本文選題：間充質干細胞 + 胚胎干細胞�。� 參考：《北京協(xié)和醫(yī)學院》2015年博士論文

【摘要】：目的：多能干細胞包括胚胎干細胞(ES)和誘導多能干細胞(iPS),作為一類具有無限自我更新和多向分化潛能的細胞群體,能進一步分化為多種類型的細胞。MSC臨床應用前景廣闊,是細胞替代治療和組織工程的首選種子細胞,是移植領域和自身性免疫疾病治療的研究熱點。探索全反式維甲酸(RA)和SB431542對胚胎干細胞(ES)和誘導多能干細胞(iPS)分化為間充質干細胞(MSC)樣細胞的影響。方法：將培養(yǎng)的鼠ES和iPS根據(jù)不同的分化條件分為對照組、SB431542。組及RA組不同濃度組,對照組為DMEM完全培養(yǎng)基,SB431542組為DMEM完全培養(yǎng)基中含10 I.tM的SB431542,RA組的DMEM完全培養(yǎng)基中含RA濃度分別為0.05、0.10、0.20、0.40 nM。分化培養(yǎng)4 d后觀察細胞形態(tài),流式細胞術檢測細胞表面標志物CDl05和干細胞抗原1(Sca-1)的表達,以CD105+細胞和Sca-1-細胞的比例確定iPS和ES向MSC分化的程度。結果：1)小鼠ES和iPS在ESGRO-2i培養(yǎng)基里生長較好。ES分化4 d后,對照組和SB431542組ES和iPS分化成內皮樣細胞,在含有不同濃度RA的培養(yǎng)基中ES和iPS可有效分化為纖維狀細胞；2)ES分化4 d后,SB431542組Sca-1+細胞比例顯著高于對照組(P0.05),CD105+細胞比例與對照組比較差異無統(tǒng)計學意義(P0.05)。在RA濃度為0.05、0.10、0.20、0.40 nM組中,ES分化的CD105+細胞比例顯著高于對照組和SB431542組(P0.05),Sca-1+細胞比例顯著高于對照組(P0.05),但與SB431542組比較顯著降低(P0.05)；RA濃度為0.20 nM組的CD105+和Sca-1+細胞比例顯著高于RA濃度為0.05、0.10、0.40 nM組(P0.05)；3)iPS分化4 d后,SB431542組CD105+細胞和Sca-1+細胞比例顯著高于對照組(P0.05)。在RA濃度為0.05、0.10、0.20、0.40 nM組中,iPS分化的CD105+細胞比例顯著高于對照組和SB431542組(P0.05),Sca-1+細胞比例顯著高于對照組(P0.05),但與SB431542組比較均顯著降低(P0.05)；RA濃度為0.20nM組Sca-1+細胞比例顯著高于RA濃度為0.05、0.10、0.40 nM組(P0.05),RA濃度為0.20 nM組CD105+細胞比例顯著高于RA濃度為0.05、0.10 nM組(P0.05),但與RA濃度為0.40 nM組差異無統(tǒng)計學意義(P0.05)。結論：利用RA可以促進多能干細胞向MSC樣細胞分化,分化方法較為簡單,分化周期較短,為多能干細胞向MSC樣細胞的分化提供了一定的方法基礎。背景：結節(jié)硬化復合物1(Tscl)和結節(jié)硬化復合物2(Tsc2)結合形成一個復合物,這個復合物可以通過哺乳動物雷帕霉素靶蛋白1(mTORC1)來調節(jié)細胞代謝和能量的穩(wěn)定,TSC1和TSC2功能的丟失可引起mTORC1活性的升高,進而引起細胞體積增大、增殖能力增強。有研究稱Tscl調節(jié)巨噬細胞M1/M2的極化,但對于Tscl對巨噬細胞的功能和自穩(wěn)的精確調控作用尚不明確,本研究我們揭示Tscl對于調節(jié)巨噬細胞的生存、生長、遷移和吞噬功能具有十分重要的作用。材料與方法：我們將由Lysozyme啟動子控制表達Cre重組酶的LysMCre鼠與Tsc1flox/flox鼠交配獲得髓系細胞特異性的Tscl條件性敲除鼠(LysMCreTsc1flox/flox);PCR鑒定鼠的基因型,western blot鑒定鼠巨噬細胞Tscl蛋白的敲除情況；巨噬細胞的凋亡和生長由流式細胞術、RT-PCR進行檢測；巨噬細胞精氨酸酶活化由QuantiChromTM arginase assay kit進行檢測；M1和M2相關的基因的mRNA的表達由RT-PCT進行檢測；巨噬細胞對T細胞活化的功能通過CD4+T細胞和巨噬細胞體外共培養(yǎng)3天后CD4+T細胞的活化進行鑒定；巨噬細胞的吞噬功能由VybrantTM phagocytosis assay試劑盒進行檢測；巨噬細胞的遷移由Transwell實驗進行檢測；脾臟細胞和淋巴結細胞Foxp3的染色是通過Treg檢測試劑盒進行。結果：Tsc1 KO鼠巨噬細胞凋亡增加、細胞變大,腹腔注射Rapa可部分抑制Tscl缺陷引起的巨噬細胞的凋亡和細胞生長；在穩(wěn)定和炎癥狀態(tài)下,Tscl缺陷的巨噬細胞在穩(wěn)定和誘導作用下M1和M2特性均較WT巨噬細胞高,抑制mTORC1部分逆轉了Tscl缺陷引起的巨噬細胞的M2和M1的變化；Tscl缺陷的巨噬細胞CCR2和CCR5趨化受體表達降低、遷移能力下降、趨化因子升高,抑制mTORC1部分逆轉了Tscl缺陷引起的巨噬細胞的趨化因子和趨化受體的變化；另外,Tscl缺陷的巨噬細胞的吞噬能力和產(chǎn)生活性氧(ROS)的能力增加；與Tsc1缺陷的巨噬細胞共培養(yǎng)的CD4+T細胞的增殖和活化功能減弱。結論：1)Tscl促進巨噬細胞的生存、抑制巨噬細胞的凋亡,并且這兩種作用是與]mTORC1的活性有關。CD71和CD98可能參與了Tsc1調節(jié)的巨噬細胞的生長；2)Tsc1促進巨噬細胞M1和M2的特性,Tsc1缺陷的巨噬細胞在穩(wěn)定狀態(tài)下,分別增高了M1和M2的特性。在LPS和IL-4刺激下,Tsc1缺陷的巨噬細胞分布增高了M1和M2的極化特性；3)Tsc1促進巨噬細胞的遷移,Tscl缺陷的巨噬細胞的CCR2和CCR5的趨化受體表達降低,CCL2趨化引起的遷移能力降低。另外,Tsc1缺陷的巨噬細胞趨化因子表達升高。Tscl抑制巨噬細胞的吞噬和ROS的產(chǎn)生,促進CD4+T細胞的活化；4) Tscl KO鼠引起自發(fā)的淋巴組織增生性的疾病。脾臟和淋巴結增大,CD4+T細胞和CD8+T細胞活化嚴重。另外,鼠的體重沒有明顯的變化。
[Abstract]:Objective : To investigate the effect of pluripotent stem cells ( ES ) on embryonic stem cells ( ES ) and differentiation of pluripotent stem cells ( ES ) into mesenchymal stem cells ( MSC ) - like cells .
( 2 ) After 4 days of ES differentiation , the proportion of Sca - 1 + cells was significantly higher than that in the control group ( P0.05 ) . In the group with RA concentration of 0.05 , 0.10 , 0.20 and 0.40 nM , the percentage of CD105 + cells differentiated in ES was significantly higher than that in the control group ( P0.05 ) , but the proportion of Sca - 1 + cells was significantly higher than that of the control group ( P0.05 ) , but the ratio of Sca - 1 + cells was significantly lower than that of the control group ( P0.05 ) ;
The percentage of CD105 + and Sca - 1 + cells at RA concentration of 0.20 nM was significantly higher than that in RA concentration of 0.05 , 0.10 , 0.40 nM ( P0.05 ) ;
3 ) The proportion of CD105 + cells and Sca - 1 + cells in SB4315group was significantly higher than that in control group ( P0.05 ) after 4 days of differentiation . The percentage of CD105 + cells in the differentiation of the cells was significantly higher than that in the control group ( P0.05 ) , but the proportion of Sca - 1 + cells was significantly higher than that of the control group ( P0.05 ) .
Conclusion : Tscl can be used to regulate the cell metabolism and energy stabilization . The results suggest that Tscl regulates the cell metabolism and energy stabilization by the mammalian rapamycin target protein 1 ( mTORC1 ) .
Apoptosis and growth of macrophages were detected by flow cytometry and RT - PCR .
The activation of macrophages was detected by the Quantizer assay kit .
The expression of mRNA of genes related to M1 and M2 was detected by RT - PCT ;
The activation of CD4 + T cells was confirmed by co - culture of CD4 + T cells and macrophages in vitro .
The phagocytic function of macrophages was detected by the vybrantTM assay kit ;
The migration of macrophages was detected by Transwell experiment ;
Results : The apoptosis and cell growth of macrophages induced by Tscl deficiency were partially inhibited by intraperitoneal injection of Rapa .
In stable and inflammatory conditions , the macrophages of Tscl - deficient macrophages were higher in both M1 and M2 than those of WT macrophages , and the inhibition of mTORC1 partially reversed the changes of M2 and M1 in macrophages caused by Tscl deficiency ;
Tscl - deficient macrophages CCR2 and CCR2 chemokine receptor expression decreased , the migration ability decreased , the chemokine increased , and the inhibition of mTORC1 partially reversed the changes of chemokine and chemokine receptors in macrophages caused by Tscl deficiency ;
In addition , the phagocytic ability of Tscl - deficient macrophages and the ability to produce reactive oxygen ( ROS ) increased ;
Conclusion : 1 ) Tscl promotes the survival of macrophages and inhibits the apoptosis of macrophages .
2 ) Tsc1 promoted the characteristics of macrophages M1 and M2 , and Tsc1 - deficient macrophages increased M1 and M2 respectively in steady state . Under the stimulation of LPS and IL - 4 , the distribution of Tsc1 - deficient macrophages increased the polarization characteristics of M1 and M2 .
3 ) Tscl promotes the migration of macrophages , the expression of CCR2 and CCR2 of Tscl - deficient macrophages decreases , and the migration ability of CCL2 tends to decrease . In addition , the expression of Tscl - deficient macrophages chemokine expression increased . Tscl inhibited the phagocytosis of macrophages and the production of ROS , and promoted the activation of CD4 + T cells .
4 ) Tscl KO mice induced spontaneous lymphoproliferative diseases . The spleen and lymph nodes increased , CD4 + T cells and CD8 + T cells were activated severely .
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R392

【共引文獻】

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1 劉曄;王育t，

本文編號：2091689