Akt1基因修饰的骨髓MSCs治疗ConA诱导的小鼠急性肝损伤的实验研究

发布时间：2018-05-02 11:02

本文选题：Akt1 + 间充质干细胞　；参考：《北京协和医学院》2014年博士论文

【摘要】：研究目的： 1.原代培养出小鼠骨髓间充质干细胞(MSCs)并鉴定； 2.构建PMSCV-Aktl-IRES-GFP逆转录病毒载体,感染MSCs,并筛选、扩增Akt过表达的MSCs; 3.体外研究过表达Akt1对小鼠MSCs生物学功能的影响； 4.观察Aktl-MSCs对不同浓度刀豆蛋白A(ConA)诱导的肝损伤模型的疗效。研究方法：取C57小鼠胫骨和股骨,利用MSCs的贴壁生长能力进行分离,并对获取的细胞进行流式鉴定和成软骨和成脂培养；构建Aktl-IRES-GFP逆转录病毒载体,感染MSCs,并筛选、扩增Aktl过表达的MSCs,并同时构建GFP-MSCs作为对照；使用RT-PCR及western blot的方法检测Aktl-MSCs的Akt1的基因、蛋白及磷酸化蛋白的表达水平；分别用浓度为10ng/ml、50ng/ml、100ng/ml IFN-γ处理Aktl-MSCs及GFP-MSCs细胞,采用四唑氮化合物(MTS)法测定MSCs的生长情况并绘制生长曲线,通过Ca2+依赖性磷脂结合蛋白/碘化丙啶(Annexin V/PI)复染,观察不同浓度IFN-γ对Aktl-MSCs及MSCs细胞凋亡的影响,并使用pcr-array对Aktl-MSCs及GFP-MSCs的凋亡信号通路进行检测,比较两者的异同；使用RT-PCR的方法检测10ng/ml IFN-γ刺激前后Aktl-MSCs及GFP-MSCs中Akt1、IL-4、IL-10、VEGF、HGF及PGE2的表达情况；使用ELISA的方法检测经浓度分别为10ng/ml、50ng/ml、100ng/ml IFN-γ刺激Aktl-MSCs及GFP-MSCs后,培养基中IL-4、IL-10、VEGF、HGF及PGE2的含量；使用Aktl-MSCs及GFP-MSCs治疗致死剂量ConA (40mg/kg)所致小鼠急性肝损伤,观察小鼠的生存情况并绘制生存曲线并了解小鼠肝脾的病理变化；使用Aktl-MSCs及GFP-MSCs治疗ConA(20mg/kg)所致的急性肝损伤模型,观察AST、ALT、TNF-α及IFN-γ的及肝脾病理变化,同时使用ELISA方法检测小鼠血清中IL-4、IL-10、VEGF、HGF及PGE2的含量并通过活体成像观察Aktl-MSCs及GFP-MSCs在ConA所致的急性肝损伤模型中向肝脏的归巢情况。研究结果：成功培养出小鼠MSCs:MSCs高表达CD29、CD44、Sca-1,而不表达造血细胞标志CD45、CD34,也不表达CD11b、CD31、FLK-1、MHC-Ⅱ类分子,成功将MSCs诱导分化成为软骨细胞和脂肪细胞;成功筛选并扩增出Akt1-MSCs, RT-PCR显示Aktl-MSCs的Aktl基因表达量明显高于GFP-MSCs(P0.01),Western blot显示Aktl-MSCs的总Aktl蛋白及磷酸化Akt(Ser473)蛋白均高于GFP-MSCs;细胞增殖及凋亡实验显示在正常培养及高浓度IFN-γ (100ng/ml)的刺激下,Aktl-MSCs显示出了增殖及抗凋亡优势,PCR-array显示Akt1-MSCs可显著抑制外源性凋亡通路；RT-PCR显示,Aktl-MSCs的Akt1、IL-4、IL-10、VEGF、HGF及PGE-2的基因表达水平均高于GFP-MSCs,在采用10ng/ml IFN-γ刺激后,Aktl-MSCs及GFP-MSCs的Akt1、IL-4、IL-10、HGF及PGE2的基因表达水平均有所上调,但Aktl-MSCs上调幅度更明显,且VEGF的基因表达水平未见明显上调；分别采用不同浓度(10ng/ml、50ng/ml、100ng/ml) IFN-γ刺激Aktl-MSCs及GFP-MSCs后,ELISA结果显示：在10ng/ml、50ng/ml IFN-γ刺激12h后,Akt1-MSCs培养基中IL-10浓度明显高于GFP-MSCs,在100ng/ml IFN-γ刺激12h后,Aktl-MSCs及GFP-MSCs的细胞因子IL-10表达水平无明显差异。在10ng/ml、50ng/ml、100ng/ml IFN-γ刺激后,细胞因子PGE-2的含量两者无明显差异,而Aktl-MSCs培养基中HGF和VEGF的浓度显著高于GFP-MSCs。由于培养基IL-4浓度过低(各组均4pg/ml),未得出可分性的数据；使用Akt1-MSCs及GFP-MSCs治疗致死剂量ConA(40mg/kg)所致的小鼠急性肝损伤,小鼠共分为5组,每组10只。24小时后,ConA40mg/kg、ConA40mg/kg+l×106Akt1-MSCs、ConA40mg/kg+1×106MSCs的三组小鼠均全部死亡,ConA40mg/kg+5×106Akt1-MSCs组存活3只、ConA40mg/kg+5×106MSCs组存活1只。使用Aktl-MSCs及GFP-MSCs治疗ConA(20mg/kg)所致的小鼠急性肝损伤模型,结果显示：与GFP-MSCs相比,Aktl-MSCs可以更好地降低小鼠血清中AST、ALT、TNF-α及IFN-γ水平和改善小鼠肝脏病理损伤情况,其中AST及IFN-γ的降低尤为显著。Akt1-MSCs治疗组小鼠血清中IL-10、VEGF、HGF及的浓度均高于GFP-MSCs治疗组小鼠,而由于小鼠血清中IL-4浓度过低(各组均4pg/ml,故未得出可分性的数据)。活体成像结果显示,第0天,ConA+Aktl-MSCs组小鼠肝脏GFP荧光强度(465nm)强于ConA+GFP-MSCs组,单纯Aktl-MSCs及GFP-MSCs组未检测出明显荧光。第7天,ConA+Aktl-MSCs组小鼠肝脏GFP荧光强度(465nm)局部有所升高,而ConA+GFP-MSCs、Akt1-MSCs及GFP-MSCs组未检测出明显荧光。第14天,Aktl-MSCs组小鼠肝脏GFP荧光强度(465nm)强于MSCs组,而ConA+Akt1-MSCs和ConA+GFP-MSCs组未检测到明显荧光。研究结论：通过本研究,我们1)原代培养出小鼠MSCs,流式细胞免疫表型鉴定符合MSCs表型表达特点,并将MSCs诱导分化成为软骨细胞和脂肪细胞；2)构建PMSCV-Akt1-IRES-GFP逆转录病毒载体,感染MSCs,并筛选、扩增出出Akt1过表达的MSCs;3) Akt1-MSCs匕相同代数的MSCs在正常培养及高浓度IFN-γ (100ng/ml)的刺激下增殖及抗凋亡能力均明显优于GFP-MSCs;4)在lOng/ml IFN-γ刺激前后,Aktl-MSCs的部分细胞因子的基因表达水平高于GFP-MSCs,且Aktl-MSCs表现出对IFN-γ刺激更强的应激能力；5)在不同浓度(lOng/ml、50ng/ml、100ng/ml) IFN-γ刺激12h后,Aktl-MSCs的旁分泌功能优于GFP-MSCs;6)5×106Aktl-MSCs可提高致死剂量ConA (40mg/kg)所致的急性肝损伤小鼠的存活率；7)Akt1-MSCs可显著改善ConA(20mg/kg)所致的小鼠急性肝损伤状况；8)在ConA(20mg/kg)所致的小鼠急性肝损伤模型和正常小鼠中,Aktl-MSCs体现出了向肝脏归巢的优势。
[Abstract]:Purpose of study :

1 . The bone marrow mesenchymal stem cells ( MSCs ) were cultured in primary culture and identified ;

2 . constructing the PMSCV - Aktl - to - GFP retroviral vector , infecting MSCs , screening and amplifying the MSCs which are overexpressed ;

3 . The effect of Akt1 on the biological function of MSCs was studied in vitro .

4 . To observe the effect of Aktl - MSCs on the model of liver injury induced by A ( Con A ) in different concentrations .

Study method :

C57 mouse tibia and femur were collected , the adherent growth ability of MSCs was separated , and the obtained cells were subjected to flow identification and chondrosis and adipogenesis culture ;
Construction of the Aktl - EGFP - GFP retroviral vector , infected MSCs , and screened , amplified Aktl over - expressed MSCs , and simultaneously constructed GFP - MSCs as control ;
The expression level of Akt1 gene , protein and phosphorylated protein of Aktl - MSCs was detected by RT - PCR and western blot .
Aktl - MSCs and GFP - MSCs were treated with a concentration of 10 ng / ml , 50 ng / ml , 100 ng / ml IFN - 纬 , and the growth curves of MSCs were determined by MTS . The effects of different concentrations of IFN - 纬 on Aktl - MSCs and the apoptosis of MSCs were observed .
The expression of Akt1 , IL - 4 , IL - 10 , VEGF , HGF and PGE2 in Akt1 , IL - 4 , IL - 10 , VEGF , HGF and PGE2 in 10 ng / ml IFN - 纬 were detected by RT - PCR .
ELISA was used to detect the levels of IL - 4 , IL - 10 , VEGF , HGF and PGE2 in culture medium after stimulation of Aktl - MSCs and GFP - MSCs with concentrations of 10 ng / ml , 50 ng / ml , 100 ng / ml IFN - 纬 .
Using Aktl - MSCs and GFP - MSCs to treat acute liver injury in mice , mice were observed and the survival curves were observed and the pathological changes of liver and spleen were observed .
The acute liver injury model induced by Con A ( 20mg / kg ) was treated with Aktl - MSCs and GFP - MSCs . The changes of AST , ALT , TNF - 伪 and IFN - 纬 and liver and spleen were observed . Meanwhile , ELISA was used to detect the levels of IL - 4 , IL - 10 , VEGF , HGF and PGE2 in serum of mice .

Results of the study :

The expression of Akt1 - MSCs was significantly higher than that of GFP - MSCs ( P0.01 ) .
The expression levels of Akt1 , IL - 4 , IL - 10 , VEGF , HGF and PGE - 2 of Aktl - MSCs were higher than that of GFP - MSCs , and the expression levels of Akt1 , IL - 4 , IL - 10 , HGF and PGE2 in Aktl - MSCs and GFP - MSCs were up - regulated after 10 ng / ml IFN - 纬 stimulation .
The levels of IL - 10 in Akt1 - MSCs were significantly higher than that of GFP - MSCs at 10 ng / ml , 50 ng / ml and 100 ng / ml IFN - 纬 . The levels of HGF and VEGF in Aktl - MSCs were significantly higher than that of GFP - MSCs .
Compared with GFP - MSCs , the levels of AST , ALT , TNF - 伪 and IFN - 纬 in serum of mice were significantly lower than that of GFP - MSCs . The results showed that the levels of AST , ALT , TNF - 伪 and IFN - 纬 in serum of mice were significantly lower than that of GFP - MSCs . In vivo imaging , the fluorescence intensity of liver GFP ( 465 nm ) was stronger than that of Con A + GFP - MSCs group , but only Aktl - MSCs and GFP - MSCs group did not detect the fluorescence . On day 7 , the fluorescence intensity of liver GFP ( 465 nm ) was stronger than that in group A + GFP - MSCs , Akt1 - MSCs and GFP - MSCs . On the 14th day , the fluorescence intensity of liver GFP ( 465 nm ) was stronger than that in MSCs group , while Con A + Akt1 - MSCs and Con A + GFP - MSCs group were not detected .

Conclusions of the study :

In this study , we cultured the MSCs in the primary culture , and the flow cytometry showed that the MSCs differentiated into chondrocytes and fat cells .
( 2 ) Construction of PMSCV - Akt1 2 - GFP retroviral vector , infected MSCs , and screened and amplified the MSCs which were expressed by Akt1 ; 3 ) MSCs of the same algebra as Akt1 - MSCs were significantly better than GFP - MSCs in normal culture and high concentration of IFN - 纬 ( 100 ng / ml ) , and the expression level of partial cytokines of Aktl - MSCs was higher than that of GFP - MSCs , and Aktl - MSCs showed stronger ability to stress IFN - 纬 ;
( 5 ) After 12 hours of IFN - 纬 stimulation at different concentrations ( lOng / ml , 50ng / ml , 100ng / ml ) , the parasecretory function of Aktl - MSCs was better than that of GFP - MSCs ; 6 ) 5 脳 106 Aktl - MSCs could increase the survival rate of mice with acute liver injury induced by Con A ( 40mg / kg ) ;
7 ) Akt1 - MSCs could significantly improve the acute liver injury in mice induced by Con A ( 20mg / kg ) .
8 ) Aktl - MSCs in mice induced by Con A ( 20 mg / kg ) showed the advantage of homing to the liver .

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.3

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