second part of the experiment, the mechanism of BMSCs improving RIAKI by "undifferentiated repair" was further studied. The results confirm the existence of Thl7/Treg cells in RIAKI rats. BMSCs can up-regulate the proportion of Treg cells, down-regulate the expression of specific ROR gamma TM RNA and up-regulate the expression of Treg cell-specific transcription factor Foxp3m RNA at the gene level. BMSCs inhibit the up-regulation of Thl7 cell-specific pro-inflammatory cytokine IL-17. BMSCs can regulate the immune imbalance of Thl7/Treg cells in RIAKI rats and inhibit inflammation, which may be due to BMSCs alleviating acute renal injury induced by rhabdomyolysis and exerting renal protection. Important mechanisms used.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R685.5;R692.5

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